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Patent 2584934 Summary

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(12) Patent Application: (11) CA 2584934
(54) English Title: NITROGEN-REGULATED SUGAR SENSING GENE AND PROTEIN AND MODULATION THEREOF
(54) French Title: GENE ET PROTEINE DE DETECTION DE SUCRE REGULES PAR L'AZOTE ET MODULATION ASSOCIEE
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 5/10 (2006.01)
  • A01H 1/00 (2006.01)
  • A01H 3/00 (2006.01)
  • C07K 16/16 (2006.01)
  • C12N 5/04 (2006.01)
  • C12N 15/29 (2006.01)
  • C12N 15/82 (2006.01)
  • G01N 33/53 (2006.01)
  • A01H 5/00 (2006.01)
  • C12Q 1/68 (2006.01)
(72) Inventors :
  • ROTHSTEIN, STEVEN (Canada)
  • BI, YONG-MEI (Canada)
(73) Owners :
  • UNIVERSITY OF GUELPH (Canada)
(71) Applicants :
  • UNIVERSITY OF GUELPH (Canada)
(74) Agent: SMART & BIGGAR
(74) Associate agent:
(45) Issued:
(22) Filed Date: 2007-04-17
(41) Open to Public Inspection: 2008-10-17
Examination requested: 2012-04-17
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data: None

Abstracts

English Abstract




The present invention relates to a nitrogen-regulated GATA
transcription factor gene required for sugar sensing and the modulation of the

expression of this gene to modulate a characteristic in a plant. The GATA
transcription factor of the present invention is involved in regulating sugar
sensing in plants and its expression is influenced by nitrogen status.
Increased expression of this or substantially similar genes can produce plants

with improved nitrogen utilization and increased yield.


Claims

Note: Claims are shown in the official language in which they were submitted.




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What is claimed is:


1. A method of modulating a characteristic in a plant cell comprising
modulating expression of a GATA transcription factor gene in the plant cell.


2. The method according to claim 1, wherein the expression of the GATA
transcription factor gene is modulated by administering, to the cell, an
effective amount of an agent that can modulate the expression levels of a
GATA transcription factor gene in the plant cell.


3. The method according to claim 1 or 2, wherein the characteristic is an
agronomic trait.


4. The method according to claim 3, wherein the characteristic is one that
is affected by nitrogen, carbon and/or sulfur metabolism, biosynthesis of
lipids, perception of nutrients, nutritional adaptation, electron transport
and/or
membrane associated energy conservation.


5. The method according to claim 3, wherein the characteristic is selected
from one or more of nitrogen utilization, yield, cell growth, reproduction,
chlorophyll synthesis, photosynthesis, nitrogen assimilation, disease
resistance, differentiation, signal transduction, gene regulation, abiotic
stress
tolerance and nutritional composition.


6. The method according to claim 5, wherein the characteristic is nitrogen
utilization.


7. The method according to any one of claims 1-6, wherein the plant cell
is a dicot, a gymnosperm or a monocot.


8. The method according to claim 7, wherein the monocot is selected
from the group consisting of maize, wheat, barley, oats, rye, millet, sorghum,




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triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass,
Tripsacum sp. and teosite.


9. The method according to claim 8, wherein the dicot is selected from the
group consisting of soybean, tobacco or cotton.


10. The method according to any one of claims 2-9, wherein the agent
enhances the expression levels of a GATA transcription factor gene in the
plant cell.


11. The method according to claim 10, wherein the modulated
characteristic is an increase or improvement in one or more of nitrogen
utilization, yield, cell growth, reproduction, photosynthesis, nitrogen
assimilation, disease resistance, differentiation, signal transduction, gene
regulation, abiotic stress tolerance and nutritional composition.


12. The method according to claim 10 or 11, wherein the agent that
enhances the expression levels of a GATA transcription factor gene in the
plant cell comprises a nucleic acid molecule encoding a GATA transcription
factor.


13. The method according to claim 12, wherein the nucleic acid molecule
comprises the sequence of the OsGATA11 gene of SEQ ID NO:1 or a
functional fragment thereof.


14. The method according to claim 12, wherein the nucleic acid molecule
comprises a sequence that hybridizes under medium stringency conditions to
the OsGATA11 gene of SEQ ID NO:1 or a functional fragment thereof.


15. The method according to claim 12, wherein the nucleic acid molecule
comprises a nucleic acid sequence derived from the nucleotide sequence of





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the OsGATA11 gene of SEQ ID NO:1 and has a nucleotide sequence
comprising codons specific for expression in plants.


16. The method according to any one of claims 1-12, wherein the agent
that can modulate the expression levels of a GATA transcription factor gene in

a plant cell comprises:
(a) a nucleotide sequence of SEQ ID NO:1 or a fragment or domain
thereof;
(b) a nucleotide sequence encoding a polypeptide of SEQ ID NO:2,
a fragment or domain thereof;
(c) a nucleotide sequence having substantial similarity to (a) or (b);
(d) a nucleotide sequence capable of hybridizing to (a), (b) or (c);
(e) a nucleotide sequence complementary to (a), (b), (c) or (d); or
(f) a nucleotide sequence that is the reverse complement of (a),
(b), (c) or (d).


17. The method according to any one of claims 1-12, wherein the agent
that can modulate the expression levels of a GATA transcription factor gene in

a plant cell comprises:
(a) a polypeptide sequence listed in SEQ ID NO:2, or a functional
fragment, domain, repeat, or chimera thereof;
(b) a polypeptide sequence having substantial similarity to (a);
(c) a polypeptide sequence encoded by a nucleotide sequence
identical to or having substantial similarity to a nucleotide sequence listed
in
SEQ ID NO:1, or a functional fragment or domain thereof, or a sequence
complementary thereto; or
(d) a polypeptide sequence encoded by a nucleotide sequence
capable of hybridizing under medium stringency conditions to a nucleotide
sequence listed in SEQ ID NO:1, or to a sequence complementary thereto.

18. The method according to any one of claims 12-17, wherein the nucleic
acid sequence is expressed in a specific location or tissue of the plant.




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19. The method according to claim 18, wherein the location or tissue is
selected from one or more of seed, epidermis, root, vascular tissue, meristem,

cambium, cortex, pith, leaf and flower.


20. The method according to claim 19, wherein the location or tissue is a
seed.


21. The method according to any one of claims 12-20, wherein the agent
that enhances the expression levels of a GATA transcription factor gene in the

plant cell comprises an expression cassette for modulating a characteristic in

a plant cell including a promoter sequence operably linked to the isolated
nucleic acid encoding a GATA transcription factor.


22. A method of producing a transgenic plant comprising:
(1) providing an isolated nucleic acid having the sequence shown in
SEQ ID NO:1; and
(2) introducing the nucleic acid into the plant, wherein the nucleic acid
is expressed in the plant.


23. The method according to claim 22 wherein the plant demonstrates an
increase or improvement in one or more of nitrogen utilization, yield, cell
growth, reproduction, photosynthesis, nitrogen assimilation, disease
resistance, differentiation, signal transduction, gene regulation, abiotic
stress
tolerance and nutritional composition.


24. The method according to claim 23 wherein the plant has an increase in
chlorophyll synthesis, seed yield and/or stress tolerance.


25. The method of any one of claims 22 or 24 wherein the nucleic acid is
introduced into the plant using a method selected from the group consisting of




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microparticle bombardment, Agrobacterium-mediated transformation, and
whiskers-mediated transformation.


26. A plant cell of the plant produced by any one of claims 22-25.


27. A use of an nucleic acid molecule comprising a nucleotide sequence
of at least 10 bases, which sequence is identical, complementary, or
substantially similar to a region of any of SEQ ID NO:1 or a functional
fragment thereof and wherein the use is selected from the group consisting of:
(i) use as a chromosomal marker to identify the location of the
corresponding or complementary polynucleotide on a native or artificial
chromosome;
(ii) use as a marker for RFLP analysis;
(iii) use as a marker for quantitative trait linked breeding;
(iv) use as a marker for marker-assisted breeding;
(v) use as a bait sequence in a two-hybrid system to identify sequence
encoding polypeptides interacting with the polypeptide encoded by the bait
sequence;
(vi) use as a diagnostic indicator for genotyping or identifying an
individual or population of individuals; and
(vii) use for genetic analysis to identify boundaries of genes or exons.

28. An antibody raised against an isolated polypeptide comprising:
(a) a polypeptide sequence of SEQ ID NO:2, or a fragment, domain,
repeat or chimera thereof;
(b) a polypeptide sequence having substantial similarity to (a);
(c) a polypeptide sequence encoded by a nucleotide sequence
identical to or having substantial similarity to a nucleotide sequence listed
in
SEQ ID NO:2, or a fragment or domain thereof, or a sequence
complementary thereto;




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(d) a polypeptide sequence encoded by a nucleotide sequence
capable of hybridizing under medium stringency conditions to a nucleotide
sequence listed in SEQ ID NO:1, or to a sequence complementary thereto; or
(e) a functional fragment of (a), (b), (c) or (d).


29. The antibody according to claim 28 wherein the polypeptide comprises
the sequence of SEQ ID NO:2 or a variant thereof having a conservative
amino acid modification.


30. An immunoassay kit comprising the antibody of claim 28 or 29 and
instructions for the use thereof.


Description

Note: Descriptions are shown in the official language in which they were submitted.



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B&P File No: 6580-346
Title: NITROGEN-REGULATED SUGAR SENSING GENE AND PROTEIN
AND MODULATION THEREOF

FIELD OF THE INVENTION

The present invention relates to methods of modulating agronomic
traits in plants by modulating the expression of a GATA transcription factor
in
the plant cells. In particular the present invention relates to methods of
improving nitrogen utilization in plants. The present invention also pertains
to
nucleic acid molecules isolated from Oryza sativa comprising nucleotide
sequences that encode proteins that are important in chlorophyll synthesis
and sugar sensing and, ultimately, can modulate nitrogen uptake and overall
carbon metabolism.
BACKGROUND OF THE INVENTION

Improvement of the agronomic characteristics of crop plants has been
ongoing since the beginning of agriculture. Most of the land suitable for crop
production is currently being used. As human populations continue to
increase, improved crop varieties will be required to adequately provide our
food and feed (Trewavas (2001) Plant Physiol. 125: 174-179). To avoid
catastrophic famines and malnutrition, future crop cultivars will need to have
improved yields with equivalent farm inputs. These cultivars will need to more
effectively withstand adverse conditions such as drought, soil salinity or
disease, which will be especially important as marginal lands are brought into
cultivation. Finally, we will need cultivars with altered nutrient composition
to
enhance human and animal nutrition, and to enable more efficient food and
feed processing. For all these traits, identification of the genes controlling
phenotypic expression of traits of interest will be crucial in accelerating
development of superior crop germplasm by conventional or transgenic
means.
A number of highly-efficient approaches are available to assist
identification of genes playing key roles in expression of agronomically-
important traits. These include genetics, genomics, bioinformatics, and


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functional genomics. Genetics is the scientific study of the mechanisms of
inheritance. By identifying mutations that alter the pathway or response of
interest, classical (or forward) genetics can help to identify the genes
involved
in these pathways or responses. For example, a mutant with enhanced
susceptibility to disease may identify an important component of the plant
signal transduction pathway leading from pathogen recognition to disease
resistance. Genetics is also the central component in improvement of
germplasm by breeding. Through molecular and phenotypic analysis of
genetic crosses, loci controlling traits of interest can be mapped and
followed
in subsequent generations. Knowledge of the genes underlying phenotypic
variation between crop accessions can enable development of markers that
greatly increase efficiency of the germplasm improvement process, as well as
open avenues for discovery of additional superior alieles.
Genomics is the system-level study of an organism's genome,
including genes and corresponding gene products - RNA and proteins. At a
first level, genomic approaches have provided large datasets of sequence
information from diverse plant species, including full-length and partial cDNA
sequences, and the complete genomic sequence of a model plant species,
Arabidopsis thaliana. Recently, the first draft sequence of a crop plant's
genome, that of rice (Oryza sativa), has also become available. Availability
of
a whole genome sequence makes possible the development of tools for
system-level study of other molecular complements, such as arrays and chips
for use in determining the complement of expressed genes in an organism
under specific conditions. Such data can be used as a first indication of the
potential for certain genes to play key roles in expression of different plant
phenotypes.
Bioinformatics approaches interface directly with first-level genomic
datasets in allowing for processing to uncover sequences of interest by
annotative or other means. Using, for example, similarity searches,
alignments and phylogenetic analyses, bioinformatics can often identify
homologs of a gene product of interest. Very similar homologs (eg. >--90%


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amino acid identity over the entire length of the protein) are very likely
orthologs, i.e. share the same function in different organisms.
Functional genomics can be defined as the assignment of function to
genes and their products. Functional genomics draws from genetics,
genomics and bioinformatics to derive a path toward identifying genes
important in a particular pathway or response of interest. Expression
analysis, for example, uses high density DNA microarrays (often derived from
genomic-scale organismal sequencing) to monitor the mRNA expression of
thousands of genes in a single experiment. Experimental treatments can
include those eliciting a response of interest, such as the disease resistance
response in plants infected with a pathogen. To give additional examples of
the use of microarrays, mRNA expression levels can be monitored in distinct
tissues over a developmental time course, or in mutants affected in a
response of interest. Proteomics can also help to assign function, by
assaying the expression and post-translational modifications of hundreds of
proteins in a single experiment.
Proteomics approaches are in many cases analogous to the
approaches taken for monitoring mRNA expression in microarray
experiments. Protein-protein interactions can also help to assign proteins to
a
given pathway or response, by identifying proteins that interact with known
components of the pathway or response. For functional genomics, protein-
protein interactions are often studied using large-scale yeast two-hybrid
assays. Another approach to assigning gene function is to express the
corresponding protein in a heterologous host, for example the bacterium
Escherichia coli, followed by purification and enzymatic assays.
Demonstration of the ability of a gene-of-interest to control a given trait
may be derived, for example, from experimental testing in plant species of
interest. The generation and analysis of plants transgenic for a gene of
interest can be used for plant functional genomics, with several advantages.
The gene can often be both overexpressed and underexpressed ("knocked
out"), thereby increasing the chances of observing a phenotype linking the
gene to a pathway or response of interest. Two aspects of transgenic


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functional genomics help lend a high level of confidence to functional
assignment by this approach. First, phenotypic observations are carried out
in the context of the living plant. Second, the range of phenotypes observed
can be checked and correlated with observed expression levels of the
introduced transgene. Transgenic functional genomics is especially valuable
in improved cultivar development. Only genes that function in a pathway or
response of interest, and that in addition are able to confer a desired trait-
based phenotype, are promoted as candidate genes for crop improvement
efforts. In some cases, transgenic lines developed for functional genomics
studies can be directly utilized in initial stages of product development.
Another approach towards plant functional genomics involves first
identifying plant lines with mutations in specific genes of interest, followed
by
phenotypic evaluation of the consequences of such gene knockouts on the
trait under study. Such an approach reveals genes essential for expression of
specific traits.
Genes identified through functional genomics can be directly employed
in efforts towards germplasm improvement by transgenic means, as
described above, or used to develop markers for identification of tracking of
alieles-of-interest in mapping and breeding populations. Knowledge of such
genes may also enable construction of superior alleles non-existent in nature,
by any of a number of molecular methods.
Rapid increases in yield over the last 80 years in row crops have been
due in roughly equal measure to improved genetics and improved agronomic
practices. In particular, in a crop like maize, the combination of high
yielding
hybrids and the use of large amounts of nitrogen fertilizer have under ideal
conditions allowed for yields of greater than 440bu/acre. However, the use of
large amounts of nitrogen fertilizer has negative side-effects primarily
around
increasing cost of this input to the farmer and cost to the environment since
nitrate pollution is a major problem in many agricultural areas contributing
significantly to the degradation of both fresh water and marine environments.
Developing crop genetics that use nitrogen more efficiently through an
understanding of the role of genotype on nitrogen use would be highly


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advantageous in reducing producer input costs as well as environmental load.
This is particularly important for a crop like corn which is grown using a
high
level of nitrogen fertilizer.
Nitrogen use efficiency can be defined in several ways, although the
simplest is yield/N supplied. There are two stages in this process: first, the
amount of available nitrogen that is taken up, stored and assimilated into
amino acids and other important nitrogenous compounds; second, the
proportion of nitrogen that is partitioned to the seed, resulting in final
yield. A
variety of field studies have been performed on various agriculturally
important crops to study this problem (Lawlor DW et al 2001 in Lea PJ, Morot-
Gaudry JF, eds. Plant Nitrogen. Berlin: Springer-Verlag 343-367; Lafitte HR
and Edmeades GO 1994 Field Crops Res 39, 15-25; Lawlor DW 2002 J Exp
Bot. 53, 773-87; Moll RH et al 1982 Agron J 74, 562-564). These
experiments have demonstrated that there is a genetic component to nitrogen
use efficiency, but have not proved satisfactory in determining which genes
are important for this process. In addition, corn breeders have generally not
targeted the maintenance of yield under limiting nitrogen fertilizer. These
types of field experiments on nitrogen use are difficult for a variety of
reasons
including a lack of uniformity of accessible nitrogen in a test field or
between
field sites under any treatment regime and the interplay of other
environmental factors that make experiments difficult to interpret.
Therefore, although there is experimental evidence for genetic variation
for this trait, it is difficult to make any conclusions from these experiments
on
what causes this variation. It should be feasible and is certainly important
to
develop methods to study this trait under field conditions in crop plants.
However, significant progress toward identifying, understanding and
manipulating important traits can be made through the use of a model system
like Arabidopsis. At the very least, these experiments will give important
clues
about potential target genes to evaluate in important field crops. In
addition,
there are also considerable genetic and genomic resources available to study
rice and this species will also be used for some of the proposed experiments
as a species more similar to corn than is Arabidopsis.


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Nitrate is the major form of available nitrogen in the field and there is
an extensive body of literature on genes involved in nitrate uptake and
reduction (Forde BG 2000 Biochimica et Biophysica Acta 1465, 219-235;
Howitt SM and Udvardi MK 2000 Biochimica et Biophysica Acta 1465, 152-
170; Stitt M et al 2002 J Exp Bot. 53, 959-70) as well as on genes involved in
other aspects of nitrogen metabolism (Lea PJ, Morot-Gaudry JF, eds. 2001
Plant Nitrogen. Berlin: Springer-Verlag; Morot-Gaudry JF 2001 Nitrogen
assimilation by plants Science Publishers Inc. NH, US). Also, it is clear that
the availability of carbon metabolites is crucial for the efficient use of
field
nitrate and there is good experimental evidence for a linkage between carbon
and nitrogen metabolism (Coruzzi GM and Zhou L 2001 Curr Opin Plant Biol.
4, 247-53). In addition, some experiments suggest that GS and GOGAT are
involved in remobilizing N from senescing organs to the sink organ
(Brouquisse R et al 2001 in Lea PJ, Morot-Gaudry JF, eds. Plant Nitrogen.
Berlin: Springer-Verlag 275-293; Yamaya T et al 2002 J Exp Bot. 53, 917-
925). However, most aspects of the regulation of these genes are still unclear
and there is still no notion of how this regulation affects nitrogen use
efficiency.
Plants can sense levels of carbon and nitrogen metabolites and
accordingly adjust growth and development. The perception mechanisms are
complex regulatory networks that control gene expression to accommodate
constant changes of nutrient-dependent cellular activities. Possession of a
sugar-sensing mechanism enables plants to turn off photosynthesis when C-
skeletons are abundant. The N-sensing mechanism enables plants to turn off
nitrate uptake and reduction when levels of reduced or organic N are high
(Coruzzi, G.M. & Zhou, L. (2001) Curr Opin Plant Biol. 4, 247-53).
Multiple sugar signal transduction pathways exist in plants. Glucose
has emerged as a key regulator of many vital processes in photosynthetic
plants such as in photosynthesis and in carbon and nitrogen metabolism
(Rolland, F., Moore, B. & Sheen, J. (2002) Plant Cell S185-S205).
Hexokinases (HXK) are an important control point for glucose metabolism.
They not only catalyze the phosphorylation of glucose but also function as a


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glucose sensor to interrelate nutrient, light and hormone signaling networks
for controlling growth and development in response to the changing
environment (Jang, J., Leon, P, Zhou, L. & Sheen, J. (1997) Plant Cell 9, 5-
19; Dai, N., Schaffer, A., Petreikov, M., Shahak, Y., Giller, Y., Ratner, K.,
Levine, A. & Granot, D. (1999) Plant Cell 11, 1253-1266; Moore, B., Zhou, L.,
Rolland, F., Hall, Q., Cheng, W., Liu, Y., Hwang, I., Jones, T. & Sheen, J.
(2003) Science 300, 332-336). In other organisms it has been shown that
hexose transport molecules also serve as sugar sensors.
Multiple N signals and sensing pathways exist as well in plants. Plants
have mechanisms to sense nitrate, the major form of nitrogen fertilizer, as a
signal for inorganic N status as well as to sense metabolites derived from
nitrate as signals for reduced or organic N status. Nitrate reductase (NR) and
nitrite reductase (NiR) are the first two enzymes in the nitrate reduction
process and their expression can be stimulated by the presence of nitrate and
modulated by other physiological factors including some nitrogenous
compounds, sucrose, light and hormone (Forde, B.G. (2000) Biochimica et
Biophysica Acta 1465, 219-235; Howitt, S.M. & Udvardi, M.K. (2000)
Biochimica et Biophysica Acta 1465, 152-170; Stitt, M., Muller, M., Matt, M.,
Gibon, Y., Carillo, P., Morcuende, R., Scheible, W. & Krapp, A. (2002) J Exp
Bot. 53, 959-970; Lea, P.J. & Morot-Gaudry, J.F. eds. 2001 Plant Nitrogen.
Berlin: Springer-Veriag; Morot-Gaudry JF 2001 Nitrogen assimilation by plants
Science Publishers Inc. NH, US).
It is clear that carbon and nitrogen metabolism is closely linked and
tightly regulated (Coruzzi, G. & Bush, D.R. (2001) Plant Physiol 125, 61-64).
The availability of carbon metabolites is crucial for efficient nitrate
utilization
and the nitrogen status is very sensitive to photosynthesis. Despite increased
knowledge of structural genes involved in carbon and nitrogen metabolism,
trans-acting factors involved in transcriptional regulation of C/N gene
expression have not been characterized.
GATA transcription factors are a group of transcriptional regulators
broadly distributed in eukaryotes. The GATA DNA binding domain normally
recognizes the consensus sequence WGATAR (W = T or A; R = G or A)


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(Lowry, J. & Atchley, W. (2000) J Mol Evol 50, 103-115). GATA motifs have
been identified in the regulatory regions of many light responsive genes
(Arguello-Astorga, G. & Herrera-Estrella, L. (1998) Annu Rev Plant Physiol
Plant Mol Biol 49, 525-555), including many genes involved in or relating to
photosynthesis such as the RBCS, CAB (chlorophyll A/B binding protein) and
GAP (glyceraldehyde-3-phosphate dehydrogenase) (Terzaghi, W.B. &
Cashmore, A.R. (1995) Annu Rev Plant Physiol Plant Mol Biol 46, 445-474;
Koch, K.E. (1996) Carbohydrate-modulated gene expression in plants. Annu
Rev Plant Physiol Plant Mol Biol 47, 509-540; Jeong, M.J. & Shih, M.C.
(2003) Biochem Biophys Res Commun 300, 555-562) as well as genes
involved in nitrate assimilation such as nitrate reductase, nitrite reductase,
and Gln synthetase (Jarai, G., Truong, H., Daniel-Vedele, F. & Marzluf, G.
(1992) Curr Genet 21, 37-41; Rastogi, R., Bate, N., Sivasankar, S &
Rothstein, S. (1997) Plant Mol Biol. 34, 465-76; Oliveira, I.C. & Coruzzi,
G.M.
(1999) Plant Physiol 121, 301-309). Some known trans-acting regulatory
proteins that globally regulate genes in N metabolism are GATA transcription
factor genes. In yeast, four global nitrogen regulatory factors GLN3, NIL1,
NIL2 and DAL80 are DNA-binding proteins that contain a single GATA zinc
finger, recognizing the consensus motif GATA (Hofman-Bang, J. (1999) Mol
Biotech 12, 35-73). In fungi, Neurospora crassa NIT2 (Tao Y and Marzluf GA
1999 Curr Genet 36, 153-158) and Aspergillus nidulans AREA (Caddick MX
Arst HN Jr Taylor LH Johnson RI Brownlee AG 1986 Cloning of the regulatory
gene areA mediating nitrogen metabolite repression in Aspergillus nidulans.
EMBO J 5, 1087-1090) are GATA transcription factor genes.
In plants, the in vivo function of GATA factors remains very poorly
defined, with the Arabidopsis genome having 30 GATA members
(Riechmann, J.L., Heard, J., Martin, G., Reuber, L., Jiang, C., Keddie, J.,
Adam, L., Pineda, 0., Ratcliffe, O.J., Samaha, R.R., Creelman, R., Pilgrim,
M., Broun, P., Zhang, J.Z., Ghandehari, D., Sherman, B.K. & Yu, G. (2000)
Science 290, 2105-2110; Reyes, J.C., Muro-Pastor, M.I. & Florencio, F.J.
(2004) Plant Physiol. 134, 1718-1732). Applicant identified the Arabidopsis
GATA transcription factor gene GNC (At5g56860) important in chlorophyll


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synthesis and sugar sensitivity previously (WO 2006/074547). In the rice
(Oryza sativa) genome, there are 28 GATA transcription factor genes, with
one gene OsGATA16 sharing similarity with the Arabidopsis GATA gene
At4g26150 (Reyes, J.C., Muro-Pastor, M.I. & Florencio, F.J. (2004) Plant
Physiol. 134, 1718-1732 and WO 2006/074547).
SUMMARY OF THE INVENTION
The inventors have isolated a new GATA transcription factor from rice,
termed OsGATAl 1, which is an ortholog of the At4g26150 gene from
Arabidopsis. The At4g26150 gene is a GNC paralog in the phylogenetic tree
of the 30 Arabidopsis GATA transcription factor genes (Reyes, J.C., Muro-
Pastor, M.I. & Florencio, F.J. (2004) Plant Physiol. 134, 1718-1732) and was
found to have overlapping function with GNC. The inventors have determined
that the expression of the OsGATAl 1 gene regulates chlorophyll synthesis,
seed yield and stress response to low nitrogen levels. Loss-of-function
mutant plants in the OsGATAl 1 gene resulted in reduced chlorophyll levels.
In particular, transgenic rice plants silencing the OsGATAl 1 gene via
RNAi, as well as transgenic plants over-expressing the rice gene, were
created. The plants transformed with the OsGATAl 1 gene had increased
chlorophyll levels and increased seed yield and had an improved stress
response to low nitrogen levels. Plants grown under high N experienced
stress after being transferred from the growth room to the greenhouse and the
transgenic plants over-expressing OsGATAl 1 responded much better to the
stress.
Sugars are central regulators of many vital processes in photosynthetic
plants, such as photosynthesis and carbon and nitrogen metabolism. This
regulation is achieved by regulating gene expression to either activate or
repress genes involved. The mechanisms by which sugars control gene
expression are not understood well. The GATA transcription factor disclosed
here is involved in regulating sugar sensing and the expression of the factor
itself is influenced by the change of the N status. Increased expression of
this
gene can produce plants with increased yield, particularly as the manipulation
of sugar signaling pathways can lead to increased photosynthesis and


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increased nitrogen assimilation and alter source-sink relationships in seeds,
tubes, roots and other storage organs.
Accordingly, the present invention relates to a method of modulating a
characteristic in a plant or plant cell comprising modulating expression of a
GATA transcription factor gene in the plant or plant cell. In an embodiment of
the invention, the expression of the GATA transcription factor gene is
modulated by administering, to the cell, an effective amount of an agent that
can modulate the expression levels of a GATA transcription factor gene in the
plant cell. In a further embodiment of the invention, the agent enhances the
expression levels of a GATA transcription factor gene in the plant cell.
The characteristic to be modulated in the plant may be any agronomic
trait of interest. In an embodiment of the invention, the characteristic is
any
that is affected by nitrogen, carbon and/or sulfur metabolism, biosynthesis of
lipids, perception of nutrients, nutritional adaptation, electron transport
and/or
membrane associated energy conservation. In a further embodiment of the
invention, the characteristic is selected from one or more of nitrogen
utilization, yield, cell growth, reproduction, photosynthesis, nitrogen
assimilation, disease resistance, differentiation, signal transduction, gene
regulation, abiotic stress tolerance and nutritional composition. In a still
further embodiment of the invention the modulated characteristic is an
increase or improvement in one or more of nitrogen utilization, yield, cell
growth, reproduction, photosynthesis, nitrogen assimilation, disease
resistance, differentiation, signal transduction, gene regulation abiotic
stress
tolerance and nutritional composition.
In a particular embodiment, the present invention relates to a method
of improving nitrogen utilization in a plant or plant cell comprising
enhancing
expression of a GATA transcription factor gene in the plant or plant cell.
Improving nitrogen utilization in a plant will allow for reduce amounts of
nitrogen fertilizer to applied to the plant with a concomitant reduction in
costs
to the farmer and cost to the environment since nitrate pollution is a major
problem in many agricultural areas contributing significantly to the
degradation
of both fresh water and marine environments.


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The plant or plant cell may be from any plant wherein one wishes to
modulate a characteristic. In an embodiment of the invention, the plant cell
is
a dicot, a gymnosperm or a monocot. In one embodiment, the dicot is
selected from the group consisting of soybean, tobacco or cotton. In a further
embodiment of the invention, the monocot is selected from maize, wheat,
barley, oats, rye, millet, sorghum, triticale, secale, einkorn, spelt, emmer,
teff,
milo, flax, gramma grass, Tripsacum sp. and teosite.
In an embodiment of the invention, the agent that enhances the
expression levels of a GATA transcription factor gene in the plant cell
comprises a nucleic acid molecule encoding a GATA transcription factor.
In an embodiment of the invention, the agent that can modulate the
expression levels of a GATA transcription factor gene in a plant cell
comprises:
(a) a nucleotide sequence of SEQ ID NO:1 or a fragment or
domain thereof;
(b) a nucleotide sequence encoding a polypeptide of SEQ ID
NO:2, a fragment or domain thereof;
(c) a nucleotide sequence having substantial similarity to (a)
or (b);
(d) a nucleotide sequence capable of hybridizing to (a), (b) or
(c) ;
(e) a nucleotide sequence complementary to (a), (b), (c) or
(d); or
(f) a nucleotide sequence that is the reverse complement of
(a), (b), (c) or (d).
In a further embodiment of the invention, the nucleic acid molecule
comprises the sequence of the OsGATAl1 gene of SEQ ID NO:1 or a
functional fragment thereof. In a still further embodiment of the invention,
the
nucleic acid molecule comprises a sequence that hybridizes under medium
stringency conditions to the OsGATAl 1 gene of SEQ ID NO:1 or a functional
fragment thereof. In another embodiment of the present invention, the nucleic
acid molecule is derived from the nucleotide sequence of the At5g56860 gene


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of SEQ ID NO:1 and has a nucleotide sequence comprising codons specific
for expression in plants.
In a further embodiment of the invention, the agent that can modulate
the expression levels of a GATA transcription factor gene in a plant cell
comprises:
(a) a polypeptide sequence listed in SEQ ID NO:2, or a
functional fragment, domain, repeat, or chimera thereof;
(b) a polypeptide sequence having substantial similarity to
(a);
(c) a polypeptide sequence encoded by a nucleotide
sequence identical to or having substantial similarity to a nucleotide
sequence listed in SEQ ID NO:1, or a functional fragment or domain
thereof, or a sequence complementary thereto; or
(d) a polypeptide sequence encoded by a nucleotide
sequence capable of hybridizing under medium stringency conditions
to a nucleotide sequence listed in SEQ ID NO:1, or to a sequence
complementary thereto.
In an embodiment of the present invention, when the agent is a nucleic
acid sequence, the nucleic acid sequence is expressed in a specific location
or tissue of the plant. The location or tissue is for example, but not limited
to,
epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf and/or
flower. In an alternative embodiment, the location or tissue is a seed.
Embodiments of the present invention also relate to use of a shuffled
nucleic acid molecule for modulating a characteristic in a plant cell, said
shuffled nucleic acid molecule containing a plurality of nucleotide sequence
fragments, wherein at least one of the fragments encodes a GATA
transcription factor and wherein at least two of the plurality of sequence
fragments are in an order, from 5' to 3' which is not an order in which the
plurality of fragments naturally occur in a nucleic acid. In a specific
embodiment, all of the fragments in a shuffled nucleic acid molecule
containing a plurality of nucleotide sequence fragments are from a single
gene. In a more specific embodiment, the plurality of fragments originate from


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at least two different genes. In a more specific embodiment, the shuffled
nucleic acid is operably linked to a promoter sequence. Another more specific
embodiment is a use of a chimeric polynucleotide for modulating a
characteristic in a plant cell, said chimeric polynucleotide including a
promoter
sequence operably linked to the shuffled nucleic acid. In a more specific
embodiment, the shuffled nucleic acid is contained within a host cell. In a
further specific embodiment of the invention the fragment encoding a GATA
transcription factor consists of or comprises:
(a) a nucleotide sequence of SEQ ID NO:1 or a fragment or
domain thereof;
(b) a nucleotide sequence encoding a polypeptide of SEQ ID
NO:2, a fragment or domain thereof;
(c) a nucleotide sequence having substantial similarity to (a)
or (b);
(d) a nucleotide sequence capable of hybridizing to (a), (b) or
(c) ;
(e) a nucleotide sequence complementary to (a), (b), (c) or
(d); or
(f) a nucleotide sequence that is the reverse complement of
(a), (b), (c) or (d).
Embodiments of the present invention also contemplate a use of an
expression cassette for modulating a characteristic in a plant cell including
a
promoter sequence operably linked to an isolated nucleic acid encoding a
GATA transcription factor. In embodiments of the invention the isolated
nucleic acid encoding a GATA transcription factor consists of or comprises:
(a) a nucleotide sequence of SEQ ID NO:1 or a fragment or
domain thereof;
(b) a nucleotide sequence encoding a polypeptide of SEQ ID
NO:2, a fragment or domain thereof;
(c) a nucleotide sequence having substantial similarity to (a)
or (b);


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(d) a nucleotide sequence capable of hybridizing to (a), (b) or
(c) ;
(e) a nucleotide sequence complementary to (a), (b), (c) or (d); or
(f) a nucleotide sequence that is the reverse complement of (a),
(b), (c) or (d).
Further encompassed within the invention is use of a recombinant
vector for modulating a characteristic in a plant cell comprising an
expression
cassette including a promoter sequence operably linked to an isolated nucleic
acid encoding a GATA transcription factor. In embodiments of the invention
the isolated nucleic acid encoding a GATA transcription factor consists of or
comprises:
(a) a nucleotide sequence of SEQ ID NO:1 or a fragment or
domain thereof;
(b) a nucleotide sequence encoding a polypeptide of SEQ ID
NO:2, a fragment or domain thereof;
(c) a nucleotide sequence having substantial similarity to (a)
or (b);
(d) a nucleotide sequence capable of hybridizing to (a), (b) or
(c) ; (e) a nucleotide sequence complementary to (a), (b), (c) or
(d); or
(f) a nucleotide sequence that is the reverse complement of
(a), (b), (c) or (d).
Also encompassed are uses of plant cells, which contain expression
cassettes, according to the present disclosure, and uses of plants, containing
these plant cells.
In one embodiment, the expression cassette is expressed throughout
the plant. In another embodiment, the expression cassette is expressed in a
specific location or tissue of a plant. In a specific embodiment, the location
or
tissue may be, for example, epidermis, root, vascular tissue, meristem,
cambium, cortex, pith, leaf, and flower. In an alternative specific
embodiment,
the location or tissue is a seed.


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Embodiments of the present invention also provide the use of seed and
isolated product from plants for modulating a characteristic in a plant cell,
which contain an expression cassette including a promoter sequence
operably linked to an isolated nucleic acid encoding a GATA transcription
factor gene according to the present invention.
In a specific embodiment, the expression vector includes one or more
elements such as, for example, but not limited to, a promoter-enhancer
sequence, a selection marker sequence, an origin of replication, an epitope-
tag encoding sequence, or an affinity purification-tag encoding sequence. In
a more specific embodiment, the promoter-enhancer sequence may be, for
example, the CaMV 35S promoter, the CaMV 19S promoter, the tobacco PR-
1a promoter, ubiquitin and the phaseolin promoter. In another embodiment,
the promoter is operable in plants, and more specifically, a constitutive or
inducible promoter. In another specific embodiment, the selection marker
sequence encodes an antibiotic resistance gene. In another specific
embodiment, the epitope-tag sequence encodes V5, the peptide Phe-His-His-
Thr-Thr, hemagglutinin, or glutathione-S-transferase. In another specific
embodiment the affinity purification-tag sequence encodes a polyamino acid
sequence or a polypeptide. In a more specific embodiment, the polyamino
acid sequence is polyhistidine. In a more specific embodiment, the
polypeptide is chitin binding domain or glutathione-S-transferase. In a more
specific embodiment, the affinity purification-tag sequence comprises an
intein
encoding sequence.
In a specific embodiment, the expression vector is a eukaryotic
expression vector or a prokaryotic expression vector. In a more specific
embodiment, the eukaryotic expression vector includes a tissue-specific
promoter. More specifically, the expression vector is operable in plants.
Embodiments of the present invention also relate to a plant modified by
a method that includes introducing into a plant a nucleic acid where the
nucleic acid is expressible in the plant in an amount effective to effect the
modification. The modification can be an increase or decrease in the one or
more traits of interest. The modification may include overexpression,


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underexpression, antisense modulation, sense suppression, inducible
expression, inducible repression, or inducible modulation of a gene. In an
embodiment of the invention the modification involved an increase or
improvement in the trait of interest, for example, nitrogen utilization.
Embodiments of the present invention provide nucleotide and amino
acid sequences isolated from Arabidopsis thaliana. Particularly, the present
invention relates to a nitrogen-regulated GATA transcription factor gene
required for sugar sensing.
Embodiments of the present invention relate to an isolated nucleic acid
comprising or consisting of a nucleotide sequence comprising:
(a) a nucleotide sequence listed in SEQ ID NO:1, or a
fragment or domain, thereof;
(b) a nucleotide sequence having substantial similarity to (a);
(c) a nucleotide sequence capable of hybridizing to (a);
(d) a nucleotide sequence complementary to (a), (b) or (c); or
(e) a nucleotide sequence which is the reverse complement
of (a), (b) or (c).
In a specific embodiment, the substantial similarity is at least about
65% identity, specifically about 80% identity, specifically 90%, and more
specifically at least about 95% sequence identity to the nucleotide sequence
listed as SEQ ID NO:1, a fragment or domain thereof.
In a one embodiment, the sequence having substantial similarity to the
nucleotide sequence of SEQ ID NO:1, a fragment or domain thereof, is from a
plant. In a specific embodiment, the plant is a dicot. In a more specific
embodiment, the dicot is selected from the group consisting of soybean,
tobacco or cotton. In another specific embodiment, the plant is a
gymnosperm. In another specific embodiment, the plant is a monocot. In a
more specific embodiment, the monocot is a cereal. In a more specific
embodiment, the cereal may be, for example, maize, wheat, barley, oats, rye,
millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax,
gramma grass, Tripsacum sp., or teosinte.


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In one embodiment the nucleic acid is expressed in a specific location
or tissue of a plant. The location or tissue is for example, but not limited
to,
epidermis, root, vascular tissue, meristem, cambium, cortex, pith, leaf, and
flower. In an alternative embodiment, the location or tissue is a seed. In
another embodiment, the nucleic acid encodes a polypeptide involved in a
function such as, for example, but not limited to, carbon, nitrogen and/or
sulfur
metabolism, nitrogen utilization, nitrogen assimilation, photosynthesis,
signal
transduction, cell growth, reproduction, disease resistance, abiotic stress
tolerance, nutritional composition, gene regulation, and/or differentiation.
In a specific embodiment, the isolated nucleic acid comprises or
consists of a nucleotide sequence capable of hybridizing to a nucleotide
sequence listed in SEQ ID NO:1 or a fragment or domain thereof. In a
specific embodiment, hybridization allows the sequence to form a duplex at
medium or high stringency. Embodiments of the present invention also
encompass a nucleotide sequence complementary to a nucleotide sequence
of SEQ ID NO:1 or a fragment or domain thereof. Embodiments of the
present invention further encompass a nucleotide sequence complementary
to a nucleotide sequence that has substantial similarity or is capable of
hybridizing to a nucleotide sequence of SEQ ID NO:1 or a fragment or domain
thereof.
In a specific embodiment, the nucleotide sequence having substantial
similarity is an allelic variant of the nucleotide sequence of SEQ ID NO:1 a
fragment or domain thereof. In an alternate embodiment, the sequence
having substantial similarity is a naturally occurring variant. In another
alternate embodiment, the sequence having substantial similarity is a
polymorphic variant of the nucleotide sequence of SEQ ID NO:1 or a fragment
or domain thereof.
In a specific embodiment, the isolated nucleic acid contains a plurality
of regions having the nucleotide sequence of SEQ ID NO:1 or exon or domain
thereof.
In a specific embodiment, the isolated nucleic acid contains a
polypeptide-encoding sequence. In a more specific embodiment, the


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polypeptide-encoding sequence contains a 20 base pair nucleotide portion
identical in sequence to a consecutive 20 base pair nucleotide portion of a
nucleic acid sequence of SEQ ID NO:1. In a more specific embodiment, the
polypeptide contains a polypeptide sequence of SEQ ID NO:2, or a fragment
thereof. In a more specific embodiment, the polypeptide is a plant
polypeptide. In a more specific embodiment, the plant is a dicot. In a more
specific embodiment, the plant is a gymnosperm. In a more specific
embodiment, the plant is a monocot. In a more specific embodiment, the
monocot is a cereal. In a more specific embodiment, the cereal may be, for
example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale,
einkorn, spelt, emmer, teff, miloflax, gramma grass, Tripsacum, and teosinte.
In one embodiment, the polypeptide is expressed throughout the plant.
In a more specific embodiment, the polypeptide is expressed in a specific
location or tissue of a plant. In a more specific embodiment, the location or
tissue may be, for example, epidermis, root, vascular tissue, meristem,
cambium, cortex, pith, leaf, and flower. In a most specific embodiment, the
location or tissue is a seed.
In a specific embodiment, the sequence of the isolated nucleic acid
encodes a polypeptide useful for generating an antibody having
immunoreactivity against a polypeptide encoded by a nucleotide sequence of
SEQ ID NO:2, or fragment or domain thereof.
In a specific embodiment, the sequence having substantial similarity
contains a deletion or insertion of at least one nucleotide. In a more
specific
embodiment, the deletion or insertion is of less than about thirty
nucleotides.
In a most specific embodiment, the deletion or insertion is of less than about
five nucleotides.
In a specific embodiment, the sequence of the isolated nucleic acid
having substantial similarity comprises or consists of a substitution in at
least
one codon. In a specific embodiment, the substitution is conservative.
Embodiments of the present invention also relate to an isolated nucleic
acid molecule comprising or consisting of a nucleotide sequence, its
complement, or its reverse complement, encoding a polypeptide including:


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(a) a polypeptide sequence of SEQ ID NO:2, or a fragment,
domain, repeat, or chimera thereof;
(b) a polypeptide sequence having substantial similarity to
(a);
(c) a polypeptide sequence encoded by a nucleotide
sequence identical to or having substantial similarity to a nucleotide
sequence of SEQ ID NO:1, or a fragment or domain thereof, or a
sequence complementary thereto;
(d) a polypeptide sequence encoded by a nucleotide
sequence capable of hybridizing under medium stringency conditions
to a nucleotide sequence of SEQ ID NO:1 or a sequence
complementary thereto; or
(e) a functional fragment of (a), (b), (c) or (d).
In another specific embodiment, the polypeptide having substantial
similarity is an allelic variant of a polypeptide sequence of SEQ ID NO:2, or
a
fragment, domain, repeat or chimera thereof. In another specific embodiment,
the isolated nucleic acid includes a plurality of regions from the polypeptide
sequence encoded by a nucleotide sequence identical to or having
substantial similarity to a nucleotide sequence of SEQ ID NO:1, or fragment or
domain thereof, or a sequence complementary thereto.
In another specific embodiment, the polypeptide is a polypeptide
sequence of SEQ ID NO:2. In another specific embodiment, the polypeptide
is a functional fragment or domain. In yet another specific embodiment, the
polypeptide is a chimera, where the chimera may include functional protein
domains, including domains, repeats, post-translational modification sites, or
other features. In a more specific embodiment, the polypeptide is a plant
polypeptide. In a more specific embodiment, the plant is a dicot. In a more
specific embodiment, the plant is a gymnosperm. In a more specific
embodiment, the plant is a monocot. In a more specific embodiment, the
monocot is a cereal. In a more specific embodiment, the cereal may be, for
example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale,
einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum, and
teosinte.


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In a specific embodiment, the polypeptide is expressed in a specific
location or tissue of a plant. In a more specific embodiment, the location or
tissue may be, for example, epidermis, root, vascular tissue, meristem,
cambium, cortex, pith, leaf, and flower. In another specific embodiment, the
location or tissue is a seed.
In a specific embodiment, the polypeptide sequence encoded by a
nucleotide sequence having substantial similarity to a nucleotide sequence of
SEQ ID NO:1 or a fragment or domain thereof or a sequence complementary
thereto, includes a deletion or insertion of at least one nucleotide. In a
more
specific embodiment, the deletion or insertion is of less than about thirty
nucleotides. In a most specific embodiment, the deletion or insertion is of
less
than about five nucleotides.
In a specific embodiment, the polypeptide sequence encoded by a
nucleotide sequence having substantial similarity to a nucleotide sequence of
SEQ ID NO:1, or a fragment or domain thereof or a sequence complementary
thereto, includes a substitution of at least one codon. In a more specific
embodiment, the substitution is conservative.
In a specific embodiment, the polypeptide sequences having
substantial similarity to the polypeptide sequence of SEQ ID NO:2 or a
fragment, domain, repeat, or chimeras thereof includes a deletion or insertion
of at least one amino acid.
In a specific embodiment, the polypeptide sequences having
substantial similarity to the polypeptide sequence of SEQ ID NO:2 or a
fragment, domain, repeat, or chimeras thereof includes a substitution of at
least one amino acid.
Embodiments of the present invention also relate to a shuffled nucleic
acid containing a plurality of nucleotide sequence fragments, wherein at least
one of the fragments corresponds to a region of a nucleotide sequence of
SEQ ID NO:1 and wherein at least two of the plurality of sequence fragments
are in an order, from 5' to 3' which is not an order in which the plurality of
fragments naturally occur in a nucleic acid. In a more specific embodiment,
all of the fragments in a shuffled nucleic acid containing a plurality of


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nucleotide sequence fragments are from a single gene. In a more specific
embodiment, the plurality of fragments originates from at least two different
genes. In a more specific embodiment, the shuffled nucleic acid is operably
linked to a promoter sequence. Another more specific embodiment is a
chimeric polynucleotide including a promoter sequence operably linked to the
shuffled nucleic acid. In a more specific embodiment, the shuffled nucleic
acid is contained within a host cell.
Embodiments of the present invention also contemplate an expression
cassette including a promoter sequence operably linked to an isolated nucleic
acid containing a nucleotide sequence including:
a) a nucleotide sequence of SEQ ID NO:1 or a fragment or
domain thereof;
(b) a nucleotide sequence encoding a polypeptide of SEQ ID
NO:2, a fragment or domain thereof;
(c) a nucleotide sequence having substantial similarity to (a)
or (b);
(d) a nucleotide sequence capable of hybridizing to (a), (b) or
(c) ;
(e) a nucleotide sequence complementary to (a), (b), (c) or
(d); or
(f) a nucleotide sequence that is the reverse complement of
(a), (b), (c) or (d).
Further encompassed within the invention is a recombinant vector
comprising an expression cassette according to embodiments of the present
invention. Also encompassed are plant cells, which contain expression
cassettes, according to the present disclosure, and plants, containing these
plant cells. In a specific embodiment, the plant is a dicot. In a more
specific
embodiment, the dicot is selected from the group consisting of soybean,
tobacco or cotton. In another specific embodiment, the plant is a
gymnosperm. In another specific embodiment, the plant is a monocot. In a
more specific embodiment, the monocot is a cereal. In a more specific
embodiment, the cereal may be, for example, maize, wheat, barley, oats, rye,


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millet, sorghum, triticale, secale, einkorn, spelt, emmer, teff, milo, flax,
gramma grass, Tripsacum and teosinte.
In one embodiment, the expression cassette is expressed throughout
the plant. In another embodiment, the expression cassette is expressed in a
specific location or tissue of a plant. In a specific embodiment, the location
or
tissue may be, for example, epidermis, root, vascular tissue, meristem,
cambium, cortex, pith, leaf, and flower. In an alternative specific
embodiment,
the location or tissue is a seed.
In one embodiment, the expression cassette is involved in a function
such as, for example, but not limited to, carbon, nitrogen and/or sulfur
metabolism, nitrogen utilization, nitrogen assimilation, photosynthesis,
signal
transduction, cell growth, reproduction, disease resistance, abiotic stress
tolerance, nutritional composition, gene regulation, and/or differentiation.
In a
more specific embodiment, the chimeric polypeptide is involved in a function
such as, nitrogen utilization, abiotic stress tolerance, enhanced yield,
disease
resistance and/or nutritional composition.
In one embodiment, the plant contains a modification to a phenotype or
measurable characteristic of the plant, the modification being attributable to
the expression of at least one gene contained in the expression cassette. In a
specific embodiment, the modification may be, for example, carbon, nitrogen
and/or sulfur metabolism, nitrogen utilization, nitrogen assimilation,
photosynthesis, signal transduction, cell growth, reproduction, disease
resistance, abiotic stress tolerance, nutritional composition, gene
regulation,
and/or differentiation.
Embodiments of the present invention also provide seed and isolated
product from plants which contain an expression cassette including a
promoter sequence operably linked to an isolated nucleic acid containing a
nucleotide sequence including:
(a) a nucleotide sequence of SEQ ID NO:1or a fragment or
domain thereof;
(b) a nucleotide sequence encoding a polypeptide of SEQ ID
NO:2, a fragment or domain thereof;


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(c) a nucleotide sequence having substantial similarity to (a)
or (b);
(d) a nucleotide sequence capable of hybridizing to (a), (b) or
(c) ;
(e) a nucleotide sequence complementary to (a), (b), (c) or
(d); or
(f) a nucleotide sequence that is the reverse complement of
(a), (b), (c) or (d) according to the present disclosure.
In a specific embodiment the isolated product includes an enzyme, a
nutritional protein, a structural protein, an amino acid, a lipid, a fatty
acid, a
polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a
steroid, a pigment, a vitamin and a plant hormone.
Embodiments of the present invention also relate to isolated products
produced by expression of an isolated nucleic acid containing a nucleotide
sequence including:
(a) a nucleotide sequence of SEQ ID NO:1, or fragment or
domain thereof;
(b) a nucleotide sequence encoding a polypeptide of SEQ ID
NO:2, or a fragment or domain thereof;
(c) a nucleotide sequence having substantial similarity to (a) or
(b);
(d) a nucleotide sequence capable of hybridizing to (a) or (b);
(e) a nucleotide sequence complementary to (a), (b), (c) or (d);
or
(f) a nucleotide sequence that is the reverse complement of (a),
(b) (c) or (d) according to the present disclosure.
In a specific embodiment, the product is produced in a plant. In
another specific embodiment, the product is produced in cell culture. In
another specific embodiment, the product is produced in a cell-free system.
In another specific embodiment, the product includes an enzyme, a nutritional
protein, a structural protein, an amino acid, a lipid, a fatty acid, a


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polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a
steroid, a pigment, a vitamin and a plant hormone.
In a specific embodiment, the product is a polypeptide containing an
amino acid sequence of SEQ ID NO:2. In a more specific embodiment, the
protein is an transcription factor.
Embodiments of the present invention further relate to an isolated
polynucleotide including a nucleotide sequence of at least 10 bases, which
sequence is identical, complementary, or substantially similar to a region of
any sequence of SEQ ID NO:1, and wherein the polynucleotide is adapted for
any of numerous uses.
In a specific embodiment, the polynucleotide is used as a chromosomal
marker. In another specific embodiment, the polynucleotide is used as a
marker for RFLP analysis. In another specific embodiment, the
polynucleotide is used as a marker for quantitative trait linked breeding. In
another specific embodiment, the polynucleotide is used as a marker for
marker-assisted breeding. In another specific embodiment, the
polynucleotide is used as a bait sequence in a two-hybrid system to identify
sequence- encoding polypeptides interacting with the polypeptide encoded by
the bait sequence. In another specific embodiment, the polynucleotide is
used as a diagnostic indicator for genotyping or identifying an individual or
population of individuals. In another specific embodiment, the polynucleotide
is used for genetic analysis to identify boundaries of genes or exons.
Embodiments of the present invention also relate to an expression
vector comprising or consisting of a nucleic acid molecule including:
(a) a nucleic acid encoding a polypeptide as listed in SEQ ID
NO:2
(b) a fragment, one or more domains, or featured regions of
SEQ ID NO:1; or
(c) a complete nucleic acid sequence listed in SEQ ID NO:1,
or a fragment thereof, in combination with a heterologous sequence.
In a specific embodiment, the expression vector includes one or more
elements such as, for example, but not limited to, a promoter-enhancer


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sequence, a selection marker sequence, an origin of replication, an epitope-
tag encoding sequence, or an affinity purification-tag encoding sequence. In
a more specific embodiment, the promoter-enhancer sequence may be, for
example, the CaMV 35S promoter, the CaMV 19S promoter, the tobacco PR-
1a promoter, ubiquitin and the phaseolin promoter. In another embodiment,
the promoter is operable in plants, and more specifically, a constitutive or
inducible promoter. In another specific embodiment, the selection marker
sequence encodes an antibiotic resistance gene. In another specific
embodiment, the epitope-tag sequence encodes V5, the peptide Phe-His-His-
Thr-Thr, hemagglutinin, or gIutathione-S-transferase. In another specific
embodiment the affinity purification-tag sequence encodes a polyamino acid
sequence or a polypeptide. In a more specific embodiment, the polyamino
acid sequence is polyhistidine. In a more specific embodiment, the
polypeptide is chitin binding domain or glutathione-S-transferase. In a more
specific embodiment, the affinity purification-tag sequence comprises an
intein
encoding sequence.
In a specific embodiment, the expression vector is a eukaryotic
expression vector or a prokaryotic expression vector. In a more specific
embodiment, the eukaryotic expression vector includes a tissue-specific
promoter. More specifically, the expression vector is operable in plants.
Embodiments of the present invention also relate to a cell comprising
or consisting of a nucleic acid construct comprising an expression vector and
a nucleic acid including a nucleic acid encoding a polypeptide as listed in
SEQ
ID NO:2, or a nucleic acid sequence listed in SEQ ID NO:1, or a segment
thereof, in combination with a heterologous sequence.
In a specific embodiment, the cell is a bacterial cell, a fungal cell, a
plant cell, or an animal cell. In a specific embodiment, the cell is a plant
cell. In
a more specific embodiment, the polypeptide is expressed in a specific
location or tissue of a plant. In a most specific embodiment, the location or
tissue may be, for example, epidermis, root, vascular tissue, meristem,
cambium, cortex, pith, leaf, and flower. In an alternate most specific
embodiment, the location or tissue is a seed. In a specific embodiment, the


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polypeptide is involved in a function such as, for example, carbon, nitrogen
and/or sulfur metabolism, nitrogen utilization, nitrogen assimilation,
photosynthesis, signal transduction, cell growth, reproduction, disease
resistance, abiotic stress tolerance, nutritional composition, gene
regulation,
and/or differentiation.
Embodiments of the present invention also relate to polypeptides
encoded by the isolated nucleic acid molecules of the present disclosure
including a polypeptide containing a polypeptide sequence encoded by an
isolated nucleic acid containing a nucleotide sequence including:
(a) a nucleotide sequence listed in SEQ ID NO:1, or an exon
or domain thereof;
(b) a nucleotide sequence having substantial similarity to (a);
(c) a nucleotide sequence capable of hybridizing to (a);
(d) a nucleotide sequence complementary to (a), (b) or (c); or
(e) a nucleotide sequence which is the reverse complement
of (a), (b) or (c);
(f) or a functional fragment thereof.
A polypeptide containing a polypeptide sequence encoded by an
isolated nucleic acid containing a nucleotide sequence, its complement, or its
reverse complement, encoding a polypeptide including a polypeptide
sequence including:
(a) a polypeptide sequence listed in SEQ ID NO:2, or a
domain, repeat, or chimeras thereof;
(b) a polypeptide sequence having substantial similarity to
(a);
(c) a polypeptide sequence encoded by a nucleotide
sequence identical to or having substantial similarity to a nucleotide
sequence listed SEQ ID NO:1, or an exon or domain thereof, or a
sequence complementary thereto;
(d) a polypeptide sequence encoded by a nucleotide
sequence capable of hybridizing under medium stringency conditions


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to a nucleotide sequence listed in SEQ ID NO:1 or to a sequence
complementary thereto; or
(e) a functional fragment of (a), (b), (c) or (d);
(f) or a functional fragment thereof.
Embodiments of the present invention contemplate a polypeptide
containing a polypeptide sequence encoded by an isolated nucleic acid which
includes a shuffled nucleic acid containing a plurality of nucleotide sequence
fragments, wherein at least one of the fragments corresponds to a region of a
nucleotide sequence listed SEQ ID NO:1, and wherein at least two of the
plurality of sequence fragments are in an order, from 5' to 3' which is not an
order in which the plurality of fragments naturally occur in a nucleic acid,
or
functional fragment thereof.
Embodiments of the present invention contemplate a polypeptide
containing a polypeptide sequence encoded by an isolated polynucleotide
containing a nucleotide sequence of at least 10 bases, which sequence is
identical, complementary, or substantially similar to a region of any of
sequences of SEQ ID NO:1, or functional fragment thereof and wherein the
polynucleotide is adapted for a use including:
(a) use as a chromosomal marker to identify the location of
the corresponding or complementary polynucleotide on a native or
artificial chromosome;
(b) use as a marker for RFLP analysis;
(c) use as a marker for quantitative trait linked breeding;
(d) use as a marker for marker-assisted breeding;
(e) use as a bait sequence in a two-hybrid system to identify
sequence encoding polypeptides interacting with the polypeptide
encoded by the bait sequence;
(f) use as a diagnostic indicator for genotyping or identifying
an individual or population of individuals; or
(g) use for genetic analysis to identify boundaries of genes or
exons.


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Embodiments of the present invention also contemplate an isolated
polypeptide containing a polypeptide sequence including:
(a) a polypeptide sequence listed SEQ ID NO:2, or exon or
domain thereof;
(b) a polypeptide sequence having substantial similarity to
(a);
(c) a polypeptide sequence encoded by a nucleotide
sequence identical to or having substantial similarity to a nucleotide
sequence SEQ ID NO:1, or an exon or domain thereof, or a sequence
complementary thereto;
(d) a polypeptide sequence encoded by a nucleotide
sequence capable of hybridizing under medium stringency conditions
to a nucleotide sequence listed in SEQ ID NO:1, or to a sequence
complementary thereto; or
(e) a functional fragment of (a), (b), (c) or (d).
In a specific embodiment, the substantial similarity is at least about
65% identity. In a more specific embodiment, the substantial similarity is at
least about 80% identity. In a most specific embodiment, the substantial
similarity is at least about 95% identity. In a specific embodiment, the
substantial similarity is at least three percent greater than the percent
identity
to the closest homologous sequence listed in any of the Sequence Listings.
In a specific embodiment, the sequence having substantial similarity is
from a plant. In a more specific embodiment, the plant is a dicot. In a more
specific embodiment, the plant is a gymnosperm. In a more specific
embodiment, the plant is a monocot. In a more specific embodiment, the
monocot is a cereal. In a more specific embodiment, the cereal may be, for
example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale,
einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum and teosinte.
In a specific embodiment, the polypeptide is expressed in a specific
location or tissue of a plant. In a more specific embodiment, the location or
tissue may be, for example, epidermis, root, vascular tissue, meristem,
cambium, cortex, pith, leaf, and flower. In another specific embodiment, the


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location or tissue is a seed. In a specific embodiment, the polypeptide is
involved in a function such as, for example, carbon, nitrogen and/or sulfur
metabolism, nitrogen utilization, nitrogen assimilation, photosynthesis,
signal
transduction, cell growth, reproduction, disease resistance, abiotic stress
tolerance, nutritional composition, gene regulation, and/or differentiation.
In a specific embodiment, hybridization of a polypeptide sequence
encoded by a nucleotide sequence identical to or having substantial similarity
to a nucleotide sequence listed in SEQ ID NO:1, or an exon or domain
thereof, or a sequence complementary thereto, or a polypeptide sequence
encoded by a nucleotide sequence capable of hybridizing under medium
stringency conditions to a nucleotide sequence listed SEQ ID NO:1, or to a
sequence complementary thereto, allows the sequence to form a duplex at
medium or high stringency.
In a specific embodiment, a polypeptide having substantial similarity to
a polypeptide sequence listed in SEQ ID NO:2, or exon or domain thereof, is
an allelic variant of the polypeptide sequence listed in SEQ ID NO:2. In
another specific embodiment, a polypeptide having substantial similarity to a
polypeptide sequence listed in SEQ ID NO:2, or exon or domain thereof, is a
naturally occurring variant of the polypeptide sequence listed in SEQ ID NO:2.
In another specific embodiment, a polypeptide having substantial similarity to
a polypeptide sequence listed in SEQ ID NO:2, or exon or domain thereof, is
a polymorphic variant of the polypeptide sequence listed in SEQ ID NO:2.
In an alternate specific embodiment, the sequence having substantial
similarity contains a deletion or insertion of at least one amino acid. In a
more
specific embodiment, the deletion or insertion is of less than about ten amino
acids. In a most specific embodiment, the deletion or insertion is of less
than
about three amino acids.
In a specific embodiment, the sequence having substantial similarity
encodes a substitution in at least one amino acid.
Also contemplated is a method of producing a plant comprising a
modification thereto, including the steps of: (1) providing a nucleic acid
which
is an isolated nucleic acid containing a nucleotide sequence including:


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(a) a nucleotide sequence listed SEQ ID NO:1, or exon or
domain thereof;
(b) a nucleotide sequence having substantial similarity to (a);
(c) a nucleotide sequence capable of hybridizing to (a);
(d) a nucleotide sequence complementary to (a), (b) or (c); or
(e) a nucleotide sequence which is the reverse complement
of (a), (b) or (c);
and (2) introducing the nucleic acid into the plant, wherein the nucleic acid
is
expressible in the plant in an amount effective to effect the modification. In
one embodiment, the modification comprises an altered characteristic in the
plant, wherein the characteristic corresponds to the nucleic acid introduced
into the plant. In other specific embodiments the characteristic corresponds
to
carbon, nitrogen and/or sulfur metabolism, nitrogen utilization, nitrogen
assimilation, photosynthesis, signal transduction, cell growth, reproduction,
disease resistance, abiotic stress tolerance, nutritional composition, gene
regulation, and/or differentiation.
In another embodiment, the modification includes an increased or
decreased expression or accumulation of a product of the plant. Specifically,
the product is a natural product of the plant. Equally specifically, the
product
is a new or altered product of the plant. Specifically, the product comprises
a
GATA transcription factor.
Also encompassed within the presently disclosed invention is a method
of producing a recombinant protein, comprising the steps of:
(a) growing recombinant cells comprising a nucleic acid construct
under suitable growth conditions, the construct comprising an expression
vector and a nucleic acid including: a nucleic acid encoding a protein as
listed
in SEQ ID NO:2, or a nucleic acid sequence listed in SEQ ID NO:1, or
segments thereof; and
(b) isolating from the recombinant cells the recombinant protein
expressed thereby.
Embodiments of the present invention provide a method of producing a
recombinant protein in which the expression vector includes one or more


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elements including a promoter-enhancer sequence, a selection marker
sequence, an origin of replication, an epitope-tag encoding sequence, and an
affinity purification-tag encoding sequence. In one specific embodiment, the
nucleic acid construct includes an epitope-tag encoding sequence and the
isolating step includes use of an antibody specific for the epitope-tag. In
another specific embodiment, the nucleic acid construct contains a polyamino
acid encoding sequence and the isolating step includes use of a resin
comprising a polyamino acid binding substance, specifically where the
polyamino acid is polyhistidine and the polyamino binding resin is nickel-
charged agarose resin. In yet another specific embodiment, the nucleic acid
construct contains a polypeptide encoding sequence and the isolating step
includes the use of a resin containing a polypeptide binding substance,
specifically where the polypeptide is a chitin binding domain and the resin
contains chitin-sepharose.
Embodiments of the present invention also relate to a plant modified by
a method that includes introducing into a plant a nucleic acid where the
nucleic acid is expressible in the plant in an amount effective to effect the
modification. The modification can be, for example, carbon, nitrogen and/or
sulfur metabolism, nitrogen utilization, nitrogen assimilation,
photosynthesis,
signal transduction, cell growth, reproduction, disease resistance, abiotic
stress tolerance, nutritional composition, gene regulation, and/or
differentiation. In one embodiment, the modified plant has increased or
decreased resistance to an herbicide, a stress, or a pathogen. In another
embodiment, the modified plant has enhanced or diminished requirement for
light, water, nitrogen, or trace elements. In yet another embodiment, the
modified plant is enriched for an essential amino acid as a proportion of a
protein fraction of the plant. The protein fraction may be, for example, total
seed protein, soluble protein, insoluble protein, water-extractable protein,
and
lipid-associated protein. The modification may include overexpression,
underexpression, antisense modulation, sense suppression, inducible
expression, inducible repression, or inducible modulation of a gene.


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The invention further relates to a seed from a modified plant or an
isolated product of a modified plant, where the product may be an enzyme, a
nutritional protein, a structural protein, an amino acid, a lipid, a fatty
acid, a
polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a
steroid, a pigment, a vitamin and a plant hormone.
The above Summary of Invention lists several embodiments of the
invention, and in many cases lists variations and permutations of these
embodiments. The Summary is merely exemplary of the numerous and
varied embodiments. Mention of one or more specific features of a given
embodiment is likewise exemplary. Such embodiment can typically exist with
or without the feature(s) mentioned; likewise, those features can be applied
to
other embodiments of the invention, whether listed in this Summary or not.
To avoid excessive repetition, this Summary does not list or suggest all
possible combinations of such features.
For purposes of summarizing the invention and the advantages
achieved over the prior art, certain objects and advantages of the invention
have been described above. Of course, it is to be understood that not
necessarily all such objects or advantages may be achieved in accordance
with any particular embodiment of the invention. Thus, for example, those
skilled in the art will recognize that the invention may be embodied or
carried
out in a manner that achieves or optimizes one advantage or group of
advantages as taught herein without necessarily achieving other objects or
advantages as may be taught or suggested herein.
Further aspects, features and advantages of this invention will become
apparent from the detailed description of the specific embodiments that
follow.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 and SEQ ID NO:1 shows the nucleic acid sequence of full
length OsGATAl 1.
Figure 2 and SEQ ID NO:2 shows the amino acid sequence of
OsGATAl 1.
Figure 3 shows the alignment of the amino acid sequence of
At4g26150 (SEQ ID NO:7) and its rice ortholog OsGATAl 1 (SEQ ID NO:2).


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Figure 4A and B shows the phenotypes of the OsGATAl 1 over-
expressing plants.
Figure 5A and B shows the chlorophyll level affected by the expression
of OsGATA11 gene.
Figure 6A and B shows the seed yield of OsGATAl 1 over-expressing
plants.
Figure 7 are pictures showing more resistant to stress in the
OsGATAl1 over-expressing plants.
DEFINITIONS
For clarity, certain terms used in the specification are defined and
presented as follows:
"Associated with / operatively linked" refer to two nucleic acid
sequences that are related physically or functionally. For example, a
promoter or regulatory DNA sequence is said to be "associated with" a DNA
sequence that codes for an RNA or a protein if the two sequences are
operatively linked, or situated such that the regulator DNA sequence will
affect
the expression level of the coding or structural DNA sequence.
A "chimeric construct" is a recombinant nucleic acid sequence in which
a promoter or regulatory nucleic acid sequence is operatively linked to, or
associated with, a nucleic acid sequence that codes for an mRNA or which is
expressed as a protein, such that the regulatory nucleic acid sequence is able
to regulate transcription or expression of the associated nucleic acid
sequence. The regulatory nucleic acid sequence of the chimeric construct is
not normally operatively linked to the associated nucleic acid sequence as
found in nature.
A "co-factor" is a natural reactant, such as an organic molecule or a
metal ion, required in an enzyme-catalyzed reaction. A co-factor is e.g.
NAD(P), riboflavin (including FAD and FMN), folate, molybdopterin, thiamin,
biotin, lipoic acid, pantothenic acid and coenzyme A, S-adenosylmethionine,
pyridoxal phosphate, ubiquinone, menaquinone. Optionally, a co-factor can
be regenerated and reused.


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A"coding sequence" is a nucleic acid sequence that is transcribed into
RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.
Specifically the RNA is then translated in an organism to produce a protein.
Complementary: "complementary" refers to two nucleotide sequences
that comprise antiparallel nucleotide sequences capable of pairing with one
another upon formation of hydrogen bonds between the complementary base
residues in the antiparaliel nucleotide sequences.
Enzyme activity: means herein the ability of an enzyme to catalyze the
conversion of a substrate into a product. A substrate for the enzyme
comprises the natural substrate of the enzyme but also comprises analogues
of the natural substrate, which can also be converted, by the enzyme into a
product or into an analogue of a product. The activity of the enzyme is
measured for example by determining the amount of product in the reaction
after a certain period of time, or by determining the amount of substrate
remaining in the reaction mixture after a certain period of time. The activity
of
the enzyme is also measured by determining the amount of an unused co-
factor of the reaction remaining in the reaction mixture after a certain
period of
time or by determining the amount of used co-factor in the reaction mixture
after a certain period of time. The activity of the enzyme is also measured by
determining the amount of a donor of free energy or energy-rich molecule
(e.g. ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine)
remaining in the reaction mixture after a certain period of time or by
determining the amount of a used donor of free energy or energy-rich
molecule (e.g. ADP, pyruvate, acetate or creatine) in the reaction mixture
after
a certain period of time.
Expression Cassette: "Expression cassette" as used herein means a
nucleic acid molecule capable of directing expression of a particular
nucleotide sequence in an appropriate host cell, comprising a promoter
operatively linked to the nucleotide sequence of interest which is operatively
linked to termination signals. It also typically comprises sequences required
for proper translation of the nucleotide sequence. The coding region usually
codes for a protein of interest but may also code for a functional RNA of


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interest, for example antisense RNA or a nontransiated RNA, in the sense or
antisense direction. The expression cassette comprising the nucleotide
sequence of interest may be chimeric, meaning that at least one of its
components is heterologous with respect to at least one of its other
components. The expression cassette may also be one that is naturally
occurring but has been obtained in a recombinant form useful for
heterologous expression. Typically, however, the expression cassette is
heterologous with respect to the host, i.e., the particular DNA sequence of
the
expression cassette does not occur naturally in the host cell and must have
been introduced into the host cell or an ancestor of the host cell by a
transformation event. The expression of the nucleotide sequence in the
expression cassette may be under the control of a constitutive promoter or of
an inducible promoter that initiates transcription only when the host cell is
exposed to some particular external stimulus. In the case of a multicellular
organism, such as a plant, the promoter can also be specific to a particular
tissue or organ or stage of development.
The term "functional fragment" as used herein in relation to a nucleic
acid or protein sequence means a fragment or portion of the sequence that
retains the function of the full length sequence.
Gene: the term "gene" is used broadly to refer to any segment of DNA
associated with a biological function. Thus, genes include coding sequences
and/or the regulatory sequences required for their expression. Genes also
include nonexpressed DNA segments that, for example, form recognition
sequences for other proteins. Genes can be obtained from a variety of
sources, including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters.
Heterologous/exogenous: The terms "heterologous" and "exogenous"
when used herein to refer to a nucleic acid sequence (e.g. a DNA sequence)
or a gene, refer to a sequence that originates from a source foreign to the
particular host cell or, if from the same source, is modified from its
original
form. Thus, a heterologous gene in a host cell includes a gene that is


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endogenous to the particular host cell but has been modified through, for
example, the use of DNA shuffling. The terms also include non-naturally
occurring multiple copies of a naturally occurring DNA sequence. Thus, the
terms refer to a DNA segment that is foreign or heterologous to the cell, or
homologous to the cell but in a position within the host cell nucleic acid in
which the element is not ordinarily found. Exogenous DNA segments are
expressed to yield exogenous polypeptides.
A "homologous" nucleic acid (e.g. DNA) sequence is a nucleic acid
(e.g. DNA) sequence naturally associated with a host cell into which it is
introduced.
Hybridization: The phrase "hybridizing specifically to" refers to the
binding, duplexing, or hybridizing of a molecule only to a particular
nucleotide
sequence under stringent conditions when that sequence is present in a
complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially"
refers to complementary hybridization between a probe nucleic acid and a
target nucleic acid and embraces minor mismatches that can be
accommodated by reducing the stringency of the hybridization media to
achieve the desired detection of the target nucleic acid sequence.
Inhibitor: a chemical substance that inactivates the enzymatic activity of
a protein such as a biosynthetic enzyme, receptor, signal transduction
protein,
structural gene product, or transport protein. The term "herbicide" (or
"herbicidal compound") is used herein to define an inhibitor applied to a
plant
at any stage of development, whereby the herbicide inhibits the growth of the
plant or kills the plant.
Interaction: quality or state of mutual action such that the effectiveness
or toxicity of one protein or compound on another protein is inhibitory
(antagonists) or enhancing (agonists).
A nucleic acid sequence is "isocoding with" a reference nucleic acid
sequence when the nucleic acid sequence encodes a polypeptide having the
same amino acid sequence as the polypeptide encoded by the reference
nucleic acid sequence.


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Isogenic: plants that are genetically identical, except that they may
differ by the presence or absence of a heterologous DNA sequence.
Isolated: in the context of the present invention, an isolated DNA
molecule or an isolated enzyme is a DNA molecule or enzyme that, by human
intervention, exists apart from its native environment and is therefore not a
product of nature. An isolated DNA molecule or enzyme may exist in a
purified form or may exist in a non-native environment such as, for example,
in a transgenic host cell.
Mature protein: protein from which the transit peptide, signal peptide,
and/or propeptide portions have been removed.
Minimal Promoter: the smallest piece of a promoter, such as a TATA
element, that can support any transcription. A minimal promoter typically has
greatly reduced promoter activity in the absence of upstream activation. In
the presence of a suitable transcription factor, the minimal promoter
functions
to permit transcription.
Modified Enzyme Activity: enzyme activity different from that which
naturally occurs in a plant (i.e. enzyme activity that occurs naturally in the
absence of direct or indirect manipulation of such activity by man), which is
tolerant to inhibitors that inhibit the naturally occurring enzyme activity.
Native: refers to a gene that is present in the genome of an
untransformed plant cell.
Naturally occurring: the term "naturally occurring" is used to describe
an object that can be found in nature as distinct from being artificially
produced by man. For example, a protein or nucleotide sequence present in
an organism (including a virus), which can be isolated from a source in nature
and which has not been intentionally modified by man in the laboratory, is
naturally occurring.
Nucleic acid: the term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or double-stranded
form. Unless specifically limited, the term encompasses nucleic acids
containing known analogues of natural nucleotides which have similar binding
properties as the reference nucleic acid and are metabolized in a manner


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similar to naturally occurring nucleotides. Unless otherwise indicated, a
particular nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g. degenerate codon substitutions) and
complementary sequences and as well as the sequence explicitly indicated.
Specifically, degenerate codon substitutions may be achieved by generating
sequences in which the third position of one or more selected (or all) codons
is substituted with mixed-base and/or deoxyinosine residues (Batzer et al.,
Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:
2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). The
terms "nucleic acid" or "nucleic acid sequence" may also be used
interchangeably with gene, cDNA, and mRNA encoded by a gene.
"ORF" means open reading frame.
Percent identity: the phrases "percent identityl" or "percent identical," in
the context of two nucleic acid or protein sequences, refers to two or more
sequences or subsequences that have for example 60%, specifically 70%,
more specifically 80%, still more specifically 90%, even more specifically
95%,
and most specifically at least 99% nucleotide or amino acid residue identity,
when compared and aligned for maximum correspondence, as measured
using one of the following sequence comparison algorithms or by visual
inspection. Specifically, the percent identity exists over a region of the
sequences that is at least about 50 residues in length, more specifically over
a
region of at least about 100 residues, and most specifically the percent
identity exists over at least about 150 residues. In an especially specific
embodiment, the percent identity exists over the entire length of the coding
regions.
For sequence comparison, typically one sequence acts as a reference
sequence to which test sequences are compared. When using a sequence
comparison algorithm, test and reference sequences are input into a
computer, subsequence coordinates are designated if necessary, and
sequence algorithm program parameters are designated. The sequence
comparison algorithm then calculates the percent sequence identity for the


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test sequence(s) relative to the reference sequence, based on the designated
program parameters.
Optimal alignment of sequences for comparison can be conducted,
e.g., by the local homology algorithm of Smith & Waterman, Adv. Appi. Math.
2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch,
J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson &
Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in
the Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, WI), or by visual inspection (see generally, Ausubel et
al., infra).
One example of an algorithm that is suitable for determining percent
sequence identity and sequence similarity is the BLAST algorithm, which is
described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). Software for
performing BLAST analyses is publicly available through the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm
involves first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match or satisfy
some positive-valued threshold score T when aligned with a word of the same
length in a database sequence. T is referred to as the neighborhood word
score threshold (Altschul et al., 1990). These initial neighborhood word hits
act as seeds for initiating searches to find longer HSPs containing them. The
word hits are then extended in both directions along each sequence for as far
as the cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M (reward score
for a pair of matching residues; always > 0) and N (penalty score for
mismatching residues; always < 0). For amino acid sequences, a scoring
matrix is used to calculate the cumulative score. Extension of the word hits
in
each direction are halted when the cumulative alignment score falls off by the
quantity X from its maximum achieved value, the cumulative score goes to
zero or below due to the accumulation of one or more negative-scoring
residue alignments, or the end of either sequence is reached. The BLAST


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algorithm parameters W, T, and X determine the sensitivity and speed of the
alignment. The BLASTN program (for nucleotide sequences) uses as defaults
a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4,
and a comparison of both strands. For amino acid sequences, the BLASTP
program uses as defaults a wordiength (W) of 3, an expectation (E) of 10, and
the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89: 10915 (1989)).
In addition to calculating percent sequence identity, the BLAST
algorithm also performs a statistical analysis of the similarity between two
sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:
5873-5787 (1993)). One measure of similarity provided by the BLAST
algorithm is the smallest sum probability (P(N)), which provides an indication
of the probability by which a match between two nucleotide or amino acid
sequences would occur by chance. For example, a test nucleic acid sequence
is considered similar to a reference sequence if the smallest sum probability
in
a comparison of the test nucleic acid sequence to the reference nucleic acid
sequence is less than about 0.1, more specifically less than about 0.01, and
most specifically less than about 0.001.
Pre-protein: protein that is normally targeted to a cellular organelle,
such as a chloroplast, and still comprises its native transit peptide.
Purified: the term "purified," when applied to a nucleic acid or protein,
denotes that the nucleic acid or protein is essentially free of other cellular
components with which it is associated in the natural state. It is
specifically in
a homogeneous state although it can be in either a dry or aqueous solution.
Purity and homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high performance
liquid chromatography. A protein that is the predominant species present in a
preparation is substantially purified. The term "purified" denotes that a
nucleic
acid or protein gives rise to essentially one band in an electrophoretic gel.
Particularly, it means that the nucleic acid or protein is at least about 50%
pure, more specifically at least about 85% pure, and most specifically at
least
about 99% pure.


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Two nucleic acids are "recombined" when sequences from each of the
two nucleic acids are combined in a progeny nucleic acid. Two sequences are
"directly" recombined when both of the nucleic acids are substrates for
recombination. Two sequences are "indirectly recombined" when the
sequences are recombined using an intermediate such as a cross-over
oligonucleotide. For indirect recombination, no more than one of the
sequences is an actual substrate for recombination, and in some cases,
neither sequence is a substrate for recombination.
"Regulatory elements" refer to sequences involved in controlling the
expression of a nucleotide sequence. Regulatory elements comprise a
promoter operatively linked to the nucleotide sequence of interest and
termination signals. They also typically encompass sequences required for
proper translation of the nucleotide sequence.
Significant Increase: an increase in enzymatic activity that is larger
than the margin of error inherent in the measurement technique, specifically
an increase by about 2-fold or greater of the activity of the wild-type enzyme
in the presence of the inhibitor, more specifically an increase by about 5-
fold
or greater, and most specifically an increase by about 10-fold or greater.
Significantly less: means that the amount of a product of an enzymatic
reaction is reduced by more than the margin of error inherent in the
measurement technique, specifically a decrease by about 2-fold or greater of
the activity of the wild-type enzyme in the absence of the inhibitor, more
specifically an decrease by about 5-fold or greater, and most specifically an
decrease by about 10-fold or greater.
Specific Binding/Immunological Cross-Reactivity: An indication that
two nucleic acid sequences or proteins are substantially identical is that the
protein encoded by the first nucleic acid is immunologically cross reactive
with, or specifically binds to, the protein encoded by the second nucleic
acid.
Thus, a protein is typically substantially identical to a second protein, for
example, where the two proteins differ only by conservative substitutions.
The phrase "specifically (or selectively) binds to an antibody," or
"specifically
(or selectively) immunoreactive with," when referring to a protein or peptide,


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refers to a binding reaction which is determinative of the presence of the
protein in the presence of a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the specified
antibodies bind to a particular protein and do not bind in a significant
amount
to other proteins present in the sample. Specific binding to an antibody under
such conditions may require an antibody that is selected for its specificity
for a
particular protein. For example, antibodies raised to the protein with the
amino
acid sequence encoded by any of the nucleic acid sequences of the invention
can be selected to obtain antibodies specifically immunoreactive with that
protein and not with other proteins except for polymorphic variants. A variety
of immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase ELISA
immunoassays, Western blots, or immunohistochemistry are routinely used to
select monoclonal antibodies specifically immunoreactive with a protein. See
Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York "Harlow and Lane"), for a description of
immunoassay formats and conditions that can be used to determine specific
immunoreactivity. Typically a specific or selective reaction will be at least
twice background signal or noise and more typically more than 10 to 100
times background.
"Stringent hybridization conditions" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization experiments such as
Southern and Northern hybridizations are sequence dependent, and are
different under different environmental parameters. Longer sequences
hybridize specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993) Laboratory
Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic
Acid Probes part I chapter 2 "Overview of principles of hybridization and the
strategy of nucleic acid probe assays" Elsevier, New York. Generally, highly
stringent hybridization and wash conditions are selected to be about 5 C
lower than the thermal melting point (Tm) for the specific sequence at a


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defined ionic strength and pH. Typically, under "stringent conditions" a probe
will hybridize to its target subsequence, but to no other sequences.
The Tm is the temperature (under defined ionic strength and pH) at
which 50% of the target sequence hybridizes to a perfectly matched probe.
Very stringent conditions are selected to be equal to the Tm for a particular
probe. An example of stringent hybridization conditions for hybridization of
complementary nucleic acids which have more than 100 complementary
residues on a filter in a Southern or northern blot is 50% formamide with 1 mg
of heparin at 42 C, with the hybridization being carried out overnight. An
example of highly stringent wash conditions is 0.1 5M NaCI at 72 C for about
minutes. An example of stringent wash conditions is a 0.2x SSC wash at
65 C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
Often, a high stringency wash is preceded by a low stringency wash to
remove background probe signal. An example medium stringency wash for a
15 duplex of, e.g., more than 100 nucleotides, is lx SSC at 45 C for 15
minutes.
An example low stringency wash for a duplex of, e.g., more than 100
nucleotides, is 4-6x SSC at 40 C for 15 minutes. For short probes (e.g., about
10 to 50 nucleotides), stringent conditions typically involve salt
concentrations
of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion
concentration (or other salts) at pH 7.0 to 8.3, and the temperature is
typically
at least about 30 C. Stringent conditions can also be achieved with the
addition of destabilizing agents such as formamide. In general, a signal to
noise ratio of 2x (or higher) than that observed for an unrelated probe in the
particular hybridization assay indicates detection of a specific
hybridization.
Nucleic acids that do not hybridize to each other under stringent conditions
are still substantially identical if the proteins that they encode are
substantially
identical. This occurs, e.g., when a copy of a nucleic acid is created using
the
maximum codon degeneracy permitted by the genetic code.
The following are examples of sets of hybridization/wash conditions
that may be used to clone nucleotide sequences that are homologues of
reference nucleotide sequences of the present invention: a reference
nucleotide sequence specifically hybridizes to the reference nucleotide


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sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at
50 C with washing in 2X SSC, 0.1% SDS at 50 C, more desirably in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with
washing in 1X SSC, 0.1% SDS at 50 C, more desirably still in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in
0.5X SSC, 0.1% SDS at 50 C, specifically in 7% sodium dodecyl sulfate
(SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in 0.1X SSC, 0.1%
SDS at 50 C, more specifically in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO4, 1 mM EDTA at 50 C with washing in 0.1X SSC, 0.1% SDS at 65 C.
A "subsequence" refers to a sequence of nucleic acids or amino acids
that comprise a part of a longer sequence of nucleic acids or amino acids
(e.g., protein) respectively.
Substantial similarity: The term "substantial similarity" in the context of
two nucleic acid or protein sequences, refers to two or more sequences or
subsequences that are substantially similar, for example that have 50%,
specifically 60%, more specifically 70%, even more specifically 80%, still
more
specifically 90%, further more specifically 95%, and most specifically 99%
sequence identity.
Substrate: a substrate is the molecule that an enzyme naturally
recognizes and converts to a product in the biochemical pathway in which the
enzyme naturally carries out its function, or is a modified version of the
molecule, which is also recognized by the enzyme and is converted by the
enzyme to a product in an enzymatic reaction similar to the naturally-
occurring
reaction.
Transformation: a process for introducing heterologous DNA into a
plant cell, plant tissue, or plant. Transformed plant cells, plant tissue, or
plants are understood to encompass not only the end product of a
transformation process, but also transgenic progeny thereof.
"Transformed," "transgenic," and "recombinant" refer to a host
organism such as a bacterium or a plant into which a heterologous nucleic
acid molecule has been introduced. The nucleic acid molecule can be stably
integrated into the genome of the host or the nucleic acid molecule can also


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be present as an extrachromosomal molecule. Such an extrachromosomal
molecule can be auto-replicating. Transformed cells, tissues, or plants are
understood to encompass not only the end product of a transformation
process, but also transgenic progeny thereof. A "non-transformed," "non-
transgenic," or "non-recombinant" host refers to a wild-type organism, e.g., a
bacterium or plant, which does not contain the heterologous nucleic acid
molecule.
Viability: "viability" as used herein refers to a fitness parameter of a
plant. Plants are assayed for their homozygous performance of plant
development, indicating which proteins are essential for plant growth.
DETAILED DESCRIPTION OF THE INVENTION
1. General Description of Trait Functional Genomics
The goal of functional genomics is to identify genes controlling
expression of organismal phenotypes, and employs a variety of
methodologies, including but not limited to bioinformatics, gene expression
studies, gene and gene product interactions, genetics, biochemistry and
molecular genetics. For example, bioinformatics can assign function to a
given gene by identifying genes in heterologous organisms with a high degree
of similarity (homology) at the amino acid or nucleotide level. Expression of
a
gene at the mRNA or protein levels can assign function by linking expression
of a gene to an environmental response, a developmental process or a
genetic (mutational) or molecular genetic (gene overexpression or
underexpression) perturbation. Expression of a gene at the mRNA level can
be ascertained either alone (Northern analysis) or in concert with other genes
(microarray analysis), whereas expression of a gene at the protein level can
be ascertained either alone (native or denatured protein gel or immunoblot
analysis) or in concert with other genes (proteomic analysis). Knowledge of
protein/protein and protein/DNA interactions can assign function by
identifying
proteins and nucleic acid sequences acting together in the same biological
process. Genetics can assign function to a gene by demonstrating that DNA
lesions (mutations) in the gene have a quantifiable effect on the organism,
including but not limited to: its development; hormone biosynthesis and


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response; growth and growth habit (plant architecture); mRNA expression
profiles; protein expression profiles; ability to resist diseases; tolerance
of
abiotic stresses; ability to acquire nutrients; photosynthetic efficiency;
altered
primary and secondary metabolism; and the composition of various plant
organs. Biochemistry can assign function by demonstrating that the protein
encoded by the gene, typically when expressed in a heterologous organism,
possesses a certain enzymatic activity, alone or in combination with other
proteins. Molecular genetics can assign function by overexpressing or
underexpressing the gene in the native plant or in heterologous organisms,
and observing quantifiable effects as described in functional assignment by
genetics above. In functional genomics, any or all of these approaches are
utilized, often in concert, to assign genes to functions across any of a
number
of organismal phenotypes.
It is recognized by those skilled in the art that these different
methodologies can each provide data as evidence for the function of a
particular gene, and that such evidence is stronger with increasing amounts of
data used for functional assignment: specifically from a single methodology,
more specifically from two methodologies, and even more specifically from
more than two methodologies. In addition, those skilled in the art are aware
that different methodologies can differ in the strength of the evidence for
the
assignment of gene function. Typically, but not always, a datum of
biochemical, genetic and molecular genetic evidence is considered stronger
than a datum of bioinformatic or gene expression evidence. Finally, those
skilled in the art recognize that, for different genes, a single datum from a
single methodology can differ in terms of the strength of the evidence
provided by each distinct datum for the assignment of the function of these
different genes.
The objective of crop trait functional genomics is to identify crop trait
genes, i.e. genes capable of conferring useful agronomic traits in crop
plants.
Such agronomic traits include, but are not limited to: enhanced yield, whether
in quantity or quality; enhanced nutrient acquisition and enhanced metabolic
efficiency; enhanced or altered nutrient composition of plant tissues used for


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food, feed, fiber or processing; enhanced utility for agricultural or
industrial
processing; enhanced resistance to plant diseases; enhanced tolerance of
adverse environmental conditions (abiotic stresses) including but not limited
to
drought, excessive cold, excessive heat, or excessive soil salinity or extreme
acidity or alkalinity; and alterations in plant architecture or development,
including changes in developmental timing. The deployment of such identified
trait genes by either transgenic or non-transgenic means could materially
improve crop plants for the benefit of agriculture.
Cereals are the most important crop plants on the planet, in terms of
both human and animal consumption. Genomic synteny (conservation of
gene order within large chromosomal segments) is observed in rice, maize,
wheat, barley, rye, oats and other agriculturally important monocots, which
facilitates the mapping and isolation of orthologous genes from diverse cereal
species based on the sequence of a single cereal gene. Rice has the
smallest (- 420 Mb) genome among the cereal grains, and has recently been
a major focus of public and private genomic and EST sequencing efforts.
To identify crop trait genes in the rice [wheat] genome controlling [trait],
genes from the rice draft genome sequence [wheat EST databases] were
prioritized based on one or more functional genomic methodologies. For
example, genome-wide expression studies of rice plants infected with rice
blast fungus (Magnaporthe grisea) were used to prioritize candidate genes
controlling disease resistance. Full-length and partial cDNAs of rice trait
gene
candidates could then be predicted based on analysis of the rice whole-
genome sequence, and isolated by designing and using primers for PCR
amplification using a commercially available PCR primer-picking program.
Primers were used for PCR amplification of full-length or partial cDNAs from
rice cDNA libraries or first-strand cDNA. cDNA clones resulting from either
approach were used for the construction of vectors designed for altering
expression of these genes in transgenic plants using plant molecular genetic
methodologies, which are described in detail below. Alteration of plant
phenotype through overexpression or underexpression of key trait genes in
transgenic plants is a robust and established method for assigning functions


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to plant genes. Assays to identify transgenic plants with alterations in
traits of
interest are to be used to unambiguously assign the utility of these genes for
the improvement of rice, and by extension, other cereals, either by transgenic
or classical breeding methods.
II. Identifying, Cloning and Sequencing cDNAs
The cloning and sequencing of the cDNAs of the present invention are
described in Example 1.
The isolated nucleic acids and proteins of the present invention are
usable over a range of plants, monocots and dicots, in particular monocots
such as rice, wheat, barley and maize. In a more specific embodiment, the
monocot is a cereal. In a more specific embodiment, the cereal may be, for
example, maize, wheat, barley, oats, rye, millet, sorghum, triticale, secale,
einkorn, spelt, emmer, teff, milo, flax, gramma grass, Tripsacum sp., or
teosinte. In a most specific embodiment, the cereal is rice. Other plants
genera include, but are not limited to, Cucurbita, Rosa, Vitis, Juglans,
Gragaria, Lotus, Medicago, Onobrychis, Trigonella, Vigna, Citrus, Linum,
Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis,
Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum,
Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus,
Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum,
Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia,
Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium,
and Triticum.
The present invention also provides a method of genotyping a plant or
plant part comprising a nucleic acid molecule of the present invention.
Optionally, the plant is a monocot such as, but not limited rice or wheat.
Genotyping provides a means of distinguishing homologs of a chromosome
pari and can be used to differentiate segregants in a plant population.
Molecular marker methods can be used in phylogenetic studies,
characterizing genetic relationships among crop varieties, identifying crosses
or somatic hybrids, localizing chromosomeal segments affecting mongenic
traits, map based cloning, and the study of quantitative inheritance (see
Plant


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Molecular Biology: A Laboratory Manual, Chapter 7, Clark ed., Springer-
Verlag, Berlin 1997; Paterson, A.H., "The DNA Revolution", chapter 2 in
Genome Mapping in Plants, Paterson, A.H. ed., Academic Press/R.G. Lands
Co., Austin, Texas 1996).
The method of genotyping may employ any number of molecular
marker analytical techniques such as, but not limited to, restriction length
polymorphisms (RFLPs). As is well known in the art, RFLPs are produced by
differences in the DNA restriction fragment lengths resulting from nucleotide
differences between alleles of the same gene. Thus, the present invention
provides a method of following segregation of a gene or nucleic acid of the
present invention or chromosomal sequences genetically linked by using
RFLP analysis. Linked chromosomal sequences are within 50 centiMorgans
(50 cM), within 40 or 30 cM, specifically within 20 or 10 cM, more
specifically
within 5, 3, 2, or 1 cM of the nucleic acid of the invention.
Ill. Traits of Interest
The present invention encompasses the identification and isolation of
polynucleotides encoding proteins involved in sugar sensing and, ultimately,
in nitrogen uptake and carbon metabolism. Altering the expression of genes
related to these traits can be used to improve or modify plants and/or grain,
as
desired. Examples describe the isolated genes of interest and methods of
analyzing the alteration of expression and their effects on the plant
characteristics.
One aspect of the present invention provides compositions and methods
for altering (i.e. increasing or decreasing) the level of nucleic acid
molecules
and polypeptides of the present invention in plants. In particular, the
nucleic
acid molecules and polypeptides of the invention are expressed constitutively,
temporally or spatially, e.g. at developmental stages, in certain tissues,
and/or
quantities, which are uncharacteristic of non-recombinantly engineered plants.
Therefore, the present invention provides utility in such exemplary
applications as altering the specified characteristics identified above.
VI. Controlling Gene Expression in Transgenic Plants


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The invention further relates to transformed cells comprising the
nucleic acid molecules, transformed plants, seeds, and plant parts, and
methods of modifying phenotypic traits of interest by altering the expression
of
the genes of the invention.
A. Modification of Coding Sequences and Adjacent Sequences
The transgenic expression in plants of genes derived from
heterologous sources may involve the modification of those genes to achieve
and optimize their expression in plants. In particular, bacterial ORFs which
encode separate enzymes but which are encoded by the same transcript in
the native microbe are best expressed in plants on separate transcripts. To
achieve this, each microbial ORF is isolated individually and cloned within a
cassette which provides a plant promoter sequence at the 5' end of the ORF
and a plant transcriptional terminator at the 3' end of the ORF. The isolated
ORF sequence specifically includes the initiating ATG codon and the
terminating STOP codon but may include additional sequence beyond the
initiating ATG and the STOP codon. In addition, the ORF may be truncated,
but still retain the required activity; for particularly long ORFs, truncated
versions which retain activity may be preferable for expression in transgenic
organisms. By "plant promoter" and "plant transcriptional terminator" it is
intended to mean promoters and transcriptional terminators that operate
within plant cells. This includes promoters and transcription terminators that
may be derived from non-plant sources such as viruses (an example is the
Cauliflower Mosaic Virus).
In some cases, modification to the ORF coding sequences and
adjacent sequence is not required. It is sufficient to isolate a fragment
containing the ORF of interest and to insert it downstream of a plant
promoter.
For example, Gaffney et al. (Science 261: 754-756 (1993)) have expressed
the Pseudomonas nahG gene in transgenic plants under the control of the
CaMV 35S promoter and the CaMV tml terminator successfully without
modification of the coding sequence and with nucleotides of the
Pseudomonas gene upstream of the ATG still attached, and nucleotides
downstream of the STOP codon still attached to the nahG ORF. Specifically,


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as little adjacent microbial sequence as possible should be left attached
upstream of the ATG and downstream of the STOP codon. In practice, such
construction may depend on the availability of restriction sites.
In other cases, the expression of genes derived from microbial sources
may provide problems in expression. These problems have been well
characterized in the art and are particularly common with genes derived from
certain sources such as Bacillus. These problems may apply to the
nucleotide sequence of this invention and the modification of these genes can
be undertaken using techniques now well known in the art. The following
problems may be encountered:
1. Codon Usage.
The specific codon usage in plants differs from the specific codon
usage in certain microorganisms. Comparison of the usage of codons within
a cloned microbial ORF to usage in plant genes (and in particular genes from
the target plant) will enable an identification of the codons within the ORF
that
should specifically be changed. Typically plant evolution has tended towards
a strong preference of the nucleotides C and G in the third base position of
monocotyledons, whereas dicotyledons often use the nucleotides A or T at
this position. By modifying a gene to incorporate specific codon usage for a
particular target transgenic species, many of the problems described below
for GC/AT content and illegitimate splicing will be overcome.
2. GC/AT Content.
Plant genes typically have a GC content of more than 35%. ORF
sequences which are rich in A and T nucleotides can cause several problems
in plants. Firstly, motifs of ATTTA are believed to cause destabilization of
messages and are found at the 3' end of many short-lived mRNAs. Secondly,
the occurrence of polyadenylation signals such as AATAAA at inappropriate
positions within the message is believed to cause premature truncation of
transcription. In addition, monocotyledons may recognize AT-rich sequences
as splice sites (see below).
3. Sequences Adjacent to the Initiating Methionine.


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Plants differ from microorganisms in that their messages do not
possess a defined ribosome-binding site. Rather, it is believed that ribosomes
attach to the 5' end of the message and scan for the first available ATG at
which to start translation. Nevertheless, it is believed that there is a
preference for certain nucleotides adjacent to the ATG and that expression of
microbial genes can be enhanced by the inclusion of a eukaryotic consensus
translation initiator at the ATG. Clontech (1993/1994 catalog, page 210,
incorporated herein by reference) have suggested one sequence as a
consensus translation initiator for the expression of the E. coli uidA gene in
plants. Further, Joshi (N.A.R. 15: 6643-6653 (1987), incorporated herein by
reference) has compared many plant sequences adjacent to the ATG and
suggests another consensus sequence. In situations where difficulties are
encountered in the expression of microbial ORFs in plants, inclusion of one of
these sequences at the initiating ATG may improve translation. In such cases
the last three nucleotides of the consensus may not be appropriate for
inclusion in the modified sequence due to their modification of the second AA
residue. Specific sequences adjacent to the initiating methionine may differ
between different plant species. A survey of 14 maize genes located in the
GenBank database provided the following results:
Position Before the Initiating ATG in 14 Maize Genes:
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1
C 3 8 4 6 2 5 6 0 10 7
T 3 0 3 4 3 2 1 1 1 0
A 2 3 1 4 3 2 3 7 2 3
G 6 3 6 0 6 5 4 6 1 5

This analysis can be done for the desired plant species into which the
nucleotide sequence is being incorporated, and the sequence adjacent to the
ATG modified to incorporate the specific nucleotides.
4. Removal of Illegitimate Splice Sites.
Genes cloned from non-plant sources and not optimized for expression
in plants may also contain motifs which may be recognized in plants as 5' or


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3' splice sites, and be cleaved, thus generating truncated or deleted
messages. These sites can be removed using the techniques well known in
the art.
Techniques for the modification of coding sequences and adjacent
sequences are well known in the art. In cases where the initial expression of
a microbial ORF is low and it is deemed appropriate to make alterations to the
sequence as described above, then the construction of synthetic genes can
be accomplished according to methods well known in the art. These are, for
example, described in the published patent disclosures EP 0 385 962 (to
Monsanto), EP 0 359 472 (to Lubrizol) and WO 93/07278 (to Ciba-Geigy), all
of which are incorporated herein by reference. In most cases it is preferable
to assay the expression of gene constructions using transient assay protocols
(which are well known in the art) prior to their transfer to transgenic
plants.
B. Construction of Plant Expression Cassettes
Coding sequences intended for expression in transgenic plants are first
assembled in expression cassettes behind a suitable promoter expressible in
plants. The expression cassettes may also comprise any further sequences
required or selected for the expression of the transgene. Such sequences
include, but are not restricted to, transcription terminators, extraneous
sequences to enhance expression such as introns, vital sequences, and
sequences intended for the targeting of the gene product to specific
organelles and cell compartments. These expression cassettes can then be
easily transferred to the plant transformation vectors described below. The
following is a description of various components of typical expression
cassettes.
1. Promoters
The selection of the promoter used in expression cassettes will
determine the spatial and temporal expression pattern of the transgene in the
transgenic plant. Selected promoters will express transgenes in specific cell
types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in
specific tissues or organs (roots, leaves or flowers, for example) and the
selection will reflect the desired location of accumulation of the gene
product.


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Alternatively, the selected promoter may drive expression of the gene under
various inducing conditions. Promoters vary in their strength, i.e., ability
to
promote transcription. Depending upon the host cell system utilized, any one
of a number of suitable promoters can be used, including the gene's native
promoter. The following are non-limiting examples of promoters that may be
used in expression cassettes.
a. Constitutive Expression, the Ubiquitin Promoter:
Ubiquitin is a gene product known to accumulate in many cell types
and its promoter has been cloned from several species for use in transgenic
plants (e.g. sunflower - Binet et al. Plant Science 79: 87-94 (1991); maize -
Christensen et al. Plant Molec. Biol. 12: 619-632 (1989); and Arabidopsis -
Callis et al., J. Biol. Chem. 265:12486-12493 (1990) and Norris et al., Plant
Mol. Biol. 21:895-906 (1993)). The maize ubiquitin promoter has been
developed in transgenic monocot systems and its sequence and vectors
constructed for monocot transformation are disclosed in the patent publication
EP 0 342 926 (to Lubrizol) which is herein incorporated by reference. Taylor
et al. (Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) that
comprises the maize ubiquitin promoter and first intron and its high activity
in
cell suspensions of numerous monocotyledons when introduced via
microprojectile bombardment. The Arabidopsis ubiquitin promoter is ideal for
use with the nucleotide sequences of the present invention. The ubiquitin
promoter is suitable for gene expression in transgenic plants, both
monocotyledons and dicotyledons. Suitable vectors are derivatives of
pAHC25 or any of the transformation vectors described in this application,
modified by the introduction of the appropriate ubiquitin promoter and/or
intron
sequences.
b. Constitutive Expression, the CaMV 35S Promoter:
Construction of the plasmid pCGN1761 is described in the published
patent application EP 0 392 225 (Example 23), which is hereby incorporated
by reference. pCGN1761 contains the "double" CaMV 35S promoter and the
tml transcriptional terminator with a unique EcoRl site between the promoter
and the terminator and has a pUC-type backbone. A derivative of pCGN1761


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is constructed which has a modified polylinker which includes Notl and Xhol
sites in addition to the existing EcoRl site. This derivative is designated
pCGN 1761 ENX. pCGN 1761 ENX is useful for the cloning of cDNA sequences
or coding sequences (including microbial ORF sequences) within its polylinker
for the purpose of their expression under the control of the 35S promoter in
transgenic plants. The entire 35S promoter-coding sequence-tml terminator
cassette of such a construction can be excised by Hindlil, Sphl, Sall, and
Xbal sites 5' to the promoter and Xbal, BamHl and BgII sites 3' to the
terminator for transfer to transformation vectors such as those described
below. Furthermore, the double 35S promoter fragment can be removed by 5'
excision with Hindlll, Sphl, Sall, Xbal, or Pstl, and 3' excision with any of
the
polylinker restriction sites (EcoRl, Notl or Xhol) for replacement with
another
promoter. If desired, modifications around the cloning sites can be made by
the introduction of sequences that may enhance translation. This is
particularly useful when overexpression is desired. For example,
pCGN1761ENX may be modified by optimization of the translational initiation
site as described in Example 37 of U.S. Patent No. 5,639,949, incorporated
herein by reference.
c. Constitutive Expression, the Actin Promoter:
Several isoforms of actin are known to be expressed in most cell types
and consequently the actin promoter is a good choice for a constitutive
promoter. In particular, the promoter from the rice Actl gene has been cloned
and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)). A 1.3kb
fragment of the promoter was found to contain all the regulatory elements
required for expression in rice protoplasts. Furthermore, numerous
expression vectors based on the Actl promoter have been constructed
specifically for use in monocotyledons (McElroy et al. Mol. Gen. Genet. 231:
150-160 (1991)). These incorporate the Actl-intron 1, Adhl 5' flanking
sequence and Adhi-intron 1(from the maize alcohol dehydrogenase gene)
and sequence from the CaMV 35S promoter. Vectors showing highest
expression were fusions of 35S and Actl intron or the Actl 5' flanking
sequence and the Actl intron. Optimization of sequences around the initiating


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ATG (of the GUS reporter gene) also enhanced expression. The promoter
expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-
160 (1991)) can be easily modified for gene expression and are particularly
suitable for use in monocotyledonous hosts. For example, promoter-
containing fragments is removed from the McElroy constructions and used to
replace the double 35S promoter in pCGN1761ENX, which is then available
for the insertion of specific gene sequences. The fusion genes thus
constructed can then be transferred to appropriate transformation vectors. In
a separate report, the rice Actl promoter with its first intron has also been
found to direct high expression in cultured barley cells (Chibbar et al. Plant
Cell Rep. 12: 506-509 (1993)).
d. Inducible Expression, PR-1 Promoters:
The double 35S promoter in pCGN1761 ENX may be replaced with any
other promoter of choice that will result in suitably high expression levels.
By
way of example, one of the chemically regulatable promoters described in
U.S. Patent No. 5,614,395, such as the tobacco PR-1a promoter, may replace
the double 35S promoter. Alternately, the Arabidopsis PR-1 promoter
described in Lebel et al., Plant J. 16:223-233 (1998) may be used. The
promoter of choice is specifically excised from its source by restriction
enzymes, but can alternatively be PCR-amplified using primers that carry
appropriate terminal restriction sites. Should PCR-amplification be
undertaken, the promoter should be re-sequenced to check for amplification
errors after the cloning of the amplified promoter in the target vector. The
chemically/pathogen regulatable tobacco PR-la promoter is cleaved from
plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104, which
is hereby incorporated by reference) and transferred to plasmid
pCGN1761ENX (Uknes et al., Plant Cell 4: 645-656 (1992)). pCIB1004 is
cleaved with Ncol and the resultant 3' overhang of the linearized fragment is
rendered blunt by treatment with T4 DNA polymerase. The fragment is then
cleaved with Hindlll and the resultant PR-la promoter-containing fragment is
gel purified and cloned into pCGN1761ENX from which the double 35S
promoter has been removed. This is accomplished by cleavage with Xhol


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and blunting with T4 polymerase, followed by cleavage with Hindlll, and
isolation of the larger vector-terminator containing fragment into which the
pCIB1004 promoter fragment is cloned. This generates a pCGN1761ENX
derivative with the PR-la promoter and the tml terminator and an intervening
polylinker with unique EcoRl and Notl sites. The selected coding sequence
can be inserted into this vector, and the fusion products (i.e. promoter-gene-
terminator) can subsequently be transferred to any selected transformation
vector, including those described infra. Various chemical regulators may be
employed to induce expression of the selected coding sequence in the plants
transformed according to the present invention, including the
benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in
U.S. Patent Nos. 5,523,311 and 5,614,395.
e. Inducible Expression, an Ethanol-Inducible Promoter:
A promoter inducible by certain alcohols or ketones, such as ethanol,
may also be used to confer inducible expression of a coding sequence of the
present invention. Such a promoter is for example the alcA gene promoter
from Aspergillus nidulans (Caddick et al. (1998) Nat. Biotechnol 16:177-180).
In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, the
expression of which is regulated by the AIcR transcription factors in presence
of the chemical inducer. For the purposes of the present invention, the CAT
coding sequences in plasmid palcA:CAT comprising a alcA gene promoter
sequence fused to a minimal 35S promoter (Caddick et al. (1998) Nat.
Biotechnol 16:177-180) are replaced by a coding sequence of the present
invention to form an expression cassette having the coding sequence under
the control of the alcA gene promoter. This is carried out using methods well
known in the art.
f. Inducible Expression, a Glucocorticoid-Inducible Promoter:
Induction of expression of a nucleic acid sequence of the present
invention using systems based on steroid hormones is also contemplated.
For example, a glucocorticoid-mediated induction system is used (Aoyama
and Chua (1997) The Plant Journal 11: 605-612) and gene expression is
induced by application of a glucocorticoid, for example a synthetic


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glucocorticoid, specifically dexamethasone, specifically at a concentration
ranging from 0.1 mM to 1mM, more specifically from 10mM to 100mM. For the
purposes of the present invention, the luciferase gene sequences are
replaced by a nucleic acid sequence of the invention to form an expression
cassette having a nucleic acid sequence of the invention under the control of
six copies of the GAL4 upstream activating sequences fused to the 35S
minimal promoter. This is carried out using methods well known in the art.
The trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et
al. (1986) Science 231: 699-704) fused to the transactivating domain of the
herpes viral protein VP16 (Triezenberg et al. (1988) Genes Devel. 2: 718-729)
fused to the hormone-binding domain of the rat glucocorticoid receptor (Picard
et al. (1988) Cell 54: 1073-1080). The expression of the fusion protein is
controlled either by a promoter known in the art or described here. This
expression cassette is also comprised in the plant comprising a nucleic acid
sequence of the invention fused to the 6xGAL4/minimal promoter. Thus,
tissue- or organ-specificity of the fusion protein is achieved leading to
inducible tissue- or organ-specificity of the insecticidal toxin.
g. Root Specific Expression:
Another pattern of gene expression is root expression. A suitable root
promoter is the promoter of the maize metallothionein-like (MTL) gene
described by de Framond (FEBS 290: 103-106 (1991)) and also in U.S.
Patent No. 5,466,785, incorporated herein by reference. This "MTL" promoter
is transferred to a suitable vector such as pCGN1761 ENX for the insertion of
a selected gene and subsequent transfer of the entire promoter-gene-
terminator cassette to a transformation vector of interest.
h. Wound-Inducible Promoters:
Wound-inducible promoters may also be suitable for gene expression.
Numerous such promoters have been described (e.g. Xu et al. Plant Molec.
Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158 (1989),
Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant
Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191-201 (1993)) and
all are suitable for use with the instant invention. Logemann et al. describe


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the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu et al.
show that a wound-inducible promoter from the dicotyledon potato (pin2) is
active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the
cloning of the maize Wipl cDNA which is wound induced and which can be
used to isolate the cognate promoter using standard techniques. Similar,
Firek et al. and Warner et al. have described a wound-induced gene from the
monocotyledon Asparagus officinalis, which is expressed at local wound and
pathogen invasion sites. Using cloning techniques well known in the art,
these promoters can be transferred to suitable vectors, fused to the genes
pertaining to this invention, and used to express these genes at the sites of
plant wounding.
i. Pith-Specific Expression:
Patent Application WO 93/07278, which is herein incorporated by
reference, describes the isolation of the maize trpA gene, which is
preferentially expressed in pith cells. The gene sequence and promoter
extending up to -1726 bp from the start of transcription are presented. Using
standard molecular biological techniques, this promoter, or parts thereof, can
be transferred to a vector such as pCGN1761 where it can replace the 35S
promoter and be used to drive the expression of a foreign gene in a pith-
specific manner. In fact, fragments containing the pith-specific promoter or
parts thereof can be transferred to any vector and modified for utility in
transgenic plants.
j. Leaf-Specific Expression:
A maize gene encoding phosphoenol carboxylase (PEPC) has been
described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using
standard molecular biological techniques the promoter for this gene can be
used to drive the expression of any gene in a leaf-specific manner in
transgenic plants.
k. Pollen-Specific Expression:
WO 93/07278 describes the isolation of the maize calcium-dependent
protein kinase (CDPK) gene which is expressed in pollen cells. The gene
sequence and promoter extend up to 1400 bp from the start of transcription.


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Using standard molecular biological techniques, this promoter or parts
thereof, can be transferred to a vector such as pCGN1761 where it can
replace the 35S promoter and be used to drive the expression of a nucleic
acid sequence of the invention in a pollen-specific manner.
2. Transcriptional Terminators
A variety of transcriptional terminators are available for use in
expression cassettes. These are responsible for the termination of
transcription beyond the transgene and correct mRNA polyadenylation.
Appropriate transcriptional terminators are those that are known to function
in
plants and include the CaMV 35S terminator, the tml terminator, the nopaline
synthase terminator and the pea rbcS E9 terminator. These can be used in
both monocotyledons and dicotyledons. In addition, a gene's native
transcription terminator may be used.
3. Sequences for the Enhancement or Regulation of Expression
Numerous sequences have been found to enhance gene expression
from within the transcriptional unit and these sequences can be used in
conjunction with the genes of this invention to increase their expression in
transgenic plants.
Various intron sequences have been shown to enhance expression,
particularly in monocotyledonous cells. For example, the introns of the maize
Adhl gene have been found to significantly enhance the expression of the
wild-type gene under its cognate promoter when introduced into maize cells.
Intron 1 was found to be particularly effective and enhanced expression in
fusion constructs with the chloramphenicol acetyltransferase gene (Callis et
al., Genes Develop. 1: 1183-1200 (1987)). In the same experimental system,
the intron from the maize bronzel gene had a similar effect in enhancing
expression. Intron sequences have been routinely incorporated into plant
transformation vectors, typically within the non-translated leader.
A number of non-translated leader sequences derived from viruses are
also known to enhance expression, and these are particularly effective in
dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic
Virus (TMV, the "W-sequence"), Maize Chlorotic Mottle Virus (MCMV), and


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Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing
expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski
et al. Plant Molec. Biol. 15: 65-79 (1990)). Other leader sequences known in
the art include but are not limited to: picornavirus leaders, for example,
EMCV
leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, 0., Fuerst, T.
R., and Moss, B. PNAS USA 86:6126-6130 (1989)); potyvirus leaders, for
example, TEV leader (Tobacco Etch Virus) (Allison et al., 1986); MDMV
leader (Maize Dwarf Mosaic Virus); Virology 154:9-20); human
immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak, D. G.,
and Sarnow, P., Nature 353: 90-94 (1991); untranslated leader from the coat
protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and
Gehrke, L., Nature 325:622-625 (1987); tobacco mosaic virus leader (TMV),
(Gallie, D. R. et al., Molecular Biology of RNA, pages 237-256 (1989); and
Maize Chlorotic Mottle Virus leader (MCMV) (Lommel, S. A. et al., Virology
81:382-385 (1991). See also, Della-Cioppa et al., Plant Physiology 84:965-
968 (1987).
In addition to incorporating one or more of the aforementioned
elements into the 5' regulatory region of a target expression cassette of the
invention, other elements peculiar to the target expression cassette may also
be incorporated. Such elements include but are not limited to a minimal
promoter. By minimal promoter it is intended that the basal promoter elements
are inactive or nearly so without upstream activation. Such a promoter has
low background activity in plants when there is no transactivator present or
when enhancer or response element binding sites are absent. One minimal
promoter that is particularly useful for target genes in plants is the Bzl
minimal promoter, which is obtained from the bronzel gene of maize. The
Bzl core promoter is obtained from the "myc" mutant Bzl-luciferase construct
pBzlLucR98 via cleavage at the Nhel site located at -53 to -58. Roth et al.,
Plant Cell 3: 317 (1991). The derived Bzl core promoter fragment thus
extends from -53 to +227 and includes the Bzl intron-1 in the 5' untranslated
region. Also useful for the invention is a minimal promoter created by use of
a
synthetic TATA element. The TATA element allows recognition of the


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promoter by RNA polymerase factors and confers a basal level of gene
expression in the absence of activation (see generally, Mukumoto (1993)
Plant Mol Biol 23: 995-1003; Green (2000) Trends Biochem Sci 25: 59-63)
4. Targeting of the Gene Product Within the Cell
Various mechanisms for targeting gene products are known to exist in
plants and the sequences controlling the functioning of these mechanisms
have been characterized in some detail. For example, the targeting of gene
products to the chloroplast is controlled by a signal sequence found at the
amino terminal end of various proteins which is cleaved during chloroplast
import to yield the mature protein (e.g. Comai et al. J. Biol. Chem. 263:
15104-15109 (1988)). These signal sequences can be fused to heterologous
gene products to effect the import of heterologous products into the
chioroplast (van den Broeck, et al. Nature 313: 358-363 (1985)). DNA
encoding for appropriate signal sequences can be isolated from the 5' end of
the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP
synthase enzyme, the GS2 protein and many other proteins which are known
to be chloroplast localized. See also, the section entitled "Expression With
Chloroplast Targeting" in Example 37 of U.S. Patent No. 5,639,949.
Other gene products are localized to other organelles such as the
mitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol. 13:
411-418 (1989)). The cDNAs encoding these products can also be
manipulated to effect the targeting of heterologous gene products to these
organelles. Examples of such sequences are the nuclear-encoded ATPases
and specific aspartate amino transferase isoforms for mitochondria. Targeting
cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad.
Sci. USA 82: 6512-6516 (1985)).
In addition, sequences have been characterized which cause the
targeting of gene products to other cell compartments. Amino terminal
sequences are responsible for targeting to the ER, the apoplast, and
extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-
783 (1990)). Additionally, amino terminal sequences in conjunction with


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carboxy terminal sequences are responsible for vacuolar targeting of gene
products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).
By the fusion of the appropriate targeting sequences described above
to transgene sequences of interest it is possible to direct the transgene
product to any organelle or cell compartment. For chloroplast targeting, for
example, the chloroplast signal sequence from the RUBISCO gene, the CAB
gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the
amino terminal ATG of the transgene. The signal sequence selected should
include the known cleavage site, and the fusion constructed should take into
account any amino acids after the cleavage site which are required for
cleavage. In some cases this requirement may be fulfilled by the addition of a
small number of amino acids between the cleavage site and the transgene
ATG or, alternatively, replacement of some amino acids within the transgene
sequence. Fusions constructed for chloroplast import can be tested for
efficacy of chloroplast uptake by in vitro translation of in vitro transcribed
constructions followed by in vitro chloroplast uptake using techniques
described by Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast
Molecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann et al. Mol.
Gen. Genet. 205: 446-453 (1986). These construction techniques are well
known in the art and are equally applicable to mitochondria and peroxisomes.
The above-described mechanisms for cellular targeting can be utilized
not only in conjunction with their cognate promoters, but also in conjunction
with heterologous promoters so as to effect a specific cell-targeting goal
under
the transcriptional regulation of a promoter that has an expression pattern
different to that of the promoter from which the targeting signal derives.
C. Construction of Plant Transformation Vectors
Numerous transformation vectors available for plant transformation are
known to those of ordinary skill in the plant transformation arts, and the
genes
pertinent to this invention can be used in conjunction with any such vectors.
The selection of vector will depend upon the specific transformation technique
and the target species for transformation. For certain target species,
different
antibiotic or herbicide selection markers may be specific. Selection markers


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used routinely in transformation include the nptll gene, which confers
resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19:
259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene,
which confers resistance to the herbicide phosphinothricin (White et al.,
Nucl.
Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl. Genet 79: 625-631
(1990)), the hph gene, which confers resistance to the antibiotic hygromycin
(Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene,
which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7):
1099-1104 (1983)), the EPSPS gene, which confers resistance to glyphosate
(U.S. Patent Nos. 4,940,935 and 5,188,642), and the mannose-6-phosphate
isomerase gene, which provides the ability to metabolize mannose (U.S.
Patent Nos. 5,767,378 and 5,994,629).
1. Vectors Suitable for Agrobacterium Transformation
Many vectors are available for transformation using Agrobacterium
tumefaciens. These typically carry at least one T-DNA border sequence and
include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)). Below, the
construction of two typical vectors suitable for Agrobacterium transformation
is
described.
a. pCIB200 and pCIB2001:
The binary vectors pCIB200 and pCIB2001 are used for the
construction of recombinant vectors for use with Agrobacterium and are
constructed in the following manner. pTJS75kan is created by Narl digestion
of pTJS75 (Schmidhauser & Helinski, J. Bacteriol. 164: 446-455 (1985))
allowing excision of the tetracycline-resistance gene, followed by insertion
of
an Accl fragment from pUC4K carrying an NPTII (Messing & Vierra, Gene 19:
259-268 (1982): Bevan et al., Nature 304: 184-187 (1983): McBride et al.,
Plant Molecular Biology 14: 266-276 (1990)). Xhol linkers are ligated to the
EcoRV fragment of PCIB7 which contains the left and right T-DNA borders, a
plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et
al., Gene 53: 153-161 (1987)), and the Xhol-digested fragment are cloned into
Sall-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example
19). pCIB200 contains the following unique polylinker restriction sites:
EcoRl,


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Sstl, Kpnl, BgIlI, Xbal, and Sall. pCIB2001 is a derivative of pCIB200 created
by the insertion into the polylinker of additional restriction sites. Unique
restriction sites in the polylinker of pCIB2001 are EcoRl, Sstl, Kpnl, Bglll,
Xbal, Sall, Mlul, Bcll, Avrll, Apal, Hpal, and Stul. pCIB2001, in addition to
containing these unique restriction sites also has plant and bacterial
kanamycin selection, left and right T-DNA borders for Agrobacterium-
mediated transformation, the RK2-derived trfA function for mobilization
between E. coli and other hosts, and the OriT and OriV functions also from
RK2. The pCIB2001 polylinker is suitable for the cloning of plant expression
cassettes containing their own regulatory signals.
b. pCIB10 and Hygromycin Selection Derivatives thereof:
The binary vector pCIB10 contains a gene encoding kanamycin
resistance for selection in plants and T-DNA right and left border sequences
and incorporates sequences from the wide host-range plasmid pRK252
allowing it to replicate in both E. coli and Agrobacterium. Its construction
is
described by Rothstein et al. (Gene 53: 153-161 (1987)). Various derivatives
of pCIB10 are constructed which incorporate the gene for hygromycin B
phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)).
These derivatives enable selection of transgenic plant cells on hygromycin
only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).
2. Vectors Suitable for non-Agrobacterium Transformation
Transformation without the use of Agrobacterium tumefaciens
circumvents the requirement for T-DNA sequences in the chosen
transformation vector and consequently vectors lacking these sequences can
be utilized in addition to vectors such as the ones described above which
contain T-DNA sequences. Transformation techniques that do not rely on
Agrobacterium include transformation via particle bombardment, protoplast
uptake (e.g. PEG and electroporation) and microinjection. The choice of
vector depends largely on the specific selection for the species being
transformed. Below, the construction of typical vectors suitable for non-
Agrobacterium transformation is described.
a. pCIB3064:


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pCIB3064 is a pUC-derived vector suitable for direct gene transfer
techniques in combination with selection by the herbicide basta (or
phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter
in operational fusion to the E. coli GUS gene and the CaMV 35S
transcriptional terminator and is described in the PCT published application
WO 93/07278. The 35S promoter of this vector contains two ATG sequences
5' of the start site. These sites are mutated using standard PCR techniques in
such a way as to remove the ATGs and generate the restriction sites Sspl and
Pvull. The new restriction sites are 96 and 37 bp away from the unique Sall
site and 101 and 42 bp away from the actual start site. The resultant
derivative of pCIB246 is designated pCIB3025. The GUS gene is then
excised from pCIB3025 by digestion with Sall and Sacl, the termini rendered
blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is
obtained from the John Innes Centre, Norwich and the a 400 bp Smal
fragment containing the bar gene from Streptomyces viridochromogenes is
excised and inserted into the Hpal site of pCIB3060 (Thompson et al. EMBO J
6: 2519-2523 (1987)). This generated pCIB3064, which comprises the bar
gene under the control of the CaMV 35S promoter and terminator for
herbicide selection, a gene for ampicillin resistance (for selection in E.
coli)
and a polylinker with the unique sites Sphl, Pstl, Hindill, and BamHl. This
vector is suitable for the cloning of plant expression cassettes containing
their
own regulatory signals.
b. pSOG19 and pSOG35:
pSOG35 is a transformation vector that utilizes the E. coli gene
dihydrofolate reductase (DFR) as a selectable marker conferring resistance to
methotrexate. PCR is used to amplify the 35S promoter (-800 bp), intron 6
from the maize Adh1 gene (-550 bp) and 18 bp of the GUS untransiated
leader sequence from pSOG10. A 250-bp fragment encoding the E. coli
dihydrofolate reductase type II gene is also amplified by PCR and these two
PCR fragments are assembled with a Sacl-Pstl fragment from pB1221
(Clontech) which comprises the pUC19 vector backbone and the nopaline
synthase terminator. Assembly of these fragments generates pSOG19 which


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contains the 35S promoter in fusion with the intron 6 sequence, the GUS
leader, the DHFR gene and the nopaline synthase terminator. Replacement
of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic
Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35
carry the pUC gene for ampicillin resistance and have Hindill, Sphl, Pstl and
EcoRl sites available for the cloning of foreign substances.
3. Vector Suitable for Chloroplast Transformation
For expression of a nucleotide sequence of the present invention in
plant plastids, plastid transformation vector pPH143 (WO 97/32011, example
36) is used. The nucleotide sequence is inserted into pPH143 thereby
replacing the PROTOX coding sequence. This vector is then used for plastid
transformation and selection of transformants for spectinomycin resistance.
Alternatively, the nucleotide sequence is inserted in pPH143 so that it
replaces the aadH gene. In this case, transformants are selected for
resistance to PROTOX inhibitors.
D. Transformation
Once a nucleic acid sequence of the invention has been cloned into an
expression system, it is transformed into a plant cell. The receptor and
target
expression cassettes of the present invention can be introduced into the plant
cell in a number of art-recognized ways. Methods for regeneration of plants
are also well known in the art. For example, Ti plasmid vectors have been
utilized for the delivery of foreign DNA, as well as direct DNA uptake,
liposomes, electroporation, microinjection, and microprojectiles. In addition,
bacteria from the genus Agrobacterium can be utilized to transform plant
cells.
Below are descriptions of representative techniques for transforming both
dicotyledonous and monocotyledonous plants, as well as a representative
plastid transformation technique.
1. Transformation of Dicotyledons
Transformation techniques for dicotyledons are well known in the art
and include Agrobacterium-based techniques and techniques that do not
require Agrobacterium. Non-Agrobacterium techniques involve the uptake of
exogenous genetic material directly by protoplasts or cells. This can be


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accomplished by PEG or electroporation mediated uptake, particle
bombardment-mediated delivery, or microinjection. Examples of these
techniques are described by Paszkowski et al., EMBO J 3: 2717-2722 (1984),
Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al.,
Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327: 70-73
(1987). In each case the transformed cells are regenerated to whole plants
using standard techniques known in the art.
Agrobacterium-mediated transformation is a specific technique for
transformation of dicotyledons because of its high efficiency of
transformation
and its broad utility with many different species. Agrobacterium
transformation typically involves the transfer of the binary vector carrying
the
foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate
Agrobacterium strain which may depend of the complement of vir genes
carried by the host Agrobacterium strain either on a co-resident Ti plasmid or
chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al.
Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector
to Agrobacterium is accomplished by a triparental mating procedure using E.
coli carrying the recombinant binary vector, a helper E. coli strain which
carries a plasmid such as pRK2013 and which is able to mobilize the
recombinant binary vector to the target Agrobacterium strain. Alternatively,
the recombinant binary vector can be transferred to Agrobacterium by DNA
transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).
Transformation of the target plant species by recombinant
Agrobacterium usually involves co-cultivation of the Agrobacterium with
explants from the plant and follows protocols well known in the art.
Transformed tissue is regenerated on selectable medium carrying the
antibiotic or herbicide resistance marker present between the binary plasmid
T-DNA borders.
Another approach to transforming plant cells with a gene involves
propelling inert or biologically active particles at plant tissues and cells.
This
technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006, and
5,100,792 all to Sanford et al. Generally, this procedure involves propelling


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inert or biologically active particles at the cells under conditions effective
to
penetrate the outer surface of the cell and afford incorporation within the
interior thereof. When inert particles are utilized, the vector can be
introduced
into the cell by coating the particles with the vector containing the desired
gene. Alternatively, the target cell can be surrounded by the vector so that
the vector is carried into the cell by the wake of the particle. Biologically
active
particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each
containing DNA sought to be introduced) can also be propelled into plant cell
tissue.
2. Transformation of Monocotyledons
Transformation of most monocotyledon species has now also become
routine. Specific techniques include direct gene transfer into protoplasts
using
PEG or electroporation techniques, and particle bombardment into callus
tissue. Transformations can be undertaken with a single DNA species or
multiple DNA species (i.e. co-transformation) and both these techniques are
suitable for use with this invention. Co-transformation may have the
advantage of avoiding complete vector construction and of generating
transgenic plants with unlinked loci for the gene of interest and the
selectable
marker, enabling the removal of the selectable marker in subsequent
generations, should this be regarded desirable. However, a disadvantage of
the use of co-transformation is the less than 100% frequency with which
separate DNA species are integrated into the genome (Schocher et al.
Biotechnology 4: 1093-1096 (1986)).
Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278
describe techniques for the preparation of callus and protoplasts from an
elite
inbred line of maize, transformation of protoplasts using PEG or
electroporation, and the regeneration of maize plants from transformed
protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and Fromm
et al. (Biotechnology 8: 833-839 (1990)) have published techniques for
transformation of A188-derived maize line using particle bombardment.
Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200
(1993)) describe techniques for the transformation of elite inbred lines of


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maize by particle bombardment. This technique utilizes immature maize
embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after
pollination and a PDS-1000He Biolistics device for bombardment.
Transformation of rice can also be undertaken by direct gene transfer
techniques utilizing protoplasts or particle bombardment. Protoplast-mediated
transformation has been described for Japonica-types and Indica-types
(Zhang et al. Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature 338:
274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types
are also routinely transformable using particle bombardment (Christou et al.
Biotechnology 9: 957-962 (1991)). Furthermore, WO 93/21335 describes
techniques for the transformation of rice via electroporation.
Patent Application EP 0 332 581 describes techniques for the
generation, transformation and regeneration of Pooideae protoplasts. These
techniques allow the transformation of Dactylis and wheat. Furthermore,
wheat transformation has been described by Vasil et al. (Biotechnology 10:
667-674 (1992)) using particle bombardment into cells of type C long-term
regenerable callus, and also by Vasil et al. (Biotechnology 11: 1553-1558
(1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle
bombardment of immature embryos and immature embryo-derived callus. A
specific technique for wheat transformation, however, involves the
transformation of wheat by particle bombardment of immature embryos and
includes either a high sucrose or a high maltose step prior to gene delivery.
Prior to bombardment, any number of embryos (0.75-1 mm in length) are
plated onto MS medium with 3% sucrose (Murashiga & Skoog, Physiologia
Plantarum 15: 473-497 (1962)) and 3 mg/I 2,4-D for induction of somatic
embryos, which is allowed to proceed in the dark. On the chosen day of
bombardment, embryos are removed from the induction medium and placed
onto the osmoticum (i.e. induction medium with sucrose or maltose added at
the desired concentration, typically 15%). The embryos are allowed to
plasmolyze for 2-3 hours and are then bombarded. Twenty embryos per
target plate is typical, although not critical. An appropriate gene-carrying
plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size


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gold particles using standard procedures. Each plate of embryos is shot with
the DuPont Biolistics helium device using a burst pressure of -1000 psi
using a standard 80 mesh screen. After bombardment, the embryos are
placed back into the dark to recover for about 24 hours (still on osmoticum).
After 24 hrs, the embryos are removed from the osmoticum and placed back
onto induction medium where they stay for about a month before
regeneration. Approximately one month later the embryo explants with
developing embryogenic callus are transferred to regeneration medium (MS +
1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection
agent (10 mg/I basta in the case of pCIB3064 and 2 mg/I methotrexate in the
case of pSOG35). After approximately one month, developed shoots are
transferred to larger sterile containers known as "GA7s" which contain half-
strength MS, 2% sucrose, and the same concentration of selection agent.
Tranformation of monocotyledons using Agrobacterium has also been
described. See, WO 94/00977 and U.S. Patent No. 5,591,616, both of which
are incorporated herein by reference. See also, Negrotto et al., Plant Cell
Reports 19: 798-803 (2000), incorporated herein by reference. For this
example, rice (Oryza sativa) is used for generating transgenic plants. Various
rice cultivars can be used (Hiei et al., 1994, Plant Journal 6:271-282; Dong
et
al., 1996, Molecular Breeding 2:267-276; Hiei et al., 1997, Plant Molecular
Biology, 35:205-218). Also, the various media constituents described below
may be either varied in quantity or substituted. Embryogenic responses are
initiated and/or cultures are established from mature embryos by culturing on
MS-CIM medium (MS basal salts, 4.3 g/liter; B5 vitamins (200 x), 5 mI/liter;
Sucrose, 30 g/liter; proline, 500 mg/liter; glutamine, 500 mg/liter; casein
hydrolysate, 300 mg/liter; 2,4-D (1 mg/mI), 2 mI/liter; adjust pH to 5.8 with
1 N
KOH; Phytagel, 3 g/Iiter). Either mature embryos at the initial stages of
culture response or established culture lines are inoculated and co-cultivated
with the Agrobacterium tumefaciens strain LBA4404 (Agrobacterium)
containing the desired vector construction. Agrobacterium is cultured from
glycerol stocks on solid YPC medium (100 mg/L spectinomycin and any other
appropriate antibiotic) for -2 days at 28 oC. Agrobacterium is re-suspended


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in liquid MS-CIM medium. The Agrobacterium culture is diluted to an OD600
of 0.2-0.3 and acetosyringone is added to a final concentration of 200 uM.
Acetosyringone is added before mixing the solution with the rice cultures to
induce Agrobacterium for DNA transfer to the plant cells. For inoculation, the
plant cultures are immersed in the bacterial suspension. The liquid bacterial
suspension is removed and the inoculated cultures are placed on co-
cultivation medium and incubated at 22 C for two days. The cultures are then
transferred to MS-CIM medium with Ticarcillin (400 mg/liter) to inhibit the
growth of Agrobacterium. For constructs utilizing the PMI selectable marker
gene (Reed et al., In Vitro Cell. Dev. Biol.-Plant 37:127-132), cultures are
transferred to selection medium containing Mannose as a carbohydrate
source (MS with 2%Mannose, 300 mg/liter Ticarcillin) after 7 days, and
cultured for 3-4 weeks in the dark. Resistant colonies are then transferred to
regeneration induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1 mg/liter
zeatin, 200 mg/liter timentin 2% Mannose and 3% Sorbitol) and grown in the
dark for 14 days. Proliferating colonies are then transferred to another round
of regeneration induction media and moved to the light growth room.
Regenerated shoots are transferred to GA7 containers with GA7-1 medium
(MS with no hormones and 2% Sorbitol) for 2 weeks and then moved to the
greenhouse when they are large enough and have adequate roots. Plants
are transplanted to soil in the greenhouse (TO generation) grown to maturity,
and the T1 seed is harvested.
3. Transformation of Plastids
Seeds of Nicotiana tabacum c.v. 'Xanthi nc' are germinated seven per
plate in a 1" circular array on T agar medium and bombarded 12-14 days after
sowing with 1 pm tungsten particles (M10, Biorad, Hercules, CA) coated with
DNA from plasmids pPH143 and pPH145 essentially as described (Svab, Z.
and Maliga, P. (1993) PNAS 90, 913-917). Bombarded seedlings are
incubated on T medium for two days after which leaves are excised and
placed abaxial side up in bright light (350-500 pmol photons/m2/s) on plates
of RMOP medium (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS
87, 8526-8530) containing 500 pg/mi spectinomycin dihydrochloride (Sigma,


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St. Louis, MO). Resistant shoots appearing underneath the bleached leaves
three to eight weeks after bombardment are subcloned onto the same
selective medium, allowed to form callus, and secondary shoots isolated and
subcloned. Complete segregation of transformed plastid genome copies
(homoplasmicity) in independent subclones is assessed by standard
techniques of Southern blotting (Sambrook et al., (1989) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor).
BamHI/EcoRI-digested total cellular DNA (Mettler, I. J. (1987) Plant Mol Biol
Reporter 5, 346-349) is separated on 1% Tris-borate (TBE) agarose gels,
transferred to nylon membranes (Amersham) and probed with 32P-labeled
random primed DNA sequences corresponding to a 0.7 kb BamHI/Hindlll
DNA fragment from pC8 containing a portion of the rps7/12 plastid targeting
sequence. Homoplasmic shoots are rooted aseptically on spectinomycin-
containing MS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-
7305) and transferred to the greenhouse.
V. Breeding and Seed Production
A. Breeding
The plants obtained via tranformation with a nucleic acid sequence of
the present invention can be any of a wide variety of plant species, including
those of monocots and dicots; however, the plants used in the method of the
invention are specifically selected from the list of agronomically important
target crops set forth supra. The expression of a gene of the present
invention
in combination with other characteristics important for production and quality
can be incorporated into plant lines through breeding. Breeding approaches
and techniques are known in the art. See, for example, Welsh J. R.,
Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY
(1981); Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy
Madison, Wisconsin (1983); Mayo 0., The Theory of Plant Breeding, Second
Edition, Clarendon Press, Oxford (1987); Singh, D.P., Breeding for
Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and
Wricke and Weber, Quantitative Genetics and Selection Plant Breeding,
Walter de Gruyter and Co., Berlin (1986).


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The genetic properties engineered into the transgenic seeds and plants
described above are passed on by sexual reproduction or vegetative growth
and can thus be maintained and propagated in progeny plants. Generally said
maintenance and propagation make use of known agricultural methods
developed to fit specific purposes such as tilling, sowing or harvesting.
Specialized processes such as hydroponics or greenhouse technologies can
also be applied. As the growing crop is vulnerable to attack and damages
caused by insects or infections as well as to competition by weed plants,
measures are undertaken to control weeds, plant diseases, insects,
nematodes, and other adverse conditions to improve yield. These include
mechanical measures such a tillage of the soil or removal of weeds and
infected plants, as well as the application of agrochemicals such as
herbicides, fungicides, gametocides, nematicides, growth regulants, ripening
agents and insecticides.
Use of the advantageous genetic properties of the transgenic plants
and seeds according to the invention can further be made in plant breeding,
which aims at the development of plants with improved properties such as
tolerance of pests, herbicides, or stress, improved nutritional value,
increased
yield, or improved structure causing less loss from lodging or shattering. The
various breeding steps are characterized by well-defined human intervention
such as selecting the lines to be crossed, directing pollination of the
parental
lines, or selecting appropriate progeny plants. Depending on the desired
properties, different breeding measures are taken. The relevant techniques
are well known in the art and include but are not limited to hybridization,
inbreeding, backcross breeding, multiline breeding, variety blend,
interspecific
hybridization, aneuploid techniques, etc. Hybridization techniques also
include the sterilization of plants to yield male or female sterile plants by
mechanical, chemical, or biochemical means. Cross pollination of a male
sterile plant with pollen of a different line assures that the genome of the
male
sterile but female fertile plant will uniformly obtain properties of both
parental
lines. Thus, the transgenic seeds and plants according to the invention can be
used for the breeding of improved plant lines, that for example, increase the


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effectiveness of conventional methods such as herbicide or pesticide
treatment or allow one to dispense with said methods due to their modified
genetic properties. Alternatively new crops with improved stress tolerance can
be obtained, which, due to their optimized genetic "equipment", yield
harvested product of better quality than products that were not able to
tolerate
comparable adverse developmental conditions.
B. Seed Production
In seed production, germination quality and uniformity of seeds are
essential product characteristics. As it is difficult to keep a crop free from
other crop and weed seeds, to control seedborne diseases, and to produce
seed with good germination, fairly extensive and well-defined seed production
practices have been developed by seed producers, who are experienced in
the art of growing, conditioning and marketing of pure seed. Thus, it is
common practice for the farmer to buy certified seed meeting specific quality
standards instead of using seed harvested from his own crop. Propagation
material to be used as seeds is customarily treated with a protectant coating
comprising herbicides, insecticides, fungicides, bactericides, nematicides,
molluscicides, or mixtures thereof. Customarily used protectant coatings
comprise compounds such as captan, carboxin, thiram (TMTD ), methalaxyl
(Apron ), and pirimiphos-methyl (Actellic ). If desired, these compounds are
formulated together with further carriers, surfactants or application-
promoting
adjuvants customarily employed in the art of formulation to provide protection
against damage caused by bacterial, fungal or animal pests. The protectant
coatings may be applied by impregnating propagation material with a liquid
formulation or by coating with a combined wet or dry formulation. Other
methods of application are also possible such as treatment directed at the
buds or the fruit.
VI. Alteration of Expression of Nucleic Acid Molecules
The alteration in expression of the nucleic acid molecules of the
present invention is achieved in one of the following ways:
A. "Sense" Suppression


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Alteration of the expression of a nucleotide sequence of the present
invention, specifically reduction of its expression, is obtained by "sense"
suppression (referenced in e.g. Jorgensen et al. (1996) Plant Mol. Biol. 31,
957-973). In this case, the entirety or a portion of a nucleotide sequence of
the present invention is comprised in a DNA molecule. The DNA molecule is
specifically operatively linked to a promoter functional in a cell comprising
the
target gene, specifically a plant cell, and introduced into the cell, in which
the
nucleotide sequence is expressible. The nucleotide sequence is inserted in
the DNA molecule in the "sense orientation", meaning that the coding strand
of the nucleotide sequence can be transcribed. In a specific embodiment, the
nucleotide sequence is fully translatable and all the genetic information
comprised in the nucleotide sequence, or portion thereof, is translated into a
polypeptide. In another specific embodiment, the nucleotide sequence is
partially translatable and a short peptide is translated. In a specific
embodiment, this is achieved by inserting at least one premature stop codon
in the nucleotide sequence, which bring translation to a halt. In another more
specific embodiment, the nucleotide sequence is transcribed but no
translation product is being made. This is usually achieved by removing the
start codon, e.g. the "ATG", of the polypeptide encoded by the nucleotide
sequence. In a further specific embodiment, the DNA molecule comprising the
nucleotide sequence, or a portion thereof, is stably integrated in the genome
of the plant cell. In another specific embodiment, the DNA molecule
comprising the nucleotide sequence, or a portion thereof, is comprised in an
extrachromosomally replicating molecule.
In transgenic plants containing one of the DNA molecules described
immediately above, the expression of the nucleotide sequence corresponding
to the nucleotide sequence comprised in the DNA molecule is specifically
reduced. Specifically, the nucleotide sequence in the DNA molecule is at
least 70% identical to the nucleotide sequence the expression of which is
reduced, more specifically it is at least 80% identical, yet more specifically
at
least 90% identical, yet more specifically at least 95% identical, yet more
specifically at least 99% identical.


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B. "Anti-sense" Suppression

In another specific embodiment, the alteration of the expression of a
nucleotide sequence of the present invention, specifically the reduction of
its
expression is obtained by "anti-sense" suppression. The entirety or a portion
of a nucleotide sequence of the present invention is comprised in a DNA
molecule. The DNA molecule is specifically operatively linked to a promoter
functional in a plant cell, and introduced in a plant cell, in which the
nucleotide
sequence is expressible. The nucleotide sequence is inserted in the DNA
molecule in the "anti-sense orientation", meaning that the reverse complement
(also called sometimes non-coding strand) of the nucleotide sequence can be
transcribed. In a specific embodiment, the DNA molecule comprising the
nucleotide sequence, or a portion thereof, is stably integrated in the genome
of the plant cell. In another specific embodiment the DNA molecule
comprising the nucleotide sequence, or a portion thereof, is comprised in an
extrachromosomally replicating molecule. Several publications describing this
approach are cited for further illustration (Green, P. J. et al., Ann. Rev.
Biochem. 55:569-597 (1986); van der Krol, A. R. et al, Antisense Nuc. Acids &
Proteins, pp. 125-141 (1991); Abel, P. P. et al., PNASroc. Natl. Acad. Sci.
USA 86:6949-6952 (1989); Ecker, J. R. et al., Proc. Natl. Acad. Sci. USANAS
83:5372-5376 (Aug. 1986)).
In transgenic plants containing one of the DNA molecules described
immediately above, the expression of the nucleotide sequence corresponding
to the nucleotide sequence comprised in the DNA molecule is specifically
reduced. Specifically, the nucleotide sequence in the DNA molecule is at
least 70% identical to the nucleotide sequence the expression of which is
reduced, more specifically it is at least 80% identical, yet more specifically
at
least 90% identical, yet more specifically at least 95% identical, yet more
specifically at least 99% identical.
C. Homologous Recombination
In another specific embodiment, at least one genomic copy
corresponding to a nucleotide sequence of the present invention is modified in
the genome of the plant by homologous recombination as further illustrated in


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Paszkowski et al., EMBO Journal 7:4021-26 (1988). This technique uses the
property of homologous sequences to recognize each other and to exchange
nucleotide sequences between each by a process known in the art as
homologous recombination. Homologous recombination can occur between
the chromosomal copy of a nucleotide sequence in a cell and an incoming
copy of the nucleotide sequence introduced in the cell by transformation.
Specific modifications are thus accurately introduced in the chromosomal
copy of the nucleotide sequence. In one embodiment, the regulatory elements
of the nucleotide sequence of the present invention are modified. Such
regulatory elements are easily obtainable by screening a genomic library
using the nucleotide sequence of the present invention, or a portion thereof,
as a probe. The existing regulatory elements are replaced by different
regulatory elements, thus altering expression of the nucleotide sequence, or
they are mutated or deleted, thus abolishing the expression of the nucleotide
sequence. In another embodiment, the nucleotide sequence is modified by
deletion of a part of the nucleotide sequence or the entire nucleotide
sequence, or by mutation. Expression of a mutated polypeptide in a plant cell
is also contemplated in the present invention. More recent refinements of this
technique to disrupt endogenous plant genes have been described (Kempin
et al., Nature 389:802-803 (1997) and Miao and Lam, Plant J., 7:359-365
(1995).
In another specific embodiment, a mutation in the chromosomal copy
of a nucleotide sequence is introduced by transforming a cell with a chimeric
oligonucleotide composed of a contiguous stretch of RNA and DNA residues
in a duplex conformation with double hairpin caps on the ends. An additional
feature of the oligonucleotide is for example the presence of 2'-O-
methylation at the RNA residues. The RNA/DNA sequence is designed to
align with the sequence of a chromosomal copy of a nucleotide sequence of
the present invention and to contain the desired nucleotide change. For
example, this technique is further illustrated in US patent 5,501,967 and Zhu
et al. (1999) Proc. Natl. Acad. Sci. USA 96: 8768-8773.
D. Ribozymes


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In a further embodiment, the RNA coding for a polypeptide of the
present invention is cleaved by a catalytic RNA, or ribozyme, specific for
such RNA. The ribozyme is expressed in transgenic plants and results in
reduced amounts of RNA coding for the polypeptide of the present invention
in plant cells, thus leading to reduced amounts of polypeptide accumulated
in the cells. This method is further illustrated in US patent 4,987,071.
E. Dominant-Negative Mutants
In another specific embodiment, the activity of the polypeptide
encoded by the nucleotide sequences of this invention is changed. This is
achieved by expression of dominant negative mutants of the proteins in
transgenic plants, leading to the loss of activity of the endogenous protein.
F. Aptamers
In a further embodiment, the activity of polypeptide of the present
invention is inhibited by expressing in transgenic plants nucleic acid
ligands,
so-called aptamers, which specifically bind to the protein. Aptamers are
preferentially obtained by the SELEX (Systematic Evolution of Ligands by
EXponential Enrichment) method. In the SELEX method, a candidate mixture
of single stranded nucleic acids having regions of randomized sequence is
contacted with the protein and those nucleic acids having an increased
affinity
to the target are partitioned from the remainder of the candidate mixture. The
partitioned nucleic acids are amplified to yield a ligand enriched mixture.
After
several iterations a nucleic acid with optimal affinity to the polypeptide is
obtained and is used for expression in transgenic plants. This method is
further illustrated in US patent 5,270,163.
G. Zinc finger proteins
A zinc finger protein that binds a nucleotide sequence of the present
invention or to its regulatory region is also used to alter expression of the
nucleotide sequence. Specifically, transcription of the nucleotide sequence is
reduced or increased. Zinc finger proteins are for example described in Beerli
et al. (1998) PNAS 95:14628-14633., or in WO 95/19431, WO 98/54311, or
WO 96/06166, all incorporated herein by reference in their entirety.
H. dsRNA


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Alteration of the expression of a nucleotide sequence of the present
invention is also obtained by dsRNA interference as described for example in
WO 99/32619, WO 99/53050 or WO 99/61631, all incorporated herein by
reference in their entirety. In another specific embodiment, the alteration of
the expression of a nucleotide sequence of the present invention, specifically
the reduction of its expression, is obtained by double-stranded RNA (dsRNA)
interference. The entirety or, specifically a portion of a nucleotide sequence
of the present invention is comprised in a DNA molecule. The size of the
DNA molecule is specifically from 100 to 1000 nucleotides or more; the
optimal size to be determined empirically. Two copies of the identical DNA
molecule are linked, separated by a spacer DNA molecule, such that the first
and second copies are in opposite orientations. In the specific embodiment,
the first copy of the DNA molecule is in the reverse complement (also known
as the non-coding strand) and the second copy is the coding strand; in the
most specific embodiment, the first copy is the coding strand, and the second
copy is the reverse complement. The size of the spacer DNA molecule is
specifically 200 to 10,000 nucleotides, more specifically 400 to 5000
nucleotides and most specifically 600 to 1500 nucleotides in length. The
spacer is specifically a random piece of DNA, more specifically a random
piece of DNA without homology to the target organism for dsRNA
interference, and most specifically a functional intron which is effectively
spliced by the target organism. The two copies of the DNA molecule
separated by the spacer are operatively linked to a promoter functional in a
plant cell, and introduced in a plant cell, in which the nucleotide sequence
is
expressible. In a specific embodiment, the DNA molecule comprising the
nucleotide sequence, or a portion thereof, is stably integrated in the genome
of the plant cell. In another specific embodiment the DNA molecule
comprising the nucleotide sequence, or a portion thereof, is comprised in an
extrachromosomally replicating molecule. Several publications describing this
approach are cited for further illustration (Waterhouse et al. (1998) PNAS
95:13959-13964; Chuang and Meyerowitz (2000) PNAS 97:4985-4990; Smith
et al. (2000) Nature 407:319-320). Alteration of the expression of a
nucleotide


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sequence by dsRNA interference is also described in, for example WO
99/32619, WO 99/53050 or WO 99/61631, all incorporated herein by
reference in their entirety.
In transgenic plants containing one of the DNA molecules described
immediately above, the expression of the nucleotide sequence corresponding
to the nucleotide sequence comprised in the DNA molecule is specifically
reduced. Specifically, the nucleotide sequence in the DNA molecule is at
least 70% identical to the nucleotide sequence the expression of which is
reduced, more specifically it is at least 80% identical, yet more specifically
at
least 90% identical, yet more specifically at least 95% identical, yet more
specifically at least 99% identical.
1. Insertion of a DNA molecule (Insertional mutagenesis)
In another specific embodiment, a DNA molecule is inserted into a
chromosomal copy of a nucleotide sequence of the present invention, or into
a regulatory region thereof. Specifically, such DNA molecule comprises a
transposable element capable of transposition in a plant cell, such as e.g.
Ac/Ds, Em/Spm, mutator. Alternatively, the DNA molecule comprises a T-
DNA border of an Agrobacterium T-DNA. The DNA molecule may also
comprise a recombinase or integrase recognition site which can be used to
remove part of the DNA molecule from the chromosome of the plant cell.
Methods of insertional mutagenesis using T-DNA, transposons,
oligonucleotides or other methods known to those skilled in the art are also
encompassed. Methods of using T-DNA and transposon for insertional
mutagenesis are described in Winkler et al. (1989) Methods Mol. Biol. 82:129-
136 and Martienssen (1998) PNAS 95:2021-2026, incorporated herein by
reference in their entireties.
J. Deletion mutagenesis
In yet another embodiment, a mutation of a nucleic acid molecule of
the present invention is created in the genomic copy of the sequence in the
cell or plant by deletion of a portion of the nucleotide sequence or regulator
sequence. Methods of deletion mutagenesis are known to those skilled in the
art. See, for example, Miao et al, (1995) Plant J. 7:359.


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In yet another embodiment, this deletion is created at random in a large
population of plants by chemical mutagenesis or irradiation and a plant with a
deletion in a gene of the present invention is isolated by forward or reverse
genetics. Irradiation with fast neutrons or gamma rays is known to cause
deletion mutations in plants (Silverstone et al, (1998) Plant Cell, 10:155-
169;
Bruggemann et al., (1996) Plant J., 10:755-760; Redei and Koncz in Methods
in Arabidopsis Research, World Scientific Press (1992), pp. 16-82). Deletion
mutations in a gene of the present invention can be recovered in a reverse
genetics strategy using PCR with pooled sets of genomic DNAs as has been
shown in C. elegans (Liu et al., (1999), Genome Research, 9:859-867.). A
forward genetics strategy would involve mutagenesis of a line displaying
PTGS followed by screening the M2 progeny for the absence of PTGS.
Among these mutants would be expected to be some that disrupt a gene of
the present invention. This could be assessed by Southern blot or PCR for a
gene of the present invention with genomic DNA from these mutants.
K. Overexpression in a plant cell
In yet another specific embodiment, a nucleotide sequence of the
present invention encoding a polypeptide is over-expressed. Examples of
nucleic acid molecules and expression cassettes for over-expression of a
nucleic acid molecule of the present invention are described above. Methods
known to those skilled in the art of over-expression of nucleic acid molecules
are also encompassed by the present invention.
In a specific embodiment, the expression of the nucleotide sequence of
the present invention is altered in every cell of a plant. This is for example
obtained though homologous recombination or by insertion in the
chromosome. This is also for example obtained by expressing a sense or
antisense RNA, zinc finger protein or ribozyme under the control of a
promoter capable of expressing the sense or antisense RNA, zinc finger
protein or ribozyme in every cell of a plant. Constitutive expression,
inducible,
tissue-specific or developmentally-regulated expression are also within the
scope of the present invention and result in a constitutive, inducible, tissue-

specific or developmentally-regulated alteration of the expression of a


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nucleotide sequence of the present invention in the plant cell. Constructs for
expression of the sense or antisense RNA, zinc finger protein or ribozyme, or
for over-expression of a nucleotide sequence of the present invention, are
prepared and transformed into a plant cell according to the teachings of the
present invention, e.g. as described infra.
VII. Polypeptides
The present invention further relates to isolated polypeptides
comprising the amino acid sequence of SEQ ID NO:2. In particular, isolated
polypeptides comprising the amino acid sequence of SEQ ID NO:2, and
variants having conservative amino acid modifications. One skilled in the art
will recognize that individual substitutions, deletions or additions to a
nucleic
acid, peptide, polypeptide or protein sequence which alters, adds or deletes a
single amino acid or a small percent of amino acids in the encoded sequence
is a "conservative modification" where the modification results in the
substitution of an amino acid with a chemically similar amino acid.
Conservative modified variants provide similar biological activity as the
unmodified polypeptide. Conservative substitution tables listing functionally
similar amino acids are known in the art. See Crighton (1984) Proteins, W.H.
Freeman and Company.
In a specific embodiment, a polypeptide having substantial similarity to
a polypeptide sequence of SEQ ID NO:2, or exon or domain thereof, is an
allelic variant of the polypeptide sequence listed in SEQ ID NO:2. In another
specific embodiment, a polypeptide having substantial similarity to a
polypeptide sequence listed in SEQ ID NO:2, or exon or domain thereof, is a
naturally occurring variant of the polypeptide sequence listed SEQ ID NO:2.
In another specific embodiment, a polypeptide having substantial similarity to
a polypeptide sequence listed SEQ ID NO:2, or exon or domain thereof, is a
polymorphic variant of the polypeptide sequence listed in SEQ ID NO:2.
In an alternate specific embodiment, the sequence having substantial
similarity contains a deletion or insertion of at least one amino acid. In a
more
specific embodiment, the deletion or insertion is of less than about ten amino


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acids. In a most specific embodiment, the deletion or insertion is of less
than
about three amino acids.
In a specific embodiment, the sequence having substantial similarity
encodes a substitution in at least one amino acid.
Embodiments of the present invention also contemplate an isolated
polypeptide containing a polypeptide sequence including
(a) a polypeptide sequence listed in SEQ ID NO:2, or exon or
domain thereof;
(b) a polypeptide sequence having substantial similarity to (a);
(c) a polypeptide sequence encoded by a nucleotide sequence
identical to or having substantial similarity to a nucleotide sequence
listed in SEQ ID NO:1, or an exon or domain thereof, or a sequence
complementary thereto;
(d) a polypeptide sequence encoded by a nucleotide sequence
capable of hybridizing under medium stringency conditions to a
nucleotide sequence listed in SEQ ID NO:1, or to a sequence
complementary thereto; or
(e) a functional fragment of (a), (b), (c) or (d).
In another specific embodiment, the polypeptide having substantial
similarity is an allelic variant of a polypeptide sequence listed in SEQ ID
NO:2,
or a fragment, domain, repeat or chimeras thereof. In another specific
embodiment, the isolated nucleic acid includes a plurality of regions from the
polypeptide sequence encoded by a nucleotide sequence identical to or
having substantial similarity to a nucleotide sequence listed in SEQ ID NO:1,
or fragment or domain thereof, or a sequence complementary thereto.
In another specific embodiment, the polypeptide is a polypeptide
sequence listed in SEQ ID NO:2. In another specific embodiment, the
polypeptide is a functional fragment or domain. In yet another specific
embodiment, the polypeptide is a chimera, where the chimera may include
functional protein domains, including domains, repeats, post-translational
modification sites, or other features. In a more specific embodiment, the
polypeptide is a plant polypeptide. In a more specific embodiment, the plant


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is a dicot. In a more specific embodiment, the plant is a gymnosperm. In a
more specific embodiment, the plant is a monocot. In a more specific
embodiment, the monocot is a cereal. In a more specific embodiment, the
cereal may be, for example, maize, wheat, barley, oats, rye, millet, sorghum,
triticale, secale, einkorn, spelt, emmer, teff, milo, flax, gramma grass,
Tripsacum, and teosinte. In another specific embodiment, the cereal is rice.
In a specific embodiment, the polypeptide is expressed in a specific
location or tissue of a plant. In a more specific embodiment, the location or
tissue is for example, but not limited to, epidermis, vascular tissue,
meristem,
cambium, cortex or pith. In a most specific embodiment, the location or tissue
is leaf or sheath, root, flower, and developing ovule or seed. In a more
specific embodiment, the location or tissue may be, for example, epidermis,
root, vascular tissue, meristem, cambium, cortex, pith, leaf, and flower. In a
more specific embodiment, the location or tissue is a seed.
In a specific embodiment, the polypeptide sequence encoded by a
nucleotide sequence having substantial similarity to a nucleotide sequence
listed in SEQ ID NO:1 or a fragment or domain thereof or a sequence
complementary thereto, includes a deletion or insertion of at least one
nucleotide. In a more specific embodiment, the deletion or insertion is of
less
than about thirty nucleotides. In a most specific embodiment, the deletion or
insertion is of less than about five nucleotides.
In a specific embodiment, the polypeptide sequence encoded by a
nucleotide sequence having substantial similarity to a nucleotide sequence
listed in SEQ ID NO:1, or fragment or domain thereof or a sequence
complementary thereto, includes a substitution of at least one codon. In a
more specific embodiment, the substitution is conservative.
In a specific embodiment, the polypeptide sequences having
substantial similarity to the polypeptide sequence listed in SEQ ID NO:2, or a
fragment, domain, repeat or chimeras thereof includes a deletion or insertion
of at least one amino acid.
The polypeptides of the invention, fragments thereof or variants thereof
can comprise any number of contiguous amino acid residues from a


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polypeptide of the invention, wherein the number of residues is selected from
the group of integers consisting of from 10 to the number of residues in a
full-
length polypeptide of the invention. Specifically, the portion or fragment of
the
polypeptide is a functional protein. The present invention includes active
polypeptides having specific activity of at least 20%, 30%, or 40%, and
specifically at least 505, 60%, or 70%, and most specifically at least 805,
90%
or 95% that of the native (non-synthetic) endogenous polypeptide. Further,
the substrate specificity (kcat/Km) is optionally substantially similar to the
native (non-synthetic), endogenous polypeptide. Typically the Km will be at
least 30%, 40%, or 50% of the native, endogenous polypeptide; and more
specifically at least 605, 70%, 80%, or 90%. Methods of assaying and
quantifying measures of activity and substrate specificity are well known to
those of skill in the art.
The isolated polypeptides of the present invention will elicit production
of an antibody specifically reactive to a polypeptide of the present invention
when presented as an immunogen. Therefore, the polypeptides of the present
invention can be employed as immunogens for constructing antibodies
immunoreactive to a protein of the present invention for such purposes, but
not limited to, immunoassays or protein purification techniques.
Immunoassays for determining binding are well known to those of skill in the
art such as, but not limited to, ELISAs or competitive immunoassays.
Embodiments of the present invention also relate to chimeric
polypeptides encoded by the isolated nucleic acid molecules of the present
disclosure including a chimeric polypeptide containing a polypeptide
sequence encoded by an isolated nucleic acid containing a nucleotide
sequence including:
(a) a nucleotide sequence listed in SEQ ID NO:1, or an
exon or domain thereof;
(b) a nucleotide sequence having substantial similarity to
(a);
(c) a nucleotide sequence capable of hybridizing to (a);


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(d) a nucleotide sequence complementary to (a), (b) or
(c); and
(e) a nucleotide sequence which is the reverse
complement of (a), (b) or (c); or
(f) a functional fragment thereof.
A polypeptide containing a polypeptide sequence encoded by an
isolated nucleic acid containing a nucleotide sequence, its complement, or its
reverse complement, encoding a polypeptide including a polypeptide
sequence including:
(a) a polypeptide sequence listed in SEQ ID NO:2, or a
domain, repeat or chimeras thereof;
(b) a polypeptide sequence having substantial similarity to
(a);
(c) a polypeptide sequence encoded by a nucleotide
sequence identical to or having substantial similarity to a nucleotide
sequence listed in SEQ ID NO:1, or an exon or domain thereof, or a
sequence complementary thereto;
(d) a polypeptide sequence encoded by a nucleotide
sequence capable of hybridizing under medium stringency conditions
to a nucleotide sequence listed in SEQ ID NO:1, or to a sequence
complementary thereto; and a functional fragment of (a), (b), (c) or (d);
or
(e) a functional fragment thereof.
The isolated nucleic acid molecules of the present invention are useful
for expressing a polypeptide of the present invention in a recombinantly
engineered cell such as a bacteria, yeast, insect, mammalian or plant cell.
The cells produce the polypeptide in a non-natural condition (e.g. in
quantity,
composition, location and/or time) because they have been genetically altered
to do so. Those skilled in the art are knowledgeable in the numerous
expression systems available for expression of nucleic acids encoding a
protein of the present invention, and will not be described in detail below.


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Briefly, the expression of isolated nucleic acids encoding a polypeptide
of the invention will typically be achieved, for example, by operably linking
the
nucleic acid or cDNA to a promoter (constitutive or regulatable) followed by
incorporation into an expression vector. The vectors are suitable for
replication and/or integration in either prokaryotes or eukaryotes. Commonly
used expression vectors comprise transcription and translation terminators,
initiation sequences and promoters for regulation of the expression of the
nucleic acid molecule encoding the polypeptide. To obtain high levels of
expression of the cloned nucleic acid molecule, it is desirable to use
expression vectors comprising a strong promoter to direct transcription, a
ribosome binding site for translation initiation, and a
transcription/translation
terminator. One skilled in the art will recognize that modifications may be
made to the polypeptide of the present invention without diminishing its
biological activity. Some modifications may be made to facilitate the cloning,
expression or incorporation of the polypeptide of the invention into a fusion
protein. Such modification are well known in the art and include, but are not
limited to, a methionine added at the amino terminus to provide an initiation
site, or additional amino acids (e.g. poly Histadine) placed on either
terminus
to create conveniently located purification sequences. Restriction sites or
termination codons can also be introduced into the vector.
In a specific embodiment, the expression vector includes one or more
elements such as, for example, but not limited to, a promoter-enhancer
sequence, a selection marker sequence, an origin of replication, an epitope-
tag encoding sequence, or an affinity purification-tag encoding sequence. In
a more specific embodiment, the promoter-enhancer sequence may be, for
example, the CaMV 35S promoter, the CaMV 19S promoter, the tobacco PR-
la promoter, the ubiquitin promoter, and the phaseolin promoter. In another
embodiment, the promoter is operable in plants, and more specifically, a
constitutive or inducible promoter. In another specific embodiment, the
selection marker sequence encodes an antibiotic resistance gene. In another
specific embodiment, the epitope-tag sequence encodes V5, the peptide Phe-
His-His-Thr-Thr, hemagglutinin, or glutathione-S-transferase. In another


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specific embodiment the affinity purification-tag sequence encodes a
polyamino acid sequence or a polypeptide. In a more specific embodiment,
the polyamino acid sequence is polyhistidine. In a more specific embodiment,
the polypeptide is chitin binding domain or glutathione-S-transferase. In a
more specific embodiment, the affinity purification-tag sequence comprises an
intein encoding sequence.
Prokaryotic cells may be used a host cells, for example, but not limited
to, Escherichia coli, and other microbial strains known to those in the art.
Methods for expressing proteins in prokaryotic cells are well known to those
in
the art and can be found in many laboratory manuals such as Molecular
Cloning: A Laboratory Manual, by J. Sambrook et al. (1989, Cold Spring
Harbor Laboratory Press). A variety of promoters, ribosome binding sites,
and operators to control expression are available to those skilled in the art,
as
are selectable markers such as antibiotic resistance genes. The type of
vector chosen is to allow for optimal growth and expression in the selected
cell type.
A variety of eukaryotic expression systems are available such as, but
not limited to, yeast, insect cell lines, plant cells and mammalian cells.
Expression and synthesis of heterologous proteins in yeast is well known (see
Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory
Press, 1982). Commonly used yeast strains widely used for production of
eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris, and
vectors, strains and protocols for expression are available from commercial
suppliers (e.g., Invitrogen).
Mammalian cell systems may be transfected with expression vectors
for production of proteins. Many suitable host cell lines are available to
those
in the art, such as, but not limited to the HEK293, BHK21 and CHO cells lines.
Expression vectors for these cells can include expression control sequences
such as an origin of replication, a promoter, (e.g., the CMV promoter, a HSV
tk promoter or phosphoglycerate kinase (pgk) promoter), an enhancer, and
protein processing sites such as ribosome binding sites, RNA splice sites,
polyadenylation sites, and transcription terminator sequences. Other animal


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cell lines useful for the production of proteins are available commercially or
from depositories such as the American Type Culture Collection.
Expression vectors for expressing proteins in insect cells are usually
derived from the SF9 baculovirus or other viruses known in the art. A number
of suitable insect cell lines are available including but not limited to,
mosquito
larvae, silkworm, armyworm, moth and Drosophila cell lines.
Methods of transfecting animal and lower eukaryotic cells are known.
Numerous methods are used to make eukaryotic cells competent to introduce
DNA such as but not limited to: calcium phosphate precipitation, fusion of the
recipient cell with bacterial protoplasts containing the DNA, treatment of the
recipient cells with liposomes containing the DNA, DEAE dextrin,
electroporation, biolistics, and microinjection of the DNA directly into the
cells.
Transfected cells are cultured using means well known in the art (see,
Kuchler, R.J., Biochemical Methods in Cell Culture and Virology, Dowden,
Hutchinson and Ross, Inc. 1997).
Once a polypeptide of the present invention is expressed it may be
isolated and purified from the cells using methods known to those skilled in
the art. The purification process may be monitored using Western blot
techniques or radioimmunoassay or other standard immunoassay techniques.
Protein purification techniques are commonly known and used by those in the
art (see R. Scopes, Protein Purification: Principles and Practice, Springer-
Verlag, New York 1982: Deutscher, Guide to Protein Purification, Academic
Press (1990). Embodiments of the present invention provide a method of
producing a recombinant protein in which the expression vector includes one
or more elements including a promoter-enhancer sequence, a selection
marker sequence, an origin of replication, an epitope-tag encoding sequence,
and an affinity purification-tag encoding sequence. In one specific
embodiment, the nucleic acid construct includes an epitope-tag encoding
sequence and the isolating step includes use of an antibody specific for the
epitope-tag. In another specific embodiment, the nucleic acid construct
contains a polyamino acid encoding sequence and the isolating step includes
use of a resin comprising a polyamino acid binding substance, specifically


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where the polyamino acid is polyhistidine and the polyamino binding resin is
nickel-charged agarose resin. In yet another specific embodiment, the nucleic
acid construct contains a polypeptide encoding sequence and the isolating
step includes the use of a resin containing a polypeptide binding substance,
specifically where the polypeptide is a chitin binding domain and the resin
contains chitin-sepharose.
The polypeptides of the present invention cam be synthesized using
non-cellular synthetic methods known to those in the art. Techniques for solid
phase synthesis are described by Barany and Mayfield, Solid-Phase Peptide
Synthesis, pp. 3-284 in the Peptides: Analysis, Synthesis, Biology, Vol.2,
Special Methods in Peptide Synthesis, Part A; Merrifield, et al., J. Am. Chem.
Soc. 85:2149-56 (1963) and Stewart et al., Solid Phase Peptide Synthesis,
2nd ed. Pierce Chem. Co., Rockford, IL (1984).
The present invention further provides a method for modifying (i.e.
increasing or decreasing) the concentration or composition of the
polypeptides of the invention in a plant or part thereof. Modification can be
effected by increasing or decreasing the concentration and/or the composition
(i.e. the ratio of the polypeptides of the present invention) in a plant. The
method comprised introducing into a plant cell with an expression cassette
comprising a nucleic acid molecule of the present invention, or an nucleic
acid
encoding a OsGATAl 1 sequence as described above to obtain a transformed
plant cell or tissue, culturing the transformed plant cell or tissue. The
nucleic
acid molecule can be under the regulation of a constitutive or inducible
promoter. The method can further comprise inducing or repressing
expression of a nucleic acid molecule of a sequence in the plant for a time
sufficient to modify the concentration and/or composition in the plant or
plant
part.
A plant or plant part having modified expression of a nucleic acid
molecule of the invention can be analyzed and selected using methods known
to those skilled in the art such as, but not limited to, Southern blot, DNA
sequencing, or PCR analysis using primers specific to the nucleic acid
molecule and detecting amplicons produced therefrom.


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In general, concentration or composition in increased or decreased by
at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to a
native control plant, plant part or cell lacking the expression cassette.
Sugars are central regulators of many vital processes in photosynthetic
plants, such as photosynthesis, carbon and nitrogen metabolism and this
regulation is achieved by regulating gene expression, either activate or
repress genes involved. The mechanisms by which sugars control gene
expression are not understood well. This GATA transcription factor disclosed
here is involved in regulating sugar sensing and the expression of the factor
itself is influenced by the change of the N status. Increased expression of
this
gene can produce plants with increased yield, particularly as the manipulation
of sugar signaling pathways can lead to increased photosynthesis and
increased nitrogen utilization and alter source-sink relationships in seeds,
tubes, roots and other storage organs.
The invention will be further described by reference to the following
detailed examples. These examples are provided for purposes of illustration
only, and are not intended to be limiting unless otherwise specified.
EXAMPLES
Standard recombinant DNA and molecular cloning techniques used
here are well known in the art and are described by J. Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory Press (2001); by T.J. Silhavy, M.L. Berman,
and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY (1984) and by Ausubel, F.M. et al.,
Current Protocols in Molecular Biology, New York, John Wiley and Sons Inc.,
(1988), Reiter, et al., Methods in Arabidopsis Research, World Scientific
Press (1992), and Schultz et al., Plant Molecular Biology Manual, Kluwer
Academic Publishers (1998).
EXPERIMENTAL BACKGROUND AND PROCEDURES
A. Determining rice and maize growth conditions under limiting nitrogen
conditions
In past experiments to study genes involved in nitrate uptake and


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assimilation, the present inventors and others have utilized growth conditions
in which nitrate was generally either present in excess or absent in its
entirety.
In the latter case, nitrate is typically added to plants grown in its absence
in
order to understand nitrate regulation of these and other genes. While this
type of extreme treatment is useful in defining some aspects of gene
regulation, it is not suitable to gain a better understanding of the effect of
nitrogen limitation. The inventors have defined conditions for Arabidopsis in
which nitrogen limits growth. This involved developing a system using
Rockwool (Hirai et al., 1995 Plant Cell Physiol 36, 1331-1339) and defining
three conditions: one where growth is maximal; one where nitrogen limits
growth to 70-75% maximal growth levels; one where there is a more severe
limitation to 30-35% maximal growth levels. The nitrogen limitation acts as a
'stress' with the amount of 'stress' easily varied by altering the
concentration
of nitrate. The inventors assay the physiological "nitrogen status" by
measuring nitrate, chlorophyll (which is often used as a reflection of
nitrogen
status under field conditions- see, e.g., Fox RH et al 2001 Agron J. 93, 590-
597; Minotti PL et al 1994 Hort Science 29, 1497-1550), amino acid levels,
and nitrate reductase and glutamine synthetase activities in order to give a
baseline in which to assess studies on mutant lines.
B. Expression profiling experiments on Arabidopsis plants under
nitrogen limitation
Transcript expression profiling can be used to test RNA levels of large
numbers of genes at the same time. Large numbers of these types of
experiments have been done in the past, and if the experimental system is
amenable, these can be used to pinpoint the "expression status" of an
organism under different conditions and to use this information to make
hypotheses on what genes and pathways are involved in various processes.
The inventors found that the more profound the difference in growth
conditions, the larger the differences in transcript profiles between the
plants
grown under these conditions and the more difficult it was to decipher which
changes were most important. The only published whole genome profiling
experiment in this area is one in Arabidopsis where an extreme change in


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nitrate levels was studied (Wang R et al 2003 Plant Physiol. 132, 556-67). In
the case of nitrogen limitations, the inventors studied the effect of growing
plants under chronic nitrogen stress as well as changes in the level of
available nitrogen. The inventors have already determined the impact on
growth of different nitrogen levels in Arabidopsis.
The effect of different nitrogen levels on the transcript profiles was
studied: where nitrogen does not limit growth. For Arabidopsis the inventors
collected 4-week old shoots grown under the different nitrogen regimes.
Three different samples were collected (biological triplicates) in order to
get
statistically significant results. The transcript profiling was done using
Arabidopsis GeneChip whole genome array (Affymetrix) to study the
transcript levels in Arabidopsis. The bioinformatic analysis necessary to
study
the considerable data produced by these experiments was performed. By
studying the effect of nitrogen limitation on the expression patterns, the
inventors can pinpoint which pathways are involved in their response to
nutrient stress
EXAMPLE 1
Materials and methods
Plant growth conditions
Peat moss and vermiculate (1:4) (SunGro Horticulture Canada Ltd. BC,
Canada) was used to grow Oryza sativa Kaybonnet plants, adding nutrient
solution with different amount of nitrate once a week till harvest. The
nutrient
solution contains 4 mM MgSO4, 5 mM KCI, 5 mM CaC12, 1 mM KH2P04, 0.1
mM Fe-EDTA, 0.5 mM MES (pH6.0), 9 p M MnSO4, 0.7 pM Zn SO4, 0.3 pM
CuSOa, 46 pM NaB4O7 and 0.2 pM (NH4) 6Mo7O2. For limiting N condition,
3mM N solution was used once a week till harvest. For sufficient N condition,
10mM N solution was used once a week for the first six week, changed to
5mM for another 6 weeks, and the changed to 3mM N solution till harvest.
Plants were grown in a growth room with 16 hr light (-400 pmolm-zs"') at 28-
30 C and 8 hr dark at 22-24 C for the first four weeks and then had one week
short-day treatment (10 hr light/14 hr dark). After that, plants were moved to
greenhouse to grow till harvest.


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Generating transgenic rice plants
The constructs for over-expressing or silencing OsGATAII were
made. T1 transgenic seeds over-expressing OsGATAl1, and silencing
OsGATAl 1 (RNAi) were analyzed.
Genotyping transgenic plants
Leaf samples were grounded in 300 NI buffer (Strategic Diagnostics
Inc. Part # 7000006). One dipstick (Strategic Diagnostics Inc. Part # 7000052)
was inserted into the tube and left for -15 minutes by which time the lines on
the sticks were clear. The appearance of one red line (control) on the strip
indicates a negative result. The appearance of two red lines (control and
test)
on the strip indicates a positive result.
Expression analysis by semi-quantitative RT-PCR
One pg total RNA extracted was used to make cDNA. Primers for
OsGATAl1 are 5'- CGTCGAGCACCAAGGGCAAATC-3' (SEQ ID NO:3) and
5'- GGATAGGGTCATGAGCAGCATGG-3' (SEQ ID NO:4). Primers for
OsTubulin are: 5'- AGGAGGATGCCGCTAACAACTTTG-3' (SEQ ID NO:5)
and 5'- AAACAGCATTGGTGATTTCAGGC-3' (SEQ ID NO:6).
Chlorophyll measurement
Total chlorophyll was measured either using the Minolta SPAD 502DL
chlorophyll meter (Tokyo, Japan), or extracted by ethanol and measured by
spectrophotometer according to Kirk (1968).
Results
Strategy to phenotype transgenic plants
The strategy for initial genetic and phenotypic analysis involved
growing 5 transgenic events from each construct under mainly limiting
nitrogen (N) condition (- 18 plants). Also some plants were grown under
sufficient N condition (- 10 plants). PMI sticks were used for genotyping to
detect the selectable marker PMI. Transgene expression levels were tested
by semi-quantitative RT-PCR. Chlorophyll level, culm length, tiller number,
panicle number, flowering time, seed yield and shoot biomass was recorded.


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Phenotypes of the OsGATAl1 over-expression plants
The OsGATAl1 gene shares - 34% similarity at protein level with the
AtGATA gene (At4g26150, Figure 3). Total chlorophyll levels were measured
when the transgenic plants were about 4-wk-old under limiting N condition. At
least two transgenic events (event 5 and 6) had significant higher chlorophyll
content from the average of PMI positive plants (3-6 plants) compared to wild
type control plants (6 plants) (Figure 4A). Those transgenic plants did have
elevated expression of the OsGATAII gene (Figure 4B). To ensure that
chlorophyll level can be affected by the expression levels of the OsGATAl1
gene, the transgenic RNAi OsGATAl 1 plants were analyzed. The expression
level of the OsGATA11 gene was significantly reduced in the transgenic RNAi
OsGATAl 1 plants (Figure 5A), and indeed, chlorophyll level was significantly
lower in those plants (Figure 5B). One event (event 6) had - 20% higher seed
yield from the average of 10 PMI positive plants compared to the average of
11 wild type control plants under limiting N condition (Figure 6A). This same
event had almost doubled seed yield from the average of 4 PMI positive
plants compared to the average of 6 wild type control plants under sufficient
N
condition (Figure 6B). Also, plants grown under high N experienced stress
after being transferred from the growth room to the greenhouse and the
transgenic plants responded much better to the stress (Figure 7).
Having now described particular embodiments of the invention by way of
the foregoing examples, which are not intended to be limiting, the invention
will now be further set forth in the following claims. Those skilled in the
art will
recognize that the claims also permit for the inclusion of equivalents beyond
the claims' literal scope.


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SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT: UNIVERSITY OF GUELPH

(ii) TITLE OF INVENTION: NITROGEN-REGULATED SUGAR SENSING GENE
AND PROTEIN MODULATION THEREOF

(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BERESKIN & PARR
(B) STREET: 40 King Street West
(C) CITY: Toronto
(D) STATE: Ontario
(E) COUNTRY: Canada
(F) ZIP: M5H 3Y2

(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: iMAC - Using Virtual PC
(C) OPERATING SYSTEM: Windows 198
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: GRAVELLE, MICHELINE
(C) REFERENCE/DOCKET NUMBER: 6580-346
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 364-7311
(B) TELEFAX: (416) 361-1398
(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1343 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rice

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

gaacttctct cccatctctt tcctcctcct cctctctgat atgtctacta tctacatgag 60
ccagctacct gctactctcc ctctaatgga gggggatcag gatcaggggc tctacccagc 120
cttccataga gcaaaggacc ctcctatctt gttccctttc atgatcgaca gcgccgtcga 180


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gcaccaaggg caaatctatg gagatcaggg cttgaggagg cagcaggttt tgggtgaatc 240
caatcaacag ttcaatgatc acatgatgat gggcggatca gatgtcttcc tcacaccgtc 300
tccgttccga ccaaccatcc aaagcatcgg cagcgacatg atccagcgat catcttatga 360
tccatacgat atcgagagta acaacaagca gcatgccaat ggatcaacca gcaagtggat 420
gtcgacgccg ccaatgaaga tgaggatcat aaggaagggg gcggcaaccg atcctgaggg 480
cggggcggtg agaaagccaa ggagaagagc acaagcgcac caggatgaga gccagcaaca 540
actgcagcaa gctttgggtg tcgttagagt gtgctcggac tgcaacacca ccaagacccc 600
cttgtggaga agtggtcctt gtggccccaa gtccctttgc aacgcgtgtg gcatcaggca 660
aaggaaggcg cggcgggcga tggccgctgc tgccaacggc ggagcggcgg tggcgccggc 720
aaagagcgtg gccgcggcgc cggtgaacaa taagccggcg gcgaagaagg agaagagggc 780
ggcggacgtc gaccggtcgc tgccgttcaa gaaacggtgc aagatggtcg atcacgttgc 840
tgctgccgtc gctgccacca agcccacggc tgctggagaa gtagtggccg ccgctccgaa 900
ggaccaagat cacgtcatcg tcgtcggtgg cgagaacgcc gccgccacct ccatgccggc 960
acagaacccg atatccaagg cggcggcgac cgccgctgcc gccgccgcct ctccggcgtt 1020
cttccacggc ctccctcgcg acgagatcac cgacgccgcc atgctgctca tgaccctatc 1080
ctgtggcctc gtccacagct agctagctag ctgatcaaaa ctagctagct actagtaccg 1140
ttaatttgat gagggcaaca accagagtac tatgtaccac tactagcaat attttgtgtg 1200
tgccttgtga tcttttgttg ttttgtgttg ttgaggagat cactagatca ggatgaagga 1260
gagatagtga tcacatgtct aaggacgaaa taaacgagaa caaactcgct agctagctac 1320
tagccgggat caggattata ttt 1343
(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 353 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rice

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ser Thr Ile Tyr Met Ser Gln Leu Pro Ala Thr Leu Pro Leu Met
1 5 10 15


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Glu Gly Asp Gln Asp Gln Gly Leu Tyr Pro Ala Phe His Arg Ala Lys
20 25 30

Asp Pro Pro Ile Leu Phe Pro Phe Met Ile Asp Ser Ala Val Glu His
35 40 45
Gln Gly Gln Ile Tyr Gly Asp Gln Gly Leu Arg Arg Gln Gln Val Leu
50 55 60
Gly Glu Ser Asn Gln Gln Phe Asn Asp His Met Met Met Gly Gly Ser
65 70 75 80
Asp Val Phe Leu Thr Pro Ser Pro Phe Arg Pro Thr Ile Gln Ser Ile
85 90 95

Gly Ser Asp Met Ile Gln Arg Ser Ser Tyr Asp Pro Tyr Asp Ile Glu
100 105 110
Ser Asn Asn Lys Gln His Ala Asn Gly Ser Thr Ser Lys Trp Met Ser
115 120 125
Thr Pro Pro Met Lys Met Arg Ile Ile Arg Lys Gly Ala Ala Thr Asp
130 135 140

Pro Glu Gly Gly Ala Val Arg Lys Pro Arg Arg Arg Ala Gln Ala His
145 150 155 160
Gln Asp Glu Ser Gln Gln Gln Leu Gln Gln Ala Leu Gly Val Val Arg
165 170 175

Val Cys Ser Asp Cys Asn Thr Thr Lys Thr Pro Leu Trp Arg Ser Gly
180 185 190
Pro Cys Gly Pro Lys Ser Leu Cys Asn Ala Cys Gly Ile Arg Gln Arg
195 200 205
Lys Ala Arg Arg Ala Met Ala Ala Ala Ala Asn Gly Gly Ala Ala Val
210 215 220

Ala Pro Ala Lys Ser Val Ala Ala Ala Pro Val Asn Asn Lys Pro Ala
225 230 235 240
Ala Lys Lys Glu Lys Arg Ala Ala Asp Val Asp Arg Ser Leu Pro Phe
245 250 255


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Lys Lys Arg Cys Lys Met Val Asp His Val Ala Ala Ala Val Ala Ala
260 265 270

Thr Lys Pro Thr Ala Ala Gly Glu Val Val Ala Ala Ala Pro Lys Asp
275 280 285
Gln Asp His Val Ile Val Val Gly Gly Glu Asn Ala Ala Ala Thr Ser
290 295 300
Met Pro Ala Gln Asn Pro Ile Ser Lys Ala Ala Ala Thr Ala Ala Ala
305 310 315 320
Ala Ala Ala Ser Pro Ala Phe Phe His Gly Leu Pro Arg Asp Glu Ile
325 330 335
Thr Asp Ala Ala Met Leu Leu Met Thr Leu Ser Cys Gly Leu Val His
340 345 350
Ser

(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
Description: Primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

cgtcgagcac caagggcaaa tc 22
(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(vi) ORIGINAL SOURCE:


CA 02584934 2007-04-17

-101-
(A) ORGANISM: Artificial Sequence
Description: Primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

ggatagggtc atgagcagca tgg 23
(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
Description: Primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

aggaggatgc cgctaacaac tttg 24
(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
Description: Primer
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

aaacagcatt ggtgatttca ggc 23
(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 352 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(vi) ORIGINAL SOURCE:


CA 02584934 2007-04-17

- 102 -

(A) ORGANISM: Arabidopsis thaliana
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Gly Ser Asn Phe His Tyr Thr Ile Asp Leu Asn Glu Asp Gln Asn
1 5 10 15
His Gln Pro Phe Phe Ala Ser Leu Gly Ser Ser Leu His His His Leu
20 25 30
Gln Gin Gln Gln Gln Gln Gln Gln His Phe His His Gln Ala Ser Ser
35 40 45

Asn Pro Ser Ser Leu Met Ser Pro Ser Leu Ser Tyr Phe Pro Phe Leu
50 55 60
Ile Asn Ser Arg Gln Asp Gln Val Tyr Val Gly Tyr Asn Asn Asn Thr
65 70 75 80
Phe His Asp Val Leu Asp Thr His Ile Ser Gln Pro Leu Glu Thr Lys
85 90 95
Asn Phe Val Ser Asp Gly Gly Ser Ser Ser Ser Asp Gln Met Val Pro
100 105 110

Lys Lys Glu Thr Arg Leu Lys Leu Thr Ile Lys Lys Lys Asp Asn His
115 120 125
Gln Asp Gln Thr Asp Leu Pro Gln Ser Pro Ile Lys Asp Met Thr Gly
130 135 140
Thr Asn Ser Leu Lys Trp Ile Ser Ser Lys Val Arg Leu Met Lys Lys
145 150 155 160
Lys Lys Ala Ile Ile Thr Thr Ser Asp Ser Ser Lys Gln His Thr Asn
165 170 175
Asn Asp Gln Ser Ser Asn Leu Ser Asn Ser Glu Arg Gln Asn Gly Tyr
180 185 190

Asn Asn Asp Cys Val Ile Arg Ile Cys Ser Asp Cys Asn Thr Thr Lys
195 200 205
Thr Pro Leu Trp Arg Ser Gly Pro Arg Gly Pro Lys Ser Leu Cys Asn
210 215 220


CA 02584934 2007-04-17

-103-
Ala Cys Gly Ile Arg Gln Arg Lys Ala Arg Arg Ala Ala Met Ala Thr
225 230 235 240
Ala Thr Ala Thr Ala Val Ser Gly Val Ser Pro Pro Val Met Lys Lys
245 250 255

Lys Met Gln Asn Lys Asn Lys Ile Ser Asn Gly Val Tyr Lys Ile Leu
260 265 270
Ser Pro Leu Pro Leu Lys Val Asn Thr Cys Lys Arg Met Ile Thr Leu
275 280 285
Glu Glu Thr Ala Leu Ala Glu Asp Leu Glu Thr Gln Ser Asn Ser Thr
290 295 300

Met Leu Ser Ser Ser Asp Asn Ile Tyr Phe Asp Asp Leu Ala Leu Leu
305 310 315 320
Leu Ser Lys Ser Ser Ala Tyr Gln Gln Val Phe Pro Gln Asp Glu Lys
325 330 335
Glu Ala Ala Ile Leu Leu Met Ala Leu Ser His Gly Met Val His Gly
340 345 350

Representative Drawing
A single figure which represents the drawing illustrating the invention.
Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(22) Filed 2007-04-17
(41) Open to Public Inspection 2008-10-17
Examination Requested 2012-04-17
Dead Application 2017-04-18

Abandonment History

Abandonment Date Reason Reinstatement Date
2016-04-18 FAILURE TO PAY APPLICATION MAINTENANCE FEE
2016-04-28 R30(2) - Failure to Respond

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $400.00 2007-04-18
Registration of a document - section 124 $100.00 2007-08-09
Maintenance Fee - Application - New Act 2 2009-04-17 $100.00 2009-03-23
Maintenance Fee - Application - New Act 3 2010-04-19 $100.00 2010-02-17
Maintenance Fee - Application - New Act 4 2011-04-18 $100.00 2011-04-04
Maintenance Fee - Application - New Act 5 2012-04-17 $200.00 2012-03-23
Request for Examination $800.00 2012-04-17
Maintenance Fee - Application - New Act 6 2013-04-17 $200.00 2013-03-20
Maintenance Fee - Application - New Act 7 2014-04-17 $200.00 2014-03-18
Maintenance Fee - Application - New Act 8 2015-04-17 $200.00 2015-03-16
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
UNIVERSITY OF GUELPH
Past Owners on Record
BI, YONG-MEI
ROTHSTEIN, STEVEN
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Representative Drawing 2008-10-01 1 20
Cover Page 2008-10-01 2 52
Abstract 2007-04-17 1 13
Description 2007-04-17 103 5,061
Claims 2007-04-17 6 197
Drawings 2007-04-17 7 390
Claims 2012-04-17 5 157
Claims 2014-01-20 9 288
Claims 2015-04-28 9 327
Description 2015-04-28 109 5,299
Correspondence 2007-05-11 1 26
Correspondence 2007-07-17 1 26
Assignment 2007-08-09 3 123
Correspondence 2008-06-13 1 70
Assignment 2007-04-17 3 117
Prosecution-Amendment 2012-04-17 7 251
Prosecution-Amendment 2012-04-17 1 45
Prosecution-Amendment 2012-10-03 1 48
Prosecution-Amendment 2013-07-24 3 109
Prosecution-Amendment 2014-01-20 13 495
Prosecution-Amendment 2014-10-28 6 334
Correspondence 2014-12-02 2 62
Correspondence 2014-12-12 1 21
Correspondence 2014-12-12 1 23
Prosecution-Amendment 2015-04-28 36 1,509
Change to the Method of Correspondence 2015-01-15 45 1,704
Examiner Requisition 2015-10-28 5 333

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