U.S. patent application number 10/413909 was filed with the patent office on 2003-10-09 for methods for making plants tolerant to glyphosate and compositions thereof.
This patent application is currently assigned to Monsanto Technology LLC. Invention is credited to Baerson, Scott R., Heck, Gregory R., Rodriguez, Damian J..
Application Number | 20030192072 10/413909 |
Document ID | / |
Family ID | 22691747 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030192072 |
Kind Code |
A1 |
Baerson, Scott R. ; et
al. |
October 9, 2003 |
Methods for making plants tolerant to glyphosate and compositions
thereof
Abstract
The methods and materials disclosed herein are directed to
glyphosate herbicide tolerance in plants. In particular, the
isolation of a glyphosate resistant EPSP synthase coding sequence
and its regulatory elements from Eleusine indica. The coding
sequence and regulatory sequences are useful to genetically
engineer plants for tolerance to glyphosate herbicide.
Inventors: |
Baerson, Scott R.; (Oxford,
MS) ; Rodriguez, Damian J.; (Lebanon, IL) ;
Heck, Gregory R.; (Crystal Lake Park, MO) |
Correspondence
Address: |
Ron Laby
HOWREY SIMON ARNOLD & WHITE, LLP
750 Bering Drive
Houston
TX
77057-2198
US
|
Assignee: |
Monsanto Technology LLC
|
Family ID: |
22691747 |
Appl. No.: |
10/413909 |
Filed: |
April 15, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10413909 |
Apr 15, 2003 |
|
|
|
09800130 |
Mar 6, 2001 |
|
|
|
60188093 |
Mar 9, 2000 |
|
|
|
Current U.S.
Class: |
800/278 ;
435/191; 435/320.1; 435/419; 536/23.2 |
Current CPC
Class: |
C12N 15/8275 20130101;
C12N 9/1092 20130101 |
Class at
Publication: |
800/278 ;
536/23.2; 435/419; 435/191; 435/320.1 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04; C12N 009/06 |
Claims
We claim
1. A DNA molecule that encodes a naturally occurring glyphosate
resistant EPSPS enzyme derived from a glyphosate tolerant plant,
wherein the glyphosate resistant EPSPS enzyme has a K.sub.m for
phosphoenolpyruvate (PEP) of less than 10 .mu.M.
2. A DNA molecule of claim 1 that encodes a naturally occurring
glyphosate resistant EPSPS enzyme derived from a glyphosate
tolerant plant, wherein the glyphosate resistant EPSPS enzyme has a
K.sub.m for PEP of less than 10 .mu.M and the K.sub.m for PEP is
not more than about twice of the K.sub.m for PEP of a naturally
occurring glyphosate sensitive EPSPS enzyme derived from a
glyphosate sensitive plant.
3. A DNA molecule of claim 2, wherein said plant is Eleusine
species.
4. A DNA molecule of claim 1, wherein said naturally occurring
glyphosate resistant EPSPS enzyme is modified by a substitution or
a deletion of at least one amino acid in a catalytic domain.
5. A DNA molecule of claim 4, wherein said substitution is selected
from the group consisting of glycine to alanine 102 and threonine
to isoleucine 103 of SEQ ID NO: 7.
6. A DNA molecule that encodes a naturally occurring glyphosate
resistant EPSPS enzyme of SEQ ID NO: 7.
7. A DNA molecule of claim 6 that encodes a naturally occurring
glyphosate resistant EPSPS enzyme of SEQ ID NO: 7, wherein the DNA
molecule is substantially homologous to SEQ ID NO: 6.
8. A recombinant DNA molecule comprising: a promoter that functions
in plant cells to cause the production of an RNA sequence, operably
linked to; a structural DNA sequence that causes the production of
an RNA sequence that encodes an EPSPS enzyme comprising the
sequence of SEQ ID NO: 7, operably linked to; a 3' non-translated
region that functions in plant cells to cause the addition of
polyadenyl nucleotides to the 3'end of the RNA sequence, wherein
the promoter is heterologous with respect to the structural DNA
sequence and selected so as to cause sufficient expression of the
polypeptide to enhance the glyphosate tolerance of a transgenic
plant cell containing said recombinant DNA molecule.
9. A recombinant DNA molecule of claim 8, wherein said structural
DNA sequence encodes a fusion polypeptide comprising an
amino-terminal chloroplast transit peptide and an EPSPS enzyme
comprising the sequence of SEQ ID NO: 7.
10. A method of producing glyphosate tolerant plants comprising the
steps of: a) inserting into the genome of a plant cell a
recombinant DNA molecule comprising: a promoter that functions in
plant cells to cause the production of a RNA sequence, operably
linked to; a structural DNA sequence that caused the production of
a RNA sequence that encodes an EPSPS enzyme having the sequence of
SEQ ID NO: 7, operably linked to; a 3' non-translated region that
functions in plant cells to cause the addition of polyadenyl
nucleotides the 3'end of the RNA sequence; where the promoter is
heterologous with respect to the structural DNA sequence and
adapted to cause sufficient expression of the polypeptide to
enhance the glyphosate tolerance of a plant cell transformed with
the DNA molecule; b) obtaining a transformed plant cell; and c)
regenerating from the transformed plant cell a genetically
transformed plant which has increased tolerance to glyphosate
herbicide.
11. A method of claim 10, wherein the structural DNA sequence
encodes a fusion polypeptide comprising an amino-terminal
chloroplast transit peptide and an EPSPS enzyme comprising the
sequence of SEQ ID NO: 7.
12. A glyphosate tolerant plant cell comprising a recombinant DNA
molecule of claim 8 or 9.
13. A glyphosate tolerant plant cell of claim 12 selected from the
group consisting of corn, wheat, rice, millet, sugarcane, barley,
oat, rye, turf grasses, asparagus, soybean, cotton, sugar beet,
oilseed rape, canola, flax, sunflower, potato, tobacco, tomato,
alfalfa, forest trees, fruit trees, ornamental annuals, and
ornamental perennials.
14. A glyphosate tolerant plant comprising the plant cells of claim
12.
15. A glyphosate tolerant plant of claim 14 selected from the group
consisting of corn, wheat, rice, millet, sugarcane, barley, oat,
rye, turf grasses, asparagus, soybean, cotton, sugar beet, oilseed
rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa,
forest trees, fruit trees, ornamental annuals, and ornamental
perennials.
16. A recombinant DNA molecule comprising: a promoter that
functions in plant cells to cause the production of an RNA
sequence, operably linked to; a structural DNA sequence that causes
the production of an RNA sequence which encodes an EPSPS enzyme
having the sequence of SEQ ID NO: 7, operably linked to; a 3'
non-translated region that functions in plant cells to cause the
addition of polyadenyl nucleotides the 3' end of the RNA sequence,
wherein the promoter is homologous with respect to the structural
DNA sequence.
17. A DNA molecule of claim 16 wherein the structural DNA sequence
encodes a fusion polypeptide comprising an amino-terminal
chloroplast transit peptide and the EPSPS enzyme comprising the
sequence of SEQ ID NO: 7.
18. A glyphosate tolerant transgenic plant cell comprising a DNA
molecule of claim 16 or 17.
19. A glyphosate tolerant transgenic plant cell of claim 18
selected from the group consisting of corn, wheat, rice, millet,
sugarcane, barley, oat, rye, turf grasses, asparagus, soybean,
cotton, sugar beet, oilseed rape, canola, flax, sunflower, potato,
tobacco, tomato, alfalfa, forest trees, fruit trees, ornamental
annuals, ornamental perennials.
20. A glyphosate tolerant transgenic plant comprising plant cells
of claim 18.
21. Glyphosate tolerant transgenic plant of claim 20 selected from
the group consisting of corn, wheat, rice, millet, sugarcane,
barley, oat, rye, turf grasses, asparagus, soybean, cotton, sugar
beet, oilseed rape, canola, flax, sunflower, potato, tobacco,
tomato, alfalfa, forest trees, fruit trees, ornamental annuals,
ornamental perennials.
22. The seed of a glyphosate tolerant transgenic plant of claim
15.
23. The seed of a glyphosate tolerant transgenic plant of claim
21.
24. A DNA molecule comprising the promoter region located 5' to the
DNA molecule of claim 3.
25. A DNA molecule comprising the chloroplast transit peptide
coding region located 5' to the DNA molecule of claim 3.
26. A DNA molecule comprising the 3' untranslated region located 3'
to the DNA molecule of claim 3.
12 1 GCGGGCGCGG AGGAGGTGGT GCTGCAGCCC ATCAAGGAGA TCTCCGGCGT 51
CGTGAAGCTG CCGGGGTCCA AGTCGCTCTC CAACCGGATC CTCCTGCTCT 101
CCGCCCTCGC CGAGGGAACA ACTGTGGTGG ATAACCTTTT AAACAGTGAG 151
GACGTCCACT ACATGCTCGG GGCCCTGAAA ACCCTCGGAC TCTCTGTGGA 201
AGCGGACAAA GCTGCCAAAA GAGCGGTAGT TGTTGGCTGT GGTGGCAAGT 251
TCCCAGTTGA GAAGGATGCG AAAGAGGAGG TGCAGCTCTT CTTGGGGAAT 301
GCTGGAACTG CAATGCGATC ATTGACAGCA GCCGTAACTG CTGCTGGAGG 351
AAATGCAACT TATGTGCTTG ATGGAGTGCC AAGAATGCGG GAGAGACCCA 401
TTGGCGACTT GGTTGTCGGA TTGAAACAGC TTGGTGCGGA TGTTGATTGT 451
TTCCTTGGCA CTGACTGCCC ACCTGTTCGT GTCAAGGGAA TCGGAGGGCT 501
ACCTGGTGGC AAGGTTAAGT TATCTGGTTC CATCAGCAGT CAGTACTTGA 551
GTGCCTTGCT GATGGCTGCT CCTTTAGCTC TTGGGGATGT GGAGATTGAA 601
ATCATTGATA AACTGATCTC CATCCCTTAT GTTGAAATGA CATTGAGATT 651
GATGGAGCGT TTTGGCGTGA AAGCAGAGCA TTCTGATAGC TGGGACAGAT 701
TCTACATCAA GGGAGGTCAA AAATACAAGT CCCCTAAAAA TGCCTACGTG 751
GAAGGTGATG CCTCAAGTGC GAGCTATTTC TTGGCTGGTG CTGCAATCAC 801
TGGAGGGACT GTGACTGTTG AAGGTTGTGG CACCACCAGT CTGCAGGGTG 851
ATGTGAAATT TGCCGAGGTA CTCGAGATGA TGGGAGCGAA GGTTACATGG 901
ACTGAAACTA GCGTAACTGT TACCGGTCCA CAACGTGAGC CATTTGGGAG 951
GAAACACCTA AAAGCTATTG ATGTTAACAT GAACAAAATG CCCGATGTCG 1001
CCATGACTCT TGCCGTGGTT GCCCTATTTG CTGATGGCCC AACTGCTATC 1051
AGAGATGTGG CTTCCTGGAG AGTAAAGGAG ACCGAGAGGA TGGTTGCAAT 1101
CCGGACTGAG CTAACAAAGC TGGGAGCGTC GGTCGAGGAA GGACTGGACT 1151
ACTGCATTAT CACACCGCCC GAGAAGCTGA ACGTAACGGC CATCGACACC 1201
TACGATGACC ACAGGATGGC CATGGCCTTC TCCCTCGCCG CCTGCGCCGA 1251
CGTGCCTGTG ACCATCCGGG ACCCCGGCTG CACCCGCAAG ACCTTCCCAG 1301
ACTACTTCGA CGTGCTGAGC ACTTTCGTCA AGAACTAA
Description
FIELD OF THE INVENTION
[0001] This invention relates in general to plant molecular biology
and plant genetic engineering for herbicide resistance and, more
particularly, to a novel glyphosate resistant
5-enolpyruvylshikimate-3-ph- osphate synthase from Eleusine indica.
Plant genetic engineering methods can be used to transfer the
glyphosate resistant 5-enolpyruvylshikimate-3- -phosphate synthase
gene isolated and purified from Eleusine indica into crop and
ornamental plants of economic importance.
BACKGROUND OF THE INVENTION
[0002] N-phosphonomethylglycine, also known as glyphosate, is a
well known herbicide that has activity on a broad spectrum of plant
species. Glyphosate is the active ingredient of Roundup.RTM.
(Monsanto Co.), a safe herbicide having a desirably short half life
in the environment. When applied onto a plant surface, glyphosate
moves systemically through the plant. Glyphosate is toxic to plants
by inhibiting the shikimic acid pathway that provides a precursor
for the synthesis of aromatic amino acids. Specifically, glyphosate
affects the conversion of phosphoenolpyruvate and 3-phosphoshikimic
acid to 5-enolpyruvyl-3-phospho- shikimic acid by inhibiting the
enzyme 5-enolpyruvyl-3-phosphoshikimate synthase (hereinafter
referred to as EPSP synthase or EPSPS). For purposes of the present
invention, the term "glyphosate" should be considered to include
any herbicidally effective form of N-phosphonomethylglycine
(including any salt thereof) and other forms that result in the
production of the glyphosate anion in planta.
[0003] Through plant genetic engineering methods, it is possible to
produce glyphosate tolerant plants by inserting into the plant
genome a DNA molecule that causes the production of higher levels
of wild-type EPSPS (Shah et al., Science 233:478-481 (1986).
Glyphosate tolerance can also be achieved by the expression of
EPSPS variants that have lower affinity for glyphosate and
therefore retain their catalytic activity in the presence of
glyphosate (U.S. Pat. No. 4,940,835, U.S. Pat. No. 5,094,945, U.S.
Pat. No. 5,633,435). Enzymes that degrade glyphosate in the plant
tissues (U.S. Pat. No. 5,463,175) are also capable of conferring
cellular tolerance to glyphosate. Such genes, therefore, allow for
the production of transgenic crops that are tolerant to glyphosate,
thereby allowing glyphosate to be used for effective weed control
with minimal concern of crop damage. For example, glyphosate
tolerance has been genetically engineered into corn (U.S. Pat. No.
5,554,798), wheat (Zhou et al. Plant Cell Rep. 15:159-163 (1995),
soybean (WO 9200377) and canola (WO 9204449).
[0004] Variants of the wild-type EPSPS enzyme are
glyphosate-resistant as a result of alterations in the EPSPS amino
acid coding sequence (Kishore et al., Annu. Rev. Biochem.
57:627-663 (1988); Schulz et al., Arch. Microbiol. 137:121-123
(1984); Sost et al., FEBS Lett. 173:238-241 (1984); Kishore et al.,
In "Biotechnology for Crop Protection" ACS Symposium Series No.
379. Eds. Hedlin et al., 37-48 (1988). These variants typically
have a higher K.sub.i for glyphosate than the wild-type EPSPS
enzyme that confers the glyphosate-tolerant phenotype, but these
variants are also characterized by a high K.sub.m for PEP that
makes the enzyme kinetically less efficient. For example, the
apparent K.sub.m for PEP and the apparent K.sub.i for glyphosate
for the native EPSPS from E. coli are 10 .mu.M and 0.5 .mu.M while
for a glyphosate-resistant isolate having a single amino acid
substitution of an alanine for the glycine at position 96 these
values are 220 .mu.M and 4.0 mM, respectively. A number of
glyphosate-resistant plant variant EPSPS genes have been
constructed by mutagenesis.
[0005] A variety of native and variant EPSPS enzymes have been
expressed in transgenic plants in order to confer glyphosate
tolerance (Singh, et al., In "Biosynthesis and Molecular Regulation
of Amino Acids in Plants", Amer Soc Plant Phys. Pubs (1992).
Examples of some of these EPSP Synthases and methods for preparing
transgenic plants resistant to glyphosate include those described
and/or isolated in accordance with U.S. Pat. No. 4,940,835, U.S.
Pat. No. 4,971,908, U.S. Pat. No. 5,145,783, U.S. Pat. No.
5,188,642, U.S. Pat. No. 5,310,667, U.S. Pat. No. 5,312,910, and
U.S. Pat. No. 6,40,497. They can also be derived from a
structurally distinct class of non-homologous EPSPS genes, such as
the naturally occurring class II EPSPS genes isolated from
Agrobacterium sp. strain CP4 as described in U.S. Pat. No.
5,633,435 and U.S. Pat. No. 5,627,061.
[0006] Eleusine indica is commonly referred to as "goose grass" and
may also be known as "yard grass". It is a common monocotyledonous
plant found world wide. As a member of the Poaceae family, the
grass family, it is related to many well known crop plants.
Eleusine indica is most closely related to the millets, that
include Sorghum bicolor (sorghum or great millet), Zea mays
(maize), Pennisetum americanum (pearl millet), Eleusine coracana
(finger millet), Setaria italica (foxtail millet), Paspalum
scrobiculatum (kodo millet), Echinochloa frumentacea (barnyhard
millet) and Eragrostis tef (teff) (Chennaveeraiah et al., In
"Chromosome engineering in plants: genetics, breeding and
evolution", Cytogenetics of Minor Millets, in Tsuchiya et al., eds
Elsevier Sci Pub Amsterdam, 613-627 (1991). Eleusine indica has
been shown to hybridize with Eleusine coracana (finger millet), an
important cultivated millet of India and East Africa
(Chennaveeraiah et al., Euphytica 2-3:489-495, (1974). Classical
plant breeding methods can be used to transfer the genes and traits
of interest from Eleusine indica into agronomic crop plants within
the family Poaceae.
SUMMARY OF THE INVENTION
[0007] In its broadest sense, the present invention herein provides
a method for plant tolerance to glyphosate herbicide by the
expression of an isolated DNA molecule encoding a naturally
occurring glyphosate resistant EPSPS enzyme. The enzyme and the DNA
is isolated from Eleusine species, more particularly Eleusine
indica (E. indica).
[0008] In the first aspect of the present invention described
herein provides a method to cause plants to be tolerant to
glyphosate herbicide by the insertion of a recombinant DNA molecule
into the nuclear genome of a plant cell, the recombinant DNA
molecule comprising:
[0009] a promoter that functions in plant cells to cause the
production of an RNA molecule; operably linked to,
[0010] a DNA molecule transcribing an RNA encoding for a
chloroplast transit peptide and a E. indica glyphosate resistant
EPSPS enzyme; operably linked to, a 3' non-translated region that
functions in plant cells to cause the polyadenylation of the 3' end
of the RNA molecule.
[0011] Typically, the promoter used in the DNA molecule is
expressed in a constitutive fashion. Examples of suitable promoters
that function effectively in this capacity include cauliflower
mosaic virus 19S promoter, cauliflower mosaic virus 35S promoter,
figwort mosaic virus 34S promoter, sugarcane bacilliform virus
promoter, commelina yellow mottle virus promoter, small subunit of
ribulose-1,5-bisphosphate carboxylase promoter, rice cytosolic
triosephosphate isomerase promoter, adenine
phosphoribosyltransferae promoter, rice actin 1 promoter, maize
ubiquitin promoter, mannopine synthase promoter and octopine
synthase promoter. A Promoter may also comprise leader sequences
and intron sequences useful in the invention.
[0012] A DNA molecule that encodes a chloroplast transit peptide
sequence can be isolated from EPSPS genes purified from various
plant species including E. indica as well as from various plant
genes whose protein products have been shown to be transported into
the chloroplast.
[0013] A DNA molecule that encodes a glyphosate resistant EPSPS
enzyme isolated from Eleusine species, more particularly from E.
indica, comprising SEQ ID NO: 7 is an object of the invention and a
DNA molecule substantially homologous to the DNA molecule isolated
from E. indica or a portion thereof identified as SEQ ID NO: 6.
[0014] The 3' non-translated region can be obtained from various
genes that are expressed in plant cells. The nopaline synthase 3'
untranslated, the 3' untranslated region from pea small subunit
Rubisco gene, the wheat heat shock protein 17.9 3' untranslated
region, the 3' untranslated region from soybean 7S seed storage
protein gene are commonly used in this capacity.
[0015] The invention also relates to a glyphosate tolerant
transgenic crop plant cell, a glyphosate tolerant crop plant and
crop plant parts, crop seeds and progeny thereof comprising the
recombinant DNA molecule of the present invention.
[0016] A DNA molecule that encodes a naturally occurring plant
derived glyphosate resistant EPSPS enzyme, wherein the glyphosate
resistant EPSPS enzyme has a K.sub.m for phosphoenolpyruvate (PEP)
of less than 10 .mu.M. More preferably, a DNA molecule that encodes
a naturally occurring plant derived glyphosate resistant EPSPS
enzyme wherein the glyphosate resistant EPSPS enzyme has a K.sub.m
for PEP of less than 10 .mu.M and the K.sub.m for PEP is not more
than about 2.times. of the naturally occurring plant derived
glyphosate sensitive EPSPS enzyme.
[0017] A DNA molecule that encodes a naturally occurring glyphosate
resistant EPSPS enzyme derived from Eleusine species, wherein the
glyphosate resistant EPSPS enzyme has a K.sub.m for
phosphoenolpyruvate (PEP) of less than 10 .mu.M. More preferably, a
DNA molecule that encodes a naturally occurring glyphosate
resistant EPSPS enzyme derived from Eleusine species, wherein the
glyphosate resistant EPSPS enzyme has a K.sub.m for PEP of less
than 10 .mu.M and the K.sub.m for PEP is not more than about
2.times. of the naturally occurring plant derived glyphosate
sensitive EPSPS enzyme.
[0018] A DNA molecule that encodes a naturally occurring glyphosate
resistant EPSPS enzyme derived from E. indica, wherein the
naturally occurring glyphosate resistant EPSPS enzyme amino acid
sequence has been modified by amino acid substitutions selected
from the group consisting of threonine to isoleucine at amino acid
position 103 and glycine to alanine at amino acid position 102.
[0019] The invention also relates to the homologous genetic
elements regulating expression of the E. indica glyphosate
resistant EPSPS gene. These elements include but are not limited to
the DNA sequences of a promoter, a 5' untranslated region, a
chloroplast transit peptide, an intron, and a 3' untranslated
region of E. indica EPSPS glyphosate resistance gene. A DNA
molecule that encodes a glyphosate resistant EPSPS enzyme purified
from the genome of Eleusine species, more particularly from E.
indica glyphosate resistant biotype provided by the ATCC deposit
#PTA-2177 is an object of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0021] FIG. 1. Eleusine indica (glyphosate tolerant) EPSP synthase
DNA sequence (SEQ ID NO: 6).
[0022] FIG. 2. Deduced amino acid sequence for the mature
protein-coding region of the Eleusine indica (glyphosate tolerant
biotype) EPSP synthase gene (SEQ ID NO: 7).
[0023] FIG. 3. Growth rates on glyphosate containing media of
transgenic E. coli on expressing the E. indica glyphosate sensitive
EPSPS enzyme and the E. indica glyphosate resistant EPSPS
enzyme.
[0024] FIG. 4. Glyphosate inhibition study comparing the EPSP
synthase activities detectable in extracts prepared from the
glyphosate-sensitive and tolerant E. indica biotypes.
[0025] FIG. 5. Plasmid map of pMON45364
[0026] FIG. 6. Plasmid map of pMON45365
[0027] FIG. 7. Plasmid map of pMON45367
[0028] FIG. 8. Plasmid map of pMON45369
[0029] FIG. 9. The amino acid sequence deduced from the cDNA
sequence of the mature EPSP synthase protein sequence derived from
the glyphosate-tolerant E. indica biotype (top row) aligned with
that of the glyphosate sensitive E. indica biotype (bottom
row).
DETAILED DESCRIPTION OF THE INVENTION
[0030] This application claims the benefit of U.S. Provisional
Application No. 60/188,093, filed Mar. 9, 2000.
[0031] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant art.
Definitions of common terms in molecular biology may also be found
in Rieger et al., Glossary of Genetics: Classical and Molecular,
5th edition, Springer-Verlag: New York, (1991); and Lewin, Genes V,
Oxford University Press: New York, (1994). The nomenclature for DNA
bases as set forth at 37 CFR .sctn. 1.822 is used. The standard
one- and three-letter nomenclature for amino acid residues is
used.
[0032] "cDNA library" refers to a collection of cDNA fragments,
each cloned into a separate vector molecule.
[0033] The term "chimeric" refers to a fusion nucleic acid or
protein sequence. A chimeric nucleic acid coding sequence is
comprised of two or more sequences joined in-frame that encode a
chimeric protein. A chimeric gene refers to the multiple genetic
elements derived from heterologous sources comprising a gene.
[0034] The phrases "coding sequence", "open reading frame", and
"structural sequence" refer to the region of continuous sequential
nucleic acid triplets encoding a protein, polypeptide, or peptide
sequence.
[0035] "Codon" refers to a sequence of three nucleotides that
specify a particular amino acid.
[0036] "Complementarity" and "complement" when referring to nucleic
acid sequences, refers to the specific binding of adenine to
thymine (or uracil in RNA) and cytosine to guanine on opposite
strands of DNA or RNA.
[0037] "Construct" refers to the heterologous genetic elements
operably linked to each other making up a recombinant DNA
molecule.
[0038] "C-terminal region" refers to the region of a peptide,
polypeptide, or protein chain from the middle thereof to the end
that carries the amino acid having a free carboxyl group.
[0039] The term "encoding DNA" refers to chromosomal DNA, plasmid
DNA, cDNA, or synthetic DNA that encodes any of the proteins
discussed herein.
[0040] The term "endogenous" refers to materials originating from
within an organism or cell.
[0041] "Endonuclease" refers to an enzyme that hydrolyzes double
stranded DNA at internal locations.
[0042] "Exogenous" refers to materials originating from outside of
an organism or cell. This typically applies to nucleic acid
molecules used in producing transformed or transgenic host cells
and plants.
[0043] "Exon" refers to the portion of a gene that is actually
translated into protein, i.e. a coding sequence.
[0044] The term "expression" refers to the transcription of a gene
to produce the corresponding mRNA.
[0045] "Fragments". A fragment of a EPSPS gene is a portion of a
full-length EPSPS gene nucleic acid that is of at least a minimum
length capable of expressing a protein with EPSPS activity.
[0046] The term "gene" refers to chromosomal DNA, plasmid DNA,
cDNA, synthetic DNA, or other DNA that encodes a peptide,
polypeptide, protein, or RNA molecule, and regions flanking the
coding sequence involved in the regulation of expression.
[0047] The term "genome" as it applies to viruses encompasses all
of the nucleic acid sequence contained within the capsid of the
virus. The term "genome" as it applies to bacteria encompasses both
the chromosome and plasmids within a bacterial host cell. Encoding
nucleic acids of the present invention introduced into bacterial
host cells can therefore be either chromosomally-integrated or
plasmid-localized. The term "genome" as it applies to plant cells
encompasses not only chromosomal DNA found within the nucleus, but
organelle DNA found within subcellular components of the cell.
Nucleic acids of the present invention introduced into plant cells
can therefore be either chromosomally-integrated or
organelle-localized.
[0048] "Glyphosate" refers to N-phosphonomethylglycine and its'
salts, Glyphosate is the active ingredient of Roundup.RTM.
herbicide (Monsanto Co.). Plant treatments with "glyphosate" refer
to treatments with the Roundup.RTM. or Roundup Ultra.RTM. herbicide
formulation, unless otherwise stated. Glyphosate as
N-phosphonomethylglycine and its' salts (not formulated
Roundup.RTM. herbicide) are components of synthetic culture media
used for the selection of bacteria and plant tolerance to
glyphosate or used to determine enzyme resistance in in vitro
biochemical assays.
[0049] "Heterologous DNA" refers to DNA from a source different
than that of the recipient cell.
[0050] "Homologous DNA" refers to DNA from the same source as that
of the recipient cell.
[0051] "Hybridization" refers to the ability of a strand of nucleic
acid to join with a complementary strand via base pairing.
Hybridization occurs when complementary sequences in the two
nucleic acid strands bind to one another.
[0052] "Identity" refers to the degree of similarity between two
nucleic acid or protein sequences. An alignment of the two
sequences is performed by a suitable computer program. A widely
used and accepted computer program for performing sequence
alignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids Res., 22:
4673-4680, 1994). The number of matching bases or amino acids is
divided by the total number of bases or amino acids, and multiplied
by 100 to obtain a percent identity. For example, if two 580 base
pair sequences had 145 matched bases, they would be 25 percent
identical. If the two compared sequences are of different lengths,
the number of matches is divided by the shorter of the two lengths.
For example, if there are 100 matched amino acids between 200 and a
400 amino acid proteins, they are 50 percent identical with respect
to the shorter sequence. If the shorter sequence is less than 150
bases or 50 amino acids in length, the number of matches are
divided by 150 (for nucleic acid bases) or 50 (for amino acids),
and multiplied by 100 to obtain a percent identity.
[0053] "Intron" refers to a portion of a gene not translated into
protein, even though it is transcribed into RNA.
[0054] "Isolated" An "isolated" nucleic acid is one that has been
substantially separated or purified away from other nucleic acid
sequences in the cell of the organism that the nucleic acid
naturally occurs, i.e., other chromosomal and extrachromosomal DNA
and RNA, by conventional nucleic acid-purification methods. The
term also embraces recombinant nucleic acids and chemically
synthesized nucleic acids.
[0055] "Native" The term "native" refers to a naturally-occurring
("wild-type") nucleic acid or polypeptide.
[0056] "N-terminal region" refers to the region of a peptide,
polypeptide, or protein chain from the amino acid having a free
amino group to the middle of the chain.
[0057] "Nucleic acid" refers to deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA).
[0058] Nucleic acid codes: A=adenosine; C=cytosine; G=guanosine;
T=thymidine. Codes used for synthesis of oligonucleotides:
N=equimolar A, C, G, and T; I=deoxyinosine; K=equimolar G and T;
R=equimolar A and G; S=equimolar C and G; W=equimolar A and T;
Y=equimolar C and T.
[0059] A "nucleic acid segment" or a "nucleic acid molecule
segment" is a nucleic acid molecule that has been isolated free of
total genomic DNA of a particular species, or that has been
synthesized. Included with the term "nucleic acid segment" are DNA
segments, recombinant vectors, plasmids, cosmids, phagemids, phage,
viruses, et cetera. "Nucleic-Acid Hybridization"; "Stringent
Conditions"; "Specific" The term "stringent conditions" is
functionally defined with regard to the hybridization of a
nucleic-acid probe to a target nucleic acid (i.e., to a particular
nucleic-acid sequence of interest) by the specific hybridization
procedure discussed in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press (1989), at 9.52-9.55.
See also, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press (1989) at 9.47-9.52, 9.56-9.58; Kanehisa,
Nucl. Acids Res. 12:203-213, (1984); and Wetmur and Davidson, J.
Mol. Biol. 31:349-370, (1968).
[0060] "Nucleotide Sequence Variants" Using well-known methods, the
skilled artisan can readily produce nucleotide and amino acid
sequence variants of EPSPS genes and proteins, respectively.
"Variant" DNA molecules are DNA molecules containing minor changes
in a native EPSPS gene sequence, i.e., changes that one or more
nucleotides of a native EPSPS gene sequence is deleted, added,
and/or substituted, such that the variant EPSPS gene encodes a
protein that retains EPSPS activity. Variant DNA molecules can be
produced, for example, by standard DNA mutagenesis techniques or by
chemically synthesizing the variant DNA molecule or a portion
thereof. Methods for chemical synthesis of nucleic acids are
discussed, for example, in Beaucage et al, Tetra. Letts.
22:1859-1862 (1981), and Matteucci et al., J. Am. Chem. Soc.
103:3185-(1981). Chemical synthesis of nucleic acids can be
performed, for example, on automated oligonucleotide synthesizers.
Such variants preferably do not change the reading frame of the
protein-coding region of the nucleic acid and preferably encode a
protein having no amino acid changes. Nucleic acid sequence
variants are most often created for the purposes of modification of
the sequence to add or delete restriction endonuclease sites or to
affect transcription or translation of the nucleic acid
molecule.
[0061] "Amino-acid substitutions", "Amino-acid variants", are
preferably substitutions of single amino-acid residue for another
amino-acid residue at any position within the protein.
Substitutions, deletions, insertions or any combination thereof can
be combined to arrive at a final construct.
[0062] "Open reading frame (ORF)" refers to a region of DNA or RNA
encoding a peptide, polypeptide, or protein.
[0063] "Operably Linked". A first nucleic-acid sequence is
"operably" linked with a second nucleic-acid sequence when the
first nucleic-acid sequence is placed in a functional relationship
with the second nucleic-acid sequence. For instance, a promoter is
operably linked to a protein-coding sequence if the promoter
effects the transcription or expression of the coding sequence.
Generally, operably linked DNA sequences are contiguous and, where
necessary to join two protein-coding regions, in reading frame.
[0064] "Overexpression" refers to the expression of a polypeptide
or protein encoded by a DNA introduced into a host cell, wherein
said polypeptide or protein is either not normally present in the
host cell, or wherein said polypeptide or protein is present in
said host cell at a higher level than that normally expressed from
the endogenous gene encoding said polypeptide or protein.
[0065] "Plant expression vector" refers to chimeric DNA molecules
comprising the regulatory elements that are operably linked to
provide the expression of a transgene product in plants.
[0066] "Plasmid" refers to a circular, extrachromosomal,
self-replicating piece of DNA.
[0067] "Polyadenylation signal" or "polyA signal" refers to a
nucleic acid sequence located 3' to a coding region that causes the
addition of adenylate nucleotides to the 3' end of the mRNA
transcribed from the coding region.
[0068] "Polymerase chain reaction (PCR)" refers to an enzymatic
technique to create multiple copies of one sequence of nucleic
acid. Copies of DNA sequence are prepared by shuttling a DNA
polymerase between two amplimers. The basis of this amplification
method is multiple cycles of temperature changes to denature, then
re-anneal amplimers, followed by extension to synthesize new DNA
strands in the region located between the flanking amplimers.
[0069] The term "promoter" or "promoter region" refers to a nucleic
acid sequence, usually found upstream (5') to a coding sequence,
that controls expression of the coding sequence by controlling
production of messenger RNA (mRNA) by providing the recognition
site for RNA polymerase and/or other factors necessary for start of
transcription at the correct site. As contemplated herein, a
promoter or promoter region includes variations of promoters
derived by means of ligation to various regulatory sequences,
random or controlled mutagenesis, and addition or duplication of
enhancer sequences. The promoter region disclosed herein, and
biologically functional equivalents thereof, are responsible for
driving the transcription of coding sequences under their control
when introduced into a host as part of a suitable recombinant
vector, as demonstrated by its ability to produce mRNA.
[0070] "Recombinant". A "recombinant" nucleic acid is made by an
artificial combination of two otherwise separated segments of
sequence, e.g., by chemical synthesis or by the manipulation of
isolated segments of nucleic acids by genetic engineering
techniques.
[0071] The term "recombinant DNA construct" or "recombinant vector"
refers to any agent such as a plasmid, cosmid, virus, autonomously
replicating sequence, phage, or linear or circular single-stranded
or double-stranded DNA or RNA nucleotide sequence, derived from any
source, capable of genomic integration or autonomous replication,
comprising a DNA molecule that one or more DNA sequences have been
linked in a functionally operative manner. Such recombinant DNA
constructs or vectors are capable of introducing a 5' regulatory
sequence or promoter region and a DNA sequence for a selected gene
product into a cell in such a manner that the DNA sequence is
transcribed into a functional mRNA that is translated and therefore
expressed. Recombinant DNA constructs or recombinant vectors may be
constructed to be capable of expressing antisense RNAs, in order to
inhibit translation of a specific RNA of interest.
[0072] "Regeneration" refers to the process of growing a plant from
a plant cell (e.g., plant protoplast or explant).
[0073] "Reporter" refers to a gene and corresponding gene product
that when expressed in transgenic organisms produces a product
detectable by chemical or molecular methods or produces an
observable phenotype.
[0074] "Restriction enzyme" refers to an enzyme that recognizes a
specific palindromic sequence of nucleotides in double stranded DNA
and cleaves both strands; also called a restriction endonuclease.
Cleavage typically occurs within the restriction site.
[0075] "Selectable marker" refers to a nucleic acid sequence whose
expression confers a phenotype facilitating identification of cells
containing the nucleic acid sequence. Selectable markers include
those that confer resistance to toxic chemicals (e.g. ampicillin
resistance, kanamycin resistance), complement a nutritional
deficiency (e.g. uracil, histidine, leucine), or impart a visually
distinguishing characteristic (e.g. color changes or fluorescence).
Useful dominant selectable marker genes include genes encoding
antibiotic resistance genes (e.g., resistance to hygromycin,
kanamycin, bleomycin, G418, streptomycin or spectinomycin); and
herbicide resistance genes (e.g., phosphinothricin
acetyltransferase). A useful strategy for selection of
transformants for herbicide resistance is described, e.g., in
Vasil, Cell Culture and Somatic Cell Genetics of Plants, Vols.
I-III, Laboratory Procedures and Their Applications Academic Press,
New York (1984).
[0076] The term "specific for (a target sequence)" indicates that a
probe or primer hybridizes under given hybridization conditions
only to the target sequence in a sample comprising the target
sequence.
[0077] "Tolerant" refers to a reduced toxic effect of glyphosate on
the growth and development of microorganisms and plants.
[0078] "Transcription" refers to the process of producing an RNA
copy from a DNA template.
[0079] "Transformation" refers to a process of introducing an
exogenous nucleic acid sequence (e.g., a vector, recombinant
nucleic acid molecule) into a cell or protoplast that exogenous
nucleic acid is incorporated into a chromosome or is capable of
autonomous replication.
[0080] "Transformed" or "transgenic" refers to a cell, tissue,
organ, or organism into that has been introduced a foreign nucleic
acid, such as a recombinant vector. A "transgenic" or "transformed"
cell or organism also includes progeny of the cell or organism and
progeny produced from a breeding program employing such a
"transgenic" plant as a parent in a cross and exhibiting an altered
phenotype resulting from the presence of the foreign nucleic
acid.
[0081] The term "transgene" refers to any nucleic acid sequence
nonnative to a cell or organism transformed into said cell or
organism. "Transgene" also encompasses the component parts of a
native plant gene modified by insertion of a nonnative nucleic acid
sequence by directed recombination.
[0082] The term "translation" refers to the production the
corresponding gene product, i.e., a peptide, polypeptide, or
protein from a mRNA.
[0083] "Vector" refers to a plasmid, cosmid, bacteriophage, or
virus that carries foreign DNA into a host organism.
[0084] "Isolated," "Purified," "Homogeneous" Polypeptides. A
polypeptide is "isolated" if it has been separated from the
cellular components (nucleic acids, lipids, carbohydrates, and
other polypeptides) that naturally accompany it or that is
chemically synthesized or recombinant. A monomeric polypeptide is
isolated when at least 60% by weight of a sample is composed of the
polypeptide, preferably 90% or more, more preferably 95% or more,
and most preferably more than 99%. Protein purity or homogeneity is
indicated, for example, by polyacrylamide gel electrophoresis of a
protein sample, followed by visualization of a single polypeptide
band upon staining the polyacrylamide gel; high pressure liquid
chromatography; or other conventional methods. Coat proteins can be
purified by any of the means known in the art, for example as
described in Guide to Protein Purification, ed. Deutscher, Meth.
Enzymol. 185, Academic Press, San Diego, 1990; and Scopes, Protein
Purification: Principles and Practice, Springer Verlag, New York,
1982.
[0085] "Labeling". There are a variety of conventional methods and
reagents for labeling polypeptides and fragments thereof Typical
labels include radioactive isotopes, ligands or ligand receptors,
fluorophores, chemiluminescent agents, and enzymes. Methods for
labeling and guidance in the choice of labels appropriate for
various purposes are discussed, e.g., in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989) and
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
(1992).
[0086] "Mature protein coding region". This term refers to the
sequence of the processed protein product of EPSPS remaining after
the chloroplast transit peptide sequence has been removed.
[0087] "Polypeptide fragments". The present invention also
encompasses fragments of an E indica EPSPS that lacks at least one
residue of a native full-length E. indica EPSPS protein, but that
specifically maintains EPSPS activity.
[0088] "Transit peptide or targeting peptide sequence". These terms
generally refer to peptide sequences that when linked to a protein
of interest directs the protein to a particular tissue, cell,
subcellular location, or cell organelle. Examples include, but are
not limited to, chloroplast transit peptides, nuclear targeting
signals, and vacuolar signals. The chloroplast transit peptide is
of particular utility in the present invention to direct expression
of the EPSPS enzyme to the chloroplast.
[0089] The term "plant" encompasses any higher plant and progeny
thereof, including monocots (e.g., corn, rice, wheat, barley,
etc.), dicots (e.g., soybean, cotton, tomato, potato, Arabidopsis,
tobacco, etc.), gymnosperms (pines, firs, cedars, etc) and includes
parts of plants, including reproductive units of a plant (e.g.,
seeds, bulbs, tubers, or other parts or tissues from that the plant
can be reproduced), fruits and flowers.
[0090] Exogenous genetic material may be transferred into a plant
by the use of a DNA vector designed for such a purpose by methods
that utilize Agrobacterium, particle bombardment or other methods
known to those skilled in the art. A particularly preferred
subgroup of exogenous material comprises a nucleic acid molecule of
the present invention. Design of such a vector is generally within
the skill of the art (Plant Molecular Biology: A Laboratory Manual,
eds. Clark, Springer, New York (1997). Examples of such plants in
to which exogenous genetic material may be transferred, include,
without limitation, alfalfa, Arabidopsis, barley, Brassica,
broccoli, cabbage, citrus, cotton, garlic, oat, oilseed rape,
onion, canola, flax, maize, an ornamental annual and ornamental
perennial plant, pea, peanut, pepper, potato, rice, rye, sorghum,
soybean, strawberry, sugarcane, sugar beet, tomato, wheat, poplar,
pine, fir, eucalyptus, apple, lettuce, lentils, grape, banana, tea,
turf grasses, sunflower, oil palm, Phaseolus etc.
[0091] The particular promoters selected for use in embodiments of
the present invention should be capable of causing the production
of sufficient expression to, in the case of the DNA molecule,
generate protein expression in vegetative and reproductive tissues
of a transformed plant. The DNA molecule will typically contain a
constitutive promoter, a structural DNA sequence encoding a
herbicide resistant enzyme, and a 3' non-translated region. A
number of constitutive promoters that are active in plant cells
have been described. Suitable promoters for constitutive expression
in plants of herbicide tolerance for the DNA molecule include, but
are not limited to, the cauliflower mosaic virus (CaMV) 35S
promoter (Odell et al. Nature 313:801-812 (1985), the Figwort
mosaic virus (FMV) 35S (Sanger et al. Plant Mol. Biol. 14:433-443
(1990), the sugarcane bacilliform virus promoter (Bouhida et al.,
J. Gen. Virol. 74:15-22 (1993), the commelina yellow mottle virus
promoter (Medberry et al., Plant J. 3:619-626 (1993), the
light-inducible promoter from the small subunit of the
ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO) (Coruzzi et al.,
EMBO J. 3:1671-1679 (1984), the rice cytosolic triosephosphate
isomerase (TPI) promoter (Xu et al. Plant Physiol. 106:459-467
(1994), the adenine phosphoribosyltransferase (APRT) promoter of
Arabidopsis (Moffatt et al. Gene 143:211-216 (1994), the rice actin
1 gene promoter (Zhong et al. Plant Sci. 116:73-84 (1996), the
mannopine synthase and octopine synthase promoters (Ni et al. Plant
J. 7:661-676 (1995), the Adh promoter (Walker et al., Proc. Natl.
Acad. Sci. U.S.A. 84: 6624-6628 (1987), the sucrose synthase
promoter (Yang et al., Proc. Natl. Acad. Sci. U.S.A. 87: 4144-4148
(1990), the R gene complex promoter (Chandler et al., The Plant
Cell 1: 1175-1183 (1989), and the chlorophyll .alpha./.beta.
binding protein gene promoter, et cetera These promoters have been
used to create DNA vectors that have been expressed in plants; see,
e.g., PCT publication WO 8402913. All of these promoters have been
used to create various types of plant-expressible recombinant DNA
vectors. Comparative analysis of constitutive promoters by the
expression of reporter genes such as the uidA
(.beta.-glucuronidase) gene from E. coli has been performed with
many of these and other promoters (Li et al. Mol. Breeding 3:1-14
(1997); Wen et al. Chinese J. of Bot. 5:102-109 (1993). Promoters
that are known or are found to cause transcription of DNA in plant
cells can be used in the present invention. Such promoters may be
obtained from a variety of sources such as plants and plant
viruses. In addition to promoters that are known to cause
transcription of DNA in plant cells, other promoters may be
identified for use in the current invention by screening a plant
cDNA library for genes that are selectively or preferably expressed
in the target tissues or cells. For the purpose of expression in
source tissues of the plant, such as the leaf, seed, root or stem,
it is preferred that the promoters utilized in the present
invention have relatively high expression in these specific
tissues. For this purpose, one may choose from a number of
promoters for genes with tissue- or cell-specific or -enhanced
expression. Examples of such promoters reported in the literature
include the chloroplast glutamine synthetase GS2 promoter from pea
(Edwards et al., Proc. Natl. Acad. Sci. U.S.A. 87: 3459-3463
(1990), the chloroplast fructose-1,6-biphosphatase (FBPase)
promoter from wheat (Lloyd et al., Mol. Gen. Genet. 225: 209-216
(1991), the nuclear photosynthetic ST-LS1 promoter from potato
(Stockhaus et al., EMBO J. 8: 2445-2451, (1989), the
serine/threonine kinase (PAL) promoter and the glucoamylase (CHS)
promoter from Arabidopsis thaliana. Also reported to be active in
photosynthetically active tissues are the ribulose-1,5-bisphosphate
carboxylase (RBCS) promoter from eastern larch (Larix laricina),
the promoter for the Cab gene, Cab6, from pine (Yamamoto et al.,
Plant Cell Physiol. 35: 773-778 (1994), the promoter for the Cab-1
gene from wheat (Fejes et al., Plant Mol. Biol. 15: 921-932 (1990),
the promoter for the Cab-1 gene from spinach (Lubberstedt et al.,
Plant Physiol. 104:997-1006 (1994), the promoter for the Cab1R gene
from rice (Luan et al., Plant Cell. 4:971-981 (1992), the pyruvate,
orthophosphate dikinase (PPDK) promoter from Zea mays (Matsuoka et
al., Proc. Natl. Acad. Sci. U.S.A. 90: 9586-9590 (1993), the
promoter for the tobacco Lhcb1*2 gene (Cerdan et al., Plant Mol.
Biol. 33:245-255 (1997), the Arabidopsis thaliana Suc2
sucrose-H.sup.+ symporter promoter (Truernit et al., Planta.
196:564-570 (1995), and the promoter for the thylakoid membrane
protein genes from spinach (PsaD, PsaF, PsaE, PC, FNR, AtpC, AtpD,
Cab, RbcS).
[0092] Other promoters for the chlorophyll .alpha./.beta.-binding
proteins may also be utilized in the present invention, such as the
promoters for LhcB gene and PsbP gene from white mustard (Sinapis
alba) (Kretsch et al., Plant Mol. Biol. 28: 219-229 (1995). A
variety of plant gene promoters that are regulated in response to
environmental, hormonal, chemical, and/or developmental signals,
also can be used for expression of RNA-binding protein genes in
plant cells, including promoters regulated by (1) heat (Callis et
al., Plant Physiol. 88:965-968 (1988), (2) light (e.g., pea RbcS-3A
promoter, Kuhlemeier et al., Plant Cell 1:471-478 (1989); maize
RbcS promoter, Schaffner et al., Plant Cell 3:997-1012 (1991); (3)
hormones, such as abscisic acid (Marcotte et al., Plant Cell
1:969-976 (1989), (4) wounding (e.g., WunI, Siebertz et al., Plant
Cell 1:961-968 (1989); or (5) chemicals, such as methyl jasminate,
salicylic acid, steroid hormones, alcohol, Safeners (Gatz, Curr.
Opin. Biotech 7:168-172 (1996), WO 9706269), or it may also be
advantageous to employ (6) organ-specific promoters (e.g., Roshal
et al., EMBO J. 6:1155-(1987); Schernthaner et al., EMBO J.
7:1249-1255 (1988); Bustos et al., Plant Cell 1:839.-853
(1989).
[0093] For the purpose of expression in sink tissues of the plant,
such as the tuber of the potato plant, the fruit of tomato, or the
seed of soybean, canola, cotton, Zea mays, wheat, rice, and barley,
it is preferred that the promoters utilized in the present
invention have relatively high expression in these specific
tissues. A number of promoters for genes with tuber-specific or
-enhanced expression are known, including the class I patatin
promoter (Bevan et al., EMBO J. 8:1899-1906 (1986); Jefferson et
al., Plant Mol. Biol. 14:995-1006 (1990), the promoter for the
potato tuber ADPGPP genes, both the large and small subunits, the
sucrose synthase promoter (Salanoubat et al.,, Gene 60:47-56
(1987); Salanoubat et al., Gene 84:181-185 (1989), the promoter for
the major tuber proteins including the 22 kD protein complexes and
proteinase inhibitors (Hannapel, Plant Physiol. 101:703-704 (1993),
the promoter for the granule bound starch synthase gene (GBSS)
(Visser et al., Plant Mol. Biol. 17:691-699 (1991), and other class
I and II patatins promoters (Koster-Topfer et al., Mol. Gen. Genet.
219:390-396 (1989); Mignery et al., Gene 62:27-44 (1988). Other
promoters can also be used to express a protein in specific
tissues, such as seeds or fruits. The promoter for
.beta.-conglycinin (Chen et al., Dev. Genet. 10:112-122 (1989) or
other seed-specific promoters such as the napin and phaseolin
promoters, can be used. The zeins are a group of storage proteins
found in Zea mays endosperm. Genomic clones for zein genes have
been isolated (Pedersen et al., Cell 29:1015-1026 (1982), and the
promoters from these clones, including the 15 kD, 16 kD, 19 kD, 22
kD, 27 kD, and gamma genes, could also be used. Other promoters
known to function, for example, in Zea mays include the promoters
for the following genes: waxy, Brittle, Shrunken 2, Branching
enzymes I and II, starch synthases, debranching enzymes, oleosins,
glutelins, and sucrose synthases. A particularly preferred promoter
for Zea mays endosperm expression is the promoter for the glutelin
gene from rice, more particularly the Osgt-1 promoter (Zheng et
al., Mol. Cell Biol. 13:5829-5842 (1993). Examples of promoters
suitable for expression in wheat include those promoters for the
ADPglucose pyrosynthase (ADPGPP) subunits, the granule bound and
other starch synthase, the branching and debranching enzymes, the
embryogenesis-abundant proteins, the gliadins, and the glutenins.
Examples of such promoters in rice include those promoters for the
ADPGPP subunits, the granule bound and other starch synthase, the
branching enzymes, the debranching enzymes, sucrose synthases, and
the glutelins. A particularly preferred promoter is the promoter
for rice glutelin, Osgt-1 gene. Examples of such promoters for
barley include those for the ADPGPP subunits, the granule bound and
other starch synthase, the branching enzymes, the debranching
enzymes, sucrose synthases, the hordeins, the embryo globulins, and
the aleurone specific proteins.
[0094] Root specific promoters may also be used. An example of such
a promoter is the promoter for the acid chitinase gene (Samac et
al., Plant Mol. Biol. 25:587-596 (1994). Expression in root tissue
could also be accomplished by utilizing the root specific
subdomains of the CaMV 35S promoter that have been identified (Lam
et al., Proc. Natl. Acad. Sci. U.S.A. 86: 7890-7894 (1989). Other
root cell specific promoters include those reported by Conkling et
al. (Plant Physiol. 93: 1203-1211 (1990).
[0095] The 5' non-translated leader sequence can be derived from
the promoter selected to express the heterologous gene sequence of
the DNA molecule of the present invention, and can be specifically
modified if desired so as to increase translation of mRNA. For a
review of optimizing expression of transgenes, see Koziel et al.,
(Plant Mol. Biol. 32:393-405 (1996). The 5' non-translated regions
can also be obtained from plant viral RNAs (Tobacco mosaic virus,
Tobacco etch virus, Maize dwarf mosaic virus, Alfalfa mosaic virus,
among others) from suitable eukaryotic genes, plant genes (wheat
and maize chlorophyll a/b binding protein gene leader), or from a
synthetic gene sequence. The present invention is not limited to
constructs wherein the non-translated region is derived from the 5'
non-translated sequence that accompanies the promoter sequence. The
leader sequence could also be derived from an unrelated promoter or
coding sequence. Leader sequences useful in context of the present
invention comprise the maize Hsp70 leader (U.S. Pat. No. 5,362,865
and U.S. Pat. No. 5,859,347), and the TMV omega element (Gallie et
al., The Plant Cell 1:301-311 (1989).
[0096] A vector or construct may also include various regulatory
elements. Intron sequences are known in the art to aid in the
expression of transgenes in monocot plant cells. Examples of such
introns include the Adh intron 1 (Callis et al., Genes and Develop.
1:1183-1200 (1987), the sucrose synthase intron (Vasil et al.,
Plant Physiol. 91:1575-1579 (1989), U.S. Pat. No. 5,955,330), first
intron of the rice actin gene (U.S. Pat. No. 5,641,876).
[0097] A vector may also include a transit peptide nucleic acid
sequence. The glyphosate target in plants, the
5-enolpyruvyl-shikimate-3-phosate synthase (EPSPS) enzyme, is
located in the chloroplast. Many chloroplast-localized proteins,
including EPSPS, are expressed from nuclear genes as precursors and
are targeted to the chloroplast by a chloroplast transit peptide
(CTP) that is removed during the import steps. Examples of other
such chloroplast proteins include the small subunit (SSU) of
Ribulose-1,5,-bisphosphate carboxylase, Ferredoxin, Ferredoxin
oxidoreductase, the light-harvesting complex protein I and protein
II, and Thioredoxin F. It has been demonstrated in vivo and in
vitro that non-chloroplast proteins may be targeted to the
chloroplast by use of protein fusions with a CTP and that a CTP
sequence is sufficient to target a protein to the chloroplast.
Incorporation of a suitable chloroplast transit peptide, such as,
the Arabidopsis thaliana EPSPS CTP (Klee et al., Mol. Gen. Genet.
210:437-442 (1987), and the Petunia hybrida EPSPS CTP (della-Cioppa
et al., Proc. Natl. Acad. Sci. USA 83:6873-6877 (1986) has been
show to target heterologous EPSPS protein sequences to chloroplasts
in transgenic plants. The production of glyphosate tolerant plants
by expression of a fusion protein comprising an amino-terminal CTP
with a glyphosate resistant EPSPS enzyme is well known by those
skilled in the art, (U.S. Pat. No. 5,627,061, U.S. Pat. No.
5,633,435, U.S. Pat. No. 5,312,910, EP 0218571, EP 189707, EP
508909, and EP 924299). Those skilled in the art will recognize
that various chimeric constructs can be made that utilize the
functionality of a particular CTP to import glyphosate resistant
EPSPS enzymes into the plant cell chloroplast.
[0098] The termination of transcription is accomplished by a 3'
non-translated DNA sequence operably linked in the chimeric vector
to the gene of interest. The 3' non-translated region of a
recombinant DNA molecule contains a polyadenylation signal that
functions in plants to cause the addition of adenylate nucleotides
to the 3' end of the RNA. The 3' non-translated region can be
obtained from various genes that are expressed in plant cells. The
nopaline synthase 3' untranslated region (Fraley et al., Proc.
Natl. Acad. Sci. 80:4803-4807 (1983), the 3' untranslated region
from pea small subunit Rubisco gene (Coruzzi et al., EMBO J.
3:1671-1679 (1994), the 3' untranslated region from soybean 7S seed
storage protein gene (Schuler et al., Nuc Acids Res. 10:8225-8244
(1982) are commonly used in this capacity. The 3' transcribed,
non-translated regions containing the polyadenylate signal of
Agrobacterium tumor-inducing (Ti) plasmid genes are also
suitable.
[0099] The aforesaid described genetic elements and other
regulatory elements of similar function may be substituted when
appropriate by those skilled in the art of plant molecular biology
to provide necessary function to the plant expression cassette. DNA
constructs for glyphosate tolerance designed for expression in
plastids will necessarily contain genetic elements that function in
plastids.
[0100] A vector may also include a screenable or scorable marker
gene. Screenable or scorable markers may be used to monitor
expression. Exemplary markers include a .beta.-glucuronidase or
uidA gene (GUS) that encodes an enzyme for that various chromogenic
substrates are known (Jefferson, Plant Mol. Biol, Rep. 5:387-405
(1987); Jefferson et al., EMBO J. 6:3901-3907 (1987); an R-locus
gene, that encodes a product that regulates the production of
anthocyanin pigments (red color) in plant tissues (Dellaporta et
al., Stadler Symposium 11:263-282 (1988); a .beta.-lactamase gene
(Sutcliffe et al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741
(1978); a gene that encodes an enzyme for that various chromogenic
substrates are known (e.g., PADAC, a chromogenic cephalosporin); a
luciferase gene (Ow et al., Science 234:856-859 (1986); a xylE gene
(Zukowsky et al., Proc. Natl. Acad. Sci. (U.S.A.) 80:1101-1105
(1983) that encodes a catechol dioxygenase that can convert
chromogenic catechols; an .alpha.-amylase gene (Ikatu et al.,
Bio/Technol. 8:241-242 (1990); a tyrosinase gene (Katz et al., J.
Gen. Microbiol. 129:2703-2714 (1983) that encodes an enzyme capable
of oxidizing tyrosine to DOPA and dopaquinone that in turn
condenses to melanin; green flourescence protein (Elliot et al.,
Plant cell Rep. 18:707-714 (1999) and an .alpha.-galactosidase.
[0101] Included within the terms "selectable or screenable marker
genes" are also genes that encode a secretable marker whose
secretion can be detected as a means of identifying or selecting
for transformed cells. Examples include markers that encode a
secretable antigen that can be identified by antibody interaction,
or even secretable enzymes that can be detected catalytically.
Secretable proteins fall into a number of classes, including small,
diffusible proteins that are detectable, (e.g., by ELISA), small
active enzymes that are detectable in extracellular solution (e.g.,
.alpha.-amylase, .beta.-lactamase, phosphinothricin transferase),
or proteins that are inserted or trapped in the cell wall (such as
proteins that include a leader sequence such as that found in the
expression unit of extension or tobacco PR-S). Other possible
selectable and/or screenable marker genes will be apparent to those
of skill in the art.
[0102] There are many methods for introducing transforming nucleic
acid molecules into plant cells. Suitable methods are believed to
include virtually any method shown effective in introducing the
nucleic acid molecules into a plant cell, such as by Agrobacterium
infection or direct delivery of nucleic acid molecules.
[0103] Four general methods for direct delivery of a gene into
cells have been described: (1) chemical methods (Graham et al.,
Virology 54:536-539 (1973); (2) physical methods such as
microinjection (Capecchi, Cell 22:479-488 (1980); electroporation
(Wong et al., Biochem. Biophys. Res. Commun. 107:584-587 (1982);
Fromm et al., Proc. Natl. Acad. Sci. (U.S.A.) 82:5824-5828 (1985);
(U.S. Pat. No. 5,384,253); and the gene gun (Johnston et al.,
Methods Cell Biol. 43:353-365 (1994); (3) viral vectors (Clapp,
Clin. Perinatol. 20:155-168 (1993); Lu et al., J. Exp. Med.
178:2089-2096 (1993); Eglitis et al., Biotechniques 6:608-614
(1988); and (4) receptor-mediated mechanisms (Curiel et al., Hum.
Gen. Ther. 3:147-154 (1992), Wagner et al., Proc. Natl. Acad. Sci.
USA 89:6099-6103 (1992).
[0104] Acceleration methods that may be used include, for example,
microprojectile bombardment and the like. One example of a method
for delivering transforming nucleic acid molecules to plant cells
is microprojectile bombardment. This method has been reviewed by
Yang et al., Particle Bombardment Technology for Gene Transfer,
Oxford Press, Oxford, England (1994). Non-biological particles
(microprojectiles) that may be coated with nucleic acids and
delivered into cells by a propelling force. Exemplary particles
include those comprised of tungsten, gold, platinum, and the like.
A particular advantage of microprojectile bombardment, in addition
to it being an effective means of reproducibly transforming
monocots, is that neither the isolation of protoplasts (Cristou et
al., Plant Physiol. 87:671-674 (1988), nor the susceptibility of
Agrobacterium infection are required. An illustrative embodiment of
a method for delivering DNA into Zea mays cells by acceleration is
a biolistics .alpha.-particle delivery system, that can be used to
propel particles coated with DNA through a screen, such as a
stainless steel or Nytex screen, onto a filter surface covered with
corn cells cultured in suspension. Gordon-Kamm et al., describes
the basic procedure for coating tungsten particles with DNA
(Gordon-Kamm et al., Plant Cell 2:603-618 (1990). The screen
disperses the tungsten nucleic acid particles so that they are not
delivered to the recipient cells in large aggregates. A particle
delivery system suitable for use with the present invention is the
helium acceleration PDS-1000/He gun is available from Bio-Rad
Laboratories (Bio-Rad, Hercules, Calif.) (Sanford et al., Technique
3:3-16 (1991).
[0105] For the bombardment, cells in suspension may be concentrated
on filters. Filters containing the cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the gun and the cells to be bombarded.
[0106] Alternatively, immature embryos or other target cells may be
arranged on solid culture medium. The cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the acceleration device and the cells to be bombarded.
Through the use of techniques set forth herein one may obtain up to
1000 or more foci of cells transiently expressing a marker gene.
The number of cells in a focus that express the exogenous gene
product 48 hours post-bombardment often range from one to ten and
average one to three.
[0107] In bombardment transformation, one may optimize the
pre-bombardment culturing conditions and the bombardment parameters
to yield the maximum numbers of stable transformants. Both the
physical and biological parameters for bombardment are important in
this technology. Physical factors are those that involve
manipulating the DNA/microprojectile precipitate or those that
affect the flight and velocity of either the macro- or
microprojectiles. Biological factors include all steps involved in
manipulation of cells before and immediately after bombardment, the
osmotic adjustment of target cells to help alleviate the trauma
associated with bombardment, and also the nature of the
transforming DNA, such as linearized DNA or intact supercoiled
plasmids. It is believed that pre-bombardment manipulations are
especially important for successful transformation of immature
embryos.
[0108] In another alternative embodiment, plastids can be stably
transformed. Method disclosed for plastid transformation in higher
plants include particle gun delivery of DNA containing a selectable
marker and targeting of the DNA to the plastid genome through
homologous recombination (Svab et al. Proc. Natl. Acad. Sci.
(U.S.A.) 87:8526-8530 (1990); Svab et al., Proc. Natl. Acad. Sci.
(U.S.A.) 90:913-917 (1993); (Staub et al., EMBO J. 12:601-606
(1993). The methods disclosed in U.S. Pat. No. 5,451,513, U.S. Pat.
No. 5,545,818, U.S. Pat. No. 5,877,402, U.S. Pat. No. 5,932479, and
WO 99/05265.
[0109] Accordingly, it is contemplated that one may wish to adjust
various aspects of the bombardment parameters in small scale
studies to fully optimize the conditions. One may particularly wish
to adjust physical parameters such as gap distance, flight
distance, tissue distance, and helium pressure. One may also
minimize the trauma reduction factors by modifying conditions that
influence the physiological state of the recipient cells and that
may therefore influence transformation and integration
efficiencies. For example, the osmotic state, tissue hydration and
the subculture stage or cell cycle of the recipient cells may be
adjusted for optimum transformation. The execution of other routine
adjustments will be known to those of skill in the art in light of
the present disclosure.
[0110] Agrobacterium-mediated transfer is a widely applicable
system for introducing genes into plant cells because the DNA can
be introduced into whole plant tissues, thereby bypassing the need
for regeneration of an intact plant from a protoplast. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA
into plant cells is well known in the art. See, for example the
methods described by Fraley et al., Bio/Technology 3:629-635 (1985)
and Rogers et al., Methods Enzymol. 153:253-277 (1987). Further,
the integration of the T-DNA is a relatively precise process
resulting in few rearrangements. The region of DNA to be
transferred is defined by the border sequences, and intervening DNA
is usually inserted into the plant genome as described (Spielmann
et al., Mol. Gen. Genet. 205:34 (1986).
[0111] Modern Agrobacterium transformation vectors are capable of
replication in E. coli as well as Agrobacterium, allowing for
convenient manipulations as described (Klee et al., In: Plant DNA
Infectious Agents, Hohn and Schell, eds., Springer-Verlag, New
York, pp. 179-203 (1985). Moreover, technological advances in
vectors for Agrobacterium-mediated gene transfer have improved the
arrangement of genes and restriction sites in the vectors to
facilitate construction of vectors capable of expressing various
polypeptide coding genes. The vectors described have convenient
multi-linker regions flanked by a promoter and a polyadenylation
site for direct expression of inserted polypeptide coding genes and
are suitable for present purposes (Rogers et al., Methods Enzymol.
153:253-277 (1987). In addition, Agrobacterium containing both
armed and disarmed Ti genes can be used for the transformations. In
those plant varieties where Agrobacterium-mediated transformation
is efficient, it is the method of choice because of the facile and
defined nature of the gene transfer.
[0112] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single genetic locus on one
chromosome. Such transgenic plants can be referred to as being
hemizygous for the added gene. More preferred is a transgenic plant
that is homozygous for the added structural gene; i.e., a
transgenic plant that contains two added genes, one gene at the
same locus on each chromosome of a chromosome pair. A homozygous
transgenic plant can be obtained by sexually mating (selfing) an
independent segregant transgenic plant that contains a single added
gene, germinating some of the seed produced and analyzing the
resulting plants for the gene of interest.
[0113] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating exogenous genes. Selfing of appropriate
progeny can produce plants that are homozygous for both exogenous
genes. Back-crossing to a parental plant and out-crossing with a
non-transgenic plant are also contemplated, as is vegetative
propagation. Descriptions of other breeding methods that are
commonly used for different traits and crops can be found in Fehr,
In: Breeding Methods for Cultivar Development, Wilcox J. ed.,
American Society of Agronomy, Madison Wis. (1987).
[0114] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation, and combinations of these
treatments (see, e.g., Potrykus et al., Mol. Gen. Genet.
205:193-200 (1986); Lorz et al., Mol. Gen. Genet. 199:178 (1985);
Fromm et al., Nature 319:791 (1986); Uchimiya et al., Mol. Gen.
Genet. 204:204 (1986); Marcotte et al., Nature 335:454-457 (1988).
Application of these systems to different plant varieties depends
upon the ability to regenerate that particular plant strain from
protoplasts. Illustrative methods for the regeneration of cereals
from protoplasts are described (Fujimura et al., Plant Tissue
Culture Letters 2:74 (1985); Toriyama et al., Theor Appl. Genet.
205:34 (1986); Yamada et al., Plant Cell Rep. 4:85 (1986); Abdullah
et al., Biotechnology 4:1087 (1986).
[0115] Other methods of cell transformation can also be used and
include but are not limited to introduction of DNA into plants by
direct DNA transfer into pollen (Hess et al., Intern Rev. Cytol.
107:367 (1987); Luo et al., Plant Mol Biol. Reporter 6:165 (1988),
by direct injection of DNA into reproductive organs of a plant
(Pena et al., Nature 325:274 (1987), or by direct injection of DNA
into the cells of immature embryos followed by the rehydration of
desiccated embryos (Neuhaus et al., Theor. Appl. Genet. 75:30
(1987).
[0116] The regeneration, development, and cultivation of plants
from single plant protoplast transformants or from various
transformed explants is well known in the art (Weissbach et al.,
In: Methods for Plant Molecular Biology, Academic Press, San Diego,
Calif., (1988). This regeneration and growth process typically
includes the steps of selection of transformed cells, culturing
those individualized cells through the usual stages of embryonic
development through the rooted plantlet stage. Transgenic embryos
and seeds are similarly regenerated. The resulting transgenic
rooted shoots are thereafter planted in an appropriate plant growth
medium such as soil.
[0117] The development or regeneration of plants containing the
foreign, exogenous gene is well known in the art. Preferably, the
regenerated plants are self-pollinated to provide homozygous
transgenic plants. Otherwise, pollen obtained from the regenerated
plants is crossed to seed-grown plants of agronomically important
lines. Conversely, pollen from plants of these important lines is
used to pollinate regenerated plants. A transgenic plant of the
present invention containing a desired exogenous nucleic acid is
cultivated using methods well known to one skilled in the art.
[0118] Methods for transforming dicots, primarily by use of
Agrobacterium tumefaciens, and obtaining transgenic plants have
been published for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No.
5,159,135, U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No.
5,569,834, U.S. Pat. No. 5,416,011, McCabe et. al., Bio/Technology
6:923 (1988), Christou et al., Plant Physiol. 87:671-674 (1988);
Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant
Cell Rep. 15:653-657 (1996), McKently et al., Plant Cell Rep.
14:699-703 (1995); and pea (Grant et al., Plant Cell Rep.
15:254-258, (1995).
[0119] Transformation of monocotyledons using electroporation,
particle bombardment, and Agrobacterium have also been reported.
Transformation and plant regeneration have been achieved in
asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA)
84:5354-5349 (1987); barley (Wan et al., Plant Physiol 104:37-48
(1994); Zea mays (Rhodes et al., Science 240:204-207 (1988),
Gordon-Kamm et al., Plant Cell 2:603-618 (1990), Fromm et al.,
Bio/Technology 8:833-839 (1990), Koziel et al., Bio/Technology 11:
194-200 (1993), Armstrong et al., Crop Science 35:550-557 (1995);
oat (Somers et al., Bio/Technology 10:1589-1594 (1992); orchard
grass (Horn et al., Plant Cell Rep. 7:469-472 (1988); rice
(Toriyama et al., Theor Appl. Genet. 205:34-(1986), Part et al.,
Plant Mol. Biol. 32:1135-1148, (1996), Abedinia et al., Aust. J.
Plant Physiol. 24:133-141 (1997), Battraw et al., Plant Mol. Biol..
15:527-538 (1990), Christou et al., Bio/Technology 9:957-962
(1991); rye (De la Pena et al., Nature 325:274-276 (1987);
sugarcane (Bower et al., Plant J. 2:409-416 (1992); tall fescue
(Wang et al., Bio/Technology 10:691-696 (1992); and wheat (Vasil et
al., Bio/Technology 10:667-674 (1992); U.S. Pat. No.
5,631,152).
[0120] Assays for gene expression based on the transient expression
of cloned nucleic acid vectors have been developed by introducing
the nucleic acid molecules into plant cells by polyethylene glycol
treatment, electroporation, or particle bombardment (Marcotte et
al., Nature 335:454-457 (1988); Marcotte et al., Plant Cell
1:523-532 (1989); McCarty et al., Cell 66:895-905 (1991); Hattori
et al., Genes Dev. 6:609-618 (1992); Goff et al., EMBO J.
9:2517-2522 (1990). Transient expression systems may be used to
functionally dissect gene constructs (see generally, Mailga et al.,
Methods in Plant Molecular Biology, Cold Spring Harbor Press
(1995). It is understood that any of the nucleic acid molecules of
the present invention can be introduced into a plant cell in a
permanent or transient manner in combination with other genetic
elements such as promoters, leaders, transit peptide sequences,
enhancers, introns, 3' nontranslated regions and other elements
known to those skilled in the art that are useful for control of
transgene expression in plants.
[0121] Eleusine indica has been shown to hybridize with Eleusine
coracana (finger millet), an important cultivated millet of India
and East Africa (Chennaveeraiah et al., Euphytica 2-3:489-495,
(1974). Classical plant breeding methods can be used to transfer
the gene and the glyphosate tolerant phenotype to crop plants
within the family Poaceae. The DNA molecules of the EPSPS
glyphosate resistance gene of E. indica (SEQ ID NO: 6) can be used
as a probe to identify other like DNA molecules by standard
methods. Oligonucleotide DNA molecules homologous or complementary
to the EPSPS glyphosate resistance gene of E. indica can be used in
a marker assisted breeding method (Simple sequence repeat DNA
marker analysis, in "DNA markers: Protocols, applications, and
overviews: (1997) 173-185, Cregan, et al., eds., Wiley-Liss NY ) to
assist in the breeding of this gene into related and heterologous
crop species.
[0122] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials that describe
specific conditions and procedures for the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.), generation of recombinant organisms and the
screening and isolating of clones, (see for example, Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press (1989); Mailga et al., Methods in Plant Molecular Biology,
Cold Spring Harbor Press (1995); Birren et al., Genome Analysis:
Detecting Genes, 1, Cold Spring Harbor, New York (1998); Birren et
al., Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, New
York (1998); Clark et al., Plant Molecular Biology: A Laboratory
Manual, Springer, New York (1997); and Innis et al., PCR Protocols:
A Guide to Methods and Applications, Academic Press: San Diego,
(1990).
[0123] Plant species containing a naturally occurring EPSPS enzyme
resistant to glyphosate have not been previously reported. The
subject of this invention is the EPSPS enzyme isolated from
Eleusine indica that has been shown to be resistant to glyphosate
and the expression of the DNA molecule encoding this EPSPS enzyme
in other plants that then confers glyphosate tolerance to those
recipient plants. The glyphosate resistant EPSPS enzyme isolated
from Eleusine indica glyphosate tolerant biotype has a novel Km
with respect to binding of PEP as compared to other plant EPSP
Synthases that have been modified for glyphosate resistance by a
single amino acid substitution of a proline to serine substitution
in the active site of the enzyme. The K.sub.m for PEP of the E.
indica glyphosate resistant enzyme is little changed from the E.
indica glyphosate sensitive EPSPS enzyme. In addition, this gene is
from a monocot plant and hence may not need nucleic sequence
modification to affect expression in transgenic monocot crop
plants. The E. indica glyphosate tolerant EPSPS enzyme amino acid
sequence can be modified by site directed mutation to include other
known substitutions.
[0124] The present invention also provides for parts of the plants
of the present invention. Plant parts, without limitation, include
seed, endosperm, ovule and pollen. In a particularly preferred
embodiment of the present invention, the plant part is a seed.
[0125] The following examples are included to demonstrate examples
of certain preferred embodiments of the invention. It should be
appreciated by those of skill in the art that the techniques
disclosed in the examples that follow represent approaches the
inventors have found function well in the practice of the
invention, and thus can be considered to constitute examples of
preferred modes for its practice. However, those of skill in the
art should, in light of the present disclosure, appreciate that
many changes can be made in the specific embodiments that are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention. All
references cited herein are hereby expressly incorporated herein by
reference.
[0126] Seeds from glyphosate tolerant Eleusine indica plants were
deposited with the American Type Culture Collection (ATCC, 10801
University Blvd, Manassas, Va., U.S.A., 20110-2209) and assigned
ATCC No. PTA-2177. The deposit will be maintained in the depository
for a period of 30 years, or 5 years after the last request, or for
the effective life of the patent, whichever is longer, and will be
replaced as necessary during that period.
EXAMPLE 1
[0127] Eleusine indica plants tolerant to glyphosate were collected
from a site near Johor, Malaysia. Glyphosate tolerant biotypes of
E. indica are identified and numbered. Seed is collected from each
biotype and planted in pots in the greenhouse. Clones are generated
for each plant by excising 10-20 tillers and transplanting these in
separate pots. Glyphosate sensitive and tolerant individual plants
are then identified by treatment with glyphosate at either 0.5 kg
active ingredient (ai)/hectare (ha) or 2.0 kg ai/ha, respectively.
A corresponding clone for glyphosate tolerant and glyphosate
sensitive E. indica biotype is left untreated. These clones are
used as the source of fresh tissue for enzyme analysis and gene
isolation.
[0128] Construction of a cDNA library from the glyphosate tolerant
E. indica biotype and the glyphosate sensitive E. indica biotype is
performed by isolating total RNA from the crown tissues. The crown
tissues are dissected from the plants, then flash-frozen with
liquid nitrogen and maintained at -80.degree. C. until needed.
Total RNAs are extracted from frozen crown samples using the RNeasy
Plant Mini Kit (cat. #74904, Qiagen Inc., Valencia, Calif.) per
manufacturer's instructions. Oligo.dT-primed first-strand cDNAs are
prepared from 5 .mu.g samples of total RNA using the Superscript
Pre-Amplification System (cat. #18089-011, Life Technologies,
Rockville, Md.) per manufacturer's instructions. Two .mu.l of
first-strand cDNA are then used to generate partial E. indica EPSP
synthase cDNAs via polymerase chain reaction using a modification
of the "touchdown PCR" technique (Don et al., Nucl. Acids Res.
19:4008, 1991). Degenerate oligonucleotide pools of SEQ ID NO: 1
and SEQ ID NO: 2 are added in a 50 .mu.l RT-PCR reaction at a final
concentration of 25 .mu.M.
1 5'-TNWSNGTNGARGCNGAYAARGT-3' (SEQ ID NO: 1)
5'-GCCATNGCCATNCKRTGRTCRTC-3' (SEQ ID NO: 2)
[0129] PCR amplifications are then performed using the Expand High
Fidelity PCR System (cat. #1 732 641, Roche Molecular Biochemicals,
Indianapolis, Ind.) per manufacturer's instructions. A thermal
profile of 94.degree. C. for 20 seconds, followed by 60.degree. C.
for 1 minute, then 72.degree. C. for 1 minute 30 seconds is used
for the initial 30 cycles with a 0.5.degree. C. decrease in
annealing temperature per cycle. This is followed by 10 additional
cycles of 94.degree. C. for 20 seconds, 45.degree. C. for 1 minute,
then 72.degree. C. for 1 minute 30 seconds.
[0130] RT-PCR products are then purified by agarose gel
electrophoresis using a QIAquick Gel Extraction Kit (cat. #28704,
Qiagen Inc., Valencia, Calif.) then directly cloned into the
pCR2.1-TOPO vector (cat. #K4500-40, Invitrogen, Carlsbad, Calif.).
The identity of the cloned RT-PCR products is confirmed by DNA
sequence analysis (ABI Prism.TM. 377, Perkin Elmer, Foster City,
Calif.).
[0131] The remainder of the 3' end of the EPSP synthase coding
region is generated using the 3' RACE System for Rapid
Amplification of cDNA Ends (cat. #18373-027, Life Technologies,
Rockville, Md.), using the gene-specific oligonucleotide of SEQ ID
NO: 3. The cDNA is prepared according to manufacturer's
instructions using 5 .mu.g of total RNA isolated from crown tissues
as previously described.
[0132] 5'-GTGAAAGCAGAGCATTCTGATAGC-3' (SEQ ID NO: 3)
[0133] PCR amplifications are conducted in 50 .mu.l reactions
including 5 .mu.l first-strand cDNA reaction, 20 picomoles of each
primer, 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, 200
.mu.M dNTPs, and 2.5 units Taq polymerase. A thermal profile of
94.degree. C. for 20 seconds, followed by 57.degree. C. for 1
minute, then 72.degree. C. for 1 minute 30 seconds is used for 35
cycles. The identity of the 3'-RACE products is confirmed by DNA
sequence analysis (ABI Prism.TM. 377, Perkin Elmer, Foster,
Calif.).
[0134] The remainder of the 5' end of the E. indica EPSP synthase
mature protein coding region is generated using the SMART RACE cDNA
Amplification Kit (cat. #K1811-1, Clontech Laboratories Inc., Palo
Alto, Calif.), using the gene-specific oligonucleotides of SEQ ID
NO: 4 and SEQ ID NO: 5. The cDNA is prepared according to
manufacturer's instructions using 150 ng of polyA+ mRNA isolated
from crown tissues using an Oligotex mRNA Midi Kit (cat. #28704,
Qiagen Inc., Valencia, Calif.).
2 (SEQ ID NO: 4) 5'-GGCTGCTGTCAATGTCGCATTGCAGTTCC-3' (SEQ ID NO: 5)
5'-CTCTTTCGCATCCTTCTCAACTGGGAACTTGC- -3'
[0135] PCR reactions are conducted as recommended by the
manufacturer, except that the Expand High Fidelity PCR System (cat.
#1 732 641, Roche Molecular Biochemicals, Indianapolis, Ind.) is
used and DMSO is included in all reactions at a final concentration
of 5.0% to facilitate the amplification of GC-rich sequences. The
synthetic DNA oligonucleotide described in SEQ ID NO: 4 is used in
the primary amplifications, then second round ("nested")
amplifications are performed using the oligonucleotide described in
SEQ ID NO: 5, with a 1 .mu.l aliquot of 1:100 dilution of the
primary PCR reactions. The identity of the 5'-RACE products is
confirmed by DNA sequence analysis (ABI Prism.TM. 377, Perkin
Elmer, Foster City, Calif.).
[0136] The significant overlap of sequences generated by RT-PCR, 3'
RACE, and 5' RACE allows for the unambiguous assembly of the
sequences into a single DNA sequence containing the entire open
reading frame for the mature protein, using the SEQMan II software
package (DNASTAR Inc., Madison, Wis.). The DNA sequence
corresponding to the mature protein-coding region of the Eleusine
indica (glyphosate tolerant biotype) EPSP synthase gene (SEQ ID NO:
6) is shown in FIG. 1.
[0137] The deduced amino acid sequence for the mature
protein-coding region of the Eleusine indica (glyphosate tolerant
biotype) EPSP synthase gene (SEQ ID NO: 7) for this protein is
shown in FIG. 2.
EXAMPLE 2
[0138] EPSP synthase enzyme from the glyphosate tolerant E. indica
biotype confers increased glyphosate tolerance in transgenic E.
coli. E. coli is useful as a heterologous expression system for
testing glyphosate resistant enzymes. The EPSP synthase mature
protein-coding regions isolated from the glyphosate tolerant and
glyphosate sensitive E. indica biotypes, can be directly compared
for their ability to confer tolerance to glyphosate in transgenic
hosts. E. coli (strain SR481) are transformed with the glyphosate
resistant EPSPS gene (Ei.EPSPS:glyR) and the glyphosate sensitive
EPSPS gene (Ei.EPSPS:glyS) purified from E. indica. The growth rate
differentials of transformed cell lines grown in the presence of
glyphosate contained in the culture medium is used as a measure of
the resistance of the EPSPS enzyme to the inhibitory effects of
glyphosate (Rogers et al., Appl. Enviro. Microbiol. 46:37-43
(1983). An appropriate E. coli expression vector that carries the
native promoter and operator sequence from the E. coli lac operon
(Dickson et al., Science 187:27-35 (1975) including the
sequence
3 (SEQ ID NO:8) 5'-AGATCTCCTAGGGCTTAATTAATTAAGCACTAGTCACACA-
GGAGGTA ATTCATATG-3'
[0139] is contained in pMON45337. This nucleotide sequence includes
1) flanking BglII and Nde1 endonuclease sites, 2) a ribosome
binding site, and 3) an unstructured region 5' to the ribosome
binding element (Balbas, P. et. al., in "Methods in Enzymology" (D.
V. Goeddel, ed.)185: 15-37, 1990). This was inserted by ligation to
facilitate expression and cloning at the ATG start codon of an open
reading frame. A multiple cloning site is positioned immediately
downstream of this Ndel site, followed by the rho-independant
transcriptional terminator element of the E. coli trpA gene (T.
Sato et al., J. Biochem. (Tokyo), 101:525-534, (1987). This vector
when it operbly contains the EPSP synthase coding sequences of the
present invention is employed for the inducible expression of
glyphosate resistant and glyphosate sensitive EPSP synthase cDNAs
in E. coli. Other commercially available inducible E. coli
expression vectors are suitable for testing the EPSP synthases from
E. indica.
[0140] DNA manipulations and transformations of E. coli are
performed according to standard procedures (Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press
(1989). To construct E. coli expression vectors carrying the EPSP
synthase mature protein coding sequences from the tolerant and
sensitive E. indica biotypes, the oligonucleotide primers of SEQ ID
NO: 9 and SEQ ID NO: 10.
4 5'-GCAATTCCATATGGCGGGCGCGGAGGAGGTGGTGCT-3' (SEQ ID NO: 9)
5'-GACTAGGAATTCTTAGTTCTTTTGACGAAAGTGCTCAGCACGTCGAAG-3', (SEQ ID NO:
10)
[0141] These sequences are employed in RT-PCR reactions to generate
expression cassettes suitable for cloning into pMON45337 cut with
the restriction enzymes Nde1 and EcoR1. RT-PCR reactions are
performed with total RNAs extracted from frozen crown samples using
the RNeasy Plant Mini Kit (cat. #74904, Qiagen Inc., Valencia,
Calif.) per manufacturer's instructions. Oligo.dT-primed
first-strand cDNAs are prepared from 5 .mu.g samples of total RNA
using the Superscript Pre-Amplification System (cat. #18089-011,
Life Technologies, Rockville, Md.) per manufacturer's instructions.
Two .mu.l of first-strand cDNA are then used to generate E. indica
EPSP synthase expression cassettes via polymerase chain reaction.
The oligonucleotides are added in 50 .mu.l RT-PCR reactions at a
final concentration of 0.4 .mu.M. PCR amplifications are then
performed using the Expand High Fidelity PCR System (cat. #1 732
641, Roche Molecular Biochemicals, Indianapolis, Ind.) per
manufacturer's instructions, using a thermal profile of 94.degree.
C. for 30 seconds, then 57.degree. C. for 2 minutes, followed by
75.degree. C. for 3 minutes, for a total of 35 cycles. The
resulting PCR products are digested with Nde I and EcoRI, then
ligated into pMON45337, resulting in the E. coli expression vectors
pMON45364 (FIG. 5) and pMON45365 (FIG. 6), which contain the mature
protein coding region for E. indica EPSP synthase isolated from the
resistant and sensitive biotype, respectively. Expression of the
two enzymes in E. coli will thus be directed by the Lac operon and
trpA gene genetic elements described above for pMON45337. The
accuracy of the cloned sequences are confirmed by DNA sequence
analysis (ABI Prism.TM. 377, Perkin Elmer, Foster, Calif.).
pMON45337, pMON45364, and pMON45365 are all transformed into the E.
coli strain SR481, an aroA-strain lacking endogenous EPSP synthase
activity (Padgette et al., Arch. Biochem. Biophys. 258:564-573
(1987).
[0142] To directly compare the glyphosate tolerance of E. coli
aroA-cells expressing the EPSP synthase gene isolated from the
glyphosate sensitive E. indica biotype with cells expressing the
EPSP synthase gene isolated from the glyphosate tolerant biotype,
growth rates are compared for the two cell lines in the presence of
increasing concentrations of glyphosate (FIG. 3). Growth rates are
also monitored for E. coli SR481 cells transformed with pMON45337
(empty vector) in the absence of glyphosate as a negative control.
Fresh overnight cultures of E. coli SR481 cells transformed with
pMON45337, pMON45364 (Ei.EPSPS:glypR), and pMON45365
(Ei.EPSPS:glypS) are grown in Terrific Broth (Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press
(1989), supplemented with 1.0 mM IPTG, 50 .mu.g/ml ampicillin, and
100 .mu.g/ml each of L-phenylalanine, L-tyrosine, and L-tryptophan.
O.D..sub.595 measurements are taken on all of the overnight
cultures to confirm similar cell densities. For E. coli SR48
1-pMON45364 and SR481-pMON45365 cells, 14 ml culture tubes (cat.#
60818-725, VWR Scientific, West Chester, Pa.) each containing 3.0
ml of minimal M9 media (Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press (1989) supplemented
with 50 .mu.g/ml ampicillin, 1.0 mM IPTG, and either 0.0, 0.5, 1.5,
or 5.0 mM glyphosate (N-phosphonomethyl glycine or a salt thereof)
are inoculated with 100 .mu.l of undiluted overnight culture per
tube. Each experimental condition is performed in triplicate to
confirm the reproducibility of the experiment. Where t=0, 12, 24,
28, 32, and 48 hours past the onset of growth in minimal media, 100
.mu.l aliquots from each tube are removed and O.D..sub.595
measurements are taken immediately. A typical result from these
analyses is shown in FIG. 3, where an approximately three-fold
increase in tolerance to glyphosate is observed due to the
expression of the EPSP synthase enzyme from the glyphosate tolerant
E. indica biotype.
EXAMPLE 3
[0143] Kinetic characterization of the E. indica
glyphosate-resistant EPSP synthase activity in plant and bacterial
extracts. Kinetic characterization of the glyphosate-resistant E.
indica enzyme are performed using both partially purified plant
extracts as well as bacterial extracts prepared from cells
expressing the cloned sequence on a suitable vector such as
pMON45365. Parameters that describe the enzyme's resistance to
glyphosate-mediated inhibition and the affinity for the substrate
phosphoenolpyruvate (PEP) are of particular interest, given that
glyphosate is a competitive inhibitor of EPSP synthase with respect
to PEP (Boocock, M. et al., FEBS Letts. 154:127-133 (1983).
[0144] Preparation of extracts and radiometric EPSP synthase assays
are performed using methods adapted from published procedures
(Padgette et al., J. Biol. Chem. 266:22364-22369 (1991). Crown
regions are dissected from whole plants, pulverized under liquid
nitrogen with a mortar and pestle, then stored at -80.degree. C.
prior to extraction. Homogenates are prepared from 0.5 g tissue per
sample in 25 ml extraction buffer (100 mM TrisCl, 10% glycerol, 1
mM EDTA, 1 mM benzamidine, 1 mM dithiothreitol, 1 mM
4-(2-aminoethyl)-benzenesulfonyl floride HCl, 0.1 mM leupeptin, pH
7.4) at 4.degree. C. using a model PT3000 Polytron homogenizer
(Brinkman Instuments Inc., Westbury, N.Y.). Debris is removed by
0.2 .mu.m filtration, then the resulting supernatant is
concentrated and desalted using an Ultrafree-15 centrifugal
filtration unit (cat. #UFV2-BGC-10, Millipore Corp., Bedford,
Mass.). Final sample volumes are approximately 0.5 ml. Protein
concentrations are determined spectrophotometrically using the
Bio-Rad protein assay reagent (cat. #500-0006, Bio-Rad
Laboratories, Hercules, Calif.). EPSPS specific activities are
determined by assaying 10 .mu.l extract at 25.degree. C. for 5-15
min. (50 .mu.l reactions include 50 mM HEPES, pH 7.0, 5 mM
potassium fluoride, 1 mM shikimate-3-phosphate, 0.5 mM
[1-.sup.14C]-phosphoenolpyruvate (29.0 mCi/mmol cyclohexylammonium
salt; #CFQ10004, Amersham Life Science, Inc., Arlington Heights,
Ill.), and 0.1 mM ammonium molybdate). Reactions are quenched with
the addition of 50 .mu.l 9:1 ethanol: 0.1 M acetic acid. Thirty
.mu. l of quenched reaction is then injected onto a Synchropak
AX100 anion exchange column (cat. #942804, P.J. Cobert Associates,
Inc., St. Louis, Mo.) equilibrated with 0.235 M potassium phosphate
buffer, pH 6.5, and eluted isocratically with the same buffer. A
model D525 radioactive flow detector (Packard Instrument Co.,
Downer's Grove, Ill.) is used to determine production of
[.sup.14C]-EPSP in the reaction.
[0145] For determination of I.sub.50 (glyphosate) values, the
assays as described, are performed in the presence of increasing
concentrations of glyphosate and the resulting activities analyzed
and plotted using GraFit version 3.0 software (Erithacus Software
Ltd., Staines, U.K.). FIG. 4 shows data generated for a typical
glyphosate inhibition study, comparing the EPSP synthase activities
detectable in extracts prepared from the glyphosate sensitive and
tolerant E. indica biotypes. These data demonstrate the difference
in sensitivity to glyphosate for the activities present in the two
biotypes, with the sensitive biotype EPSPS activity having an
I.sub.50 (glyphosate) of approximately 3.0 .mu.M and the tolerant
biotype EPSPS approximately 16 .mu.M.
[0146] Activities of the enzymes extracted from several different
tolerant and sensitive E. indica individuals are compared in the
presence and absence of 1.6 .mu.M glyphosate (Table 1), showing
similar sensitivity to glyphosate and very low plant-to-plant
variation exhibited among individuals from the same biotype.
5TABLE 1 Percent-maximal EPSPS activity in extracts from different
E. indica individuals assayed in the presence 1.6 uM glyphosate
EPSPS Activity, biotype-individual (% maximal @ 1.6 uM glyphosate)
Sensitive - #1 51.6 Sensitive - #2 55.6 Sensitive - #3 57.4
Tolerant - #1 76.3 Tolerant - #2 86.0 Tolerant - #3 82.4
[0147] Similar analyses are performed using extracts prepared from
E. coli strain SR481 cells expressing the cloned E. indica
sensitive and resistant EPSPS enzymes from the expression vectors
pMON45365 and pMON45364, respectively. Fresh bacterial overnight
cultures are grown at 37.degree. C. in Terrific Broth, (Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press (1989), supplemented with 50 .mu.g/ml ampicillin and 100
.mu.g/ml each of L-phenylalanine, L-tyrosine, and L-tryptophan.
Overnight cultures are used to inoculate large-scale cultures
containing the same media at a 1:100 dilution, then grown to an
O.D..sub.595 of 0.6 at 37.degree. C. with vigorous shaking. The
culture is inducted by the addition of IPTG to 1.0 mM final
concentration then incubation is continued for an additional 4
hours. Cells are then pelleted by centrifugation at 10,000.times.g
for 5 minutes, then washed twice in ice-cold 0.9% NaCl. Excess wash
buffer is removed by aspiration, then pellets are flash-frozen in
liquid nitrogen and stored at -80.degree. C. prior to use.
Bacterial extracts are prepared using the same extraction buffer
used for plant extracts, with 3 ml buffer added per gram of
pelleted cells. Cells are lysed using a French press (model #
J5-598A, American Instrument Co., Silver Springs, Md.), then
extracts are centrifuged at 14,000.times.g for 10 minutes to remove
cell debris. The supernatants are then desalted using a PD-10
column (cat.# 17-0851-01, Amersham Pharmacia Biotech Inc.,
Piscataway, N.J.). EPSP synthase assays are performed as described
above for plant-derived extracts. Table 2 illustrates a typical
result for bacterial extracts expressing the sensitive and
resistant E. indica EPSP synthase enzymes assayed in the presence
and absence of 1.6 .mu.M glyphosate. These data indicate that
similar inhibition kinetics are obtained when these results are
compared to the respective activities detected in plant
extracts.
6TABLE 2 EPSPS activity inaroA - bacterial extracts transformed
with pMON45364 or pMON45365 assayed in the presence and absence of
glyphosate activity % maximal activity sample (nMol/min/mg protein)
(1.6 uM glyphosate) SR481(pMON45364) -glyphosate 34.3 +glyphosate
24.9 72.6 SR481(pMON45365) -glyphosate 27.1 +glyphosate 14.4
53.1
EXAMPLE 4
[0148] The use of the glyphosate resistant EPSP synthase gene from
E. indica (Ei.EPSPS:glypR) to develop glyphosate tolerant crop
plants involves the construction of plant transformation vectors
that include the appropriate combination of genetic elements
necessary to direct adequate expression levels in target tissues.
For monocotyledonous crop plants, a monocot vector that utilizes a
plant expression cassette that contains a promoter (P) and first
intron (I) from the rice (Os) actin gene (P-Os.Act1/I-Os.Act1 (U.S.
Pat. No. 5,641,876), plastid transit peptide sequence (TS) from the
Arabidopsis thaliana (At) EPSP synthase gene (TS-At.EPSPS:CTP)
(Klee et al. Mol. Gen. Genet. 210:437-442), a E. indica glyphosate
resistant EPSPS coding sequence, and the
polyadenylation/termination (T) region from the Agrobacterium
tumefaciens nopaline synthase gene (T-AGRTU.nos), would be an
appropriate choice. This expression cassette may be combined with a
second transgene expression cassette by plant breeding, plant
transformation, or by joining in a DNA construct that comprises a
plant DNA virus promoter, for example, the cauliflower mosaic virus
(CaMV) 35S promoter containing a tandem duplication of the enhancer
region, operably connected to a Zea mays Hsp70 intron, operably
connected to a nucleic acid sequence encoding an Arabidopsis
thaliana EPSPS chloroplast transit peptide sequence, operably
connected to a E. indica glyphosate resistant EPSPS coding
sequence, operably connected to a nopaline synthase transcriptional
terminator. Other combinations of genetic elements are known and
those skilled in the art of plant molecular biology can easily
construct plant expression vectors that will express the E. indica
EPSPS glyphosate resistant enzyme at sufficient levels to confer
glyphosate the transformed plant.
[0149] For specific dicotyledonous species, a plant expression
vector that utilizes the promoter and 5' untranslated region
(including intron I) of the plant elongation factor 1.alpha. gene
(Elf.alpha.-A1) as described in U.S. Pat. No. 5,177,011 or more
specifically, dicot vector of the present invention which utilizes
the Arabidopsis thaliana Elf.alpha.-A1 promoter and intron sequence
(P-At.Elf1a/I-At.Elf1a), (Axelos et al., Mol. Gen. Genet. 219:
106-112 (1989); Genbank accession #U63815), the chloroplast transit
peptide from the Arabidopsis thaliana EPSP synthase gene
(TS-At.EPSPS:CTP2), and the polyadenylation/3' termination region
from the Pisum sativum ribulose-1,5-bisphosphate carboxylase gene
(T-Ps.RbcS:E9). This expression cassette may be combined with a
second transgene expression cassette by plant breeding, plant
transformation, or by joining in a DNA construct that comprises a
plant DNA virus promoter, for example, the Figwort mosaic virus
(FMV) 34S promoter, operably connected to a nucleic acid sequence
encoding an Arabidopsis thaliana EPSPS chloroplast transit peptide
sequence, operably connected to a E. indica glyphosate resistant
EPSPS coding sequence, operably connected to a nopaline synthase
transcriptional terminator. Other combinations of genetic elements
are known and those skilled in the art of plant molecular biology
can easily construct plant expression vectors that will express the
E. indica EPSPS glyphosate resistant enzyme at sufficient levels to
confer glyphosate tolerance to the transformed plant.
[0150] To construct monocotyledonous and dicotyledonous plant
expression vectors carrying the DNA encoding the EPSP synthase
mature protein coding sequence isolated from the glyphosate
tolerant E. indica biotype (SEQ ID NO. 6, FIG. 1), the
oligonucleotide primers of SEQ ID NO: 11 and SEQ ID NO: 12 are
employed in PCR reactions to generate an expression cassette
suitable for direct cloning into a monocot vector and a dicot
vector.
7 5'-GCAATTCGCATGCCGGGCGCGGAGGAGGTGGTGCT-3' (SEQ ID NO: 11)
5'-GACTAGGAATTCTTAGTTCTTTTGACGAAAGTGCTCAGCACGTCGAAG-3' (SEQ ID NO:
12)
[0151] The PCR reactions are performed using 300-500 ng of
pMON45364 plasmid DNA as template to amplify the E. indica EPSP
synthase mature protein coding region, flanked by Sph1 and EcoR1
restriction cleavage sites. The oligonucleotides are added in 50
.mu.l PCR reactions at a final concentration of 0.4 .mu.M. PCR
amplifications are then performed using the Expand High Fidelity
PCR System (cat. #1 732 641, Roche Molecular Biochemicals,
Indianapolis, Ind.) per manufacturer's instructions, using a
thermal profile of 94.degree. C. for 30 seconds, then 57.degree. C.
for 2 minutes, followed by 75.degree. C. for 3 minutes, for a total
of 20-35 cycles. The resulting PCR products are digested with Sph1
and EcoR1, then ligated into pMON45366 and pMON45368 resulting in
the plant expression vectors pMON45367 (FIG. 7) and pMON45369 (FIG.
8), respectively. The accuracy of the cloned sequences are
confirmed by DNA sequence analysis (ABI Prism.TM. 377, Perkin
Elmer, Foster City, Calif.).
EXAMPLE 5
[0152] Transgenic corn can be produced by particle bombardment
transformation methods as described in U.S. Pat. No. 5,424,412. The
plant expression vector (pMON45367) contains the glyphosate
resistant E. indica EPSPS mature protein coding sequence in an
expression cassette suitable for expression in monocot plants. The
pMON45367 plasmid DNA is digested with Not1 and Pme1 restriction
endonucleases to complete digestion. The 3.3 kb expression cassette
is agarose gel purified, then bombarded into embryogenic corn
tissue culture cells using a Biolistic.RTM. (Dupont, Wilmington,
Del.) particle gun with purified isolated DNA fragment. Transformed
cells are selected on glyphosate (N-phosphonomethyl glycine and its
salts) containing media and whole plants are regenerated then grown
under greenhouse conditions. Fertile seed is collected, planted and
the glyphosate tolerant phenotype is back crossed into commercially
acceptable corn germplasm by methods known in the art of corn
breeding (Sprague et al., Corn and Corn Improvement .sub.3rd
Edition, Am. Soc. Agron. Publ (1988).
[0153] Transgenic corn plants can be produced by an Agrobacterium
mediated transformation method. A disarmed Agrobacterium strain C58
(ABI) harboring a binary vector (pMON45367) is used for all the
experiments. The pMON45367 is transferred into Agrobacterium by a
triparental mating method (Ditta et al., Proc. Natl. Acad. Sci.
77:7347-7351). Liquid cultures of Agrobacterium are initiated from
glycerol stocks or from a freshly streaked plate and grown
overnight at 26.degree. C.-28.degree. C. with shaking
(approximately 150 rpm) to mid-log growth phase in liquid LB
medium, pH 7.0 containing 50 mg/l kanamycin, 50 mg/l streptomycin
and spectinomycin and 25 mg/l chioramphenicol with 200 .mu.M
acetosyringone (AS). The Agrobacterium cells are resuspended in the
inoculation medium (liquid CM4C) and the density is adjusted to
OD.sub.660 of 1. Freshly isolated Type II immature HiII.times.LH198
and HiII corn embryos are inoculated with Agrobacterium containing
pMON45367 and co-cultured 2-3 days in the dark at 23 .degree. C.
The embryos are then transferred to delay media (N6
1-100-12/micro/Carb 500/20 .mu.M AgNO3) and incubated at 28
.degree. C. for 4 to 5 days. All subsequent cultures are kept at
this temperature. Coleoptiles are removed one week after
inoculation. The embryos are transferred to the first selection
medium (N61-0-12/Carb 500/0.5 mM glyphosate). Two weeks later,
surviving tissue are transferred to the second selection medium
(N61-0-12/Carb 500/1.0 mM glyphosate). Subculture surviving callus
every 2 weeks until events can be identified. This will take 3
subcultures on 1.0 mM glyphosate. Once events are identified, bulk
up the tissue to regenerate. For regeneration, callus tissues are
transferred to the regeneration medium (MSOD 0.1 .mu.M ABA) and
incubated for two weeks. The regenerating calli are transferred to
a high sucrose medium and incubated for two weeks. The plantlets
are transferred to MSOD media in culture vessel and kept for two
weeks. Then the plants with roots are transferred into soil.
[0154] Three R.sub.0 plants are regenerated for any given
transgenic event. These three plants are expected to be near
isogenic because they are thought to be derived from a single
transgenic plant cell. Thus, one plant is used as a non-sprayed
control and the remaining two plants are treated with glyphosate
(as Roundup.RTM. herbicide). Plants are most effectively treated
with glyphosate at V2-V6 stage. Glyphosate (as Roundup.RTM.
herbicide) is administered through the use of a linear track
sprayer set to deliver a 16, 32 or 64 oz./A rate of glyphosate.
Vegetative tolerance to the glyphosate is visually evaluated a week
after spray based on a scale of 0 to 5 (0=No observable/vegetative
effect of glyphosate; 1=Chlorosis observed; 2=Advanced chlorosis,
minor necrosis; 3=Advanced chlorosis, moderate necrosis; 4=Advanced
chlorosis, severe necrosis; 5=No live tissue remaining). The
R.sub.0 plants produced are allowed to self, then R.sub.1 plants
are screened using spray applications of glyphosate and the rating
system as described for the R.sub.0 screen. An increase in
whole-plant tolerance to the herbicide, as compared to
non-transgenic control plants, is used to assess the utility of the
E. indica EPSP synthase enzyme for the generation of glyphosate
tolerance in planta.
EXAMPLE 6
[0155] Immature embryos of wheat (Triticum aestivum L) cultivar
Bobwhite are isolated from the immature caryopsis 13-15 days after
pollination, and cultured on CM4C (Table 3) for 3-4 days. The
embryos showing active cell division, but no apparent callus
formation are selected for Agrobacterium infection.
8TABLE 3 Supplemental Components in Basal Media.sup.1 Components
CM4 CM4C MMS.2C MMS0 2,4-D (mg/l) 0.5 0.5 0.2 -- Pichloram
(mg/l).sup.2 2.2 2.2 Maltose (g/l) 40.0 40.0 40.0 40.0 Glutamine
(g/l) 0.5 0.5 Magnesium Chloride (g/l) 0.75 0.7 Casein Hydrolysate
(g/l) 0.1 0.1 MES (g/l) 1.95 1.95 1.95 Ascorbic Acid (mg/l).sup.2
100.0 100.0 100.0 Gelling Agent (g/l).sup.3 2(P) 2(P) 2(G) 2(G)
.sup.1All media contain basal salts (MS basal salts) and vitamins
(MS vitamins) from Murashige and Skoog (1962). The pH in each
medium is adjusted to 5.8. .sup.2Filter-sterilized and added to the
medium after autoclaving. .sup.3Phytagel .TM. (P) or Gelrite .RTM.
(G).
[0156] A disarmed Agrobacterium strain C58 (ABI) harboring a binary
vector of interest (pMON45367) is used for all the experiments. The
pMON45367 is transferred into Agrobacterium by a triparental mating
method (Ditta et al., Proc. Natl. Acad. Sci. 77:7347-7351). Liquid
cultures of Agrobacterium are initiated from glycerol stocks or
from a freshly streaked plate and grown overnight at 26.degree.
C.-28.degree. C. with shaking (approximately 150 rpm) to mid-log
phase (OD.sub.660=1-1.5) in liquid LB medium, pH 7.0 containing 50
mg/l kanamycin, 50 mg/l streptomycin and spectinomycin and 25 mg/l
chloramphenicol with 200 .mu.M acetosyringone (AS). The
Agrobacterium cells are resuspended in the inoculation medium
(liquid CM4C) and the density is adjusted to OD.sub.660 of 1. The
immature embryos cultured in CM4C medium are transferred into
sterile petri plates (16.times.20 mm) or wells of a 6-well cell
culture plate (Costar Corporation, Cambridge, Mass.) containing 10
ml of inoculation medium per petri plate or 5 ml per cell culture
cluster plate. An equal amount of the Agrobacterium cell suspension
is added such that the final concentration of Agrobacterium cells
is an OD.sub.600 of 0.5. In most experiments, pluronic F68 is added
to the inoculation mixture at a final concentration of 0.01%. The
ratio between the Agrobacterium and immature embryos is about 10
ml: 20-200 IEs. The inoculation is allowed to proceed at 23.degree.
C.-26.degree. C. from 5-60 minutes.
[0157] After the inoculation period, the remaining Agrobacterium
cells are removed from the explants by using vacuum aspiration
equipment. A piece of sterile Whatman No. 1 filter paper (to fit
the size of the petri plate) is placed in each of 60.times.15 or
60.times.20 mm petri dishes. Two hundred .mu.l of sterile water is
placed in the middle of the filter paper. After 2-3 minutes, the
inoculated immature embryos are placed in the plates. Usually,
20-50 explants are grouped as one stack (about 1 cm in size and
60-80 mg/stack), with 4-5 stacks on each plate. The plates are
immediately covered with Parafilm.RTM. and then co-cultivated in
the dark at 24.degree. C.-26.degree. C. for 2-3 days.
[0158] The co-cultivated PCIEs are transferred CM4C+500 mg/l
carbenicillin medium (delay medium) at dark. After 7 days on the
delay medium, the immature embryos are transferred to CM4C
supplemented with 2 mM glyphosate and 500 mg/l carbenicillin for
selection for one week. Then calli are transferred to MMS0.2C+0.1
mM glyphosate +250 mg/l carbenicillin medium for 2 weeks under
light for further selection. Embryogenic calli are transferred to a
second regeneration medium MMS0C with lower glyphosate
concentration (0.02 mM) and 500 mg/L carbenicillin for plant
regeneration. Those embryogenic calli are transferred onto fresh
medium every two weeks. Regenerated plantlets are transferred to
Sundae cups (Sweetheart Cup Company, Chicago, Ill.) containing the
second regeneration medium for further growth and selection. When
roots are well established from transgenic plants the plants are
transferred to soil for further evaluation.
EXAMPLE 7
[0159] Novel glyphosate-resistant EPSP synthases can be designed
based on the E. indica glyphosate resistant EPSPS. The amino acid
sequence deduced from the cDNA sequence shows that two amino acid
substitutions distinguish the mature EPSP protein sequence derived
from the glyphosate-tolerant E. indica biotype (top row, FIG. 9)
from that of the glyphosate sensitive E. indica biotype EPSPS
protein sequence (bottom row, FIG. 9). The substitution of a serine
for a proline at position 107 of the E. indica EPSPS amino acid
sequence and in the corresponding amino acid position in both
higher plant and bacterial EPSP synthase enzymes is known to result
in the enzyme having resistance to glyphosate (Padgette et al., J.
Biol. Chem. 266:22364-22369 (1991); U.S. Pat. No. 4,535,060). All
catalytic domain single amino acid substitution EPSP synthase
variants characterized to date that exhibit increased tolerance to
glyphosate have a higher K; for glyphosate, but also have an
increase in the apparent K.sub.m for PEP and reduced V.sub.max,
thereby lowering the catalytic efficiency (V.sub.max/K.sub.m) of
the enzyme (Kishore et al., Annu. Rev. Biochem. 57:627-663 (1988);
glyphosate (Padgette et al., J. Biol. Chem. 266:22364-22369 (1991).
In contrast, the E. indica glyphosate resistant EPSP synthase (E.
indica glypR) exhibits a high affinity for PEP, while retaining
significant catalytic efficiency in the presence of glyphosate
(TABLE 4). The engineered petunia (Petunia hybrida) and corn (Zea
mays) glyphosate resistant (glypR) variants that in studies by
others (U.S. Pat. No. 5,866,774; U.S. Pat. No. 6,040,497) have
shown to confer a high level of glyphosate tolerance in transgenic
plants were assayed for affinity for PEP and inhibition by
glyphosate. All of the naturally occurring wild type (wt) Z. mays
glyphosate sensitive (Z. mays glypS), wild type P. hybrida
glyphosate sensitive (P. hybrida glypS) and the E. indica glypS and
E. indica glypR wild type EPSPS enzymes have very similar K.sub.m
values for PEP. The single amino acid substitutions engineered into
the catalytic domain of P hybrida EPSPS enzyme drastically
increases the K.sub.m for PEP. The single amino acid substitution
found in this domain in the naturally occurring variant of E.
indica glypR was found to not have a major effect on the K.sub.m
indicating that this enzyme will continue to function well in the
plant chloroplast. It required a double mutation in the Z. mays
EPSPS enzyme to achieve a low K.sub.m for PEP.
9TABLE 4 Comparison of the apparent K.sub.m for PEP and apparent
K.sub.i for glyphosate of the E. indica glyphosate resistant EPSPS
with other known plant EPSPS modified for glyphosate resistance.
EPSPS enzyme K.sub.m PEP (.mu.M) K.sub.i Glyphosate (.mu.M) E.
indica glypS 5 0.05 E. indica glypR (Pro-Ser) 7 1 Z. mays glypS
(wt) 5 0.2 Z. mays glypR (Thr-Ile, Pro-Ser) 5 60 P. hybrida glypS
(wt) 5 0.4 P. hybrida glypR (Pro-Ser) 44 3 P. hybrida glypR
(Gly-Ala) 200 2000 P. hybrida glypR (Gly-Ala, Pro- 340 8500
Ser)
[0160] K.sub.m(PEP) determinations for the different enzymes are
performed at saturating shikimate-3-phosphate (S3P) concentrations,
which is determined according to standard methods (Fersht, Enzyme
Structure and Mechanism, W.H. Freeman and Co., Ltd., San Francisco,
Calif., 1977). A series of PEP concentrations are tested, such that
the final range of concentrations spans one order of magnitude
above and below the experimentally determined K.sub.m. K.sub.i
(glyphosate), with respect to PEP, is determined in a similar
manner, at saturating S3P concentrations, except that velocity vs.
[PEP] is determined for a range of glyphosate concentrations (Orsi
in "Methods in Enzymology" (Purich, ed.), vol. 63, pg. 159-183,
1979). Calculations, graphical representation, and statistical
analysis of enzyme kinetic data are performed using GraFit version
3.0 software (Erithacus Software Ltd., Staines, U.K.).
[0161] It is anticipated by this study that transgenic plants
resistant to glyphosate are made by transformation with an E.
indica glyphosate resistant EPSPS gene construct that can include
additional modification of the naturally occurring amino acid
sequence. These changes are be made by site-directed mutagenesis of
the codons of DNA sequence to incorporate other known amino acid
substitutions in glyphosate resistant plant EPSPSs, such as the
threonine to isoleucine substitution at 103 (U.S. Pat. No.
6,040,497), and glycine to alanine substitution at 102 (U.S. Pat.
No. 5,188,642) in the catalytic domain of the E. indica EPSPS amino
acid seqeunce. Furthermore, it is anticipated that the catalytic
domain of the E. indica EPSPS, as well as other plant EPSP
Synthases, can be modified to the amino acid sequence of the
catalytic domain of the Agrobacterium strain CP4 glyphosate
resistant EPSPS (U.S. Pat. No. 5,633,435) by the same methods. This
modification will result in the plant derived EPSPS possessing
similar PEP binding and glyphosate resistance as the CP4 glyphosate
resistant EPSPS that has been used in cotton, corn, canola,
soybeans, potato, wheat, sugarbeet and other agronomically
important crop plant to impart plant tolerance to glyphosate.
Modification of plant EPSP Synthases to the CP4 EPSPS catalytic
domain sequence may comprise the deletion of an amino acid and the
substitution of other amino acids. In particular the deletion of
the amino acid at 107 of the E. indica EPSPS sequence (FIG. 2) or
the same relative amino acid position in other plant EPSP Synthases
that can in addition to the deletion, include substitutions of an
alanine for a glycine at 102, glycine for alanine at 104, cysteine
for methionine at 105, methionine for alanine at 110, or glycine
for alanine at 111. Previous random and site-directed mutations in
the conserved region (catalytic domain) of bacterial and plant
EPSPSs have shown that modifications that increase the K.sub.i for
glyphosate while keeping the K.sub.m for PEP low are important for
an enzyme that is useful for genetically modifying plants for
glyphosate tolerance (U.S. Pat. No. 5,866,775).
EXAMPLE 8
[0162] E. indica EPSPS regulatory sequences can be isolated by any
number of methods known to those of skill in the art for genomic
library preparation. For genomic libraries of the present
invention, E. indica genomic DNA is isolated by a CsCl purification
protocol according to Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates); or by a CTAB purification method
(Rogers et al., Plant Mol. Biol., 5:69, 1988). Reagents are
available commercially (see, for example Sigma Chemical Co., St.
Louis, Mo.). The genomic DNA libraries are prepared according to
manufacturer instructions (Genome Walker.TM., CloneTech
Laboratories, Inc, Palo Alto, Calif.). In separate reactions,
genomic DNA is subjected to restriction enzyme digestion overnight
at 37.degree. C. with the following blunt-end endonucleases: EcoRV,
Sca1, Dra1, PvuII, or Stu1 (CloneTech Laboratories, Inc. Palo Alto,
Calif.). The reaction mixtures are extracted with
phenol:chloroform, ethanol precipitated, and resuspended in
Tris-EDTA buffer (10 mM Tris-.HCI, pH 8.0, 1 mM EDTA). The purified
blunt-ended genomic DNA fragments are then ligated to the Genome
Walker.TM. adapters and ligation of the resulting DNA fragments to
adapters were done according to the manufacturer's protocol. After
ligation, each reaction is heated treated (70.degree. C. for 5 min)
to terminate the reaction and then diluted 10-fold in Tris-EDTA
buffer. One .mu.l of each respective ligation is then amplified in
a 50 .mu.l reaction according to manufacturer's recommended
protocol using an adaptor-specific oligonucleotide (supplied by
manufacturer) and an E. indica EPSP synthase gene-specific
oligonucleotide, such as SEQ ID NO 4. One .mu.l of each primary
reaction is diluted 50-fold and 1 .mu.l of this dilution is then
amplified in a secondary amplification using a "nested"
adaptor-specific oligonucleotide (supplied by manufacturer) and a
"nested" gene-specific oligonucleotide such as SEQ ID NO 5. PCR
products, representing 5' regions of the E. indica EPSP synthase
gene are then purified by agarose gel electrophoresis using a
QIAquick Gel Extraction Kit (cat. #28704, Qiagen Inc., Valencia,
Calif.) then directly cloned into the pCR2.1-TOPO vector (cat.
#K4500-40, Invitrogen, Carlsbad, Calif.). The identity of the
cloned PCR products is confirmed by DNA sequence analysis (ABI
Prism.TM. 377, Perkin Elmer, Foster City, Calif.). The same E.
indica Genome Walker.TM. libraries and methods that are used to
isolate the E. indica EPSP synthase 5' region can be used to
isolate the 3' region of E. indica EPSP synthase gene, by
substituting gene-specific primers (first round SEQ ID NO: 13 and
second round SEQ ID NO: 14) that anneal to the 3' end of the gene.
Amplification products are cloned and verified as for the 5' end of
the E. indica EPSP synthase gene.
10 5'-TGCAATCCGGACTGAGCTAACAAAGC-3' (SEQ ID NO: 13)
5'-ACTGCATTATCACACCGCCCGAGAAG-3' (SEQ ID NO: 14)
[0163] The translation initiation codon is determined for the E.
indica EPSP synthase gene by inspection, anticipating an initiation
codon approximately 63 codons upstream of the start of the mature
protein codon region, based on comparison to the maize EPSP
synthase gene. Primers are then designed to amplify approximately
2.5 kb of the 5' region beginning at the initiation codon. These
primers incorporate restriction sites for cloning into expression
vectors, for example, placing a EcoR1 site in the 5' end of
promoter region the E. indica EPSP synthase gene and an Nco1 site
incorporating the translation start. Such primers are added in a 50
.mu.l RT-PCR reaction at a final concentration of 25 .mu.M with 50
ng of E. indica genomic DNA. PCR amplifications are then performed
using the Expand High Fidelity PCR System (cat. #1 732 641, Roche
Molecular Biochemicals, Indianapolis, Ind.) per manufacturer's
instructions. A thermal profile of 94.degree. C. for 30 seconds,
followed by 60.degree. C. for 30seconds, then 72.degree. C. for 3
minutes is used for thirty cycles. This is followed by a cycle of
72.degree. C. for 3 minutes. The gel purified amplification product
is then digested with Pst1 and Nco1.
[0164] The 3' end of the E. indica EPSP synthase gene is amplified
using two gene specific primers (SEQ ID NO: 15 and SEQ ID NO: 16)
which incorporate a BamH1 site immediately downstream of the
translation stop codon and a Pst1 approximately 650 bases
downstream of the translation stop codon. The product is amplified
as for the 5' end of the E. indica EPSP synthase gene except an
extension time of 1 minute is used. The gel purified product is
digested with BamH1 and Pst1.
11 (SEQ ID NO: 15) 5'-CTAAGGATCCTCTGTGCCTGCCTCATGAAGAGAGT- T-3'
(SEQ ID NO: 16) 5'-TGATCTGCAGGCAAGTGTCTTA- CCCTTACCCTTCTG-3'
[0165] The 5' (EcoR1/Nco1 fragment) regulatory and 3' (BamH1/Pst1
fragment) regulatory regions of the E. indica EPSP synthase gene
can be ligated to a compatibly digested vector and a coding region
to generate a transgene capable of expressing a transcript under
control of E. indica EPSP synthase gene regulatory elements. An
example of such is a coding region would be the A. fumefaciens
strain CP4 EPSP synthase gene (U.S. Pat. No. 5,633,435) expressed
under the control of the E. indica regulatory sequences.
[0166] The basal expression of the E. indica EPSP synthase gene
promoter may be modified to enhance its expression. Methods known
to those of skill in the art can be used to insert enhancing
elements (for example, subdomains of the CaMV 35S promoter, Benfey
et. al, 1990 EMBO J. 9: 1677-1684) into the E. indica EPSP synthase
gene 5' sequence to generate a promoter which encompasses the
temporal and spatial expression of the E. indica EPSP synthase gene
but have quantitatively higher levels of expression. Similarly,
tissue specific modifications of the E. indica EPSP synthase 5'
region expression can be accomplished with elements determined to
specifically activate or repress gene expression (for example,
pollen specific elements, Eyal et al., 1995 Plant Cell 7:
373-384).
[0167] From the foregoing, it will be seen that this invention is
one well adapted to attain all the end and object herein above set
forth together with advantages that are obvious and that are
inherent to the invention.
[0168] The embodiments described above are provided to better
elucidate the practice of the present invention. Many possible
embodiments may be made of the invention without departing from the
scope thereof, it should be understood that these embodiments are
provided for illustrative purposes only, and are not intended to
limit the scope of the invention.
[0169] It will be understood that certain features and
subcombinations are of utility and may be employed without
reference to other features and subcombinations. This is
contemplated by and is within the scope of the claims. All
publications and published patent documents cited in this
specification are incorporated herein by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
16 1 22 DNA Artificial sequence fully synthetic DNA primer 1 tnw
sng tng arg cng aya arg t 22 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 2 23
DNA Artificial sequence fully synthetic DNA primer 2 gcc atn gcc
atn ckr tgr tcr tc 23 Ala Xaa Ala Xaa Xaa Xaa Xaa 1 5 3 24 DNA
Artificial sequence fully synthetic DNA primer 3 gtg aaa gca gag
cat tct gat agc 24 Val Lys Ala Glu His Ser Asp Ser 1 5 4 29 DNA
Artificial sequence fully synthetic DNA primer 4 ggc tgc tgt caa
tgt cgc att gca gtt cc 29 Gly Cys Cys Gln Cys Arg Ile Ala Val 1 5 5
32 DNA Artificial sequence fully synthetic DNA primer 5 ctc ttt cgc
atc ctt ctc aac tgg gaa ctt gc 32 Leu Phe Arg Ile Leu Leu Asn Trp
Glu Leu 1 5 10 6 1338 DNA Eleusine indica 6 gcgggcgcgg aggaggtggt
gctgcagccc atcaaggaga tctccggcgt cgtgaagctg 60 ccggggtcca
agtcgctctc caaccggatc ctcctgctct ccgccctcgc cgagggaaca 120
actgtggtgg ataacctttt aaacagtgag gacgtccact acatgctcgg ggccctgaaa
180 accctcggac tctctgtgga agcggacaaa gctgccaaaa gagcggtagt
tgttggctgt 240 ggtggcaagt tcccagttga gaaggatgcg aaagaggagg
tgcagctctt cttggggaat 300 gctggaactg caatgcgatc attgacagca
gccgtaactg ctgctggagg aaatgcaact 360 tatgtgcttg atggagtgcc
aagaatgcgg gagagaccca ttggcgactt ggttgtcgga 420 ttgaaacagc
ttggtgcgga tgttgattgt ttccttggca ctgactgccc acctgttcgt 480
gtcaagggaa tcggagggct acctggtggc aaggttaagt tatctggttc catcagcagt
540 cagtacttga gtgccttgct gatggctgct cctttagctc ttggggatgt
ggagattgaa 600 atcattgata aactgatctc catcccttat gttgaaatga
cattgagatt gatggagcgt 660 tttggcgtga aagcagagca ttctgatagc
tgggacagat tctacatcaa gggaggtcaa 720 aaatacaagt cccctaaaaa
tgcctacgtg gaaggtgatg cctcaagtgc gagctatttc 780 ttggctggtg
ctgcaatcac tggagggact gtgactgttg aaggttgtgg caccaccagt 840
ctgcagggtg atgtgaaatt tgccgaggta ctcgagatga tgggagcgaa ggttacatgg
900 actgaaacta gcgtaactgt taccggtcca caacgtgagc catttgggag
gaaacaccta 960 aaagctattg atgttaacat gaacaaaatg cccgatgtcg
ccatgactct tgccgtggtt 1020 gccctatttg ctgatggccc aactgctatc
agagatgtgg cttcctggag agtaaaggag 1080 accgagagga tggttgcaat
ccggactgag ctaacaaagc tgggagcgtc ggtcgaggaa 1140 ggactggact
actgcattat cacaccgccc gagaagctga acgtaacggc catcgacacc 1200
tacgatgacc acaggatggc catggccttc tccctcgccg cctgcgccga cgtgcctgtg
1260 accatccggg accccggctg cacccgcaag accttcccag actacttcga
cgtgctgagc 1320 actttcgtca agaactaa 1338 7 445 PRT Eleusine indica
7 Ala Gly Ala Glu Glu Val Val Leu Gln Pro Ile Lys Glu Ile Ser Gly 1
5 10 15 Val Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu
Leu 20 25 30 Leu Ser Ala Leu Ala Glu Gly Thr Thr Val Val Asp Asn
Leu Leu Asn 35 40 45 Ser Glu Asp Val His Tyr Met Leu Gly Ala Leu
Lys Thr Leu Gly Leu 50 55 60 Ser Val Glu Ala Asp Lys Ala Ala Lys
Arg Ala Val Val Val Gly Cys 65 70 75 80 Gly Gly Lys Phe Pro Val Glu
Lys Asp Ala Lys Glu Glu Val Gln Leu 85 90 95 Phe Leu Gly Asn Ala
Gly Thr Ala Met Arg Ser Leu Thr Ala Ala Val 100 105 110 Thr Ala Ala
Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly Val Pro Arg 115 120 125 Met
Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu 130 135
140 Gly Ala Asp Val Asp Cys Phe Leu Gly Thr Asp Cys Pro Pro Val Arg
145 150 155 160 Val Lys Gly Ile Gly Gly Leu Pro Gly Gly Lys Val Lys
Leu Ser Gly 165 170 175 Ser Ile Ser Ser Gln Tyr Leu Ser Ala Leu Leu
Met Ala Ala Pro Leu 180 185 190 Ala Leu Gly Asp Val Glu Ile Glu Ile
Ile Asp Lys Leu Ile Ser Ile 195 200 205 Pro Tyr Val Glu Met Thr Leu
Arg Leu Met Glu Arg Phe Gly Val Lys 210 215 220 Ala Glu His Ser Asp
Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln 225 230 235 240 Lys Tyr
Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser 245 250 255
Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr Val Thr 260
265 270 Val Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe
Ala 275 280 285 Glu Val Leu Glu Met Met Gly Ala Lys Val Thr Trp Thr
Glu Thr Ser 290 295 300 Val Thr Val Thr Gly Pro Gln Arg Glu Pro Phe
Gly Arg Lys His Leu 305 310 315 320 Lys Ala Ile Asp Val Asn Met Asn
Lys Met Pro Asp Val Ala Met Thr 325 330 335 Leu Ala Val Val Ala Leu
Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp 340 345 350 Val Ala Ser Trp
Arg Val Lys Glu Thr Glu Arg Met Val Ala Ile Arg 355 360 365 Thr Glu
Leu Thr Lys Leu Gly Ala Ser Val Glu Glu Gly Leu Asp Tyr 370 375 380
Cys Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Ala Ile Asp Thr 385
390 395 400 Tyr Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala
Cys Ala 405 410 415 Asp Val Pro Val Thr Ile Arg Asp Pro Gly Cys Thr
Arg Lys Thr Phe 420 425 430 Pro Asp Tyr Phe Asp Val Leu Ser Thr Phe
Val Lys Asn 435 440 445 8 56 DNA Artificial sequence fully
synthetic DNA leader sequence 8 agatctccta gggcttaatt aattaagcac
tagtcacaca ggaggtaatt catatg 56 9 36 DNA Artificial sequence fully
synthetic DNA sequence 9 gca att cca tat ggc ggg cgc gga gga ggt
ggt gct 36 Ala Ile Pro Tyr Gly Gly Arg Gly Gly Gly Gly Ala 1 5 10
10 48 DNA Artificial sequence fully synthetic DNA sequence 10 gac
tag gaa ttc tta gtt ctt ttg acg aaa gtg ctc agc acg tcg aag 48 Asp
Glu Phe Leu Val Leu Leu Thr Lys Val Leu Ser Thr Ser Lys 1 5 10 15
11 35 DNA Artificial sequence Fully synthetic DNA sequence 11 gca
att cgc atg ccg ggc gcg gag gag gtg gtg ct 35 Ala Ile Arg Met Pro
Gly Ala Glu Glu Val Val 1 5 10 12 48 DNA Artificial sequence Fully
synthetic DNA primer 12 gac tag gaa ttc tta gtt ctt ttg acg aaa gtg
ctc agc acg tcg aag 48 Asp Glu Phe Leu Val Leu Leu Thr Lys Val Leu
Ser Thr Ser Lys 1 5 10 15 13 26 DNA Artificial sequence fully
synthetic DNA sequence 13 tgc aat ccg gac tga gct aac aaa gc 26 Cys
Asn Pro Asp Ala Asn Lys 1 5 14 26 DNA Artificial sequence fully
synthetic DNA sequence 14 act gca tta tca cac cgc ccg aga ag 26 Thr
Ala Leu Ser His Arg Pro Arg 1 5 15 36 DNA Artificial sequence fully
synthetic DNA sequence 15 cta agg atc ctc tgt gcc tgc ctc atg aag
aga gtt 36 Leu Arg Ile Leu Cys Ala Cys Leu Met Lys Arg Val 1 5 10
16 36 DNA Artificial sequence fully synthetic DNA sequence 16 tga
tct gca ggc aag tgt ctt acc ctt acc ctt ctg 36 Ser Ala Gly Lys Cys
Leu Thr Leu Thr Leu Leu 1 5 10
* * * * *