Note: Descriptions are shown in the official language in which they were submitted.
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ENHANCED EXPRESSION IN PLANTS
USIDIG NON-TRANSLATED LEADER SEQUENCES
This application is a divisional application of copending
J Canadian Application Serial No.: 2,169,854, filed September l, 1994.
FIELD OF THE INVENTION
The present invention is related to the genetic engineering of
plants. In particular, the present invention relates to recombinant
expression systems easing non-translated leader sequences derived
from heat shock proteins for the enhanced expression of proteins in
plants.
BACKGROUND OF THE INVENTION
Recombinant genes for producing proteins in plants comprise in
sequence the following operably linked elements: a promoter which
l~ functions in plants, a structural gene encoding the target protein,
and a non-translated region which also functions in plants to cause
the addition of polyadenylated nucleotides to the RNA sequence.
Much scientific effort has been directed to the improvement of these
recombinant plant genes in order to achieve the expression of larger
amounts of the target protein.
One advantage of higher levels of expression is that fewer
numbers of transgenic plants would need to be produced and screened
in order to recover plants which produce agronomically significant
quantities of the target protein. High level expression of the
2J target protein often leads to plants which exhibit commercially
important properties.
Improved recombinant plant genes have been generated by using
stronger promoters, such as promoters from plant viruses. Further
improvements in expression have been obtained in gene constructs by
placing enhancer sequences 5' to the promoter. Still further
improvements have been achieved, especially in monocot plants, by
gene constructs which have introns in the non-translated leader
positioned between the promoter and the structural gene coding
sequence. For example, Callis et al (1987) Genes and Development,
Vol. l, pp. 1183-1200, reported that the presence of alcohol
dehydrogenase-1 (Adh-1) introns or Bronze-1 introns resulted in
higher levels of expression. Dietrich et al (1987) reported that
the length of the
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5' non-translated leader was important for gene expression in
protoplasts. Mascarenkas et al. (1990) reported a 12-fold and 20-fold
enhancement of CA'r expression by use of the Adh-1 intron.
Expression of~recombinant plant genes may also be improved by
the optimization of the non-translated leader sequences. These leader
sequences are by definition located at the 5' end of the mRNA and are
untranslated. The leader sequence is further defined as that portion of
the mRNA molecule which extends from the 5' CAP site to the AUG
protein translation initiation codon. This region of the mRNA plays a
critical role in translation initiation and in the regulation of gene
expression. For most eukaryotic mRNAs, translation initiates with the
binding of the CAP binding protein to the mRNA cap. This is then
followed by the binding of several other translation factors, as well as
the 43S ribosome pre-initiation complex. This complex travels down the
mRNA molecule while scanning for a AUG initiation codon in an
appropriate sequence context. Once this has been found and with the
addition of the 60S ribosomal subunit, the complete 80S initiation
complex initiates protein translation (Pain 1986; Moldave 1985; Kozak
1986). A second cla:>s of mRNAs have been identified which possess
translation initiation features different from those described above.
Translation from these mRNAs initiates in a CAP-independent manner
and is believed to initiate with the ribosome binding to internal portions
of the leader sequence (Sonenberg 1990; Carrington and Freed 1990;
Jackson et al. 1990).
The efficiency of translation initiation is determined by features
of the 5' mRNA leader sequence, and presumably this ultimately affects
the levels of gene expression. By optimizing the leader sequence, levels
of gene expression can be maximized. In plant cells most studies have
investigated the use of plant virus leaders for their effects on plant gene
expression (Gallie et al. 1987; Jobling and Gehrke 1987; Skuzeski et al.
1990). The most significant increases in gene expression have been
reported using the Tobacco Mosaic Virus Omega (TMV) leader
sequence. When compared v~~ith other viral leader sequences, such as
the Alfalfa Mosaic Virus RNA 4 (AMV) leader, two to three fold
improvements in the levels of gene expression have been observed using
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the TMV Omega leader sequence (Gallie et al. 1987; Skuzeski et al.
1990). Larger increases in gene expression have been observed when
comparisons were made with an artificial non-native leader sequence.
No consensus regulatory sequences have been identified within the
TMV leader sequence.
Like the TMV leader sequence, most 5' untranslated leader
sequences are very A,U rich and are predicted to lack any significant
secondary structure. One of the early steps in translation initiation is
the relaxing or unwinding of the secondary mRNA structure (Sonenberg
1990). Messenger RI\fA leader sequences with negligible secondary
structure may not require this additional unwinding step and may
therefore be more accessible to the translation initiation components.
Introducing sequences which can form stable secondary structures
reduces the level of gene expression (Kozak 1988; Pelletier and
Sonenberg 1985). The ability of a leader sequence to interact with
translational components may play a key role in affecting levels of
subsequent gene expression.
In the search for leader sequences with improved properties,
genes coding for heat shock proteins were scrutinized. Regulation of
heat shock genes has been shown to occur at the transcriptional and
translational level (Baumann et al. 1987; Kimpel and Key, 1985). Heat
shock genes may be induced and expressed in response to hyperthermic
stress (Key et al. 1981), as well as in response to other environmental
conditions. During heat shock there is preferential translation of heat
shock mRNAs (Storti et al. 1980). The translational control has been
shown to be determined by the 5' untranslated leader sequence
(McGarry and Lindqtust 1985). A heat shock mRNA leader sequence
operably linked to the mRNA of a non-heat shock gene would facilitate
translation during heat shock conditions (Klemenz et al. 1985). The
specific aspects of this regulation are not known. The heat shock
mRNA 5' leader sequence may be more efficient at initiating
translation, or may contain a particular structural feature that allows
preferential translation during heat shock. Whatever the mechanism,
the characteristics oi'the heat shock mRNA leader sequence may also
provide an improvement to gene expression during non-heat shock
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conditions.
This invention makes a significant contribution to the art by
providing non-translated leader sequences for use in genetic constructs
which enhance gene expression in plants. The 5' non-translated leader
. sequences described herein provide for a significant increase in
expression over other non-translated leader sequences which have been
previously employed by those skilled in the art.
SUMMARY OF THE INVENTION
It is a feature of one embodiment of the present invention to provide
an isolated DNA molecule which comprises:
(a) a promoter which functions in plant cells to cause the
production of an RNA sequence;
which is operably linked to
(b) a non-translated leader sequence derived from a heat shock
protein, wherein said non-translated leader sequence is heterologous to
said promoter;
which is operably linked to
(c) a structural DNA sequence, wherein said structural DNA
seuqence is heterologous to said non-translated leader sequence;
which is operably linked to
(d) a 3' non-translated sequence that functions in plant cells to
cause the termination of transcription and the addition of
polyadenylated ribonucleotides to the 3' end of the transcribed mRNA
sequence.
It is a feature of another embodiment of this invention to provide a
method for enhancing gene expression in plants which comprises:
(a) transforming plant cells with a DNA molecule which
comprises:
(i) a promoter region which functions in plant cells to
cause the production of an RNA sequence;
which is operably linked to
(ii) a non-translated leader sequence derived from a
heat shock protein, wherein said non-translated leader sequence is
heterologous to said promoter,
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which is operably linked to
I;iii) a structural DNA sequence,wnereinsaid
structural DNA sequence is heterologous to said non-translated leader
sequence;
which is operably linked to
(iv) a 3' non-translated DNA sequence which
functions in plant cells to cause the termination of transcription and the
addition of polyadenylated ribonucleotides to the 3' end of the
transcribed mRNA sequence;
(b) selecting said plant cells which have been transformed;
(c) regenerating said plant cells to provide a differentiated
plant; and
(d) selecting a transformed plant which expresses said
structural gene.
Yet another embodiment of the present invention provides a
transformed plant which contains a DNA molecule which comprises:
(a) a promoter which functions in plant cells to cause the
production of an RNA sequence;
which is operably linked to
(b) a non-translated leader sequence derived from a heat shock
protein, wherein said non-translated leader sequence is heterologous to
said promoter;
which is operably linked to
(c) a structural DNA sequence, wherein said structurla DNA
sequence is heterologous to said non-translated leader sequence;
which is operably linked to
(d) a 3' non-translated sequence that functions in plant cells to
cause the termination of transcription and the addition of
polyadenylated ribo;nucleotides to the 3' end of the transcribed mRNA
sequence.
Other aspects, and advantages of the present invention
will be apparent to those skilled in the art from the following description,
Example, and claims.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the petunia HSP70 leader sequence (SEQ ID
NO. 1, SEQ ID N0. 2, SEQ ID N0.3, and SEQ ID N0.4).
Figure 2 illustrates the soybean HSP17.9 leader sequence (SEQ
ID N0.5 and SEQ ID N0.6).
Figure 3 illustrates the maize HSP70 leader sequence (SEQ ID
N0.7, SEQ ID N0.8, SEQ ID N0.9, and SEQ ID NO.10).
Figure 4 illustrates the AMV leader sequence (SEQ ID NO.11 and
SEQ ID N0.12).
Figure 5 illustrates the TMV leader sequence (SEQ ID N0.13 and
SEQ ID N0.14).
Figure 6 illustrates the AMV-B leader sequence (SEQ ID N0.15
and SEQ ID N0.16).
Figure 7 illustrates the TMV-B leader sequence (SEQ ID N0. 17
and SEQ ID N0.18).
Figure 8 illustrates the Soybean HSP17.9 -B leader seuence
(SEQ ID N0.19 and SEQ ID N0. 20).
Figure 9 illustrates the Petunia HSP70 -B leader sequence (SEQ
ID N0.21 and SEQ ID N0.22).
Figure 10 illustrates pMON755.
Figure 11 illustrates pMON8796.
Figure 12 illustrates pMON772.
Figure 13 illustrates pMON10871.
Figure 14 illustrates pMON10086.
Figure.l5 illustrates pR'ION10028.
DETAILED DESCRIPTION OF THE INVENTION
Enhanced gene expression in plants is herein provided by the use
of 5' non-translated leader sequences derived from heat shock proteins
in genetic constructs. Plant gene expression employing vectors
containing same may be evaluated in order to determine whether or not
said expression is in fact enhanced by the use of a heat shock 5' non-
translated leader sequence during non-heat shock conditions.
Heat shock proteins are proteins which are induced in response to
a particular stress-related event. The heat shock response is not
CA 02565423 2006-10-20
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limited to plants, and. has been noted in organisms as diverse as
Drosophila, Eschericjaia coli, Sczccharomyces cereUisiae, and humans.
The particular stress-related event is also not solely limited to an
increase in temperature as the name "heat shock protein" would
suggest. Other stress-related events which induce heat shock proteins
include, for example, an exposure to ethanol, arsenite, heavy metals,
amino acid analogues, glucose starvation, calcium ionophores, and a
number of other treatments.
Heat shock proteins are typically designated as HSPX,
wherein X is a number which reflects the molecular weight of the protein
in question. Suitable heat shock proteins from which 5' non-translated
leader sequences could be isolated include but are not limited to HSP70
from petunia, HSP17.3, 17.5, 17.9, 18.5, and 26 from soybean, and
HSP18, 22, 27, G5, 68, 70, 72, 77, 78, 79, 85, and 87 from maize. Said 5'
non-translated leader sequences are selected such that the leader
sequences provide for enhanced expression in plants. In addition, those
of skill in the art would recognize that certain optimizations of the 5'
non-translated leader sequences disclosed herein may in fact be made
such that the expression levels may be altered. These optimizations
may involve changes in the nucleotide sequence of the leader such that
a change in the secondary structure results therefrom. It is speculated
that the secondary structure of the leader is required for the
enhancement of expression; the specific nucleotide sequence of the
leader is important insofar as the secondary structure is concerned.
Therefore, the.leader sequence may in fact tolerate modifications in the
nucleotide sequence which do not result in changes in the secondary
structure. These changes would not affect the resulting expression
levels, and are in fact contemplated by the present invention.
Preferred for the practice of the present invention are those 5'
non-translated leader' sequences selected from the group consisting of
petunia HSP 70 , soybean HSP17.9 and the Maize HSP70.
The 5' non-translated leader sequences from plant heat shock
genes have been shovsm to regulate gene expression during heat shock
conditions. The mechanism for this selective or enhanced expression
may in fact extend to non-heat shock conditions, thereby providing a
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means for selectively increasing plant gene expression. Several plant
heat shock 5' non-translated leader sequences have been ev aluated for
their effect on plant gene expression during non-heat shock conditions.
5' non-translated leader sequences were tested in dicot and monocot
species using both transient and stable plant transformation assays.
The 5' non-translated leader sequence may be isolated from a
gene expressing a known heat shock protein by methods kno'vn to those
of skill in the art, or alternatively, may be synthesized from a known
sequence. In the practice of the instant invention, all leaders were
generated as synthetic oligonucleotides and were tested with several
different genes to show that the enhanced expression was general and
not gene specific. Data demonstrating this is included in the Example.
Nucleic acid sequences which contain a 5' non-translated
sequence may also be obtained by using the specific 5' non-translated
sequences disclosed herein as probes. These obtained sequences could
then be evaluated for enhanced expression in plants.
The nucleotide sequence of the 5' non-translated leader sequence
may be modified at the 5' and 3' ends to facilitate cloning. This may be
accomplished by site-directed mutagenesis, using the method described
by Kunkel (1985), and may provide different restriction sites as needed.
Various oligonucleotide primers may be used to modify the 5' and 3'
ends. Multilinkers may be utilized, which facilitate ordered assembly of
the heterologous DNA sequence. Sequencing of the respective 5'
non-translated leader sequence may be performed by the method of
Singer and Coulsora, Proc. Nat'l Acad. Sci. 74: 5463-54G? (1977) using a
Sequenase ~~ produca, according to the manufacturer's instructions.
Expression levels of the various constructs may be evaluated by
comparing the level of expression ~zth that obtained using a known
leader sequence, such as, for example, those 5' non-translated leader
sequences obtained from TMV- Omega and AMV as discussed
previously, v~~herein the baseline of expression is that obtained using the
kno~;~n leader sequence. Furthermore, the instant invention embraces
the additional feature that the enhanced expression of genes using the 5'
non-translated leader sequences occurs during non- heat shock
conditions.
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The use of 5' non-translated leader sequences may result in
overall expression levels which vary from gene to gene. This variability
may in fact be due to a number of reasons including but not limited to
the efficiency of expression of a particular gene. For example, the
expression of a Bacillus thuringiensis toxin gene using the 5' non-
translated leader sequences may be lower than the expression of an
ACC deaminase gene using the same general construct as is taught by
the instant invention. One postulated explanation for the cause of lower
'expression is the possible presence of fortuitous transcription
processing sites, which could produce aberrant forms of the Bacillus
thuringiensis mRNA transcript as is discussed in Koziel et al., WO
93/07278. These aberrantly processed transcripts may be non-
functional in a plant, in terms of producing an insecticidal protein.
Possible processing sites include polyadenylation sites, intron splicing
sites, transcriptional i~ermination signals, and transport signals. The
fortuitous occurrence of such processing sites in a coding region might
complicate the expression of that gene in transgenic hosts, which may
include improper processing in plants.
A series of plasmids or vectors may be constructed, wherein the
vectors would each contain a different heat shock 5' non-translated
leader sequence fused to a particular reporter or structural gene. The
level of reporter gene activity would then be measured and compared
with the activity of vectors which contained previously described plant
~~irus leader sequences such as the TMV and AMV leader sequences
pre~~iously described. Two dicot heat shock 5' leader sequences, the
petunia HSP70 (Winter et al. 1988) and soybean HSP17.9 (Raschke et
al. 1988 ) leader sequences were shown to increase levels of gene
expression in a dicot system. In addition, the maize HSP70 5' leader
sequence was shown 'to increase levels of gene expression in maize cells
(a monocot system) (pMON9508 - Rochester et al. 198G).
Disclosed in the Example herein is the evaluation of three heat
shock leaders in various constructs for their effect on plant gene
expression. The 5' non-translated leader sequences employed were the
petunia HSP70 (Winter et al. 1988), the soybean HSP17.9 (Raschke et
al. 1988) and the maize HSP70 (pMON9508 - Rochester et al. 1986)
CA 02565423 2006-10-20
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5' non-translated leaders. Comparisons for effects on plant gene
expression were made to the AMV and TMV plant viral leader
sequences.
The 5' non-translated leader sequence for the soybean (Raschke
et al. 1988) and maize (Rochester et al. 1986) heat shock mRNAs was
derived using published information detailing the start of transcription
and translation for each heat shock gene. The start of translation is
known for the petunia HSP70 mRNA. However, the start of
transcription has not been determined. A start site was therefore
chosen (base 144 - Winter et al. 1988) based on the putative TATA box
(bases 108 - 115, Winter et al. 1988; Joshi 1987) and from preliminary,
unpublished experiments performed in order to determine the
transcriptional start site. The TMV and AMV viral leaders were also
constructed using synthetic oligonucleotides which contained the
consensus 5' and 3' sequences as represented in the Example below.
It is understood that the particular nucleotide and/ or amino acid
sequences disclosed herein are representative in the sense that
equivalent genes or portions thereof may be obtained and/ or generated
pursuant to this disclosure. By equivalent it is meant that said gene or
portion thereof would function in a manner substantially the same as
the gene disclosed herein, and would provide a benefit or particular
characteristic to a plant in substantially the same manner.
A structural DANA sequence encoding a particular gene of interest
may be inserted into a plant transformation vector. A gene is defined as
an element or combination of elements that are capable of being
expressed in a cell, either alone or in combination with other elements.
In general, a gene comprises (from the 5' to the 3' end): (1) a promoter
region which may include a 5' non-translated leader sequence capable of
functioning in plant cells; (2) a structural gene or structural DNA
sequence which codes for the desired protein; and (3) a 3' non-translated
region, which typically causes the termination of transcription and the
polyadenylation of the 3' region of the RNA sequence. Each of these
elements is operably linked to the adjacent element. A gene comprising
the above elements may be inserted by standard recombinant DNA
methods into a plant transformation vector. Some or all of the elements
CA 02565423 2006-10-20
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of the gene may be present, with additional or remaining elements added
to the vector if necessary. Additionally, the plant transformation vector
may be constructed «vith all of the elements present except for the
structural gene, which may then be added at an appropriate time by
known methods.
The segment of DNA referred to as the promoter is responsible
for the regulation of the transcription of DNA into mRNA. A number of
promoters which function in plant cells are known in the art and may
be employed in the practice of the present invention. These promoters
may be obtained from a variety of sources such as plants or plant
viruses, and may include but are not limited to promoters isolated from
the caulimovirus group such as the cauliflower mos~ac virus 35S
promoter (CaMV35S), the enhanced cauliflower mosaic virus 35S
promoter (CaIVIVe35S) , the figwort mosaic virus full-length transcript
promoter (FMV), and. the promoter isolated from the chlorophyll a/b
binding protein as is known in the art. Other useful promoters include
promoters which are capable of expressing the replicase enzyme in an
inducible manner or in a tissue-specific manner in certain cell types in
which the infection is known to occur. For example, the inducible
promoters from phenylalanine ammonia lyase, chalcone synthase,
hydro~.yproline rich gMycoprotein, extensin, pathogenesis-related proteins
(e.g. PR-la) , and wound-inducible protease inhibitor from potato would
be useful.
Alternate promoters, such as the promoter from glutamine
synthetase for expression in vascular tissues or promoters from
epidermal cells, coulf~ be used to express the protein in certain cell types.
The patatin promoter could be used to express the protein in the tuber.
The particular promoter selected is preferably capable of causing
sufficient expression ~of the structural gene to which it is operably linked
to result in the production of a suitable amount of the respective
protein, but not so much as to be detrimental to the cell in which it is
expressed. The promoters selected should be capable of functioning in
tissues including but not limited to epidermal, vascular, and mesophyll
tissues. The actual choice of the promoter is not critical, as long as it
has sufficient transcriptional activity to accomplish the expression of
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the structural gene.
The non-translated leader sequence can be derived from any
suitable source and may be specifically modified to increase the
translation of the mRNA. The 5' non-translated region may be obtained
from the promoter selected to express the gene, the native leader
sequence of the gene or coding region to be expressed, viral RNAs,
suitable eucaryotic genes, or a synthetic gene sequence. Specifically,
the 5' non-translated leader sequence may be heterologous to the
promoter employed in the construct, for example, the non-translated
leader sequence may be derived from an unrelated promoter as
described. The present invention is not limited to the constructs
presented in the following Example.
The structural DNA sequence which codes for the structural gene
may be isolated from a particular source using methods known to those
of skill in the art as discussed earlier in this section. Other modifications
to this gene may also be made, including modifications to the 5' or 3'
termini of the structural gene, such as, for example, the introduction of
an initiation codon at the 5' end. Such structural genes may in fact be
heterologous to the 5' non-translated leader sequence. Suitable
structural genes which may be employed in the practice of the present
invention include those structural genes selected from the group
consisting of ACC deaminase, PLRV replicase, viral coat proteins,
EPSP synthase or other genes conferring herbicide tolerance, selectable
marker genes, genes affecting carbohydrates or oils, and genes affecting
carotenoids or, other nutritional components produced in plants. In
addition, expression of antisense genes may also be employed, such as
ACC synthase, or genes conferring nematode resistance.
The termination region or 3' non-translated region which is
employed is one which will cause the termination of transcription and
the addition of polyadenylated ribonucleotides to the 3' end of the
transcribed mRNA sequence. The termination region or 3' non-
translated region will be additionally one of convenience. The
termination region may be native with the promoter region, native with
the structural gene, or may be derived from another source, and
preferably include a terminator and a sequence coding for
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polyadenylation. Suitable 3' non-translated regions of the chimeric plant
gene include but are not limited to:
(1) the 3' transcribed, non-translated regions containing the
polyadenylate signal of Agrobczcterium tumor-inducing (~) plasmid
genes, such as the nopaline synthase (NOS) gene, (2) plant genes like
the soybean 7S storage protein genes and the pea small subunit of the
ribulose 1,5-bisphosphate carboxylase-oxygenase (ssRUBISCO) E9
gene and the like.
In developing ithe expression construct, the various components
of the expression construct or fragments thereof will normally be
inserted into a convenient cloning vector which is capable of replication
in a bacterial host, such as E. coli. Numerous vectors exist that have
been described in the literature. After each cloning, the vector may be
isolated and subjected to further manipulation, such as restriction,
insertion of new fragments, ligation, deletion, resection, insertion, in vitro
mutagenesis, addition of polylinker fragments, and the like, in order to
provide a vector which will meet a particular need. Once the construct
is completed, it may ithen be transferred to an appropriate vector for
further manipulation. in accordance with the manner of transformation
of the plant cell.
A variety of techniques are available for the introduction of the
genetic material into or transformation of the plant cell host. However,
the particular manner of introduction of the plant vector into the host is
not critical to the practice of the present invention. Any method which
provides for efficient transformation may be employed as is known and
practiced by those of skill in the art. In addition to transformation using
plant transformation vectors derived from the tumor-inducing (~) or
root-inducing (Ri) plasmids ofAgrobacterium, alternative methods could
be used to insert the DNA constructs of the present invention into plant
cells. Such methods may include, for example, the use of liposomes,
electroporation, chemicals that increase the free uptake of DNA, DNA
delivery via microprojectile bombardment, microinjection, and
transformation using; viruses or pollen.
A plant transformation vector preferably includes all of the
necessary elements for transformation of plant cells. 'I~pical plant
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cloning vectors comprise selectable marker genes, scoreable marker
genes, T-DNA borders, cloning sites, appropriate bacterial genes to
facilitate the identification of transformants, broad host range
replication and mobilization functions, and other elements as desired.
The structural gene may be inserted into any suitable plant
transformation vector for transformation into the desired plant species.
Suitable plant transformation vectors include those derived from a Ti
plasmid ofAdarobacterium tumefaciens, in addition to thdse disclosed, for
example, by Herrera-Estrella (1983), Bevan (1984), HIee (1985) and
Fraley (1983).
Selectable marker genes may be used to select for those cells
which have become transformed. Conveniently, the marker employed
may be resistance to an antibiotic, such as kanamycin, 6418,
hygromycin, streptomycin, and ~he like. Other markers could be
employed in addition to or in the alternative, such as, for example, a
gene coding for herbicide tolerance such as tolerance to glyphosate,
sulfonylurea, phosphinothricin, or bromoxynil. Additional means of
selection could also be employed. The particular marker employed will
be one «~hich will allow for the selection of transformed cells as opposed
to those cells which were not transformed. Depending on the number of
different host species one or more markers may be employed, where
different conditions of selection would be used to select the different host,
and would be known to those of skill in the art.
Plant transformation vectors containing the 5' non-translated
leader sequence which is operably linked to a structural gene may be
used to transform plants of the Solanaceae family. An Agrobdcterium-
mediated transformation protocol is known to be effective in
transforming members of the Solanaceae family. When an
Agrobdcterium-mediated transformation is used, the desired
transformation vector is mobilized into a suitableAgrobacterium strain.
The ABI Agrobacterium strain is described for exemplary purposes. The
desired transformation vector is mobilized into an ABI A~arobacterium
strain by the triparental mating system using the helper plasmid
pRK2013 (Ditto et al. 1980). The binary ABI strain is the
chloramphenicol resistant derivative ofAgrobaeterium tumefaciens
CA 02565423 2006-10-20
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A208 which carries t:he disarmed Ti plasmid pTiC58 (Koncz and Schell
1986). The Ti plasmid does not carry the T-DNA phvtohormone genes
and the strain is therefore unable to cause crown gall disease. The
disarmed Ti plasmid provides the trfA gene functions required for
autonomous replication of the vector after conjugation into the ABI
strain. When the plant tissue is incubated with the
ABI:aransformation vector conjugate, the vector is transferred to the
plant cells by the uir functions encoded by the disarmed pTiC58
plasmid. The pTiC58 Ti plasmid does not transfer to the plant cells, but
remains in the Agrobacterium. Either single- or double-border
transformation vectors can be delivered to the plant by A.grobacterium.
Single border vectors open at the right T-DNA border region, and the
entire vector sequence is inserted into the host plant chromosome. The
right border is lost during transfer and integration. In a double border
1 ~ vector, DNA between the right and left borders is inserted into the plant
chromosome, thereby delivering only the chimeric genes of interest to
the chromosome. The remainder of the vector, and the border
sequences are lost during the transfer and integration.
Transformation and regeneration protocols for members of the
Solanaceae family are known in the art. After the tomato or potato
plant has been transformed and after transformed callus has been
identified, the transformed callus tissue is regenerated into whole plants.
Any known method of regeneration of potato plants can be used in this
invention.
For tomato, the transformation protocol described in McCormick
et dl. ( 19S6 ~ may generally be employed. The regeneration of plants
from either single plant protoplasts or various explants is well known in
the art. See, for example, Methods for Plant Molecular Biolow, A.
W eissbach and H. Weissbach, eds., Academic Press, Inc., San Diego, CA
(1988). This regeneration and growth process includes the steps of
selection of transformant cells and shoots, rooting the transformant
shoots and growth of the plantlets in soil. The regeneration of plants
transformed by Agrobr~cteriurn from leaf explants can be achieved as
described by Horsch et cal., Science 227:1229-1231 (1985). In this
procedure, transformants are grown in the presence of a selection agent
CA 02565423 2006-10-20
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and in a medium that induces the regeneration of shoots in the plant
species being transformed as described by Fraley et dl., Proc. Nat'l.
Acad. Sci. U.S.A., 80:4803 (1983). This procedure typically produces
shoots within 2 to 4 months and these transformant shoots are then
transferred to an. appropriate root-inducing medium containing the
selective agent and an antibiotic to prevent bacterial grov~~th.
Transformant shoots that are rooted in the presence of the selective
agent to form plantlets are then transplanted to soil or other media to
-allow the production of roots. These procedures vary depending on the
particular plant species employed, such variations being well known in
the art.
The invention also provides plant cells, the genome of v~~hich
comprises an expression cassette comprising the 5' non-translated
leader sequence of the present invention, wherein said 5' non-translated
leader sequence functions in such a way as to provide enhanced
expression of the structural gene to which the 5' non-translated leader
sequence is operabl.y linked. Whole plants comprising such cells will
have the features or benefits provided by the expression of the
structural gene which is operably linked to said 5' non-translated leader
sequence. Such plants may be monocots or dicots, and may include but
are not limited to plants belonging to families selected from the group
consisting of Solanaceae, Graminae, Cucurbitaceae, Caricaceae,
Dioscoreacea, Leguminosae, Compositae, and Chenopodiaceae.
A plant of the present invention containing the desired structural
gene may be cultivated using methods known to those of skill in the art.
A transformed plant of the present invention thus is capable of
expressing the structural gene and exhibits the particular trait thereby.
The presence of the particular structural gene or gene product in the
transformed plant may be determined by any suitable method known to
3 0 those of shill in the art. Included in these methods are Southern,
Northern, and Western Blot techniques, ELISA, and various bioassays.
The transformed plant capable of expressing the structural gene may
then be assayed for the determination of the particular activity.
The following Example is provided to better elucidate the practice
of the present invention and should not be interpreted in any way as to
CA 02565423 2006-10-20
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limit the scope of the present invention. Those skilled in the art will
recognize that various modifications can be made to the methods, 5'
non-translated leader sequences, and genes described herein while not
departing from the spirit and scope of the present invention.
~~A~,l PLE
Each 5' leader oligonucleotide complementary pair was originally
subcloned into the plasmid vector pMON755. The plasmid pMON755 is
a pUC119 (Vieira and Messing 1987) based vector which contains the
CaMV enhanced 35S promoter (e35S - Kay et al. 1987), the 13-
glucuronidase gene (C'~US, pRAJ275, Clontech Laboratories, Inc.) and
the nopaline synthase 3' termination sequence (NOS 3'- Fraley et al.
1983). In addition, pMON755 contains a Stul blunt end restriction
enzyme side at the start of transcription fron the CaMV promoter
(Guilley et al. 1982) and a Ncol site at the start of translation for the
GUS gene (Jefferson et al. 1986). Synthetic oligonucleotides were
designed as complimentary pairs which when annealed would generate a
blunt 5' end, and would generate a 5' overhang at the 3' end which is
compatible with and can be ligated to a DNA fragment restricted with
Ncol. Each leader was also synthesized to contain the four nucleotides
ACAC at the 5' end. These four nucleotides are the naturally occurring
bases dom~nstream of the start of CaMV transcription (Guilley et al.
1982 ) and were provided with each oligonucleotide to provide similar
sequence context at the start of transcription for each leader construct.
Similarly, a consensus sequence was used at the 3' end of the
oligonucleotide to provide similar and near optimum sequence conte~-t at
the start of translation (Kozak 1986).
Plasmid pMON 7 55 was digested with Ncol (Boehringer
Manheim) and Stul (New England Biolabs) according to manufacturer
directions. Complimentary synthetic oligonucleotide pairs were
annealed and subcloned into pMON755. Each vector was identical
except for the leader sequence used. The soybean HSP17.9 heat shock
leader was constructed from one complimentary oligonucleotide pair.
However, due to the long length and limitations of oligonucleotide
synthesis, the petunia arid maize HSP70 leaders were constructed from
CA 02565423 2006-10-20
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two pairs of complimentary oligonucleotides. For either the maize or
petunia HSP leaders, two oligonucleotides were synthesized and
annealed to generate Fragment 1. Similarity, two additional
oligonucleotides were used to create Fragment 2. For cloning, Fragment
1 and Fragment 2 were legated with previously digested pMON755 as
described below. Legations were performed using 25 pmol of each
annealed oligonucl~eotide pair with 200 ngs of digested pMON755.
Legations were performed according to manufacturer's specifications
(New England Biolabs). The E. coli host MM294 (Talmadge and Gilbert
1980) was rendered.competent (Sambrook et al. 1989) and transformed
with the legation mix. Transformed cells were selected by plating the
cells on LB media (Sambrook et al. 1989) containing 100p.g1m1
carbenicillin (Sigma Chemical Company). Presence of the synthetic 5'
leader was confirmed by restriction enzyme analysis. Leader sequences
was verified from double stranded template DNA (prepared via Amorese
mini-prep procedure from the Genesis 2000 DNA Analysis System,
Application #8) using standard sequencing procedures (USB
Sequenas ~t kit).
The constructs containing leader sequences were evaluated using
a tobacco protoplast transient assay. TXD tobacco suspension cell
protoplasts were electroporated with CsCI purified (Sambrook et al.
1989) plasmid DNA. Transformations were performed in triplicate and
each transformation included an internal control plasmid. The control
plasmid contained a different reporter gene and was used to correct for
variability in the transformation and extraction procedures. For the
GUS evaluations, tree luciferase expression vector pMON8796 was used
as the internal control. Other published plant luciferase vectors such as
pD0432 (Ow et al. 1986) or pCaMVLN (Callis et al. 1987) could be used.
pMON8796 is a pUC119 derivative (Vieira and Messing 1987) similar
to pMON755 containing the e35S CaMV promoter, the luciferase (LUX)
gene lDe Wet et al. 1.987) and the NOS 3'. For each transformation,
25pg of plasmid DNA was used with 5ug of the internal control plasmid.
TXD cells were grown in TXD media which contained 4.3 gll
Murashige and Skoog salts (Gibco), 3% sucrose, 0.2 g/1 inositol, 0.13 g/1
asparagine, 4 ug/ml of PCPA( p-chlorophenoxyacetic acid), 5 nglml of
CA 02565423 2006-10-20
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kinetin, 1.3 mg/1 nicotinic acid, 0.25 mil thiamine, 0.25 mg/1 pyridoxine
HCL, and 0.25 mg/l calcium pantothenate at a pH of 5.8. Fifty mls of
TXD cells were maintained in a 250 ml flask, in the dark at 25°C,
shaking at 140 rpm. Cells were sub-cultured every 3-4 days by adding 9
mls of cells to 41 mls of fresh TXD media. For protoplast preparation,
16 mls of a 2 day old culture was added to 40m1s of fresh TXD medium.
After approximately 24 hours cells were spun down in 50 ml sterile
centrifuge tubes at :200 x g for 5 minutes. The supernatant was
removed and saved ;as conditioning media.
Forty mls of protoplast isolation media (7.35 g/1 calcium chloride,
1 g/1 sodium acetate, and 45 g/1 mannitol pH 5.8), containing the
following enzyme mixture 0.5°'o BSA (Sigma Fraction V), 40p113-
mercaptoethanol, 0.5% cellulase 'RS' (Onazuka RS Yakult Honsha Co.,
LTD). 0.5% *Rhozyme (Genecor HP-150), and 0.02% Y-23 pectolyase
(Seishin Pharmaceutical Co., LTD) was then added to each tube, mixed
with protoplasts using a wide bore pipette, and transferred to 100 x
25mm petri dishes (10 ml/plate). The plates were parafilmed and
incubated at 26-28°C on a rotary shaker at 50-60 rpm for one hour in
the light. Digestion was monitored by observation through an inverted
microscope. After digestion was complete the protoplasts were
transferred back into 50 ml sterile centrifuge tubes using 10 ml pipettes
with standard tips. The protoplasts were spun down at 200 x g for 5
minutes. The supernatant discarded and the protoplasts gently
resuspended in 20 mls protoplast isolation media. The protoplasts were
spun down and then resuspended in 20 mls of electroporation buffer (EB
- 0.02 g/1 KH2P04, 0.115 g/1 Na2HP04, r .5 g/1 NaCI, and 36.4 g/1
Mannitol pH 7.2). The protoplasts were counted using a
hemocytometer and yields were determined. Protoplasts were spun
down again and resuspended in EB to a density of 2x106 cells per ml and
held on ice.
Electroporations were performed using a BioRad Gene Pulse f~
electroporation system (Gene Pulser and Capacitance Extender).
Protoplasts (0.4 ml) were mixed with plasmid DNA (diluted to 0.4m1 with
EB) and added to a 0.8 ml cuvette (BioRad 0.4 cm gap). The protoplasts
and DNA were mixed by gently inverting the cuvette twice and then
*Trade-mark
CA 02565423 2006-10-20
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electroporated at 150 volts at a capacitance of 500 Farads. The
transformed protoplasts were placed on ice for 10 minutes then allotved
to warm to room temperature for 10 minutes. Protoplasts were
resuspended in 7m.1 of T~ media containing 0.4M mannitol plus one-
s fifth volume of conditioning media (previously described) and transferred
to 100 x 25mm petri dishes. The protoplasts were then incubated in
light at 26-28°C. After 20-24 hours the protoplasts were collected by
centrifugation and the media was removed. The pellet was resuspended
in 250.1 extraction buffer (0.1M KP04 pH 7.8, lOmM DTT, 1mM
NaEDTA, 5% glycerol). Cells were lysed for assay by freeze-thawing
between dry ice and a 3?°C water bath. Cell debris was removed by
centrifugation for 5 minutes at 16,000 x g. GUS activity was
determined from 5 ~1 of cell extract according to the methods of
Jefferson et al. (1987) using 2 mM MUG in the previously described
extraction buffer. Fluorescence was measured using a Hoescht DNA
Fluorometer (Model TKO 100). A methylumbelliferone (Sigma)
standard curve was generated using a lp.m solution. GUS activity was
calculated as pmol MU/minute/ml extract. To determine luciferase
activ ity 5~t1 of cell e~.~tract was added to 200 ~.l assay buffer (25mM
Tricine pH 7.8, lSm:M MgCl2, 5mM ATP, 0.5 mg/ml BSA) in a
luminometer cuvette (Analytical Luminescence Laboratories). The
cuvette was placed in a luminometer (Berthold Instruments Model
LB9500) and reaction started with the addition of 100 ~1 0.5mM
luciferin (Analytical Luminescence Laboratories). Peak light emission
was measured over a 10 second interval. Five luciferase assays were
performed per extract. Luciferase activity was calculated as the
average relative light units/ml extract. For the comparison of different
leader constructs expression results are presented as a ratio of
activities for the experimental and control gene, i.e. GUS/LUX (pmol
MUimin per average peak light units). Comparisons were made with the
Ah~TV leader; a leader which had previously been used to optimize gene
expression in plants (:Barton et al. 1987, Jobling and Gehrke 1987,
McCabe et al. 1988). Results are shown in Table 1 below:
CA 02565423 2006-10-20
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Table 1
Leader Effects on Transient Levels Of GUS Expression
in Tobacco Protoplast Cells
pMON Leader GUS / LUX Relative Expression
766 AIVIV 6.17 +/- 1.0x
0.4
769 TMV 15.4 +/- 2.5x
1.2
1'1711 Soy HSP17.9 21.9 +/- 3.5x
1.8
11715 Pet HSF70 22.3 +/- 3.6x
3.1
As indicated in Table 1 above, the level of gene expression using a
heat shock leader sequence was greater than expression levels from the
pre~zously described. viral leader sequences (Skuzeski et al. 1990). To
show that this leader sequence effect was not specific for the GUS gene,
a series of vectors were constructed which contained 5' leader sequence
fusions to the luciferase, ACC deaminase, and the Bacillus thuringinesis
v. kurstahi coding sequences (Ow et al. 1986, Klee et al. 1991, and
Wong et al. 1992, respectively). To generate the luciferase vectors, the
GUS coding sequence was replaced with the luciferase coding sequence
from pMON772. The luciferase coding sequence was subcloned as a
Ncol to BamHl fragment using standard digestion and ligation
protocols. Similarily, the B.t.k. expression plasmids were constructed
using a I~TCOl/BamHl fragment isolated from pMON10871.
The ACC dearninase expression vectors were constructed as
follows: Plasmid pMON 10866, which contains the P-FMV GUS NOS
3' gene. vas digested with the restriction endonucleases Stul and Bgl2.
New heat shock and control leader oligonucleotides were synthesized
and subcloned into the digested pMON10866. These new leaders
(Figures 6, 7, 8, and 9~) are essentially identical to the previously
described leaders except they contain modifications at their respective
3' overhangs to allow cloning into a Bgl2 restriction site. For the petunia
I-ISP r 0 leader, only a new fragment 2 was synthesized (See Figure 9);
the previously described Fragment 1 (Figure 1) was used here as well for
the ACC deaminase cloning. The resulting plasmids were restricted
CA 02565423 2006-10-20
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with the endonucleases Bgl2 and BamHl. The ACC deaminase gene,
isolated as a BamHl fragment from pMON10028, was subcloned into
the leader plasmids. The resulting plasmids contained the leader of
interest fused to the ACC deaminase gene driven by the FMV promoter
(Richins et al., 1987).
Tobacco protoplast transformations were performed with these
leader luciferase vectors as previously described. An internal control
GUS expression plasmid, pMON755, was included to correct for
variations in the assay. Comparable GUS expression vectors such as
pBI121 (Clontech haboratories, Inc.) could also be used. Comparisons
axe again presented as LUX/GUS ratios and are included in Table 2
belo~.v:
Table 2
Leader Effects on Transient Levels Of Luciferase
Txpression in Tobacco Protoplast
PMOh :Leader LUX/GUS Relative Levels
778 AMV 3.1 +I- 0.2 1.0x
781 TMV 8.5 +/- 0.5 2.7x
11715 Soy HSP17.9 14.6 +/- 0.3 4.7x
11721 Pet HSP70 12.6 +/- 0.8 4.1x
Levels~of luciferase expression in tobacco protoplasts were
greatest when using the heat shock leader sequences. The heat shock
leader sequence constructs again gave levels of expression higher then
the constructs which contained the plant viral leader sequences.
ACC deaminase evaluations were performed using the tobacco
protoplast transient assay. Electroporated protoplasts were
resuspended in 0.4 ml 0.1 M Tris-HCl pH7.8, 5mM Na2EDTA, lOmM
DTT and 10 Co glycerol. Cells were extracted by freeze-thaw as
previously described. ACC deaminase activity was determined by
quantitating levels of alpha-lcetobutryate following incubation of the
enzyme with the substrate ACC (Honma and Shimomura, 1978).
CA 02565423 2006-10-20
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0.05m1 of tobacco cell extract was added to 0.05m1 of a solution
containing 0.2 M Tris-HCl pH 7.8 and 0.1 M amino cyclopropane-1-
carboxvlic acid (ACC;i. This reaction mix was incubated at 37° C for 30
minutes and then terminated with the addition of 0.9m1 of 0.56N HCI.
To this solution was added 0.15 ml of 0.1% dinitrophenyl hydrazine in 2I~T
HCl. The samples were then incubated for 15 minutes at 25°C.
Follo~~ing this period, 1.0 ml of 2N NaOH was added to the samples.
Samples were allowed to sit for 15 minutes at 25°C to allow the
color to
stabilize, then were zr~easured for absorbance at O.D. 54o using a
spectrophotometer. The luciferase vector pMON8796 ~~as used as the
internal control for the ACC deaminase electroporations. ACC
deaminase transient assay results are presented as the average of 4
electroporations and are shown in Table 3 below:
Table 3
Leader Effects on Transient Levels Of ACC Deaminase
Expression in Tobacco Protoplast
pMON 5' Leader ACC Deaminase/LUX RelativeLevels
18426 A:MV 0.83 +/- 0.12 1.0x
1842 ~ TMV 1.02 +/- 0.15 1.2x
18419 Soy HSP17.9 1.45 +/- 0.33 1.8x
1011 G I'c~t HSP70 1.45 +/- 0.25 1.8x
Results from the luciferase and ACC deaminase experiments
corroborate the earlier GUS findings, showing that the plant HSP 5'
leader sequences can in fact increase plant gene expression to levels
greater then that observed with previously described leader sequences.
In addition, these results show that the heat shock leader sequence
effect on plant gene expression extends beyond one particular coding
sequence.
The tobacco transient assays was also used for evaluating the 5'
leader effect on expression of the B.t.k. gene. The luciferase expressing
plasmid pMON772 was included as an internal control. Luciferase
CA 02565423 2006-10-20
-24-
expression levels were used to standardize loadings for westei~ analysis
of the B.t.k. protein. The electroporated protoplast were resuspended in
extraction buffer (0.1M KP04, 5% glycerol, 1mM EDTA, lOmM DTT).
One half of the resuspended cells were used for luciferase assays using
~ the procedure previously described. To the remaining cell sample was
added an equal vohn:ne of 2x SDS Loading buffer (125mM Tris-HCL
pH7.0, 4% SDS, 20°h glycerol, 10% 13 -mercapthoethanol, 4 mg/ml
phenol red) followed by boiling for 5 minutes. Equivalent amounts of
samples were loaded onto a 10% SDS-PAGE gel based on luciferase
activities. Seperated proteins were then transferred to nitrocellulose
membrane using a Hoeffer Transfer Appartus as per the
manufacturers instructions. The membrane was incubated overnight
at 4°C in 5% dry milk / TBST (10 mM Tris, pHB, 150 mM NaCI,
0.1°'0
*Tween-20). To hybridize the membrane the incubations were done at
room temperature with gentle agitation. The primary B.t.k. antibody
was bound by incubating the membrane in a 1:2000 dilution of the
rabbit serum in TBST for 18 hr. This was followed by three 10-min
washes in TBST. The secondary reagent was bound by incubating the
membrane with 5 p.C of 1'sI-labelled protein G in 20 ml of TBST for 30
min. The membrane was washed three times for 10 min each with 0.3%
*Triton X-100 followed by three washings 0.1 % Triton X-100 and then
exposed to film. Levels of protein expression were determined using a
densitometer. Results are as follows:
Tzhle 4
Leader Effect On Transient Levels Of B.t. Expression
In Tobacco Protoplast
pMON Leader Relative Area Relative Value
1174 AMV 881.7 +/- 12.21.0x
1175 TMV 1194.0 +/- 1.4x
2.8
1176 HSP17.9 1197.0 +/- 1.4x
2.4
117 ~9 Pet HSP70 933.7 +/- 31.91.1x
*Trade-mark
CA 02565423 2006-10-20
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The results from the B.t.k. analysis did not reveal significant
increases in expression as a result of the HSP leaders. However,
expression was at least equivalent to the AMV and TMV leaders which
have been described as preferred leaders for maximizing plant gene
expression. Inability to significantly enhance B.t.k. expression may be a
result of other constraints on B.t.k. expression separate from effects
dependent upon the .5' leader. This may include fortuitous transcription
processing sites, polyadenylation sites, intron splicing sites,
transcriptional termination signals, and transport signals. The
fortuitous occurrence of such processing sites in a coding region may in
fact complicate the expression of that gene in transgenic hosts, which
may include improper processing in plants.
In a similar manner a maize heat shock 5' leader was tested for
its effect on maize gene expression. The maize HSP70 leader was
subcloned as synthetic oligonucleotides in an identical fashion to the
dicot leader sequences. The maize HSP70 leader was fused with the
GUS and luciferase coding sequences. Leader sequence analysis was
performed in a monocot transient assay system.
Maize BMS f,I3lack Mexican Sweet- ATCC #54022) suspension
cell protoplasts were transformed with the maize HSP70 leader
constructs and with 'the previously described viral leader sequence
constructs. Maize B:MS cells were maintained and prepared for
protoplast transformation as described by Fromm (Fromm et al. 1987)
with the following exceptions. The BMS media used was as follov~s, MS
salts (Gibco),_2 mg~L 2,4-D, 0.25mg/L thiamine HCI, 1mM asparagine,
20g/L sucrose, 100mg/L inositol, l.3mg/L nicotinic acid, 0.25mg/L
p3~ridoxine HCl and calcium pantothenate, pH 5.8. BMS lines were
subcultured every other day by transfer of 25m1s suspended cells into
40m1s liquid media in 250m1s Erlenmeyer flasks. Lines were maintained
in the dark or very low light, at 28°C, and at a shaker speed of 120-
150
rpm. Protoplasts were isolated one day following subculturing (Fromm
et al. 1987). One gram of fresh cell weight of BMS cells was digested
with 10 mls of enzyme mixture. A protoplast concentration of 3 x 106
cells /ml was used for the electroporation. Electroporations were
performed as described for tobacco. Following electroporation cells were
CA 02565423 2006-10-20
-26-
placed on ice for 10 minutes then transferred to a 100 x 25 mm petri
dish and allowed to sit at room temperature for 10 minutes. Eight mls
of protoplast growth media (Fromm et al. 1987) were then added to the
cells. Cells were incubated 20 - 24 hours at 26° in the dark. Cells
were
harvested and extracted as described for tobacco transformations with
the following exceptions. The extraction buffer also included 1mM
phenylmethylsulfonylfluoride, 1 mM benzamidine, and 5 mM -
am.inocaproic acid. GUS assays were performed using 25 ~1 of extract.
Luciferase assays were performed using 40 ~tl of extract «kith PEG -
8000 (25 mg/ml) added to the luciferase assay buffer. As before, results
are expressed as a ratio of experimental reporter gene levels to internal
control reporter gene levels and are provided in Tables 5 and 6 below:
T able 5
Leader Effect On Transient Levels Of GUS Gene Expression
In Maize BMS Cell Protoplast
pMON Leader Average GUSlLUX Relative Ex,~ression
766 AMV 0.4 +/- 0.1 1.0x
769 TMV 0.4 +/- 0.1 1.0x
11714 Mz HSP70 9.1 +/- 3.0 22.8x
11711 Soy I-ISP17.9 14.0 +/- 0.1 35.0x
11715 Pet HSP'70 6.0 +/- 0.1 lS.Ox
35
CA 02565423 2006-10-20
-27-
Table 6
Leader Effect On Transient Levels Of Luciferase Gene Expression
In Maize BMS Cell Protoplast
pMON Leader Average LUZ/GUS Relative Expression
778 AMV 6.9 +/- 1.1 1.0x
781 TMV 6.8 +/- 2.2 1.0x
11720 Maize HSP70 86.2 +/- 2.5 12.5x
11718 Soy HSF'17.9 47.4 +/- 3.0 6.9x
11721 Pet HSP70 21.3 +/- 4.5 3.0x
As observed in the dicot system, the use of a heat shock leader
sequence in a monocot system greatly improved the level of monocot
gene expression over that obtained with the plant virus leader
sequences as shown :in Tables 5 and 6 above.
All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled in the art
to which this invention pertains.
From the foregoing, it will be seen that this invention is one well
adapted to attain all the ends and objects hereinabove set forth together
with advantages that are obvious and that are inherent to the
invention. It will be 'understood that certain features and sub-
combinations are of utility and can be employed without reference to
other features and sub-combinations. This is contemplated by and is
within the scope of the claims. Because many possible embodiments
can be made of the invention without departing from the scope thereof,
it is to be understood that all matter herein set forth or shown in the
accompanying drawings is to be interpreted as illustrative and not in a
Limiting sense.
CA 02565423 2006-10-20
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Monsanto Company
(B) STREET: 800 North Lindbergh Boulevard
(C) CITY: St. Louis
(D) STATE: Missouri
(E) COUNTRY: United States of America
(F) POSTAL CODE (ZIP): 63167
(G) TELEPHONE: .(314)694-3131
(H) TELEFAX: (314)694-5435
(ii) TITLE OF INVENTION: Enhanced Expression in Plants Using
Non-translated Leader Sequences
(iii) NUMBER OF SEQUENCES: 22
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE;. Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version #1.25 (EPO)
(2) INFORMATION FOR SEQ II) NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNES:>: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ACACAGAAAA ATTTGCTACA TTG.TTTCACA AACTTCAAAT ATTATTCATT TATTT 55
(2) INFORMATION FOR SEQ II) N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucle:Lc acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
CA 02565423 2006-10-20
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
CTGACAAATA AATGAATAAT ATTTGAAGTT TGTGAAACAA TGTAGCAAAT TTTTCTGTGT 60
(2) INFORMATION FOR SEQ ID N0:3:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
{D} TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA {genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GTCAGCTTTC AAACTCTTTG TTTCTTGTTT GTTGATTGAG AATAC 45
(2} INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
CATGGTATTC TCAATCAACA AACAAGAAAC AAAGAGTTTG AAAG 44
(2) INFORMATION FOR SEQ ID NO: S:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 71 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic}
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: S:
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ACACAGAAAC ATTCGCAAAA ACAAAATCCC AGTATCAAAA TTCTTCTCTT TTTTTCATAT 60
TTCGCAAAGA C 71
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs
(B) TYPE: nucle_Lc acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DtdA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CATGGTCTTT GCGAAATATG AAAAAAAGAG AAGAATTTTG ATACTGGGAT TTTGTTTTTG 60
CGAATGTTTC TGTGT 75
(2) INFORMATION FOR SEQ IL) N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNES~~: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
ACACTCTCTC GCCTGAGAAA AAA1?vATCCAC GAACCAATTT CTCAGCAACC AGCAGCACG 59
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS~: double
( D ) TOPOLOGY : l.i.near
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIFT'ION: SEQ ID N0:8:
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CAGGTCGTGC TGCTGGTTGC TGAGAAATTG GTTCGTGGAT TTTTTTTCTC AGGCGAGAGA 60
GTGT 64
(2) INFORMATTON FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: 'DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
ACCTGTGAGG GTTCGAAGGA AGTAGCAGTG TTTTTTGTTC CTAGAGGAAG AGC 53
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CATGGCTCTT CCTCTAGGAA CA?u~AAACAC TGCTACTTCC TTCGAACCCT CA 52
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DN'A (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ACACGTTTTT ATTTTTAATT TTCTTTCAAA TACTTCCATC 40
CA 02565423 2006-10-20
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(2) INFORMATION FOR SEQ .CD N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
CATGGATGGA AGTATTTGAA AGAAAATTAA AAATAAAAAC GTGT 44
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
ACACGTATTT TTACAACAAT TACCAACAAC AACAAACAAC AAACAACATT ACAATTACTA 60
TTTACAATTA CAC 73
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 77 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
CATGGTGTAA TTGTAAATAG TAATTGTAAT GTTGTTTGTT GTTTGTTGTT GTTGGTAATT 60
GTTGTAAAAA TACGTGT 77
CA 02565423 2006-10-20
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(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: BNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
ACACGTTTTT ATTTTTAATT TTCTTTCAAA TACTTCCATA 40
(2) INFORMATION FOR SEQ :ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: :Linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
GATCTATGGA AGTATTTGAA AGAAAATTAA AAATAAAAAC GTGT 44
(2) INFORMATION FOR SEQ TD NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
AcACGTATTT TTACAACAAT TACCAACAAC AACAAACAAC AAACAACATT ACAATTACTA 60
TTTACAATTA CAA 73
(2) INFORMATION FOR SEQ ID N0:18:
CA 02565423 2006-10-20
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 77 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNES,S: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIP'PION: SEQ ID N0:18: ,
GATCTTGTAA TTGTAAATAG TAA'rTGTAAT GTTGTTTGTT GTTTGTTGTT GTTGGTAATT 60
GTTGTAAAAA TACGTGT 77
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DtdA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
ACACAGAAAC ATTCGCAAAA ACAAAATCCC AGTATCAAAA TTCTTCTCTT TTTTTCATAT 60
TTCGCAAAGA TTTAAAAA 7g
(2) INFORMATION FOR SEQ II) N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 82 base pairs
( B ) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
GATCTTTTTA AATCTTTGCG AAATATGAAA AAAAGAGAAG AATTTTGATA CTGGGATTTT 60
GTTTTTGCGA ATGTTTCTGT GT g2
CA 02565423 2006-10-20
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(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
GTCAGCTTTC AAACTCTTTG TTTCTTGTTT GTTGATTGAG AATATTTAAA AA 52
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
GATCTTTTTA AATATTCTCA ATCAACAAAC AAGAAACAAA GAGTTTGAAA G 51
