Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF PRODUCING PLANTS WHICH ARE TOLERANT OR RESISTANT TO
HERBICIDES
The present invention relates inter alia, to a method of producing plants
which are
tolerant or resistant to herbicides and in particular to the production of
transgenic plants
which exhibit substantial resistance or substantial tolerance to herbicides
when compared
with non transgenic like plants.
Plants which are substantially "tolerant" to a herbicide when they are
subjected to it provide
a dose/response curve which is shifted to the right when compared with that
provided by
to similarly subjected non tolerant like plants. Such dose/response curves
have "dose" plotted
on the x-axis and "percentage kill", "herbicidal effect" etc. plotted on the y-
axis. Tolerant
plants will require more herbicide than non tolerant like plants in order to
produce a given
herbicidal effect. Plants which are substantially "resistant" to the herbicide
exhibit few, if
any, necrotic, lytic, chlorotic or other lesions when subjected to the
herbicide at
concentrations and rates which are typically employed by the agrochemical
community to
kill weeds in the field. Plants which are resistant to a herbicide are also
tolerant of the
herbicide. The terms "resistant" and "tolerant" are to be construed as
"tolerant and/or
resistant" within the context of the present application.
The herbicides of particular relevance to the present invention are those
which are
2o capable in vitro of inhibiting 4-Hydroxy-phenylpyruvate dioxygenase (HPPD
or 4HPPD)
enzymes. Such herbicides have been disclosed, such as the isoxazoles described
especially in
the French Patent Applications 95 06800 and 95 13570 and especially
isoxaflutole, a
selective maize herbicide, diketonitriles such as those described in European
Applications 0
496 630, 0496 631, in particular 2-cyano-3-cyclopropyl-1-(2-SOZCH3-4-CF3-
phenyl)propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SOZCH3-4-
2,3Cl2phenyl)propane-
1,3-dione, triketones described in European Applications 0 625 505 and 0 625
508, in
particular sulcotrione, mesotrione (BSI-proposed), pyrazolynate and
pyrazoxyfen. Known
genes capable of providing for tolerance to these herbicides are those which
encode HPPD
enzymes.
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According to the present invention there is provided a method of making plants
which are resistant or tolerant to herbicides which - in vitro - inhibit 4-
hydroxyphenylpyruvate dioxygenase (4HPPD) comprising the steps of:
(i) transforming plant material with a polynucleotide comprising a region
encoding a
phytoene desaturase {PDS);
(ii) regenerating the thus transformed material into morphologically normal
plants.
The region comprised by the polynucleotide may have the sequence depicted in
SEQ ID No.
1, or may be a sequence which is complementary to one which when incubated at
a
temperature of between 55 and 60°C in 0.3 strength citrate buffered
saline containing 0.1%
SDS followed by rinsing at the same temperature with 0.3 strength citrate
buffered saline
containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID No.
1.
It is preferred that the phytoene desaturase is of bacterial origin such as
that depicted in SEQ
ID No. 1 and being derived from Erwinia uredovora, and/or in particular is one
which does
not require plastoquinone 9 as a co-factor. The desaturase may, however be of
plant origin,
such as especially of monocotyledonous or dicotyledonous plants, especially of
Arabidopsis
or of Umbelliferae, such as, for example, the carrot (Daucus carotta). It can
be native or
possibly mutated while at the same time fundamentally retaining a property of
herbicidal
tolerance against HPPD inhibitors, such as herbicides of the isoxazoles family
such as the
Balance T"'' Herbicide or triketones. The herbicide resistant plants produced
by the above
2o method may be selected through their resistance to herbicides which in
vitro, inhibit 4HPPD.
In may however, be further preferred that the polynucleotide encoding the
phytoene
desaturase further comprises a selectable marker gene to facilitate the
selection of
regenerated transformats. Suitable selectable marker genes include; resistance
to antibiotics
such as kanamycin, hygromycin and gentamycin; resistance to further herbicides
such as
glyphosate based herbicides; resistance to toxins such as eutypine.
Other forms of selection are also available such as hormone based selection
systems such as
the Multi Auto Transformation (MAT) system of Hiroyrasu Ebinuma et al. 1997.
PNAS Vol.
94 pp2117-2121; visual selection systems which use the known green
flourescence protein, (3
glucoronidase, mannose isomerase, xylose isomerase and 2-DOG.
3o The plant material may be, or may have been, further transformed with a
polynucleotide
comprising a region encoding a protein capable of providing the plant material
with
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resistance or tolerance to herbicides, insects, desiccation and/or fungal,
bacterial or viral
infections, or with a polynucleotide capable of encoding proteins which
provide for
improved quality traits such as increased yield, altered starch quality and/or
increased
nutrient content.
The protein encoding sequences within the polynucleotide are bounded by plant
operable promoters and terminators. Such promoters and terminators, which are
per se not
germane to the invention, are well known to the skilled man and include, for
example, the
CaMV35S, FMV35S, NOS, OCS and E9 (derived from the small subunit of RUBISCO)
promoters and terminators, or the promoter and terminator of a gene of alpha-
tubulin (EP-A
652,286). Preferably, recourse is made to a promoter regulation sequence which
favours the
over-expression of the coding sequence, such as, for example, that comprising
at least one
histone promoter such as described in EP-A-507,698.
According to the invention, it is equally possible to use, in association with
the
promoter regulation sequence, other regulation sequences which are situated
between the
promoter and the coding sequence, such as transcriptional or translational
enhancers such as,
for example, tobacco etch virus (TEV) translation activator described in
International Patent
application, PCT publication number W087/07644 which is incorporated herein by
reference, or of transit peptides, either single, or double, and in this case
possibly separated
by an intermediate sequence, that is to say comprising, in the transcription
direction, a
2o sequence coding for a transit peptide of a plant gene coding for a plastid
localization enzyme,
a part of the sequence of the N-terminal mature part of a plant gene coding
for a plastid
localization enzyme, then a sequence coding for a second transit peptide of a
plant gene
coding for a plastid localization enzyme, formed by a part of the sequence of
the N-terminal
mature part of a plant gene coding for a plastid localization enzyme, such as
described in EP-
A-508,909.
The plant material may have been, or may subsequently be - further transformed
with
a polynucleotide comprising a region encoding a protein capable of providing
the plant with
resistance or tolerance to herbicides, insects, desiccation and/or fungal,
bacterial or viral
infections, or with a polynucleotide capable of encoding proteins which
provide for
3o improved quality traits such as increased yield, altered starch quality
and/or increased
nutrient content.
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The protein capable of providing for herbicide resistance may be selected from
the
group consisting of glyphosate oxido-reductase (GOX), 5-enol-pyruvyl-3-
phosphoshikimate
synthetase (EPSPS), phosphinothricin acetyl transferase (PAT), hydroxyphenyl
pyruvate
dioxygenase (HPPD), glutathione S transferase (GST), cytochrome P450, Acetyl-
COA
carboxylase (ACCase), Acetolactate synthase (ALS), protoporphyrinogen oxidase
(PROTOX), dihydropteroate synthase, polyamine transport proteins, superoxide
dismutase
(SOD), bromoxynil nitrilase, the product of the tfdA gene obtainable from
Alcaligenes
eutrophus, and known mutagenised or otherwise modified variants of the said
proteins.
As indicated above, the polynucleotide with which the plant material may be
t o transformed may comprise 5' of the protein encoding regions regions which
encode: (i) a
peptide which is capable of targeting the translation products of the regions
to plastids such
as chloroplasts, mitochondria, other organelles or plant cell walls; and/or
(ii) non-translated
translational enhancing sequences.
The polynucleotide may be codon-optimised, or otherwise altered to enhance at
least
transcription once it is incorporated into plant material. Thus the
polynucleotide used to
transform the material may be modified in that mRNA instability encoding
motifs and/or
fortuitous splice regions may be removed, or plant preferred codons may be
used so that
expression of the thus modified polynucleotide in a plant yields substantially
similar protein
having a substantially similar activity/function to that obtained by
expression of the
2o unmodified polyriucleotide in the organism in which the protein encoding
regions of the
unmodified polynucleotide are endogenous, with the proviso that if - in
respect of the
herbicide resistance conferring regions - the thus modified polynucleotide
comprises plant
preferred codons, the degree of identity between the protein encoding regions
within the
modified polynucleotide and like protein encoding regions endogenously
contained within
the said plant and encoding substantially the same protein is less than about
70%.
Transformation techniques are well known and include particle mediated
biolistic
transformation, Agrobacterium-mediated transformation, protoplast
transformation
(optionally in the presence of polyethylene glycols); sonication of plant
tissues, cells or
protoplasts in a medium comprising the polynucleotide or vector; micro-
insertion of the
3o polynucleotide or vector into totipotent plant material (optionally
employing the known
silicon carbide "whiskers" technique), electroporation and the like.
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The invention still further provides morphologically normal fertile (or male
sterile) whole
plants regenerated from the material mentioned in the paragraph immediately
preceding the
last and the progeny of such plants, the seed of such plants and progeny, and
parts of such
plants and progeny. The transformed inventive plants include small grain
cereals, oil seed
crops, fibre plants, fruit, vegetables, plantation crops and trees.
Particularly preferred such
plants include soybean, cotton, tobacco, sugarbeet, oilseed rape, canola,
flax, sunflower, potato,
tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat,
turf grass, forage
grass, sugar cane, pea, field bean, rice, pine, poplar, apple, grape, citrus
and nut plants.
The transformed plants of the invention have tolerance or resistance to
certain
to herbicides such as the isoxazoles described especially in French Patent
Applications 9506800
and 95 13570 and especially of 4-[4-CF3-2-(methylsulphonyl)benzoyl]-5-
cyclopropylisoxazole, and especially isoxaflutole, a selective maize
herbicide, the
diketonitriles such as those described in EP-A-496,630 and EP-A-496,631, in
particular 2-
cyano-3-cyclopropyl-1-(2-SOZCH3-4-CF3-phenyl)propane-1,3-dione and 2-cyano-3-
cyclopropyl-1-(2-SOZCH3-4-2,3-C12-phenyl)propane-1,3-dione, and the triketones
described
in EP-A-625,505 and EP-A-625,508, in particular sulcotrione, mesotrione (BSI-
proposed),
pyrazolynate and pyrazoxyfen.
The invention further includes a morphologically normal fertile (or male
sterile)
whole plant resulting from the method of the invention, the progeny of such
plants, the seed
of such plants and progeny, and parts of such plants and progeny.
The invention still further provides the use of a polynucleotide comprising a
region
encoding a phytoene desaturase in the production of plant material which is
resistant or
tolerant to herbicides which - in vitro - inhibit the enzyme 4-HPPD.
The invention still further provides a method of selectively controlling weeds
in a
field, the field comprising weeds and crop plants, the method comprising
application to the
field of a herbicide which - in vitro - is capable of inhibiting the enzyme 4-
HPPD,
characterised in that the plants have been transformed with and express the
coding regions of
a polynucleotide comprising a sequence encoding a phytoene desaturase.
It is particularly preferred that the phytoene desaturase encoding sequence is
that
3o which is depicted in SEQ ID No. 1, or is complementary to one which when
incubated at a
temperature of between 55 and 60°C in 0.3 strength citrate buffered
saline containing 0.1
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SDS followed by rinsing at the same temperature with 0.3 strength citrate
buffered saline
containing 0.1 % SDS still hybridises with the sequence depicted in SEQ ID No.
1. The
herbicide may be selected from the group consisting of mesotrione (BSI-
proposed),
pyrazolynate and pyrazoxyfen, Balance, sulcotrione etc. The field may be
treated with a
pesticide selected from the group consisting of a fungicide, insecticide and
nematicide, either
prior to or post application to the field of the herbicide.
The invention will now be described by way of the following non-limiting
example,
figure and the Sequence Listing in which:
SEQ ID No. 1 is the sequence of the phytoene desaturase (dehydrogenase) gene
isolated from
t o Erwinia uredovora. The person skilled in the art will recognise that any
phytoene desaturase
gene may be used in the production of plants having resistance/tolerance to
the herbicides
described above.
SEQ ID No. 2 is the protein encoded by SEQ ID No 1.
SEQ ID No.3 is the polynucleotide sequence encoding the pea rubisco small
subunit transit
15 peptide.
SEQ IN No. 4 is the amino acid sequence encoded by SEQ ID No. 3.
Figure 1 is the structure of plasmid pYPEIT4 carrying the Erwinia uredovora
crtI gene with
the transit peptide sequence (depicted as TP) of the pea rubisco small
subunit.
EXAMPLE
Production of plants tolerant to herbicides capable of inhibiting the enzyme 4-
HPPD ire
vitro.
The PDS gene (crtI) was cloned from Erwinia uredovora, a non-green
phytopathogenic
bacterial rot, and over-expressed in transgenic tobacco and tomato using a
plasmid
containing the CaMV 35S promoter and a chloroplast transit peptide (pYPIET4)
(Misawa et
al., 1993). Homozygous tomato lines over-expressing the crtI gene were
obtained as were
tobacco plants containing the same construct.
Construction of nlasmid pYPIET4 carr~;~ the tp-crtlgene
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Recombinant DNA techniques were performed using standard methods. A DNA
sequence
coding for the transit peptide (TP) in the precursor of the ribulose-1,5-
bisphosphte
carboxylase (Rubisco) small subunit of pea was isolated from plasmid pSNIF83
(Schreier et
al., 1985) as a 204 by HindIII-Sphl fragment, whose Spgl site contains the tp
processing site.
Plasmid pCRT-1 (Fraser et al (1992) J.Biol.Chem 267 19891-19895) carrying the
intact
phytoene desaturase gene (crtI) of Erwinia uredovora was digested with BamHl
and
HindIII, and a 1.57 kb BamHl-HindIII fragment can-ying the truncated crtl gene
was isolated.
The above 204 by HindIII-Sphl TP fragment was ligated with a 76bp synthesized
fragment
which carries the reading frame from the cohesive end for the Sphl site
containing the crtl
initiation codon to that of the BamHl site, and with the 1.57 kb BamHl-HindIII
fragment.
The desired 1.84 kb Hindlll fragment carrying the tp-crtl chimeric gene was
isolated, filled in
with Klenow enzyme, and ligated into the Smal-Sacl site of a 10.9 kb fragment
removing the
(3-glucuronidase gene from the binary vector bB/121 (purchased from Clontech
laboratories).
Thus, the desired plasmid pYPEIT4 was created, shown in Figure 1. The
initiation codons
for the transit peptide and the intact Crtl are underlined. This HindIII
fragment carrying the
tp-crtI gene is surrounded by the CaMV 3 55 promoter and the NOS terminator of
the binary
vector pB1121 in order to lead to sufficient expression in the tissues of
transgenic tobacco
and tomato plants. As a control, plasmid pBICAR4 was constructed which carries
an intact
crtl gene without tp surrounded by the CaMV 355 promoter and the NOS
terminator. The
2o plasmid pYPEIT4 was introduced into tobacco and tomato material by known
techniques and
the material then regenerated into intact plants, again by known techniques.
Tolerance of Tomato Plants transformed with crtI gene to Mesotrione and
Isoxaflutole
Homozygous seed of tomato plants cv. Ailsa Craig, derived from 'wild type'
(i.e. un-
transformed) and plants transformed with the crtl gene from Erwinina
uredovora, (see
above) were sown in a peat-based compost in 3 inch pots and transferred to the
glasshouse.
Plants were grown at 20/16 degrees day/night temperature under a 16 hour
photoperiod for
approximately 4 weeks prior to post-emergence treatment of four replicates
with mesotrione
or isoxaflutole (Balance ~'~'' Herbicide) at the 3 leaf stage. The chemicals
were suspended in
water and applied, via a track sprayer at a spray volume of 200 litres per
hectare, at rates
ranging from 1 to 500 grammes active ingredient per hectare (g a.i./ha), as
shown in Table 1.
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The plants were left to grow for a further 25 days and then assessed visually
for herbicidal
damage compared to untreated 'control' plants. Typical phytotoxic symptoms
observed were
extreme chlorosis/bleaching and necrosis of leaves and new growth. The results
from this
test are given in Table 1 below where the '% Damage/Phytotoxicity' scores
represent the
mean of the visual assessment from each of the four treatment replicates.
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Table 1
Chemical Rate ~ % Damage/Phytotoxicity
(g a.i./ha) (25 days after
treatment)
Wild Type Transformed
(Un-Transformed)(crtI )
Mesotrione 1 25 0
3 29 0
11 50 9
33 81 49
Isoxaflutole 1 11 2
(Balance''' S 15 4
)
15 19 6
SO 21 x
150 34 19
500 65 31
As can be seen, plants transformed with the crtI gene which expresses the
bacterial PDS from
Erwinia uredovora, demonstrate elevated tolerance to mesotrione and
isoxaflutole compared
to wild type, un-transformed tomatoes. For example, 11 g a.i./ha of mesotrione
caused 50%
phytotoxicity to wild type tomatoes but only 9% injury is observed in the
transformed
plants. Similarly, wild type plants are significantly more damaged by 500 g
a.i.lha of
isoxaflutole than those containing the crtI gene.
to The skilled man will recognise that the invention is not limited to that
described above. For
example, plants other than tomato and tobacco may be transformed with a gene
encoding a
PDS enzyme, whether derived from a bacterial source or otherwise.
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SEQUENCE LISTING
<110> ZENECA LIMITED
<120> METHOD OF PRODUCING PLANTS WHICH ARE TOLERANT OR
RESISTANT TO HERBICIDES
<130> PPD50336W0
<140>
<141>
<150> 9807818.1
<151> 1998-04-09
<160> 9
<170> PatentIn Ver. 2.0
<210> 1
<211> 1493
<212> DNA
<213> Erwin ia
uredovora
<220>
<221> CDS
<222> (15). .(1493)
<400> 1
taaagagcga ctacatgaaa ccaactacg gtaattggt gcaggcttcggt 50
MetLys ProThrThr ValIleGly AlaGlyPheGly
1 5 10
ggc ctg gca ctggcaatt cgtctacaa getgcgggg atccccgtctta 98
Gly Leu Ala LeuAlaIle ArgLeuGln AlaAlaGly IleProValLeu
15 20 25
ctg ctt gaa caacgtgat aaacccggc ggtcggget tatgtctacgag 146
Leu Leu Glu GlnArgAsp LysProGly GlyArgAla TyrValTyrGlu
30 35 40
gat cag ggg tttaccttt gatgcaggc ccgacggtt atcaccgatccc 194
Asp Gln Gly PheThrPhe AspAlaGly ProThrVal IleThrAspPro
45 50 55 60
agt gcc att gaagaactg tttgcactg gcaggaaaa cagttaaaagag 242
Ser Ala Ile GluGluLeu PheAlaLeu AlaGlyLys GlnLeuLysGlu
65 70 75
tat gtc gaa ctgctgccg gttacgccg ttttaccgc ctgtgttgggag 290
Tyr Val Glu LeuLeuPro ValThrPro PheTyrArg LeuCysTrpGlu
80 85 90
tca ggg aag gtctttaat tacgataac gatcaaacc cggctcgaagcg 338
Ser Gly Lys ValPheAsn TyrAspAsn AspGlnThr ArgLeuGluAla
i 95 100 105
cag att cag cag ttt aat ccc cgc gat gtc gaa ggt tat cgt cag ttt 386
Gln Ile Gln Gln Phe Asn Pro Arg Asp Val Glu Gly Tyr Arg Gln Phe
110 115 120 .
ctg gac tat tca cgc gcg gtg ttt aaa gaa ggc tat cta aag ctc ggt 434
Leu Asp Tyr Ser Arg Ala Val Phe Lys Glu Gly Tyr Leu Lys Leu Gly
125 130 135 140
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actgtccct tttttatcg ttcagagac atgcttcgcgcc gcacct caa 482
ThrValPro PheLeuSer PheArgAsp MetLeuArgAla AlaPro Gln
145 150 155
ctggcgaaa ctgcaggca tggagaagc gtttacagtaag gttgcc agt 530
LeuAlaLys LeuGlnAla TrpArgSer ValTyrSerLys ValAla Ser
160 165 170
tacatcgaa gatgaacat ctgcgccag gcgttttctttc cactcg ctg 578
TyrIleGlu AspGluHis LeuArgGln AlaPheSerPhe HisSer Leu
175 180 185
ttggtgggc ggcaatccc ttcgccacc tcatccatttat acgttg ata 626
LeuValGly GlyAsnPro PheAlaThr SerSerIleTyr ThrLeu Ile
190 195 200
cacgcgctg gagcgtgag tggggcgtc tggtttccgcgt ggcggc acc 674
HisAlaLeu GluArgGlu TrpGlyVal TrpPheProArg GlyGly Thr
205 210 215 220
ggcgcatta gttcagggg atgataaag ctgtttcaggat ctgggt ggc 722
GlyAlaLeu ValGlnGly MetIleLys LeuPheGlnAsp LeuGly Gly
225 230 235
gaagtcgtg ttaaacgcc agagtcagc catatggaaacg acagga aac 770
GluValVal LeuAsnAla ArgValSer HisMetGluThr ThrGly Asn
290 245 250
aagattgaa gccgtgcat ttagaggac ggtcgcaggttc ctgacg caa 818
LysIleGlu AlaValHis LeuGluAsp GlyArgArgPhe LeuThr Gln
255 260 265
gccgtcgcg tcaaatgca gatgtggtt catacctatcgc gacctg tta 866
AlaValAla SerAsnAla AspValVal HisThrTyrArg AspLeu Leu
270 275 280
agccagcac cctgccgcg gttaagcag tccaacaaactg cagact aag 914
SerGlnHis ProAlaAla ValLysGln SerAsnLysLeu GlnThr Lys
285 290 295 300
cgcatgagt aactctctg tttgtgctc tattttggtttg aatcac cat 962
ArgMetSer AsnSerLeu PheValLeu TyrPheGlyLeu AsnHis His
305 310 315
catgatcag ctcgcgcat cacacggtt tgtttcggcccg cgttac cgc 1010
HisAspGln LeuAlaHis HisThrVal CysPheGlyPro ArgTyr Arg
320 325 330
gagctgatt gacgaaatt tttaatcat gatggcctcgca gaggac ttc 1058
GluLeuIle AspGluIle PheAsnHis AspGlyLeuAla GluAsp Phe
335 340 345
tcactttat ctgcacgcg ccctgtgtc acggattcgtca ctggcg cct 1106
SerLeuTyr LeuHisAla ProCysVal ThrAspSerSer LeuAla Pro
350 355 360
gaaggttgc ggcagttac tatgtgttg gcgccggtgccg cattta ggc 1154
GluGlyCys GlySerTyr TyrValLeu AlaProValPro HisLeu Gly
365 370 375 380
accgcgaac ctcgactgg acggttgag gggccaaaacta cgcgac cgt 1202
ThrAlaAsn LeuAspTrp ThrValGlu GlyProLysLeu ArgAsp Arg
385 390 395
atttttgcg taccttgag cagcattac atgcctggctta cggagt cag 1250
IlePheAla TyrLeuGlu GlnHisTyr MetProGlyLeu ArgSer Gln
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400 405 410
ctggtc acgcaccggatg tttacgccg tttgatttt cgcgaccag ctt 1298
LeuVal ThrHisArgMet PheThrPro PheAspPhe ArgAsp'Gln Leu
415 920 425
aatgcc tatcatggctca gccttttct gtggagccc gttcttacc cag 1346
AsnAla TyrHisGlySer AlaPheSer ValGluPro ValLeuThr Gln
430 435 440
agcgcc tggtttcggccg cataaccgc gataaaacc attactaat ctc 1394
SerAla TrpPheArgPro HisAsnArg AspLysThr IleThrAsn Leu
495 950 455 460
tacctg gtcggcgcaggc acgcatccc ggcgcaggc attcctggc gtc 1442
TyrLeu ValGlyAlaGly ThrHisPro GlyAlaGly IleProGly Val
465 470 475
atcggc tcggcaaaagcg acagcaggt ttgatgctg gaggatctg att 1490
IleGly SerAlaLysAla ThrAlaGly LeuMetLeu GluAspLeu Ile
480 485 490
tga 1993
<210> 2
<211> 492
<212> PRT
<213> Erwinia uredovora
<900> 2
Met Lys Pro Thr Thr Val Ile Gly Ala Gly Phe Gly Gly Leu Ala Leu
1 5 10 15
Ala Ile Arg Leu Gln Ala Ala Gly Ile Pro Val Leu Leu Leu Glu Gln
20 25 30
Arg Asp Lys Pro Gly Gly Arg Ala Tyr Val Tyr Glu Asp Gln Gly Phe
35 40 95
Thr Phe Asp Ala Gly Pro Thr Val Ile Thr Asp Pro Ser Ala Ile Glu
50 55 60
Glu Leu Phe Ala Leu Ala Gly Lys Gln Leu Lys Glu Tyr Val Glu Leu
65 70 75 80
Leu Pro Val Thr Pro Phe Tyr Arg Leu Cys Trp Glu Ser Gly Lys Val
85 90 95
Phe Asn Tyr Asp Asn Asp Gln Thr Arg Leu Glu Ala Gln Ile Gln Gln
100 105 110
Phe Asn Pro Arg Asp Val Glu Gly Tyr Arg Gln Phe Leu Asp Tyr Ser
115 120 125
Arg Ala Val Phe Lys Glu Gly Tyr Leu Lys Leu Gly Thr Val Pro Phe
130 135 140
Leu Ser Phe Arg Asp Met Leu Arg Ala Ala Pro Gln Leu Ala Lys Leu
145 150 155 160
Gln Ala Trp Arg Ser Val Tyr Ser Lys Val Ala Ser Tyr Ile Glu Asp
165 170 175
Glu His Leu Arg Gln Ala Phe Ser Phe His Ser Leu Leu Val Gly Gly
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180 185 190
Asn Pro Phe Ala Thr Ser Ser Ile Tyr Thr Leu Ile His Ala Leu Glu
195 200 205
Arg Glu Trp Gly Val Trp Phe Pro Arg Gly Gly Thr Gly Ala Leu Val
210 215 220
Gln Gly Met Ile Lys Leu Phe Gln Asp Leu Gly Gly Glu Val Val Leu
225 230 235 240
Asn Ala Arg Val Ser His Met Glu Thr Thr Gly Asn Lys Ile Glu Ala
295 250 255
Val His Leu Glu Asp Gly Arg Arg Phe Leu Thr Gln Ala Val Ala Ser
260 265 270
Asn Ala Asp Val Val His Thr Tyr Arg Asp Leu Leu Ser Gln His Pro
275 280 285
Ala Ala Vai Lys Gln Ser Asn Lys Leu Gln Thr Lys Arg Met Ser Asn
290 295 300
Ser Leu Phe Val Leu Tyr Phe Gly Leu Asn His His His Asp Gln Leu
305 310 315 320
Ala His His Thr Val Cys Phe Gly Pro Arg Tyr Arg Glu Leu Ile Asp
325 330 335
Glu Ile Phe Asn His Asp Gly Leu Ala Glu Asp Phe Ser Leu Tyr Leu
340 345 350
His Ala Pro Cys Val Thr Asp Ser Ser Leu Ala Pro Glu Gly Cys Gly
355 360 365
Ser Tyr Tyr Val Leu Ala Pro Val Pro His Leu Gly Thr Ala Asn Leu
370 375 380
Asp Trp Thr Val Glu Gly Pro Lys Leu Arg Asp Arg Ile Phe Ala Tyr
385 390 395 400
Leu Glu Gln His Tyr Met Pro Gly Leu Arg Ser Gln Leu Val Thr His
905 410 415
Arg Met Phe Thr Pro Phe Asp Phe Arg Asp Gln Leu Asn Ala Tyr His
420 425 430
Gly Ser Ala Phe Ser Val Glu Pro Val Leu Thr Gln Ser Ala Trp Phe
435 940 445
Arg Pro His Asn Arg Asp Lys Thr Ile Thr Asn Leu Tyr Leu Val Gly
450 455 460
Ala Gly Thr His Pro Gly Ala Gly Ile Pro Gly Val Ile Gly Ser Ala
465 470 475 480
Lys Ala Thr Ala Gly Leu Met Leu Giu Asp Leu Ile
485 490
<210> 3
<211> 209
<212> DNA
<213> Pisum sativum
<220>
CA 02321965 2000-08-28
WO 99/53081 PCT/GB99/01059
-5-
<221>
CDS
<222>
(1)..(204)
<900>
3
atggettct atgatatcc tcttcgget gtgacaacagtc agccgt gcc 48
MetAlaSer MetIleSer SerSerAla ValThrThrVal SerArg Ala
1 5 10 15
tctaggggg caatccgcc gcagtgget ccattcggcggc ctcaaa tcc 96
SerArgGly GlnSerAla AlaValAla ProPheGlyGly LeuLys Ser
20 25 30
atgactgga ttcccagtg aagaaggtc aacactgacatt acttcc att 144
MetThrGly PheProVal LysLysVal AsnThrAspIle ThrSer Ile
35 40 45
acaagcaat ggtggaaga gtaaagtgc atgaaaccaact acggta att 192
ThrSerAsn GlyGlyArg ValLysCys MetLysProThr ThrVal Ile
50 55 60
ggtgcaggc ttc 204
GlyAlaGly Phe
65
<210> 4
<211> 68
<212> PRT
<213> Pisum sativum
<400> 4
Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala
1 5 10 15
Ser Arg Gly Gln Ser Ala Ala Val Ala Pro Phe Gly Gly Leu Lys Ser
20 25 30
Met Thr Gly Phe Pro Val Lys Lys Val Asn Thr Asp Ile Thr Ser Ile
35 40 45
Thr Ser Asn Gly Gly Arg Val Lys Cys Met Lys Pro Thr Thr Val Ile
50 55 60
Gly Ala Gly Phe
65
