Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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0~ FIBER TRANSCRIPTIOMAL F~CTORS
.
CROSS~REFERENCE TO R~LATED APPLICA~IONS
This application is a continuation in part of United
States application Serial No. 08/487,087 filed June 7, 1995,
and a continuation in part of United States application
Serial No. 08/480,178, filed June 7, 1995.
INTRODUCTION
.I Technical Field
This in~ention relates to methods of using in vitro
constructed DNA transcription or expression cassettes cAp~hle of
directing fiber-tissue transcription of a DNA sequence of interest
in plants to produce fiber cells having an altered phenotype, an~
to methods of providing for or modifying various characteristics
of cotton fiber. The invention is exemplified by methods of using
cotton fiber promoters for altering the phenotype of cotton fiber,
and cotton fibers produced by the method.
~ackaround
In general, genetic engineering techniques have been directed
to modifying the phenotype of individual prokaryotic and
eukaryotic cells, especially in culture. Plant cells have
more intransigent than other eukaryotic cells, due not only to a
lack of suitable vector systems but also as a -esult of the
different goals involved. For many applications, it is desirable
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to be able to control gene expression at a particular stage in the
growth of a plant or in a particular plant part. For this
purpose, regulatory se~uences are re~uired which afford the
desired initiation of transcription in the appropriate cell types
and/or at the appropriate time in the plant's development without
having serious detrimental effects on plant development and
productivity. It is therefore of interest to be able to isolate
se~uences which can be used to provide the desired regulation of
transcription in a plant cell during the growing cycle of the host
plant.
One aspect of this interest is the ability to change the
phenotype of particular cell types, such as differentiated
epi~rm~l cells that originate in fiber tissue, i.e. cotton fiber
cells, so as to provide for altered or improved aspects of the
mature cell type. Cotton is a plant of great commercial
significance. In addition to the use of cotton fiber in the
production of textiles, other uses of cotton include food
preparation with cotton seed oil and ~n;~l feed derived from
cotton seed husks.
Despite the importance of cotton as a crop, the breeding and
genetic engineering of cotton f iber phenotypes has taken place at
a relatively slow rate because of the absence of reliable
promoters for use in selectively effecting changes in the
phenotype of the fiber. In order to effect the desired phenotypic
changes, transcription initiation regions capable of initiating
transcription in fiber cells during development are desired.
Thus, an important goal of cotton bioengineering research is the
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ac~uisition of a reliable promoter which would permit expression
of a protein selectively in cotton fiber to affect such ~ualities
as fiber strength; length, color and dyability.
Relev~nt T-; terature
Cotton fiber-specific promoters are discussed in PCT
publications WO 94/12014 and WO 95/08914, and John and Crow, Proc.
Natl. Acad. Sci. USA, 89:5769-5773, 1992. cDNA clones that are
pre~erelltial]y expressed in cotton fiber have been isolated. One
of the clones isolated corresponds to mRNA and protein that are
highest during the late primary cell wall and early secondary cell
wall s~lthesis stages. John and Crow, supra.
In ~n;m~l.c, the ras superfamily is subdivided into the
subfamiiies ~as whizh is involved in con~rolling cel~ growt~ and
15 divisioll~ rab/YPT members which control secretory processes, and
rho which is involved in control of cytoskeletal organization
(Bourne et a]., (1991) Nature 349: 117-127), and number of
homologous genes have now been identified in plants (for a review,
see Terryn et al., (1993) Plant Mol. Biol. 22: 143-152). None
have been found for the important ras subfamily, all but one of
the genes identified have been members of the rab/YPTl subfamily,
and there is only one recent report of the cloning of a rho gene
in pea (Yang and Watson(1993) Proc. Natl. Acad. Sci. USA 90:
8732-8736).
Little work has been done to characterize the functions of
these genes in plants, although one recent report has shown that a
small G protein from Arabidopsis can functionally complement a
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mutant form in yeast involved in vesicle trafficking, suggesting a
similar function for the plant gene (Bednarek et al., (1994) Plant
Physiol 104: 591-596).
In An; m~ two members of the rho subfamily, called Rac and
Rho, have been shown to be involved in the regulation of actin
organization (for a review, see Downward, (1992) Nature 359: 273-
274).
Racl has been shown to mediate growth factor-induced membrane
ruffling by influencing microf;~ nt alignment on the plasma
membrane (Ridley et al, (1992) Cell 70: 401-410), whereas RhoA
regulates the formation of actin stress fibers associated with
focal adhesions (Ridley and Hall, (1992) Cell 70: 389-399).
In yeast, the CDC42 gene codes for a rho-type protein which
also regulates actin organization involved in the establ;~m~t of
cell polarity required for the localized deposition of chitin in
the bud scar (Adams et al., (1990) J Cell Biol 111: 131-143.
Disruption of gene function, either by temperature shifts
with a CDC42-temperature-sensitive mutant in yeast (Adams et al.,
1990), or by micro-injection into fibroblasts of mutant Rac or Rho
proteins exibiting a ~o~in~nt negative phenotype (Ridley et al.,
1992; Ridley and Hall, 1992), leads to disorganization of the
actin network.
In plants, control of cytoskeletal organization is poorly
understood in spite of its importance for the regulation of
patterns of cell division, expansion, and subsequent deposition of
secondary cell wall polymers. The cotton fiber represents an
excellent system for studying cytoskeletal organization. Cotton
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fibers are single cells in which cell elongation and secondary
wall deposition can be studied as distinct events. These fibers
develop synchronously within the boll following anthesis, and each
fiber cell elongates for about 3 weeks, depositing a thin primary
wall (~e;nPrt and Delmer, (1984) Plant Physiol. 59: 1088-1097;
Basra and Mali}c, (1984) Int Rev of Cytol 89: 65-113). At the time
of transition to s~co~ y wall cellulose synthesis, the fiber
cells undergo a synchronous shift in the patterll of cortical
microtubule and cell wall microfibril alignments, events which may
be regulated upstream by the organization of actin (Seagull,
(1990) Protoplasma 159: 44-59; and (1992) In: Proceedings of the
Cotton Fiber Cellulose Conference, National Cotton Council of
America, M~mrh;c RN, pp 171-192.
Agrobacterium-mediated cotton transformation is described in
Umbeck, United States Patents Nos. 5,004,863 and 5,159,135 and
cotton transformation by particle bombardment is reported in WO
92/15675, published September 17, 1992. Transformation of
Brassica has been described by Radke et al. (Theor. Appl. Genet.
(1988) 75;685-694; Plant Cell Reports (1992) 11:499-505.
- - - SUMMARY OF THE lNV~ ON
Novel DNA constructs and methods for their use are described
which are capable of directing transcription of a gene of interest
in cotton fiber, particularly early in fiber development and
j 25 during secondary cell wall development. The novel constructs
include a vector comprising a transcriptional and translational
initiation region obtA;n~hle from a gene expressed in cotton fiber
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and methods of using constructs including the vector for altering
fiber phenotype. Both the endogenous 3' regions and 5~ regions
may be important in directing efficient transcription and
translation.
Three promoters are provided from genes involved in the
regulation of cotton fiber development. One, Racl3, is from a
protein in cotton which codes for an ~n; m~ 1 Rac protein homolog.
Racl3, shows highly-Pnh~nced expression during fiber development.
This pattern of expression correlates well with the timing of
reorganization of the cytoskeleton, suggesting that the Racl3
cotton gene may, like its ~n, mA 1 counterpart, be involved in the
signal transduction pathway for cytoskeletal organization. Racl3
is a gene that is moderately expressed during fiber devel~rm~nt
tl~rn; n~ on at 9 dpa and shutting down a~o~imately 24 dpa. It is
maximally expressed between 17-21 dpa developing fiber.
Another promoter from a cotton protein is designated 4-4.
The 4-4 mRNA accumulates in fiber cells at day 17 post anthesis
and continues towards fiber maturity, which occurs at 60 days or
so post anthesis. Data demonstrates that the 4-4 promoter r~A;n~
very active at day 35 post anthesis.
Also provided is a promoter from a lipid transfer protein
(hereinafter sometimes referred to as "Ltp") which is
preferentially expressed in cotton fiber.
The methods of the present invention include transfecting a
host plant cell of interest with a transcription or expression
cassette comprising a cotton fiber promoter and generating a plant
which is grown to produce fiber having the desired phenotype.
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Constructs and methods of the subject invention thus find use in
modulation oE endogenous fiber products, as well as production of
exogenous products and in modifying the phenotype of fiber and
fiber produc~s. The constructs also find use as molecular probes.
In particular, constructs and methods for use in gene expression
in cotton embryo tissues are considered herein. By these methods,
novel c~tton plants and cotton plant parts, such as modified
cotton fibers, may be obt~;ne~.
Also provided are constructs and methods of use relating to
modification of color phenotype in cotton fiber. Such constructs
contain sequences for expression of genes involved in the
production o~ colored compounds, such as anthocyanins, mel An i n or
indigo, and also may contain se~uences which prcvide for targeting
of the ~ene products to particular locations in the plant cell,
such as plastid organelles, or vacuoles. Plastid targeting is of
particular interest for expression of genes involved in aromatic
amino acid biosynthesis pathways, while vacuolar targeting is of
particular interest where the precursors required in synthesis of
the pigment are present in vacuoles.
Of particular interest are plants producing fibers which are
color, that is, with pigment produced in the fiber by the plant
during fiber development, as opposed to fibers which are harvested
and dyed or otherwise pigmented by separate processing. Fibers
from a plant producing such colored fiber may be used to produce
colored yarns and/or ~abric which have not been subjected to any
dyeing process. While naturally colored cotton has been available
from various domesticated and wild type cotton varieties, the
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instant application provides cotton fiber has a color produced by
the expression of a genetically engineered protein.
Thus, the application provides constructs and methods of use
relating to modification of color phenotype in cotton fiber. Such
constructs contain sequences for expression of genes involved in
the production of colored compounds, such as melanin or indigo,
and also contain seguences which provide for targeting of the gene
products to particular locations in the plant cell, such as
plastid organelles, or vacuoles. Plastid targeting is of
particular interest for expression of genes involved in the
aromatic amino acid biosynthesis pathways, while vacuolar
targeting is of particular interest where the precursors reguired
in synthesis of the pigment are present in vacuoles.
15DESCRIP~ION OF ~H~ PRAWINÇS
Figure 1 shows the DNA sequence encoding the structural
protein from cDNA 4-4.
Figure 2 shows the sequence to the promoter construct
pCGN5606 made using genomic DNA from 4-4-6 genomic clone.
20Figure 3 shows the sequence to the 4-4 promoter construct
pCGN5610.
Figure 4 shows the cDNA sequence encoding the Racl3 gene
expressed in cotton fiber.
Figure 5 shows the sequence the promoter region from the
racl3 gene.
Figure 6 shows a restriction map for pCGN4735.
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Figure 7 shows the sequence of the Ltp promoter region from a
cotton fiber specific lipid transfer protein gene.
Figure 8 shows the arrangement of a binary vectors pCGN5148
and pCGN5616 for plant transformation to express genes for ~el ~ni n
synthesis and indigo synthesis, respectively.
Fiyure 9 provides the results of color measu~ ts taken
from fibers of the control Coker 130 cotton used in transformation
using color constructs.
Figure 10 shows the results of measurements made of color of
plants transformed by the pCGN5148 construct to express genes for
melanin synthesis.
Fi~ure 11 shows the results of measurements taken of the
color o~ plants transformed by the pCGN5149 construct to express
genes for m~l ~n; n synthesis.
Figure 12 shows the results of measurements made of color of
plants transformed to express genes for indigo synthesis, using
construct pCGN5616.
Fi~ure :L3 shows control measurements made of naturally
colored cotton plan~s which are produced by non-transgenic colored
cotton plants.
DETAIL13~) DESCR:CPTION OF THE lNV15~ lON
In accordance with the subject invention, novel constructs
and methods are described, which may be used provide for
transcription of a nucleotide se~uence of interest in cells of a
plant host, preferentially in cotton fiber cells to produce cotton
fiber having an altered color phenotype.
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Cotton fiber is a differentiated single epi~ermAl cell of the
outer integument of the ovule. It has four distinct growth
phases; initiation, elongation (primary cell wall synthesis),
secondary cell wall synthesis, and maturation. Initiation of
fiber development appears to be triggered by hormones. The
primary cell wall is laid down during the elongation phase,
lasting up to 25 days postanthesis (DPA). Synthesis of the
seCon~ry wall c~mm~nces prior to the cessation of the elongation
phase and continues to approximately 40 DPA, forming a wall o~
almost pure cellulose.
The constructs for use in such cells may include several
~orms, depen~;ng upon the intended use of the construct. Thus,
the constructs include vectors, transcriptional cassettes,
expression cassettes and plasmids. The transcriptional and
translational initiation region (also sometimes referred to as a
~promoter,"), preferably comprises a transcriptional initiation
regulatory region and a translational initiation regulatory region
of untranslated 5~ sequences, "ribosome b;n~;ng sites,"
responsible for binding mRNA to ribosomes and translational
initiation. It is preferred that all of the transcriptional and
translational functional elements of the initiation control region
are derived from or obt~in~hle from the same gene. In some
embodiments, the promoter will be modified by the addition of
sequences, such as enhancers, or deletions of nonessential and/or
undesired sequences. By '~obt~;n~hle" is intended a promoter
having a DNA sequence sufficiently similar to that of a native
promoter to provide ~or the desired specificity of transcription
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of a DNA sequence of interest. It includes natural and synthetic
sequences as well as sequences which may be a combination of
synthetic and natural sequences.
Cotton fiber transcriptional initiation regions chosen for
cotton fiber modification may include the 4-4, racl3 and Ltp
cotton fiber pxomoter regions provided herein.
A transcriptional cassette for transcription of a ~ucleotide
sequence of interest in cotton fiber will include in the direction
of transcription, the cotton fiber transcriptional initiation
region, a DNA sequence of interest, and a transcriptional
t~rm; n~ tion region functional in the plant cell. When the
cassette provides for the transcription and translation of a DNA
sequence of interest it is considered an e~le~sion cassette. One
or more introns may be also be present.
Other sequences may also be present, including those encoding
transit peptides and secretory leader sequences as desired.
Fiber-tissue transcription initiation regions of this
invention are, preferably, not readily detectable in other plant
tissues. Transcription initiation regions capable of initiating
transcription in other plant tissues and/or at other stages of
fiber development, in addition to the foregoing, are acceptable
insofar as such regions provide a significant expression level in
cotton fiber at the defined periods of interest and do not
negative.ly interfere with the plant as a whole, and, in
particular, do not interfere with the development of fiber and/or
fiber-related parts.
.
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Downstream from, and under the regulatory control of, the
cotton fiber transcriptional/translational initiation control
region is a nucleotide se~uence of interest which provides for
modification of the phenotype of fiber. The nucleotide sequence
may be any open reA~; ng frame encoding a polypeptide of interest,
for example, an enzyme, or a sequence complementary to a genomic
sequence, where the genomic sequence may be an open re~; n~ ~rame,
an intron, a noncoding leader sequence, or any other se~uence
where the complementary sequence inhibits transcription, messenger
RNA processing, for example, splicing, or translation. The
nucleotide se~uences of this invention may be synthetic, naturally
derived, or combinations thereof. Dep~n~;ng upon the nature of
the DNA se~uence of interest, it may be desirable to synthesize
the se~uence with plant preferred codons. The plant preferred
codons may be determined from the codons of highest fre~uency in
the proteins expressed in the largest amount in the partic~lar
plant species of interest. Phenotypic modification can be
achieved by modulating production either of an endogenous
transcription or translation product, for example as to the
amount, relative distribution, or the like, or an exogenous
transcription or translation product, for example to provide for a
novel function or products in a transgenic host cell or tissue.
Of particular interest are DNA sequences encoding expression
products associated with the development of plant fiber, including
genes involved in metabolism of cytok;n;n~, auxins, ethylene,
abscissic acid, and the like. Methods and compositions for
modulating cytokinin expression are described in United States
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Patent No. 5,177,307, which disclosure is hereby incorporated by
reference. Alternatively, various genes, from sources including
other eukaryotic or prokaryotic cells, including bacteria, such as
those from Agrobacterium t7~nefaciens T-DNA auxin and cytokinin
S biosynthetic gene products, for example, and m~mm~l S, ~or example
interferons, may be used.
Other phenotypic modifications include modification of the
color o~ cotton fibers. Of interest are genes involved in
production o~ m~l ~n; n and genes involved in the production of
indigo. Melanins are dark brown pigments found in ~n;m~l S, plants
and microorg~n;sm~ any of which may serve as a source for
sequences for insertion into the constructs of the present
invention. Specific examples include the tyrosinase gene which
can be cloned ~rom Streptomyces antibioticus. The 0RF438 encoded
protein in S. antibioticus also is necessary for melanin
production, and may provide a copper donor function. In addition,
a tyrosinase gene can be isolated from any organism which makes
l~n;n. The gene can be isolated from human hair, ~m~lAnocytes or
melanomas, cuttle ~ish and red roosters, among others. See, for
example, EP ~pplication No. 89118346.9 which discloses a process
for producing melanins, their precursors and derivatives in
microorg~n;sm~. Also, See, Bernan et al. Gene (1985) 37:101-110;
and della-Cioppa et al . Bio/Tec~lology (1990) 80634-638.
Indigo may be obtained by use of genes encoding a mono-
oxygenase such as xylene oxygenase which oxidizes toluene andxylene to (methyl) benzyl alcohol and also transforms indole to
indigo. Cloning of the xylene oxygenase gene and the nucleotide
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and amino acid sequences are described in unexamined Japanese
Patent Application Kokai:2-119777, published May 7, 1990. A
dioxygenase such as naphthalene dioxygenase which also converts
indole to indigo finds use; the naphthalene dioxygenase gene nahA
is described in Science (1983) 222: 167. For cloning, nucleotide
sequence in characterization of genes encoding naphthalene
dioxygenase of Pse~ mo~A~ putida. See, Kurkela et al. Gene
(1988) 73:355-362. A tryptoph~n~ce gene seguence can be used in
conjunction with an oxygenase to increase the amount of indole
available for conversion to indigo. Sources of tryptop~n~e gene
sequences include E. coli (see, for example, Deeley et al. (1982)
J. Bacteriol . 151 :942-951).
Plastid targeting sequences (transit peptides) are available
from a number of plant nuclear-encoded plastid proteins, such as
the small subunit (SSU) o~ ribulose bisphosphate carboxylase,
plant fatty acid biosynthesis related genes including acyl carrier
protein (ACP), stearoyl-ACP desaturase, $-ketoacyl-ACP synthase
and acyl-ACP thioesterase, or LHCPII genes. The encoding sequence
~or a transit peptide which provides for transport to plastids may
include all or a portion of the encoding sequence for a particular
transit peptide, and may also contain portions of the mature
protein encoding sequence associated with a particular transit
peptide. There are numerous examples in the art of transit
peptides which may be used to deliver a target protein into a
plastid organelle. The particular transit peptide encoding
sequence used in the instant invention is not critical, as long as
delivery to the plastid is obtained.
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As an alternative to using transit peptides to target pigment
.
synthesis proteins to plastid organelles, the desired constructs
may be used to transform the plastid genome directly. In this
instance, promoters capable of providing for transcription of
genes in plant plastids are desired. Of particular interest is
the use of a T7 promoter to provide for high levels of
transcription. Since plastids do not contain an ~~ iate
polymerase for transcription from the T7 promoter, T7 polymerase
may be expressed from a nuclear construct and targeted to plastids
using transit peptides as described above. (See McBride et al.
(1994) Proc. Nat. Acad. Sci. 91:7301-7305; see also cop~n~ing US
patent application entitled "Controlled Expression of Transgenic
Constructs in Plant Plastidsn, serial no. 08/472,719, filed June
~ 6, 1995, and copen~;n~ US patent application SN 08/167,638, filed
December 14, 1993 and PCT/US94/14574 filed December 12, 1994.)
Tissue speci~ic or developmentally regulated promoters may be
useful for e~ression of the T7 polymerase in order to limit
expression to the appropriate tissue or stage of development.
Targeting of melanin synthesis genes to vacuoles is also of
20 interest in plant tissues which accumulate the tyrosine substrate
involved in melanin synthesls in vacuoles. The protein signal for
targeting to vacuoles may be provided from a plant gene which is
normally transported across the rough endoplasmic reticulum, such
as the 32 amino acid N-t~rm; n~l region of the
25 metallocarboxypeptidase inhibitor gene from tomato (Martineau et
al. (1991) Mol. Gen. Genet. 228 :281-286). In addition to the
J signal sequence, vacuolar targeting constructs also encode a
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vacuolar localization signal (VLS) positioned at the carboxy
tPrm;nll~ of the encoded protein. Appropriate signal se~uences and
VLS regions may be obt~;ne~ from various other plant genes and may
be similarly used in the constructs of this invention. Numerous
vacuolar targetting peptides are known to the art, as are reviewed
in Chrispeels et al., Cell (1992) 68:613-616.
The Maize Al gene which encodes a dihydroflavonol reductase,
an enzyme of the anthocyanin pigmentation pathway is one such
gene. In cells that express the Al gene, dihydrokempferol is
converted to 2-8 alkylleucopelargonidin, which may be further
metabolized to pelargonidin pigment by endogenous plant enzymes.
Other anthocyanin or flavonoid type pigments may also be of
interest for modification of cotton cell fibers, and have been
suggested for use in plant flowers (for a review of plant flower
color, see van Tunen et al., Plant Biotechnology Series, Volume 2
(1990) Developmental Regulation of Plant Gene Expression, D.
Grierson ed.). Anthocyanin is produced by a progression of steps
from cellular phenylalanine pools. The R anc C1 genes are maize
regulatory proteins which are active by positively affecting
upstream steps in the anthocyanin biosynthesis from these pools.
The R gene is described in Perot and Cone (1989) Nucl. Acids Res.,
17:8003, and the C1 gene is described in Paz-Ares et al (1987)
EMBO, 6:3553-3558. Lloyd et al. (1992) Science, 258:1773-1775
discussed both genes.
Although cotton fibers in commercially grown varieties are
primarily white in color, other naturally occurring cotton
varieties have brown or r~ h-brown fibers. Additionally, a
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cotton line cont~;n;ng green colored fibers has been identified.
Cotton lines providing such fibers are available from various
sources, including the BC variety cottons (BC Cotton Inc., Box
8656, Bakersfield, CA 93389) and Fox Fibre cottons (Natural
Cotton Colors, Inc., P.O. Box 791, Wasco, CA 93280).
The existence of such colored cotton lines suggests that the
precursors required for the anthocyanin pigment pathway~ a~e
present in cotton fibers cells, thus allowing further color
phenotype modifications. Thus, the maize R and C1 genes could be
used in enhancing the levels of of anthocyanin produced in fiber
cells. As the R and C1 proteins are proteins with a positive
control at the regulatory level on anthocyanin pigment precursor
biosynthesis, these proteins are expressed in the nucleus, and not
targetted to plastids or vacuoles.
For some applications, it is of interest to modify other
aspects of the fiber. For example, it is of interest to modify
various aspects of cotton fibers, such as strength or texture of a
fiber. Thus, the appropriate gene may be inserted in the
constructs of the invention, including genes for PHB biosynthesis
(see, Peoples et al. ~. Biol. Chem. (1989) 264: 15298-15303 and
Ibid. 15293-15397; Saxena, Plant Molecular Biolo~y (1990) 15:673-
683, which discloses cloning and sequencing of the cellulose
synthase catalytic subunit gene; and Bowen et al. PN~S (1992)
89:519-523 which discloses chitin synthase genes of .~h~rQmyces
cerevisiae and Candida albicans. Various constructs and methods
are disclosed for the use of hormones to effect changes to fiber
quality in cop~n~l ng US patent application entitled "Cotton
-
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Modification Using Ovary-Tissue Transcriptional factors", serial
no. 08/397,652 filed February 2, 1995, the teachings of which are
incorporated herein by reference.
Transcriptional cassettes may be used when the transcription
of an anti-sense seguence is desired. When the expression of a
polypeptide is desired, expression cassettes providing for
transcription and translation of the DNA seguence of interest will
be used. Various changes are of interest; these changes may
include modulation (increase or decrease) of formation of
particular saccharides, hormones, enzymes, or other biological
parameters. These also include modifying the composition of the
final fiber that is changing the ratio and/or amounts of water,
solids, fiber or sugars. Other phenotypic properties of interest
for modification include response to stress, org~ni~cmc,
herbicides, brushing, growth regulators, and the like. These
results can be achieved by providing for reduction of expression
of one or more endogenous products, particularly an enzyme or
cofactor, either by producing a transcription product which is
complementary (anti-sense) to the transcription product of a
native gene, so as to inhibit the maturation and/or expression of
the transcription product, or by providing for expression of a
gene, either endogenous or exogenous, to be associated with the
development of a plant fiber.
The t~rm;n~tion region which is employed in the expression
cassette will be primarily one of convenience, since the
t~rm;n~tion regions appear to be relatively interchangeable. The
term;nAtion region may be native with the transcriptional
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initia~ion region, may be native ~ith the DNA sequence of
interest, may be deri~ed from another source. The t~rmin~tion
region may be naturally occurring, or wholly or partially
synthetic. Convenient t~rmin~tion regions are available ~rom the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase t~rm;n~tion regions. In some ~mh~;m~nts~ it
may be desired to use the 3' t~rm;n~tion region native to the
cotton fiber transcription initiation region used in a particular
construct.
As described herein, in some instances additional nucleotide
sequences will be present in the constructs to provide for
targeting of a particular gene product to speci~ic cellular
locations. '.For example, where coding se~n~C for synthesis of
aromatic colored pigments are used in a construct, particularly
coding se~uences for enzymes which have as their substrates
aromatic compounds such tyrosine and indole, it is preferable to
include sequences which provide for delivery of the enzyme into
plastids, such as an SSU transit peptide seguence. Also, for
synthesis of pigments derived from tyrosine, such as melanin,
targeting to the vacuole may provide for enhanced color
modifications
For melanin production, the tyrosinase and ORF438 genes from
Streptomyces antibioticus (Bermc~n et al. (1985) 37:101-110) are
provided in cotton fiber cells for expression from a 4-4 and Rac13
promoter. In Streptomyces, the ORF438 and tyrosinase proteins are
expressed from the same promoter region. For expression from
constructs in a transgenic plant genome, the coding regions may be
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provided under the regulatory control of separate promoter
regions. The promoter regions may be the same or different for
the two genes. Alternatively, coordinate expression of the two
genes from a single plant promoter may be desired. Constructs ~or
expression of the tyrosinase and ORF438 gene products from 4-4 and
rac promoter regions are described in detail in the following
examples. Additional promoters may also be desired, for example
plant viral promoters, such as CaMV 35S, can be used for
constitutive expression of one of the desired gene products, with
the other gene product being expressed in cotton fiber tissues
from the 4-4 and rac promoter.
Similarly, other constitutive promoters may also be useful in
certain applications, for example the mas, Mac or DoubleMac,
promoters described in United States Patent No. 5,106,739 and by
Comai et al., Plant Mol. Biol. (1990) 15:373-381J. When plants
comprising multiple gene constructs are desired, ~or example
plants expressing the melanin genes, ORF438 and tyrosinase, the
plants may be obtained by co-transformation with both constructs,
or by transformation with individual constructs followed by plant
breeding methods to obtain plants expressing both of the desired
genes.
A variety of techniques are available and known to those
skilled in the art for introduction of constructs into a plant
cell host. These techniques include transfection with DNA
employing A. tumefaciens or A. rhizogenes as the transfecting
agent, protoplast fusion, injection, electroporation, particle
acceleration, etc. For transformation with Agrobacterium,
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plasmids can be prepared in E. coli which contain DNA homologous
; with the Ti-plasmid, particularly T-DNA. The plasmid may or may
not be capab:Le of replication in Agrobacterium, that is, it may or
may not have a broad spectrum prokaryotic replication system such
' 5 as does, for example, pRK290, dep~n~;n~ in part upon whether the
transcription cassette is to be integrated into the Ti-plasmid or
to be ret~;n~ on an independent plasmid. The Agrobacterium host
will contain a plasmid having the vir genes necessary for transfer
of the T-DNA to the plant cell and may or may not have the
complete T-DNA. At least the right border and fre~uently both the
right and left borders of the T-DNA of the Ti- or Ri-plasmids will
be joined as flanking regions to the transcription construct. The
use of T-DNA for transformation of plant cells has received
extensive study and is amply described in EPA Serial No. 120,516,
Hoekema, In: The Binary Plant Vector System Offset-drukkerij
Kanters B.V., Alblasserdam, 1985, Chapter V, Knauf, et al.,
Genetic Anal~sis of Host Range Expression by Agrobacterium, In:
Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A.
ed., Springer-Verlag, NY, 1983, p. 245, and An, et al., EMBO J.
(1985) 4:277-284.
For infection, particle acceleration and electroporation, a
disarmed Ti-plasmid lacking particularly the tumor genes found in
; the T-DI~A region) may be introduced into the plant cell. By means
of a helper plasmid, the construct may be transferred to the A.
tumefaciens and the resulting transfected org~n;.~m used for
transfecting a plant cell; explants may be cultivated with
transformed A. tumefaciens or A. rhizogenes to allow for transfer
21
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of the transcription cassette to the plant cells. Alternatively,
to enhance integration into the plant genome, t~rm; n~l repeats of
transposons may be used as borders in conjunction with a
transposase. In this situation, expression of the transposase
should be inducible, so that once the transcription construct is
in~egrated into tXe genome, lt should be relatively stably
integrated. Transgenic plant cells are then placed in an
appropriate selective medium for selection of transgenic cells
which are then grown to callus, shoots grown and plantlets
generated from the shoot by growing in rooting medium.
To confirm the presence of the transgenes in transgenic cells
and plants, a Southern blot analysis can be performed using
methods known to those skilled in the art. ~.e~sion products of
the transgenes can be detected in any of a variety of ways,
depending upon the nature of the product, and include ;mmllne
assay, enzyme assay or visual inspection, for example to detect
pigment formation in the appropriate plant part or cells. Once
transgenic plants have been obtained, they may be grown to produce
fiber having the desired phenotype. The fibers may be harvested,
and/or the seed collected. The seed may serve as a source for
growing additional plants having the desired characteristics. The
terms transgenic plants and transgenic cells include plants and
cells derived from either transgenic plants or transgenic cells.
The various sequences provided herein may be used as
molecular probes for the isolation of other se~uences which may be
useful in the present invention, for example, to obtain related
transcriptional initiation regions from the same or different
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plant sources. Related transcriptional initiation regions
obtA; n~hle from the sequences provided in this invention will show
at least about 60% homology, and more preferred regions will
on~trate an even greater percentage of homology with the
probes. Of particular importance is the ability to obtain related
transcription initiation control regions having the t;m;n~ and
tissue parameters described herein. For example, using the probe
4-4 and rac, at least 7 additional clones, have b~n identified,
but not further characterized. Thus, by employing the techniques
described in this application, and other techniques known in the
art (such as Maniatis, et al., Molecular Cloning,- A Laborato~y
M~n7~ 7 (Cold Spring Harbor, New York) 1982), other transcription
! initiation regions capable of directing cotton fiber transcription
as described in this invention may be det~rm;n~ The constructs
can also be used in conjunction with plant regeneration systems to
obtain plant cells and plants; thus, the constructs may be used to
modify the phenotype of fiber cells, to provide cotton fibers
which are colored as the result of genetic engineering to
heretofor unavailable hues and/or intensities.
Various varieties and lines of cotton may find use in the
described methods Cultivated cotton species include Gossypium
hirsutuin and G. babadense (extra-long stable, or Pima cotton),
which evolved in the New World, and the Old World crops G.
herbaceum and G. arboreum.
Color phenotypes can be assessed by the use of a colorimeter,
an instrument which is already used to provide objective
measurements of the color of cotton samples. A colorimeter uses a
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combination of light sources and filters to make various estimates
of a samples colors, sometimes referred to as tristimulus values.
In the past such estimtes have been used to calculate a value
(Hunter's + b, described below) indicating the degree of
yellowness of a cotton sample. The yellowness and reflectance
(from Rd, the degree of lightness or darkness of the samples) has
been used to provide cotton color measurements for grading. Tests
are typically conducted by exposing the face of a sample to a
controlled light source. A typical color chart showing how the
official grade stAn~rds relate to Rd and+ b measurements is shown
in Cotton, RJ Kohel and CF Lewis, Editors #24 in AGRONOMY Series-
American Soc. A~ IOL1~ (see Fig. 12-6).
Various colorimeter methods can be so used to quantify color
and express it numerically. The Munsell method, devised by the
American artist A.. Munsell, uses a classification system of paper
color chips assorted according to their hue (Munsell Hue),
lightness (Munsell Value), and saturation (Munsell Chroma) for
visual comparison with a specimen color.
Other methods for expressing color numerically have been
developed by an international organization concerned with light
and color, the Commission Internationale de l'Eclairage (CIE),
having a Central Bureau located at Kegelgasse 27, A-1030 Vienna,
AUSTRIA. The two most widely known of these methods are the Yxy
color space, devised in 1931 based on the tristimulus value XYZ,
as defined by CIE, and the L*a*b* color space, devised in 1976 to
provide more uniform color differences in relation to visual
differences. Color spaces* such as these are now used throughout
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the world for color c~mml~n;cation. The Hunter Lab color space was
developed in 1948 by R.S. Hunter as a uniform color space which
could be read directly from a photoelectric colorimeter
(tristi~nulus method).
-~ 5 The L*C~h color space uses the same diagram as the L*a*b*
color space, but uses cylindrical coordinates instead of
rectangular coordinates. In this color space, L* indicates
lightne.ss and is the same as the L* of the L*a*b* color space, C*
is chroma, and h is the hue angle. The value of chroma C is 0 at
I0 the center and increases according to the distance from the
center. Hue angle is defined as starting at the +a axis of the
L*a*b* .space, and is expressed in degrees in a counterclockwise
rotation. Thus, relative to the L*a*b* space, 0~ and 360- would
be at the +a* line, 90 would be +b*, 180- would be -a* and 270-
would be -b*~
All of the above methods can be used to obtain precise
measurements of a cotton fiber color phenotype.
.. .
EXPERIMENTAL
The following examples are offered by way of illustration and
not by limitation.
~xam~le 1
cDNA libraries
Tissue ~re~aration for cDNA svnthesis
Leaf and root tissue were isolated from 8 inch tall
greenhouse grown seedlings and ;m~e~;ately frozen in liquid
nitrogen. Flowers were collected at the rapidly ~X~An~; ng 3 day
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preanthesis stage and also frozen. Seed was collected from 21 day
postanthesis locules which had been removed from the boll and
frozen entire in liquid nitrogen. Once frozen, the fiber was
removed from the seed and the denuded seed used for RNA isolation.
All fibers were removed from the seed under liguid nitrogen and
the ~iber was ground to a powder prior to RNA isolation. Fibers
were ~rom bolls which had been tagged at anthesis.
~NA and RNA Mani~ulations
The lambda ZapII~ cDNA library system of Stratagene was used
for screening, and was prepared from cDNA derived from poly-A+
mRNA isolated from fibers of Gossypium hirsutum cultivar Acala SJ-
2. The fibers were isolated from bolls harvested at approximately
21 dpa using field-grown plants in Israel.
Total RNA was isolated from 21 dpa seeds (G. hirsutum cv
Coker 130 from which the fiber had been removed) using the method
of Hughes and Galau ((1988) Plant Mol Biol Reporter, 6:253-257.)
All other RNAs were prepared according to Hall et al . ( (1978),
Proc Natl Acad Sci USA 75: 3196-3200), with the following
modifications. After the second 2M LiCl wash, the pellet was
dissolved in 1/10 original volume of 10 mM Tris pH7.5 and brought
to 35mM potassium acetate pH6.5 and 1/2 volume EtOH was added
slowly. The mixture was placed on ice for 15 minutes and then
centrifuged at 20,000 x g for 15 minutes at 4~C. The potassium
acetate concentration was brought to 0.2M, 2 1/2 volumes EtOH
added and the RNA placed at -20~C for several hours. The
precipitate was centrifuged at 12,000 x g for 30 minutes at 4~C
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and the pellet was resuspended in diethylpyrocarbonate-treated
water. Poly-A~~ RNA was prepared from total mRNA utilizing an
oligo(dT)-cellulose kit (Becton Dickenson) and following the
m~nllf~cturer's protocol.
Cotton genomic DNA was prepared as follows. Four grams of
young cotton leaf tissue (cv Coker 130) was gro~nd to a powder in
N2 and placed in an Oak Ridge tube with 0.4g polyvinylpyrolidone
and 20m]s extraction buffer (200mM Ches/NaOH ph9.1, 200mM NaCl,
100mMEDl'A/NaOH pH9.0, 2% SDS, 0.5% Na deoxycholate, 2~ Nonidet NP-
40, 20mM B-mercaptoethanol) was added to sample, gently mixed and
incubated at 65~C in a shaking water bath for 10 minutes. 7.0 mls
of 5M potassium acetate pH6.5 was added and carefully mixed.
Incubation was carried out on ice for 30 minutes with gentle
mixing every 5 minutes. The sample was centrifuged for 20 minutes
at 21,000 x g and the supernatant was filtered through Miracloth
into another tube and centrifuged as before. The supernatant was
again filtered through Miracloth into 15 mls of room temperature
isopropanol in an Oak Ridge tube. After gentle mixing, the sample
was incubated at room temperature for 10-60 minutes until the DNA
precipitated. The DNA was spooled and allowed to air dry before
being resuspended in 4 mls of TE on ice for 1 hour. CsCl was
added to 0.97g/ml final concentration and 300 ul 10mg/ml ethidium
bromide was also added before filling VTi80 ~uick seal tubes. The
sample was centrifuged overnight at 225,000 x g overnight. The
DNA was extracted with water saturated butanol and enough water
was added to bring the volume to 4 mls before ~; ng 2 volumes
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EtOH. The DNA was spooled, air dried and resuspended in 200 ul
sterile water.
Northe~n ~n~l Southern ~n;~lvsis
For Northerns, 10ug of total RNA was isolated from various
tissues, separated by electro~horesis in 1.2% agarose-formaldehyde
gels and transfered onto Nytran Plus m~,~l~les (Schleicher and
Schuell). Hybridization conditions consisted of a solution
cont~; n; n~ 50% formamide(v/~), 5xSSC, O . 1% SDS, 5mM EDTA, 10X
10 Denhardts solution, 25mM sodium phosphate pH6.5 and 250 ug/ml
carrier DNA. Washes were performed in 2xSSC, 0.1% SDS at 42~C 3
times for 30 minutes each time.
Cotton genomic DNA (12ug) was digested with various
restriction endonucleases, electrophoresed in 0.9~ agarose gels
and blotted onto Nytran Plus membranes. Hybridization and filter
washing conditions for both the 3' speci~ic and full-length cDNA
insert probes were as described for Northern analysis.
Probes derived from 3'-untranslated regions were synthesized
via oligonucleotide primers from the Racl3 cDNA, corresponding to
bases 600-619 and 843-864 (Figure 4). Each set of primers was
used in a polymerase chain reaction to synthesize copies of 3'-
specific DNA sequences. These sequences were used as templates in
the generation of single-stranded, 32P-labeled probes off the
ntisense strand in a polymerase chain reaction. The full-length
cDNA inserts for Racl3 were used as templates for double stranded,
random primed probes using the Prime-It kit (Stratagene).
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~ Example 2
Isolation of CDNA Clones from Cotton
cDNA ~o the 4-4 clone was isolated from the cotton ~iber
library described above, and shown to express in fiber but not
other tissues. This seguence was not related to any known
protein" Only 400 kb of encoding sequence was present in this
clone, so the library was rescreened using the cDNA to obtain
full-length clones. The ~ull-length encoding se~uence is provided
in Figure 1.
By comparing sequences of random cDNA clones against various
sequence data banks via BLAST, a National Center for Biotechnology
Information service, a clone, designated #105, was found to have
an encoding sequence related to that o~ a reported lipid transfer
protein.
Another clone was seguenced which showed high homology to
~n;m~l Rac proteins. This clone, designated Rac, was not guite
full-length, and the library was re-screened using this initial
Rac DNA segment as probe. Of approximately 130,000 primary
plaques screened, 56 screened positive; of these, 14 clones were
isolated and sequenced. Of these 14 clones, 12 showed identical
sequence homology to the original Rac clone and one of these cDNA
clones encoded a full length cDNA and received the name Racl3.
Figure 4 shows the cDNA sequence encoding the Racl3 gene expressed
in cotton fiber.
One other partial-length cDNA clone, designated Rac9, was
clearly related, but distinct in DNA and amino acid sequence from
r Racl3. Re-screening of 150,000 plaques resulted in the isolation
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of 36 positive clones of which only two clones corresponded to the
Rac9 sequence (both full-length clones), the r~m~in~er being
Racl3. These results suggest that cotton contains genes for at
least two distinct Rac proteins. Based upon the fre~uency of
clone isolation, Racl3 is relatively highly-expressed and Rac9
less so in cotton fibers at 21 days post-anthesis (dpa), the age
at which polyA+ mRNA was isolated for library construction.
Comparisons of the deduced amino acid se~uence of Racl3 with
other small G-proteins showed that the cotton Rac proteins are
very closely related to the Rhol protein sequence deduced from a
cDNA clone isolated recently from pea (Yang and Watson, supra).
After the pea Rhol, m~mm~l ian Rac proteins show the highest
homology with the cotton Rac proteins. Other proteins of the rho
subf~mily, such as the yeast CDC42 and human RhoA, are also
clearly related to the cotton Rac genes. By contrast, the other
small G-proteins of the Rab/YPT subfamily isolated from plants
such as the example shown of the tobacco RAB5 protein, as well as
the human Ras proteins, are least homologous to the cotton Rac
proteins of all the small G-proteins compared. The cotton and pea
proteins, as well as the mammalian Racs, all have pI's above 9,
whereas those of other rho and ras proteins are in the range of
5.0-6.5.
Example 3
~x~ression of Cotton Fiber Genes in Develo~in~ Fibers
Expression of the Racl3 and 4-4 genes was assessed using
mRNA prepared from various cotton tissues and from fibers at
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different stages of development. Blots were hybridized with
probes derived from untranslated regions of Ltp, Racl3 and 4-4
genes. The gene for Racl3 exhibits highly-~nh~nced expression in
fibers; virtually no detectable mRNA is present in leaves, roots,
or flower parts, even under conditions of extended developmen~
time. Racl3 expression is detected in seeds at an age that
corresponds to the highest expression levels observed in fiber
tissue derived from seeds of this same age. The pattern of Racl3
expression in fibers is very dependent upon the developmental
stage. Expression lS very low during the stage of primary wall
synthesis (0-14 dpa, see M~;ne~t and Delmer, 1977), re~cheC a
maximum duri~g the transition to secondary wall synthesis (about
15-18 dpa), and declining during the stage of m~ l s~on~ y
wall cellulose synthesis (about 24-28 dpa).
4-4 mRN~ is begins to accumulate in fiber cells only at day
17 post anthesis and continues through at least day 35 post
anthesis. Levels peak at day 21 and remain high. 4-4 mRNA is not
detected in other cotton tissues, and is not detected in fiber
tissue before onset at 17 days post anthesis.
The #105 lipid trans~er protein cDNA clone was used as a
probe against cotton tissue and in a cotton fiber northern. The
northern showed that the cotton fiber Ltp is highly expressed in
cotton fiber. The mRNA that codes for this protein is expressed
throughout fiber development at extremely high level. Northern
blots indicate that this mRNA is expressed at 5 dpa and is
continually expressed at a high level at 40 dpa.
.
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Example 4
G~nomic DN~
cDNA for both the 4-4 and Racl3 was used to probe for
genomic clones. For both, full length genomic DNA was obtained
from a library made using the lambda dash 2 vector from
Stratagene~, which was used to construct a genomic DNA library
from cotton variety Coker 130 (Gossypium hirsutum cv. coker 130),
using DNA obt~;n~ from g~rm;nAting seedlings.
The cotton genomic library was probed with a 3'-specific Ltp
probe and 6 genomic phage candidates were identified and puri~ied.
Figure 7 provides an approximately 2 kb sequence of the Ltp
promoter region which is ; mm~ tely 5' to the Ltp encoding
region.
Six genomic phage clones from the cotton genomic library
were identified using a 3'-specific probe for the Ltp mRNA. This
was done to select the promoter from the Ltp gene that is
maximally expressed in cotton fiber from the family of Ltp genes
in cotton. The Ltp promoter is active throughout the fiber
development period.
Example 5
PreDaration of 4-4 Promoter Constructs
~CGN5606
The pCGN5606 promoter construct col,l~rises the 4-4 cotton
fiber expression cassette in a first version, version I (Figure
2). The sequences from ntl to 65 and nt 5,494 to 5,547 correspond
to fragments of the pBluescriptII polylinker where this cassette
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is cloned. Uni9ue restriction enzyme sites present in these
regions flan]cing the cassette allow the cloning of the fiber
expression cassette into binary vectors including the pCGN 5138
and 1547 series.
The se~uences from nt57 to 5,494 are cont~;ne~ in a l~mh~
phage clone of a cotton Coker 130 genomic library. This lambda
genomic clone was given the designation 4-4(6).
The region from nt 65 to nt 4,163 corresponds to the 5'
flanking region of the 4-4(6) gene. At nt 4,163 there is a NcoI
restriction site se~uence that corresponds to the first codon of
the 4-4 (6)0RF.
The region from nucleotide 4,163 to 4,502 corresponds to part
of the 4-4 (6)0RF. The seguence from nt 4,502 to 4,555 is a
synthetic po:Lylinker oligonucleotide that contains uni~ue target
sites for the restriction enzymes EcoRI, SmaI, SalI, NheI and
BglII. This fragment from nt4,163 to 4,555 is a stuffer fragment
and is left in place to facilitate the monitoring of cloning
manipulations.
The genes to be expressed in cotton fiber cells using this
cassette can be cloned between the NcoI restriction site and any
of the polylinker sites. This operation will replace the stuffer
fragment with the gene of interest. The region from nt 4,555to
5,494 corresponds to the 940 nucleotides downstream of the stop
codon and constitute the 3' flanking region of the 4-4 (6) gene.
There is a unigue AscI restriction enzyme site at nt 5483.
pCGN5610
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The pCGN5610 construct is a second version of a 4-4 cotton
fiber expression cassette, version II, which is a modified version
of pCGN5606. The two versions of the 4-4 cotton ~iber expression
cassette are designed to allow the cloning of t~n~m arrays of two
fiber cassettes in one binary plasmid. The differences with
respect to pCGN5606 are very minor and described below.
The XbaI restriction site in the region of nt 1 to 65 has
been deleted by stAn~d cloning manipulations.
The polylinker region is in the reverse orientation of pCGN5606.
There is a unique XbaI restriction enzyme site at nt5484. The
se~uences from ntl to 57 and nt 5,494 to 5,518 of pCGN5610
correspond to fragments of the pBluescriptII polylinker where this
cassette is cloned. Unigue restriction enzyme sites present in
these regions allow the cloning of the fiber expression cassette
into binary vectors of the pCGN 5138 and 1547 series.
The sequences from nt57 to 5,494 are contdined a lambda
phage clone of a Coker 130 genomic library. This clone is
described in my notebook as 1~mh~ genomic clone 4-4(6). The
region from nt 57 to nt 4,155 corresponds to the 5' flanking
region. At nt 4,155 there is a NcoI restriction site seguence
that corresponds to the first codon of the 4-4 ORF.
The region from nucleotide 4,156 to 4,500 corresponds to part of
the 4-4 ORF. This fragment from nt4,156 to 4,550 is a stuffer
fragment and is left in place to facilitate the monitoring of
cloning manipulations. The sequence from nt 4,500 to 4,550 is a
synthetic polylinker oligonucleotide cont~;n'ng unique target
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sites for the restriction enzymes BglII, NheI, SalI, SmaI and
EcoRI.
The genes to be expressed in cotton fiber cells using this
cassette can be cloned between the NcoI restriction site and any
of the polylinker sites. This operation replaces the stuffer
fragment with the gene of interest. The region from nt 4,550 to
5,494 corresponds to the 940 nucleotides downstream of ~he stop
I ~ codon and constitute the 3' flanking region of the 4-4 (6) gene.
~xam~le 6
Pre~aration of Racl3 Promoter Constructs
Genomic clone
From a genomic clone designated 15-1, mapping was done with
restricl:ion endonucleases. The largest fragment with the Rac13
coding region was identified. Theis was a Pst fragment, and when
subcloned in the Bluescript~ KS+ vector (BSKS+; Stratagene) was
named pCGN4722. The insert had a length of 9.2 kb.
The region of the Pst fragment with the Racl3 co~;ng sequence
was identified. DNA sequence was determined for approximately 1.7
; 20 kb 5' of the start codon and approximately 1.2 kb 3' of the stop
i codon. The entire Rac coding region (exons and introns) was
conveniently flanked by Ndel sites.
pCGN4722 was dlgested with Xbal, and a 2.7 kb fragment was
removed. Religation gave pCGN4730, which was then digested with
Ndel, dropping out a 1.7 kb fragment cont~;n;ng the entire Rac
coding region. Religation yielded pCGN4731.
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A polylinker region was created using overlapping synthetic
oligonucleotides which were PCR'ed using primers homologous to the
5' and 3' ends of the resynthesized section. The resulting
product was digested with EcooRl and Hind III and ligated into
BSKS+ at the EcoRl and Hind III sites. The resulting plasmid was
designated pCGN4733.
pCGN4731 and pCGN4633 were digested with Ndel and the Ndel
fragment cont~;n;ng the synthesized polylinker region from
pCGN4733 was dropped in the Ndel site of 4731, giving pCGN4734.
This last plasmid was digested with Sal and Xba, and so was
pCGN5133. pCGN5133 was the 9.2 kb pst fragment in BSKS+ where the
polylinker sites flanking the insert were altered to different
sites for ease of manipulation. The fragment from pCGN4734 was
then placed into the equivalent site of pCGN5143, giving pCGN4735.
A sequence for approximately 3 kb of the promoter construct
pCGN4735 is provided in Figure 5. The resynthesized sequence
falls between the Ndel sites located at bases 1706 and 1898 o~ the
seguences. Thus, the sequence in Figure 5 includes approximately
1.7 kb 5' to the Ndel site 5' to the resynthesized polylinker
region. There is a roughly 2.5 kb sequence 5' from this sequence
which is not provided in Figure 5, relative to the total 9.2 kb
insert. The sequence o~ Figure 5 also includes approximately 1.1
kb 3' to the 3' Ndel site. Approximately 3 kb which is most 3' in
the Racl3 insert is not provided in Figure 5. A map for pCGN4735
is provided in Figure 6.
~mnle 7
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~ Piament Svnthesis Genes
Melanin
A binary construct for plant transformation to express gene.s
for mel ~n; n synthesis is prepared as follows. The m~l ~ni n genes
were originally isolated from the cnmmnn soil bacterium
StreptQmyces antibioticus (R~rn~n et al. (1985) 34:101-110).
M~l An; n prod~ction is composed of a two gene system. The first
gene, tyrA, encodes the catalytic unit responsible for the
polymerization of the amino acid tyrosine, the primary substrate,
and is termed tyrosinase The second gene, 0RF438, is responsible
for b; n~; ng copper and delivering copper to the tyrosinase and
activating the enzyme. Expression of both the ORF438 and tyrA
genes ensures maximal tyrosinase activity.
The genes for both 0RF438 and tyrA were fully re-synthesized
with respect to their DNA seauence. This was performed as the
initial DNA se~uence isolated from Streptomyces has a very high
guanine and cytosine (G+C) DNA content. Thus, the 0RF438 and t~yA
genes were re-synthesized to appear more "plant like" (reduced G+C
content) with respect to plant pre~erred codons encoding their
corresponding amino acids.
Tn~; ao
Indigo production involves conversion of the amino acid
tryptophan, the primary substrate, into indole which is then
converted into indoxyl. Molecules of indoxyl spontaneously
convert to indigo in the presence of oxygen. A two gene system
. was used to affect indigo production in fiber cells. The first
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gene (tna) was obtained from the bacterium E. coli and encodes
the enzyme tryptoph~n~e. The designation tna stands for the gene
encoding trypto~n~e f~rom E. coli, an enzyme which converts
tryptophan to indole (Stewart et al., (1986) J Bacteriol 166:217-
5 223).
The pig designation is used for the encoding sesIuence to the
protein for indigo production from ~hodococcus, which produces
indigo from indole (Hart et al., (1990) J Gen Microbiol 136:1357-
1363). Both tna and pig were obtained by PCR. Tryptoph~nAce is
10 responsible for the conversion of tryptophan to indole, while the
second gene (pig) encodes an indole oxygenase enzyme responsible
for the conversion of indole to indoxyl. Both these bacterial
genes were utilized in their native form.
~ m~?le 8
Constructs for Taraetina Pi~m~nt Synthes;s Genes
For plastid targeting, the constructs contain a fragment of
the tobacco ribulose bisphosphate carbo~lase small subunit gene
encoding the transit peptide and 12 amino acids of the mature
20 protein (Tssu) positioned in r~;n~ frame with the a~lo~riate
encoding sequence.
For vacuolar targeting of the mel~qn; n synthesis genes,
constructs include a fragment of the metallocarboxypeptidase
;nh;h;tor gene, encoding the entire 32 amino acid N-t~rm;nll~
25 signal peptide of that protein plus 6 amino acids of the mature
protein (CPI+6) (Martineau et al., supra), positioned in r~;~-l;ng
frame with the appropriate encoding se~uences. In addition to the
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signal peptide, a sequence encoding a vacuolar localization signal
(VLS) is inserted 3' of the protein encoding se~uence.
C~nstructs which contain encoding sequences for bacterial
~enes involved in biosynthesis of pigmented compounds and
5 sequences for directing transport of the encoded proteins into
! plastids or vacuoles are prepared as follows.
Me~;~n; n
The re-synthesized 0RF438 and tyrA genes were treated in two
distinct ways dep~n~; ng on which compartment in the fiber cell the
~inal protein products would be localized. One ch;m~ric
gene/plant binary construct (designated pCGN5148) cont~;n~ the
genes target~d to the ~iber cell plastids. To do this, 12 amino
acids of a gene for the small subunit of carboxylase (SSU) plus
the original 54 amino acid SSU transit peptide were fused to the
amino t~rm; ni of both the ORF438 and tyrA gene products
respectively. These peptide seguences allow the 0RF438 and tyrA
gene products (proteins) to be efficiently targeted to the
plastid. This targeting was initiated as the plastid is the site
of tyrosine production within the fiber cell.
; The second chimeric gene/plant binary construct (designated
pCGN5149) contained the 0RF438 and tyrA genes targeted to the
vacuole within the fiber cell. Based on information from other
biological systems, it was postulated that the fiber cell vacuole
may contain a high concentration of tyrosine for melanin
polymerization. Both the 0RF438 and tryA genes contain the 29
amino acid signal peptide from a tomato cArh~xypeptidase ; nh; h; tor
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(CPI) protein as amino t~rm;n~l gene ~usions to direct these
proteins to the endoplasmic reticulum (ER) secretory system o~ the
fiber cell.
In addition, the tyrA gene has an 8 amino acid vacuolar
targeting peptide (VTP) from CPI fused at the carboxy t~rm;ntlc 50
that the mature copper-activated tyrosinase will eventually be
targeted to the vacuole of the fiber cell. Both the ORF438 and
tyrA proteins also had potential glycosylation sites l~,lov~d via
site-directed mutagenesis of the ORF438 and tyrA genes
respectively. Potential plant cell glycosylation of these
proteins upon their expression in fiber cells could result in
tyrosinase inactivation, hence L~.w~dl of potential glycosylation
sites was ~e~meA necessary.
15 Tn-l; ao
The only modification to the indigo genes was the fusion of
the tobacco SSU transit peptide encoding DNA sequences onto the
amino term; n~ 1 region of both the tna and pig genes to affect the
localization o~ both the tryptophanase and indole oxygenase
proteins to the fiber cell plastid. These are the same exact gene
fusions that were made for the plastid-directed proteins for
melanin production in construct 5148. The tna and pig gene
products were targeted to the ~iber cell plastid as that is the
primary site o~ tryptophan synthesis.
Ex;~mnle 9
~nression C~n~tructs
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Melanin.
The modified genes for both the plastid and vacuolar targeted
ORF438 and tyrosinase proteins were placed into a fiber expression
cassette to be "switched" on during development of the cotton
fiber cell. The "switch" (promoter) utilized for the m~l~n;n
constructs was 4-4. The modified ORF438 and tyrA genes were
cloned into the 4-4 promoter cassette and these ~h;m~riC genes
i then inserted into a binary plasmid to create plasmids pCGN5148
and pCGN5149, cont~; n; ng the modi~ied genes for plastid and
vacuolar targeted ORF438 and tyrosinase proteins, respectively.
These binary plasmids also contain genetic det~rm;n~nts for their
stable maintenance in E. coli and Agrobacterium and also contain a
~-h;meric gene for plant cell e~pression of the bacterial kanamycin
resistance gene. ~his kanamycin reslstance marker allows for the
selection of transformed versus non-transformed cotton cells when
plant hypocotyl or leaf segments are infected with Agrobacterium
cont~;n;ng the binary plasmids.
A block diagram of the plasmid pCGN5149, having vacuolor
targetting sequences, is shown in Figure 8. Plasmid pCGN5148 (not
shown) is constructed the same as 5149, only pCGN5148 has plastid-
targetting sequences.
Tn~; ao
As with the mel ~n; n genes, the plastid-directed tna and pig
genes were placed in the fiber-specific 4-4 promoter cassette and
these ~;m~ric genes subseguently inserted into a binary plasmid
~ .
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to create plasmid pCGN5616. A block diagram of plasmid pCGN5616
is shown in Figure 8.
~nthr~cy;~n; n
A construct has been prepared for the expression of the maize
R and CI genes in developing cotton fiber. These genes are known
to be responsible for the production of Anthocyanin pigments by
acting in a regulatory manner to turn on the chalcone pathway for
production of anthocyanins (red spectrum colors). The R and CI
genes were placed under the control of the Racl3 promoter
cassette. A binary plasmid designated pCGN4745 (not shown),
contains both the R and CI genes each under control of the Racl3
promoter.
~am~le 10
Cotton Tr~n~formation
~nlAnt Preparation
Coker 315 seeds are surface disinfected by placing in 50%
Clorox (2.5% sodium hypochlorite solution) for 20 minutes and
rinsing 3 times in sterile distilled water. Following surface
sterilization, seeds are germin~ted in 25 x 150 sterile tubes
cont~;n;n~ 25 mls 1/2 x MS salts: 1/2 x B5 vitAm;n~: 1.5% glucose:
0.3~ gelrite. Seedlings are g~rm;n~ted in the dark at 28~C for 7
days. On the seventh day seedlings are placed in the light at
28~2~C.
Coc~lltiv;~t;on ~n~l Plant Reaeneration
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Single colonies of A. tumefaciens strain 2760 cont~in;ng
binary plasmids pCGN2917 and pCGN2926 are transferred to 5 ml of
MG/L broth and grown overnight at 30~C. Bacteria cultures are
~ diluted to 1 x 108 cells/ml with MG/L just prior to cocultivation.
'I 5 Hypocotyls are excised from eight day old see~l;ngs, cut into 0.5-
0.7 cm sections and placed onto tohAc~s feeder plates (Horsch et
al. 1985). Feeder plates are prepared one day before use by
plating 1.0 ml tobacco suspension culture onto a petri plate
contA; n; ng Callus Initiation Medium CIM without antibiotics (MS
salts. B5 vit~m;nS: 3 % glucose: 0.1 mg/L 2,4-D: 0.1 mg/L kinetin:
0.3~ gelrite, pH adjusted to 5 . 8 prior to autoclaving). A sterile
filter paper disc (Whatman #l) was placed on top of the feeder
¦ cells prior to use. After all sections are prepared, each section
was dipped into an A. tume~aciens culture, blotted on sterile
paper towels and returned to the tobacco feeder plates.
Following two days of cocultivation on the feeder plates,
hypocotyl sections are placed on fresh Callus Initiation Medium
contA;n;ng 75 mg/L kanamycin and 500 mg/L carbenicillin. Tissue
was incubated at 28+2~C, 30uE 16:8 light:dark period for 4 weeks.
i 20 At four weeks the entire explant was transferred to fresh callus
initiation meclium containing antibiotics. After two weeks on the
second pass, the callus was removed from the explants and split
between Callus Initiation Medium and Regeneration Medium (MS
salts: 40m~ KNO3: 10 mM NH4Cl:B5 vitAm;n.C 3% glucose:0.3%
gelrite:400 mg/L carb:75 mg/L kanamycin).
Embryogenic callus was identified 2-6 months following
initiation and was subcultured onto fresh regeneration medium.
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Embryos are selected for g~rm;n~tion, placed in static liquid
Embryo Pulsing Medium (Stewart and Hsu medium: 0.01 mg/l NAA: 0.01
mg/L kinetin: 0.2 mg/L GA3) and incubated overnight at 30~C. The
embryos are blotted on paper towels and placed into Magenta boxes
cont~in;n~ 40 mls of Stewart and Hsu medium solidified with
Gelrite. G~rm;n~ting embryos are maintA;n~ at 28~2~C 50 uE m~2s~
16:8 photoperiod. Rooted plantlets are transferred to soil and
established in the greenhouse.
Cotton growth conditions in growth chambers are as follows:
16 hour photoperiod, temperature of approximately 80-85~, light
intensity of approximately 500~Einsteins. Cotton growth
conditions in greenhouses are as follows: 14-16 hour photoperiod
with light intensity of at least 400~Einsteins, day temperature
90-95~F, night temperature 70-75~F, relative humidity to
approximately 80%.
pl~nt ~n~l Ysis
Flowers from greenhouse grown Tl plants are tagged at
anthesis in the greenhouse. Squares (cotton flower buds),
flowers, bolls etc. are harvested from these plants at various
stages of development and assayed for enzyme activity. GUS
fluorometric and histochemical assays are performed on hand cut
sections as described in co-pending application filed for
Martineau et al ., supra . For fiber color characteristics, plants
are visually inspected, or northern or western analysis can be
performed, if necessary.
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~x~m~le 11
Ex~ression of Tr~n~enic Piqment Svnthesis Genes
i
Melanin
~ 5 Plants that exhibited resistance to the kanamycin selectable
marker via a leaf assay and corresron~; n~ Western analysis were
considered transformed. Transgenic fiber was collected from
individual plant transformants at different stages of fiber
development and analyze in two ways. One was to analyze fiber at
a single developmental time point for each transgenic cotton plant
to compare tyrosinase expression between transgenic events. The
other was to screen developing fiber ~rom selected plants to
analyze the t; m; ng of tyrosinase expression under the control o~
the fiber-specific 4-4 promoter, by Western blots using antisera
prepared against purified tyrosinase protein.
For the plastid-targeted construct pCGN5148 9 of 13 events
screened for tyrosinase expression were positive, while 13 of the
16 transformed vacuolar-targeted construct pCGN5149 events which
were screened were positive. Expression level in the fiber in
tyrosinase positive plants is approximately 0.1-0.5~ fiber cell
protein. Clearly, the cotton fiber cells comprising the DNA color
constructs DNA produce the necessary proteins re~uired for
synthesis of a pigment.
Visually, the lint from the tyrosinase positive events
exhibits color to varying degrees, while plants that do not
express the enzyme do not exhlbit any color. Colorimeter
measurements of cotton fiber taken from control Coker 130 plants
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and plants from various events transformed with pCGN5148 are
provided in Figures 9 and 10, respectively.
Fiber from pCGN5148 (plastid-directed) plants ~mo~trates a
bluish-green color phenotype. One event, 5148-50-2-1 included
cotton fiber cells (linters) which were colored and which had an
negative a* value less than - 8.0, as measured on the L*a*b* color
space. Coker 130 cotton fiber cells do not typically ~ qtrate
a negative a* value.
These colored cotton cells also had a color located on the
L*C*h color space with a relatively high hue angle value h,
greater than 135-. Normal Coker 130 fibers have a similar value
which is not greater than about 90 as measured by this method.
Results of colorimeter measurements of cotton fiber taken
from plants transformed with pCGN5149 are provided in Figure 11.
Fiber from plants expressing tyrosinase from construct pCGN5149
(vacuolar-targetted) tends to have a light brown phenotype.
~n~; ao
Resistance to the kanamycin selectable marker via leaf assay
and western analysis was again the criterion for designating a
plant as transformed by pCGN5616. Transgenic fiber was collected
from individual plant transformants at different stages of fiber
development. The transgenic developing fiber is screened from
selected plants to analyze the t;m;ng of tna and pig gene
expression under the control of the ~iber-specific 4-4 promoter
and fiber is also analyzed at a single developmental time point
for each transgenic cotton plant for comparison of both
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tryptop~n~ce and indole oxygenase expression between transgenic
events, by using Western blots with antisera prepared against the
tryptop~n~e and indole oxygenase proteins.
For the indigo events, 15 of 24 screened plants were positive
for expression of both the tryptorh~nAce and indole oxygenase
enzymes. Expression levels in the fiber of these proteins is
between 0.05-0.5% fiber cell protein. Approximately half of these
transformants are expressing both genes in the fiber resulting in
a very faint light blue color phenotype. Visually, there is a
faint blue color in the majority of these positive events,
particularly in 20-30 dpa fiber in the unopened boll. Results of
colorimeter measurements of cotton fiber taken from various events
of plants transformed with pCGN5616 are provided in Figure 12.
Many of these events had relatively low a* values (less than 2)
with elevated b* values (greater than 10), as measured on the
L*a*b* color space. Similarly, several 5149 events also measured
with an a* value less than 2 while maint~;n;ng a b* value greater
than 10.
.
BC Cotton
Colorimeter measurements taken on naturally colored fiber
from four separate BC cotton lines is provided in Figure 13.
The above results demonstrate that the color phenotype of a
transgenic cotton fiber cell can be altered by expressing pigment
synthesis genes. The transgenic cotton fiber cells include both a
pigment synthesizing protein, and pigment produced by the pigment
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synthesizing protein. As shown from the results of Figures 9
through 13, expression of a pigment gene of interest can result in
cotton fiber cells in which the synthesis of pigments combined
with appropriate targeting sequences results in modification of
color phenotype in the selected plant tissue, yielding colored
cotton fiber by expression from a genetically engineered
construct.
All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application are specifically and
individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some
detail, by way of illustration and example for purposes of clarity
and underst~n~; ng, it will be readily apparent to those of
ordinary skill in the art that certain changes and modifications
may be made thereto, without departing from the spirit or scope of
the appended claims.
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