U.S. patent application number 15/356597 was filed with the patent office on 2017-03-09 for isolated polypeptides and polynucleotides encoding same for generating plants with increased cuticlar water permeability.
This patent application is currently assigned to The State of Israel, Ministry of Agriculture & Rural Development, Agricultural Research Organizat. The applicant listed for this patent is The State of Israel, Ministry of Agriculture & Rural Development, Agricultural Research Organizat. Invention is credited to Ran HOVAV, Arthur A. SCHAFFER.
Application Number | 20170066803 15/356597 |
Document ID | / |
Family ID | 36060427 |
Filed Date | 2017-03-09 |
United States Patent
Application |
20170066803 |
Kind Code |
A1 |
SCHAFFER; Arthur A. ; et
al. |
March 9, 2017 |
ISOLATED POLYPEPTIDES AND POLYNUCLEOTIDES ENCODING SAME FOR
GENERATING PLANTS WITH INCREASED CUTICLAR WATER PERMEABILITY
Abstract
An isolated polynucleotide is provided. The isolated
polynucleotides comprising a nucleic acid sequence encoding a
polypeptide having an amino acid sequence at least 88% homologous
to SEQ ID NO: 22, the polypeptide being capable of increasing a
cuticular water permeability of a plant expressing same. Also
provided are methods of generating plants expressing such
polypeptides which can be used for producing dehydrated plants or
cuticular covered portions thereof.
Inventors: |
SCHAFFER; Arthur A.;
(Hashmonaim, IL) ; HOVAV; Ran; (Yashresh,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The State of Israel, Ministry of Agriculture & Rural
Development, Agricultural Research Organizat |
Rishon-LeZion |
|
IL |
|
|
Assignee: |
The State of Israel, Ministry of
Agriculture & Rural Development, Agricultural Research
Organizat
Rishon-LeZion
IL
|
Family ID: |
36060427 |
Appl. No.: |
15/356597 |
Filed: |
November 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13902885 |
May 27, 2013 |
9497909 |
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15356597 |
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11663151 |
Dec 29, 2008 |
8481809 |
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PCT/IL2005/001000 |
Sep 19, 2005 |
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13902885 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8273 20130101;
A23V 2002/00 20130101; A23L 27/63 20160801; A01H 5/08 20130101;
A23L 19/09 20160801; C07K 14/415 20130101 |
International
Class: |
C07K 14/415 20060101
C07K014/415; A23L 27/60 20060101 A23L027/60; A23L 19/00 20060101
A23L019/00; C12N 15/82 20060101 C12N015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 19, 2004 |
IL |
164125 |
Claims
1. A processed product comprising DNA of a cultivated tomato plant
having a genome comprising an introgression derived from a wild
Lycopersicon hirsutum said introgression consisting of a portion of
chromosome 4 of said Lycopersicon hirsutum smaller than a
chromosomal fraction extending from telomeric marker TG464 to
centromeric marker, CT61 and includes TG464 and the nucleic acid
sequence encoding cwp1, said introgression being capable of
increasing cuticular water permeability of the fruit of the
cultivated tomato plant.
2. The processed product of claim 1 being a tomato paste.
3. The processed product of claim 1 being a ketchup.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/902,885 filed on May 27, 2013, which is a
division of U.S. patent application Ser. No. 11/663,151 filed on
Dec. 29, 2008, now U.S. Pat. No. 8,481,809, which is a National
Phase of PCT Patent Application No. PCT/IL2005/001000 filed on Sep.
19, 2005, which claims the benefit of priority of Israel Patent
Application No. 164125 filed on Sep. 19, 2004.
[0002] The contents of all of the above documents are incorporated
by reference as if fully set forth herein.
SEQUENCE LISTING STATEMENT
[0003] The ASCII file, entitled 68179SequenceListing.txt, created
on Nov. 17, 2016, comprising 201,216 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
[0004] The present invention relates to polynucleotides and
polypeptides for increasing cuticular water permeability of a plant
expressing same. More particularly the present invention relates to
genetically modified plants capable of producing dehydrated fruits,
such as tomato.
[0005] Aerial portions of higher plants are covered with a
continuous extracellular layer of cuticle. The cuticle is a polymer
matrix which is mostly composed of cutin monomers (primarily
short-chain hydroxylated fatty acids) and various amounts of
cuticular waxes (solvent-soluble lipids). Both the cutin and the
wax components vary greatly in amount and composition between
different plant species and plant organs (Holloway, 1982). Although
the components and structure of plant cuticle as well as the
biological and genetic regulation of its biosynthesis has been
extensively investigated (Kolattukudy, 1980; Koornneef et al.,
1989; Blee and Schuber, 1993; Arts et al., 1996; Negruk et al.,
1996; Millar et al., 1997; Todd et al., 1999; Yaphremov et al.,
1999; Flebig et al., 2000; Pruitt et al., 2000; Wellesen et al.,
2001 Hooker et al., 2002; Chen et al., 2003; Kuns and Samuels,
2003; Kurata et al., 2003; Aharoni et al., 2004; Schnurr et at.
2004;), the mechanisms controlling the differentiation and/or
function of the cuticle remain to be characterized.
[0006] The tomato fruit cuticle is a thin layer with a 4-10 micron
thickness with two unique structural properties (Wilson and
Sterling, 1976). First, the cutin deposits are arranged in a
laminar structure--the layers are assembled in parallel to the
epidermis cells. The second property of the tomato fruit cuticle is
that it does not contain any stomata, pores or channels. As a
result, the water permeability of the tomato skin is very low and
the fully ripe tomato fruit retains its water content. The water
permeability of a number of other cuticles lacking stomata
(astomatous) and the mechanism of water transport across them have
been the subjects of numerous investigations (Schonherr, 1976a;
Schonherr and Schmidt, 1979; Riederer and Schreiber, 2001).
Apparently, both the cutin and wax components have an integrated
effect against water loss from the organ. In some cases, the
thickness of the cuticular layer is inversely proportional to
diffusion of water across cuticular membranes (Lownds et al.,
1993). However, frequently the cuticular wax component is primary
in affecting plant water permeability. For example, removal of the
epicuticular wax layer from tomato fruit cuticles by organic
solvents increased their water permeability by a factor of 300 to
500, as evidenced by rapid plant dehydration (Schonherr, 1976b).
Additional evidence for the role of cuticular waxes as a
transpiration barrier in tomato fruits is the recently reported
gene encoding the enzyme very-long-chain-fatty acid (VLFA)
.beta.-ketoacyl-CoA synthase (LeCER6, Vogg et al., 2004). This gene
plays an important role in the synthesis of VFLA which are a major
component in fruit cuticular wax. A loss of function mutation in
this gene led to the reduction of n-alkanes and aldehydes with
chain lengths beyond C.sub.30 in both leaf and fruit waxes. Tomato
fruits with the LeCER6 mutation were characterized with a 4-fold
increase in water permeability. Another factor affecting water
permeability of tomato fruit cuticle is the presence of cracking on
the cuticular surface. Fruit cracking has received much research
attention (Cotner et al., 1969; Voisey et al., 1970; peet, 1992;
peet and willits, 1995). Tomato fruits are affected by three main
types of cracking: i) Concentric cracking (coarse cracking); ii)
Radial cracking (splitting); and iii) Cuticle cracking (russeting)
(Bakker, 1988). The first two types of cracking are deep and
extended fissures that penetrate through the fruit pericarp and in
addition to water loss also allow pathogen penetration and fruit
decomposition.
[0007] Unlike radial or concentric cracks, cuticle cracks are
superficial micro fissures of the cuticle that are generally
confined to the cuticle and do not penetrate to the pericarp cells.
The causes and circumstances leading to fruit cracking in tomatoes
are mostly unclear and may be related to cuticular layer thickness
(Emmons and Scott, 1998), shape of the underlying epidermis cells
(Conter et al., 1969; Emmons and Scott, 1998), fruit shape
(Considine and Brown, 1981), fruit size (Koske et al., 1980; Emmons
and Scott, 1997), relative humidity around the fruit (Young, 1947;
Tukey, 1959), strong foliage pruning (Ehret et al., 1993) and the
tensile strength and extensibility of the epidermis (Bakker,
1988).
[0008] The occurrence of cracks in tomato fruit also has a
significant genetic component, which is mainly expressed upon gene
transfer from wild species of Lycopersicon. Fulton et al. (2000)
described a trait, "Epidermal reticulation" (Er), and, using an
advanced backcross QTL analysis strategy (with the wild type L.
parviflorum) reported four QTLs affecting it. Cuticlar cracks also
have been reported in Lycopersicon fruit derived from crosses of L.
esculentum and other wild species such has L. hirsutum (WO 0113708)
and L. penellii (Monforte et al., 2001).
[0009] Cracks in fruit cuticle, particularly extreme cracks which
are visually evidenced as epidermal relticulation, due to the
development of a suberized coating along the fissure (Monforte et
al., 2001), are generally considered to be negative phenomenon due
to the esthetic damages that lower fruit value (Tukey, 1959), as
well as due to the loss of fruit moisture content. However, the
economic potential of fruits that dehydrate while whole and while
still attached to the vine, is high. Dehydrated tomato products
comprise an important portion of the tomato industry. The
production of tomato pastes, ketchup, and other processed tomato
products is dependant on the energy-requiring steps of dehydration.
In addition, "sun-dried" tomato fruit are prepared in a drying
process which consists of dehydrating cut tomato fruit either in
the sun or in drying ovens. Both sun-drying and oven drying may
lead to losses in food quality. For example, levels of ascorbic
acid, one of the major nutritional contributions of tomatoes in the
human diet, decrease significantly in response to sun-drying or
oven-drying (Ojimelukwe, 1994). Furthermore, the necessity to cut
the tomato fruit in half before the drying process does not allow
for the production of whole dried tomato fruit.
[0010] The present inventor has previously described dehydrated
tomatoes having reduced water content using classical genetic
breeding techniques (WO 01/13708). It is appreciated that the
classical genetic breeding techniques are limiting to gene transfer
within species or between closely related species of the same
genus. Also, classical breeding is characterized by relatively
large introgressions which include other undesirable genes closely
linked to the gene of interest.
[0011] Introgressed cultivated tomato plants have been previously
described by Eshed and Zamir (1985) having a genetic background
(Introgression line IL4-4, i.e., resulting from an introgression
extending from telomeric marker TG464 to centromeric marker CT50;
ca20 cM) which may be associated with undesired traits. Similarly,
Monforte et al. (2001) have described tomato plants having a
similar genetic background derived from L. hirsutum [sub-near
introgression lines TA1468, TA1469, TA1476 which span from, and
including, TG464 to CT173 (approximately. 10 cM)] and which display
numerous effects, including undesirable effects.
[0012] There is thus a widely recognized need for and it would be
highly advantageous to have genetically modified plants with
increased cuticular water permeability which are devoid of the
above limitations.
SUMMARY OF THE INVENTION
[0013] According to one aspect of the present invention there is
provided an isolated polynucleotide comprising a nucleic acid
sequence encoding a polypeptide having an amino acid sequence at
least 88% homologous to SEQ ID NO: 22, the polypeptide being
capable of increasing a cuticular water permeability of a plant
expressing same.
[0014] According to further features in preferred embodiments of
the invention described below, the nucleic acid sequence is as set
forth in SEQ ID NO: 21 or 23.
[0015] According to still further features in the described
preferred embodiments the amino acid sequence is as set forth in
SEQ ID NO: 22.
[0016] According to another aspect of the present invention there a
nucleic acid construct comprising the isolated polynucleotide.
[0017] According to still further features in the described
preferred embodiments the nucleic acid construct further comprising
a promoter operably linked to the nucleic acid sequence.
[0018] According to another aspect of the present invention there a
host cell comprising the nucleic acid construct.
[0019] According to another aspect of the present invention there a
genetically modified plant comprising the isolated
polynucleotide.
[0020] According to another aspect of the present invention there
an oligonucleotide capable of specifically hybridizing with the
isolated polynucleotide
[0021] According to another aspect of the present invention there
is provided an isolated polypeptide comprising an amino acid
sequence at least 88% homologous to SEQ ID NO: 22, the polypeptide
being capable of increasing a cuticular water permeability of a
plant expressing same.
[0022] According to yet another aspect of the present invention
there is provided an antibody capable of specifically recognizing
the polypeptide.
[0023] According to yet another aspect of the present invention
there is provided a cultivated tomato plant having a genome
comprising an introgression derived from a wild Lycopersicon spp.
the introgression comprising a portion of chromosome 4 of the
Lycopersicon spp. smaller than a chromosomal fraction extending
from telomeric marker TG464 to centromeric marker CT173, the
introgression being capable of increasing cuticular water
permeability of the cultivated tomato plant.
[0024] According to still another aspect of the present invention
there is provided a method of producing a dehydrated fruit of a
crop plant, the method comprising genetically modifying the plant
to express a polypeptide having an amino acid sequence at least 30%
homologous to SEQ ID NO: 22, the polypeptide being capable of
increasing a cuticular water permeability of a plant expressing
same.
[0025] According to still further features in the described
preferred embodiments the method further comprising:
[0026] allowing the fruit to dehydrate on the plant; and
subsequently
[0027] collecting the dehydrated fruit.
[0028] According to still further features in the described
preferred embodiments the method further comprising:
removing the fruit from the crop plant prior to dehydration
thereof; and subsequently allowing the fruit to dehydrate.
[0029] According to an additional aspect of the present invention
there is provided a genetically modified seed comprising an
isolated polynucleotide comprising a nucleic acid sequence encoding
a polypeptide having an amino acid sequence at least 30% homologous
to SEQ ID NO: 22, the polypeptide being capable of increasing a
cuticular water permeability of a plant expressing same.
[0030] According to yet an additional aspect of the present
invention there is provided a genetically modified fruit comprising
an isolated polynucleotide comprising a nucleic acid sequence
encoding a polypeptide having an amino acid sequence at least 30%
homologous to SEQ ID NO: 22, the polypeptide being capable of
increasing a cuticular water permeability of a plant expressing
same.
[0031] According to still further features in the described
preferred embodiments the nucleic acid sequence is as set forth in
SEQ ID NO: 21, 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54 or 56.
[0032] According to still further features in the described
preferred embodiments the amino acid sequence is as set forth in
SEQ ID NO: 22, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,
51, 53, 55 or 57.
[0033] According to still an additional aspect of the present
invention there is provided a genetically modified plant expressing
a polypeptide having an amino acid sequence at least 30% homologous
to SEQ ID NO: 22, the polypeptide being capable of increasing a
cuticular water permeability of the plant.
[0034] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
polynucleotides and polypeptides being capable of increasing
cuticular water permeability of a plant expressing same and by
providing genetically modified plants for producing dehydrated
fruits.
[0035] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0036] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0037] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0038] In the drawings:
[0039] FIGS. 1a-1b are graphs showing the effect of cwp (PUT)
genotype on dehydration rate in population 2148 (FIG. 1a) and
population 2149 (FIG. 1b). In the population 2148 the trait of
dehydration behaves as a completely dominant trait while in 2149 it
behaves as a partially dominant trait. Fruit were picked when
red-ripe and allowed to dehydrate at ambient room temperature and
weighed at approximately daily intervals. Data are expressed as Log
% weight. The superscripts HH, HE and EE indicate the genotypes of
the segregating plants.
[0040] FIGS. 2a-2c show fine mapping of CWP gene. FIG. 2a-CAPS
marker analysis of the TG464 molecular marker. Genomic DNA was
extracted from 20 F.sub.2 individuals segregating for dehydration
rate. PCR analysis was performed using the appropriate primers for
TG464 marker which showed polymorphism between the two parental
species. PCR products were cleaved with HinF1 endonuclease
restriction site enzyme, and electrophoresed on 2% agarose gel. The
+ or - signs indicate the presence or absence of microfissures and
the dehydrating condition. E--L. esculentum. H--L. hirsutum.
.M--HindIII/EcorI lambda marker (Fermentas Cat. No. SM0191) FIG.
2b-Genetic linkage map (in cM) of the chromosomal region of CWP
oriented relative to the position of the centromere. Lycopersicon
penellii introgression lines IL4.3 and IL4.4 (Eshed and Zamir,
1995) are indicated. The hatched bar represents the L. hirsutum
segment in the near-isogenic line that was used as the dehydrating
donor parent in this analysis. FIG. 2c-Magnification of the
chromosomal segment flanking the Cwp gene.
[0041] FIGS. 3a-3b show physical positioning of CWP gene. FIG.
3a-Genetically ordered contiguous BACs creating a bridge between
CT61 and TG464 CAPS markers, and phenotypic analysis of the
recombinants and the characterization of the recombinants according
to polymorphisms of the sequenced BAC ends. Each recombinant
genotype is represented by a bar divided into hatches (L. hirsutum
genotype) and empty (L. esculentum genotype) segments. FIG.
3b-Magnification of the three crossover events in BAC 37B8. The
three crossover events are those of the first three recombinants
presented in FIG. 3a.
[0042] FIG. 4 illustrates the 15 kb introgression from L. hirsutum
which includes the Cwp gene. The sequence was analyzed for
homologous open reading frames using the NCBI program TBLAST. Three
homologous sequences were identified and the direction of each of
the open reading frames is indicated by arrows.
[0043] FIGS. 5a-5b are graphs showing expression analysis of the
PUT (FIG. 5a) and the DBP (FIG. 5b) genes in developing ovaries and
fruitlets of tomato. Expression was measured on extracted cDNA as
described in the Methods section using an On-line quantitative PCR
and is expressed relative to the expression of the actin gene in
each sample. Ov, ovary; 5 and 15 days after anthesis; IG, immature
green, MG, mature green; B, breaker stage. Hatched bars are the
Cwp.sup.HH genotypes and solid bar is the Cwp.sup.EE genotypes.
[0044] FIG. 6 is a graph showing expression analysis of the PUT
gene in 15 day fruitlets of tomato genotypes. HH, Cwp.sup.HH
genotype; HE, heterozygous Cwp.sup.HE genotype; EE, Cwp.sup.EE
genotype. The three genotypes were selected from a segregating
heterozygous population. IL4.4 represents the L. pennellii
introgression line IL4.4 (Eshed and Zamir, 1985) which contains the
L. pennellii homologue of PUT. M82 is the recurrent L. esculentum
parent of the IL 4.4.
[0045] FIGS. 7a-7b show transgenic tomato plants (T.sub.0)
expressing the PUT gene from the wild tomato species Solanum
habrochaites S. (previously Lycopersicon hirsutum Mill.) under the
35S constitutive promoter. FIG. 7a shows binocular photographs
presenting the intact surface of the fruit of the wild type MP1
tomato line (W.T.), and the micro-fissured transgenic fruit
(Mp1-4). FIG. 7b show drying rate comparison between a wild type
MP1 tomato line (W.T.) and another independent transgenic T.sub.0
plant (MP 1-1). Fruit were picked-up at mature red developing stage
and were placed at room temperature (15-25.degree. C.). Pictures
are from the beginning of the experiments (T.sub.0) and after 7
days of drying (T.sub.7).
[0046] FIGS. 8a-8b show the effect of the PUT transgene copy number
on microfissure severity (scale between 1 to 5, FIG. 8a) and weight
loss percentage of the fruit (after 14 days at room temperature,
FIG. 8b). Measurements were collected from 2 independent transgenic
(T.sub.1) segregating populations (16 individuals from each
population). Each graph shows the mean (the horizontal line at the
middle of each diamond), the 95% of confidence limit (the vertical
edge of the diamond), and the scattering extent of individuals from
each copy numbers group. The difference between groups is
significant when base of one group triangle is not congruent to the
triangle base of the other group. Statistics carried out by JMP
program.
[0047] FIGS. 9a-9b show a comparison between transgenic tomato
individuals (T.sub.1 generation) expressing no copies, analogous to
wild type, and two copies of the PUT gene from the wild tomato
species Solanum habrochaites S. FIG. 9a-Scanning electron
micrograph presenting the intact surface of the fruit from an
individual with no copies of the PUT gene (0 copies) and the
micro-fissured fruit of an individual with two copies of the
transgene. FIG. 9b-Drying rate comparison between an individual
with no copies of the PUT gene (0 copies) and an individual with
two copies (2 copies). Fruit were picked-up at mature red
developing stage and were placed at room temperature (15-25.degree.
C.). Pictures are from the beginning of the experiments (T.sub.0)
and after 7 days of drying (T.sub.7).
[0048] FIGS. 10a-10b are dendrograms depicting conservation of CWP1
and CWP2 and related sequences from monocot and dicot species (SEQ
ID NOs. 21, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54 and 56). These sequences were retrieved from the EST TIGR
database based on sequence homology to CWP1. Percentage homology to
CWP1 is indicated above. FIG. 10a-conservation at the amino acid
level. FIG. 10b-conservation at the nucleic acid level.
[0049] FIGS. 11a-11c show multiple alignment between different
protein members of the CWP1 family (SEQ ID NOs: 22, 25, 27, 29, 31,
33, 34, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57) of the
present invention generated by the ClustalW software of
EMBL-EBI.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0050] The present invention is of isolated polynucleotides and
polypeptides which can be used for increasing cuticular water
permeability of plants. Specifically, the present invention can be
used to produce dehydrated fruit, such as tomato fruit.
[0051] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0052] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0053] The development of tomato varieties capable of being
naturally dehydrated while still attached to the vine, without the
accompaniment of degradative processes leading to fruit breakdown
is highly valuable, to many fruit industries, such as the tomato
industry.
[0054] PCT Publ. No. WO 01/13708 to Schaffer teaches the generation
of dehydrated tomatoes having reduced cuticular water content using
classical genetic breeding techniques (WO 01/13708). It is
appreciated that the classical genetic breeding techniques are
limiting to gene transfer within species or between closely related
species of the same genus. Also, classical breeding is
characterized by relatively large introgressions which include
other undesirable genes closely linked to the gene of interest.
[0055] Introgressed cultivated tomato plants have been previously
described by Eshed and Zamir (1985) having a genetic background
(Introgression line IL4-4, i.e., resulting from an introgression
extending from telomeric marker TG464 to centromeric marker CT50;
ca20 cM) which may be associated with undesired traits. Similarly,
Monforte et al. (2001) have described tomato plants having a
similar genetic background derived from L. hirsutum (sub near
introgression line (NIL) which spans from TG464 to CT173 (>10
cM). In the latter study the relatively large introgression is
accompanied by undesirable horticultural traits, including traits
of brix-yield, total yield, and fruit weight.
[0056] While reducing the present invention to practice the present
inventors uncovered a single gene cwp1 (also termed put, used
interchangeably herein) which is capable of increasing cuticular
water permeability of a plant expressing same. As is illustrated
hereinbelow and in the Examples section which follows, the present
inventors identified the inheritance pattern of the trait of fruit
dehydration derived from L. hirsutum as a single major gene. Using
a map-based positional cloning strategy, the present inventors
cloned a gene from the wild tomato species L. hirsutum that
increases the cuticular water permeability (CWP) of the mature red
tomato fruit and leads to the dehydration of the intact fruit.
[0057] The present inventors showed that the wild species allele
for cwp allows for expression of the gene in developing tomato
fruit while the standard cultivated L. esculentum allele is not
expressed and may be considered a null allele. They further showed
that there is an allele dosage effect at the expression level and
the heterozygous HE genotype is characterized by approximately half
the expression as the homozygous genotype with two alleles from the
wild species.
[0058] Bioinformatic analysis showed that cwp1 encodes a protein
with no known biological function. This gene may contribute to
breeding programs for new tomato products, as well as for other
crops, as it controls water loss through the cuticle. Furthermore,
the structural phenotype of micro-fissures associated with this
gene indicates a role for cwp in fruit cuticle development.
Expression of cwp1 gene under the 35S promoter in cultivated tomato
induced the formation of microfissures in the expanding fruit,
supporting the suggested role of this gene in regulation of
cuticular water permeability. Southern blot analysis uncovered an
additional tomato homolog cwp2. Interestingly, this homologue maps
to tomato chromosome 2-1 where there is a reported QTL for tomato
fruit epidermal reticulation (Frary et al, 2004). Developing fruit
of the solanaceous cultivated pepper (Capsicum annum) also express
a cwp homologue highly similar (87%) to the Lecwp1 gene in its
epidermal tissue and pepper fruit are characterized by the
horticultural problem of post-harvest water loss, as well as by the
desirable trait of fruit dehydration in paprika cultivars.
Therefore it is likely that homologues of the CWP gene may also
contribute to cuticular modification and water permeability.
[0059] These results indicate that the expression of the cwp gene
leads to a structurally modified cuticle (based on weight and TEM)
which presumably undergoes fissuring during fruit expansion due to
reduction in elasticity. However, this phenomenon is observed only
in fruit with a highly developed fruit cuticle such as the
astomatous thick skinned cultivated tomato and is not apparent in
fruit of the wild species, with their characteristic thinner
cuticle. The deposition of cuticular components during cultivated
tomato fruit development undergoes a surge during the transition
from the immature to the mature green stage (Baker, 1982) and it is
reasonable that this coincides with the observation of the
microfissure phenotype. Without being bound by theory, it is
suggested that the genetic trait of a relatively impervious fruit
cuticle was a positive development in the evolution and
domestication process of cultivated tomatoes, allowing for the
stability of the ripening and harvested fruit. The genetic control
of the trait of dehydration indicates a selection procedure for the
null Cwp at some stage of evolution and domestication of the crop.
Phylogenetic analysis (FIGS. 10a-10b) indicates that the CWP genes
of the present invention belong to a larger family of genes, which
may be used for controlling cuticular water permeability in a broad
range of crop plants.
[0060] Thus, according to one aspect of the present invention there
is provided an isolated polynucleotide comprising a nucleic acid
sequence encoding a polypeptide having an amino acid sequence at
least about 30%, at least about 40%, at least about 50%, at least
about 55%, at least about 60%, at least about 65%, at least about
70%, at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 91%, at least about 92%, at least
about 93%, at least about 94%, at least about 95%, at least about
96%, at least about 97%, at least about 98%, at least about 99% or
100% homologous to SEQ ID NO: 22, the polypeptide being capable of
increasing a cuticular water permeability of a plant expressing
same.
[0061] As used herein the phrase "cuticular water permeability"
refers to the ability of the cuticle to inhibit water evaporation
from a cuticle-surrounded plant tissue (aerial tissues of the
plant), such as the fruit. It is appreciated that increased
cuticular water permeability will result in dehydration of the
cuticle surrounded tissue, as a result of enhanced evaporation.
[0062] As used herein the phrase "increasing cuticular water
permeability" refers to at least about 5%, at least about 10%, at
least about 15%, at least about 20%, at least about 30%, at least
about 40%, at least about 50%, increase in cuticular water
permeability as compared to plants of similar parental cultivar or
genotype not expressing same.
[0063] Methods of determining cuticular water permeability are well
known in the art and described in length in the Examples section
which follows (e.g fissure severity and weight loss percentage of
the fruit. See Example 5 of the Examples section which follows. In
addition, methods for measuring cuticular water permeability also
include, but are not limited to, measurements of water diffusion
across isolated fruit skin, measurement of polar pore size and
hydrodynamic permeability (Schonherr, 1976). These functional
assays together with the structural guidelines provided herein,
allow the identification of functional homologs for the
polynucleotides and polypeptides of the present invention.
[0064] Homology (e.g., percent homology) can be determined using
any homology comparison software, including for example, the BlastP
software of the National Center of Biotechnology Information (NCBI)
such as by using default parameters.
[0065] Identity (e.g., percent homology) can be determined using
any homology comparison software, including for example, the BlastN
software of the National Center of Biotechnology Information (NCBI)
such as by using default parameters.
[0066] As used herein the phrase "an isolated polynucleotide"
refers to a single or double stranded nucleic acid sequences which
is isolated and provided in the form of an RNA sequence, a
complementary polynucleotide sequence (cDNA), a genomic
polynucleotide sequence and/or a composite polynucleotide sequences
(e.g., a combination of the above).
[0067] As used herein the phrase "complementary polynucleotide
sequence" refers to a sequence, which results from reverse
transcription of messenger RNA using a reverse transcriptase or any
other RNA dependent DNA polymerase. Such a sequence can be
subsequently amplified in vivo or in vitro using a DNA dependent
DNA polymerase.
[0068] As used herein the phrase "genomic polynucleotide sequence"
refers to a sequence derived (isolated) from a chromosome and thus
it represents a contiguous portion of a chromosome.
[0069] As used herein the phrase "composite polynucleotide
sequence" refers to a sequence, which is at least partially
complementary and at least partially genomic. A composite sequence
can include some exonal sequences required to encode the
polypeptide of the present invention, as well as some intronic
sequences interposing therebetween. The intronic sequences can be
of any source, including of other genes, and typically will include
conserved splicing signal sequences. Such intronic sequences may
further include cis acting expression regulatory elements.
[0070] According to one preferred embodiment of this aspect of the
present invention, the nucleic acid sequence of the above-described
isolated polynucleotide of the present invention is as set forth in
SEQ ID NO: 21, 23, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54 or 56.
[0071] According to another preferred embodiment of this aspect of
the present invention, the amino acid sequence of the encoded
polypeptide of the present invention is as set forth in SEQ ID NO:
22, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55
or 57.
[0072] The isolated polynucleotides of this aspect of the present
invention can be qualified using a hybridization assay by
incubating the isolated polynucleotides described above in the
presence of oligonucleotide probe or primer under moderate to
stringent hybridization conditions.
[0073] As used herein the term "oligonucleotide" refers to a
single-stranded or double-stranded oligomer or polymer of
ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics
thereof. This term includes oligonucleotides composed of naturally
occurring bases, sugars, and covalent internucleoside linkages
(e.g., backbone), as well as oligonucleotides having non-naturally
occurring portions, which function similarly to respective
naturally occurring portions.
[0074] Oligonucleotides designed according to the teachings of the
present invention can be generated according to any oligonucleotide
synthesis method known in the art, such as enzymatic synthesis or
solid-phase synthesis. Equipment and reagents for executing
solid-phase synthesis are commercially available from, for example,
Applied Biosystems. Any other means for such synthesis may also be
employed; the actual synthesis of the oligonucleotides is well
within the capabilities of one skilled in the art and can be
accomplished via established methodologies as detailed in, for
example: Sambrook, J. and Russell, D. W. (2001), "Molecular
Cloning: A Laboratory Manual"; Ausubel, R. M. et al., eds. (1994,
1989), "Current Protocols in Molecular Biology," Volumes I-III,
John Wiley & Sons, Baltimore, Md.; Perbal, B. (1988), "A
Practical Guide to Molecular Cloning," John Wiley & Sons, New
York; and Gait, M. J., ed. (1984), "Oligonucleotide Synthesis";
utilizing solid-phase chemistry, e.g. cyanoethyl phosphoramidite
followed by deprotection, desalting, and purification by, for
example, an automated trityl-on method or HPLC.
[0075] The oligonucleotide of the present invention is of at least
17, at least 18, at least 19, at least 20, at least 22, at least
25, at least 30 or at least 40, bases specifically hybridizable
with polynucleotide sequences of the present invention.
[0076] Moderate to stringent hybridization conditions are
characterized by a hybridization solution such as containing 10%
dextrane sulfate, 1 M NaCl, 1% SDS and 5.times.10.sup.6 cpm
.sup.32P labeled probe, at 65.degree. C., with a final wash
solution of 0.2.times.SSC and 0.1% SDS and final wash at 65.degree.
C. and whereas moderate hybridization is effected using a
hybridization solution containing 10% dextrane sulfate, 1 M NaCl,
1% SDS and 5.times.10.sup.6 cpm .sup.32P labeled probe, at
65.degree. C., with a final wash solution of 1.times.SSC and 0.1%
SDS and final wash at 50.degree. C.
[0077] Using hybridization methodology, the present inventors were
able to isolate cwp2, another tomato homolog of cwp1, which is
mapped to a reported QTL for tomato fruit epidermal reticulation
(Frary et al, 2004), supporting its role in cuticular water
permeability.
[0078] Thus, the present invention encompasses nucleic acid
sequences described hereinabove; fragments thereof, sequences
hybridizable therewith, sequences homologous thereto, sequences
encoding similar polypeptides with different codon usage, altered
sequences characterized by mutations, such as deletion, insertion
or substitution of one or more nucleotides, either naturally
occurring or man induced, either randomly or in a targeted
fashion.
[0079] Since the polynucleotide sequences of the present invention
encode previously unidentified polypeptides, the present invention
also encompasses novel polypeptides or portions thereof, which are
encoded by the isolated polynucleotides and respective nucleic acid
fragments thereof described hereinabove.
[0080] Thus, the present invention also encompasses polypeptides
encoded by the polynucleotide sequences of the present invention.
The amino acid sequences of these novel polypeptides are set forth
in SEQ ID NO: 22, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55 or 57.
[0081] The present invention also encompasses homologues of these
polypeptides, such homologues can be at least about 70%, at least
about 75%, at least about 80%, at least about 81%, at least about
82%, at least about 83%, at least about 84%, at least about 85%, at
least about 86%, at least about 87%, at least about 88%, at least
about 89%, at least about 90%, at least about 91%, at least about
92%, at least about 93%, at least about 93%, at least about 94%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98%, at least about 99%, or more say 100% homologous to SEQ
ID NO: 22.
[0082] The present invention also encompasses fragments of the
above described polypeptides and polypeptides having mutations,
such as deletions, insertions or substitutions of one or more amino
acids, either naturally occurring or man induced, either randomly
or in a targeted fashion.
[0083] Amino acid sequence information of the polypeptides of the
present invention can be used to generate antibodies, which
specifically bind to the polypeptides of the present invention. For
example, such antibodies can be directed to amino acid sequence
coordinates 55-160 of SEQ ID NO: 22. Sequence coordinates 55-160
include the majority of conserved sequences and motifs of the
multiple comparison analysis (FIG. 11). Due to high sequence
homology in this amino acid sequence region, such antibodies are
expected to be cross-reactive to the various polypeptides the
present invention (e.g., SEQ ID NOs. 22, 25, 27, 29, 31, 33, 35,
37, 39, 41, 43, 45, 47, 49, 51, 53, 55 and 57).
[0084] Polynucleotide and polypeptide sequences of the present
invention can be used to generate plants with increased cuticular
water permeability.
[0085] For example, genetically modified plants can be generated by
expressing in the plant an isolated polynucleotide of the present
invention.
[0086] As used herein the term "plant" refers to a crop plant
(whole plant or a portion thereof, e.g., fruit, seed) such as a
monocot or dicot crop plant, as well as other plants coniferous
plants, moss or algae, in which increased cuticular water
permeability is commercially desired. Preferably, the plant of the
present invention produces fruits which dehydration is of
commercial value. Examples of such plants include, but are not
limited, to tomato, grapes, pepper, apples, peach, apricot, dates,
figs, eggplants, onion, strawberries, cucurbits, hay plants, forage
plants, spice plants, herb plants and others.
[0087] To express exogenous polynucleotides in plant cells, a
polynucleotide sequence of the present invention is preferably
ligated into a nucleic acid construct suitable for plant cell
expression. Such a nucleic acid construct includes a cis-acting
regulatory region such as a promoter sequence for directing
transcription of the polynucleotide sequence in the cell in a
constitutive or inducible manner. The promoter may be homologous or
heterologous to the transformed plant/cell.
[0088] Preferred promoter sequences which can be used in accordance
with this aspect of the present invention are fruit specific or
seed specific promoters.
[0089] For example, the novel promoter sequence of the cwp1 gene
(or functional fragments thereof) may be preferably used in the
nucleic acid constructs of the present invention (SEQ ID NO:
58).
[0090] Other examples of fruit specific promoters are described in
U.S. Pat. No. 4,943,674.
[0091] Other promoters which can be used in accordance with this
aspect of the present invention are those that ensure expression
only in specified aerial exposed organs of the plant, such as the
leaf, tuber, seed, stem, flower or specified cell types such as
parenchyma, epidermal, trichome or vascular cells.
[0092] Preferred promoters enhancing expression in seeds include
the phas promoter (Geest et al., Plant Mol. Biol. 32:579-588
(1996)); the GluB-1 promoter (Takaiwa et al., Plant Mol. Biol.
30:1207-1221 (1996)); the gamma-zein promoter (Torrent et al. Plant
Mol. Biol. 34:139-149 (1997)), and the oleosin promoter (Sarmiento
et al., The Plant Journal 11:783-796 (1997)).
[0093] Other promoter sequences which mediate constitutive,
inducible, tissue-specific or developmental stage-specific
expression are disclosed in WO 2004/081173.
[0094] The nucleic acid construct can be, for example, a plasmid, a
bacmid, a phagemid, a cosmid, a phage, a virus or an artificial
chromosome. Preferably, the nucleic acid construct of the present
invention is a plasmid vector, more preferably a binary vector.
[0095] The phrase "binary vector" refers to an expression vector
which carries a modified T-region from Ti plasmid, enable to be
multiplied both in E. coli and in Agrobacterium cells, and usually
comprising reporter gene(s) for plant transformation between the
two boarder regions. A binary vector suitable for the present
invention includes pBI2113, pBI121, pGA482, pGAH, pBIG, pBI101
(Clonetech), pPI, and pBIN PLUS (see Example 5 of the Examples
section which follows) or modifications thereof.
[0096] The nucleic acid construct of the present invention can be
utilized to transform a host cell (e.g., bacterial, plant) or
plant.
[0097] As used herein, the terms "transgenic" or "transformed" are
used interchangeably referring to a cell or a plant into which
cloned genetic material has been transferred.
[0098] In stable transformation, the nucleic acid molecule of the
present invention is integrated into the plant genome, and as such
it represents a stable and inherited trait. In transient
transformation, the nucleic acid molecule is expressed by the cell
transformed but not integrated into the genome, and as such
represents a transient trait.
[0099] There are various methods of introducing foreign genes into
both monocotyledonous and dicotyledonous plants (Potrykus, I.
(1991). Annu Rev Plant Physiol Plant Mol Biol 42, 205-225;
Shimamoto, K. et al. (1989). Fertile transgenic rice plants
regenerated from transformed protoplasts. Nature (1989) 338,
274-276).
[0100] The principal methods of the stable integration of exogenous
DNA into plant genomic DNA includes two main approaches:
[0101] (i) Agrobacterium-Mediated Gene Transfer.
[0102] See: Klee, H. J. et al. (1987). Annu Rev Plant Physiol 38,
467-486; Klee, H. J. and Rogers, S. G. (1989). Cell Culture and
Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant
Nuclear Genes, pp. 2-25, J. Schell and L. K. Vasil, eds., Academic
Publishers, San Diego, Calif.; and Gatenby, A. A. (1989).
Regulation and Expression of Plant Genes in Microorganisms, pp.
93-112, Plant Biotechnology, S. Kung and C. J. Arntzen, eds.,
Butterworth Publishers, Boston, Mass.
[0103] (ii) Direct DNA Uptake.
[0104] See, e.g.: Paszkowski, J. et al. (1989). Cell Culture and
Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant
Nuclear Genes, pp. 52-68, J. Schell and L. K. Vasil, eds., Academic
Publishers, San Diego, Calif.; and Toriyama, K. et al. (1988).
Bio/Technol 6, 1072-1074 (methods for direct uptake of DNA into
protoplasts). See also: Zhang et al. (1988). Plant Cell Rep 7,
379-384; and Fromm, M. E. et al. (1986). Stable transformation of
maize after gene transfer by electroporation. Nature 319, 791-793
(DNA uptake induced by brief electric shock of plant cells). See
also: Klein et al. (1988). Bio/Technology 6, 559-563; McCabe, D. E.
et al. (1988). Stable transformation of soybean (Glycine max) by
particle acceleration. Bio/Technology 6, 923-926; and Sanford, J.
C. (1990). Biolistic plant transformation. Physiol Plant 79,
206-209 (DNA injection into plant cells or tissues by particle
bombardment). See also: Neuhaus, J. M. et al. (1987). Theor Appl
Genet 75, 30-36; and Neuhaus, J. M. and Spangenberg, G. C. (1990).
Physiol Plant 79, 213-217 (use of micropipette systems). See U.S.
Pat. No. 5,464,765 (glass fibers or silicon carbide whisker
transformation of cell cultures, embryos or callus tissue). See
also: DeWet, J. M. J. et al. (1985). "Exogenous gene transfer in
maize (Zea mays) using DNA-treated pollen," Experimental
Manipulation of Ovule Tissue, G. P. Chapman et al., eds., Longman,
New York-London, pp. 197-209; and Ohta, Y. (1986). High-Efficiency
Genetic Transformation of Maize by a Mixture of Pollen and
Exogenous DNA. Proc Natl Acad Sci USA 83, 715-719 (direct
incubation of DNA with germinating pollen).
[0105] The Agrobacterium-mediated system includes the use of
plasmid vectors that contain defined DNA segments which integrate
into the plant genomic DNA. Methods of inoculation of the plant
tissue vary depending upon the plant species and the Agrobacterium
delivery system. A widely used approach is the leaf-disc procedure,
which can be performed with any tissue explant that provides a good
source for initiation of whole-plant differentiation (Horsch, R. B.
et al. (1988). "Leaf disc transformation." Plant Molecular Biology
Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht). A
supplementary approach employs the Agrobacterium delivery system in
combination with vacuum infiltration. The Agrobacterium system is
especially useful for in the creation of transgenic dicotyledenous
plants.
[0106] There are various methods of direct DNA transfer into plant
cells. In electroporation, the protoplasts are briefly exposed to a
strong electric field, opening up mini-pores to allow DNA to enter.
In microinjection, the DNA is mechanically injected directly into
the cells using micropipettes. In microparticle bombardment, the
DNA is adsorbed on microprojectiles such as magnesium sulfate
crystals or tungsten particles, and the microprojectiles are
physically accelerated into cells or plant tissues.
[0107] Following stable transformation, plant propagation occurs.
The most common method of plant propagation is by seed. The
disadvantage of regeneration by seed propagation, however, is the
lack of uniformity in the crop due to heterozygosity, since seeds
are produced by plants according to the genetic variances governed
by Mendelian rules. In other words, each seed is genetically
different and each will grow with its own specific traits.
Therefore, it is preferred that the regeneration be effected such
that the regenerated plant has identical traits and characteristics
to those of the parent transgenic plant. The preferred method of
regenerating a transformed plant is by micropropagation, which
provides a rapid, consistent reproduction of the transformed
plants.
[0108] Micropropagation is a process of growing second-generation
plants from a single tissue sample excised from a selected parent
plant or cultivar. This process permits the mass reproduction of
plants having the preferred tissue and expressing a fusion protein.
The newly generated plants are genetically identical to, and have
all of the characteristics of, the original plant. Micropropagation
allows for mass production of quality plant material in a short
period of time and offers a rapid multiplication of selected
cultivars with preservation of the characteristics of the original
transgenic or transformed plant. The advantages of this method of
plant cloning include the speed of plant multiplication and the
quality and uniformity of the plants produced.
[0109] Micropropagation is a multi-stage procedure that requires
alteration of culture medium or growth conditions between stages.
The micropropagation process involves four basic stages: stage one,
initial tissue culturing; stage two, tissue culture multiplication;
stage three, differentiation and plant formation; and stage four,
greenhouse culturing and hardening. During stage one, the tissue
culture is established and certified contaminant-free. During stage
two, the initial tissue culture is multiplied until a sufficient
number of tissue samples are produced to meet production goals.
During stage three, the newly grown tissue samples are divided and
grown into individual plantlets. At stage four, the transformed
plantlets are transferred to a greenhouse for hardening where the
plants' tolerance to light is gradually increased so that they can
continue to grow in the natural environment.
[0110] Transient transformation can be effected by any of the
direct DNA transfer methods described above or by viral infection
using modified plant viruses.
[0111] Viruses that have been shown to be useful for the
transformation of plant hosts include cauliflower mosaic virus
(CaMV), tobacco mosaic virus (TMV), and baculovirus (BV).
Transformation of plants using plant viruses is described in, for
example: U.S. Pat. No. 4,855,237 (bean golden mosaic virus, BGMV);
EPA 67,553 (TMV); Japanese Published Application No. 63-14693
(TMV); EPA 194,809 (BV); EPA 278,667 (BV); and Gluzman, Y. et al.
(1988). Communications in Molecular Biology: Viral Vectors, Cold
Spring Harbor Laboratory, New York, pp. 172-189. The use of
pseudovirus particles in expressing foreign DNA in many hosts,
including plants, is described in WO 87/06261.
[0112] Construction of plant RNA viruses for the introduction and
expression of non-viral exogenous nucleic acid sequences in plants
is demonstrated by the above references as well as by: Dawson, W.
O. et al. (1989). A tobacco mosaic virus-hybrid expresses and loses
an added gene. Virology 172, 285-292; French, R. et al. (1986)
Science 231, 1294-1297; and Takamatsu, N. et al. (1990). Production
of enkephalin in tobacco protoplasts using tobacco mosaic virus RNA
vector. FEBS Lett 269, 73-76.
[0113] If the transforming virus is a DNA virus, one skilled in the
art may make suitable modifications to the virus itself.
Alternatively, the virus can first be cloned into a bacterial
plasmid for ease of constructing the desired viral vector with the
foreign DNA. The virus can then be excised from the plasmid. If the
virus is a DNA virus, a bacterial origin of replication can be
attached to the viral DNA, which is then replicated by the
bacteria. Transcription and translation of the DNA will produce the
coat protein, which will encapsidate the viral DNA. If the virus is
an RNA virus, the virus is generally cloned as a cDNA and inserted
into a plasmid. The plasmid is then used to make all of the plant
genetic constructs. The RNA virus is then transcribed from the
viral sequence of the plasmid, followed by translation of the viral
genes to produce the coat proteins which encapsidate the viral
RNA.
[0114] Construction of plant RNA viruses for the introduction and
expression in plants of non-viral exogenous nucleic acid sequences,
such as those included in the construct of the present invention,
is demonstrated in the above references as well as in U.S. Pat. No.
5,316,931.
[0115] In one embodiment, there is provided for insertion a plant
viral nucleic acid, comprising a deletion of the native coat
protein coding sequence from the viral nucleic acid, a non-native
(foreign) plant viral coat protein coding sequence, and a
non-native promoter, preferably the subgenomic promoter of the
non-native coat protein coding sequence, and capable of expression
in the plant host, packaging of the recombinant plant viral nucleic
acid, and ensuring a systemic infection of the host by the
recombinant plant viral nucleic acid. Alternatively, the native
coat protein coding sequence may be made non-transcribable by
insertion of the non-native nucleic acid sequence within it, such
that a non-native protein is produced. The recombinant plant viral
nucleic acid construct may contain one or more additional
non-native subgenomic promoters. Each non-native subgenomic
promoter is capable of transcribing or expressing adjacent genes or
nucleic acid sequences in the plant host and incapable of
recombination with each other and with native subgenomic promoters.
In addition, the recombinant plant viral nucleic acid construct may
contain one or more cis-acting regulatory elements, such as
enhancers, which bind a trans-acting regulator and regulate the
transcription of a coding sequence located downstream thereto.
Non-native nucleic acid sequences may be inserted adjacent to the
native plant viral subgenomic promoter or the native and non-native
plant viral subgenomic promoters if more than one nucleic acid
sequence is included. The non-native nucleic acid sequences are
transcribed or expressed in the host plant under control of the
subgenomic promoter(s) to produce the desired products.
[0116] In a second embodiment, a recombinant plant viral nucleic
acid construct is provided as in the first embodiment except that
the native coat protein coding sequence is placed adjacent to one
of the non-native coat protein subgenomic promoters instead of
adjacent to a non-native coat protein coding sequence.
[0117] In a third embodiment, a recombinant plant viral nucleic
acid construct is provided comprising a native coat protein gene
placed adjacent to its subgenomic promoter and one or more
non-native subgenomic promoters inserted into the viral nucleic
acid construct. The inserted non-native subgenomic promoters are
capable of transcribing or expressing adjacent genes in a plant
host and are incapable of recombination with each other and with
native subgenomic promoters. Non-native nucleic acid sequences may
be inserted adjacent to the non-native subgenomic plant viral
promoters such that said sequences are transcribed or expressed in
the host plant under control of the subgenomic promoters to produce
the desired product.
[0118] In a fourth embodiment, a recombinant plant viral nucleic
acid construct is provided as in the third embodiment except that
the native coat protein coding sequence is replaced by a non-native
coat protein coding sequence.
[0119] Viral vectors are encapsidated by expressed coat proteins
encoded by recombinant plant viral nucleic acid constructs as
described hereinabove, to produce a recombinant plant virus. The
recombinant plant viral nucleic acid construct or recombinant plant
virus is used to infect appropriate host plants. The recombinant
plant viral nucleic acid construct is capable of replication in a
host, systemic spread within the host, and transcription or
expression of one or more foreign genes (isolated nucleic acid) in
the host to produce the desired protein.
[0120] In addition to the above, the nucleic acid molecule of the
present invention can also be introduced into a chloroplast genome
thereby enabling chloroplast expression. A technique for
introducing exogenous nucleic acid sequences to the genome of the
chloroplasts is known. This technique involves the following
procedures. First, plant cells are chemically treated so as to
reduce the number of chloroplasts per cell to about one. Then, the
exogenous nucleic acid is introduced into the cells preferably via
particle bombardment, with the aim of introducing at least one
exogenous nucleic acid molecule into the chloroplasts. The
exogenous nucleic acid is selected by one ordinarily skilled in the
art to be capable of integration into the chloroplast's genome via
homologous recombination, which is readily effected by enzymes
inherent to the chloroplast. To this end, the exogenous nucleic
acid comprises, in addition to a gene of interest, at least one
nucleic acid sequence derived from the chloroplast's genome. In
addition, the exogenous nucleic acid comprises a selectable marker,
which by sequential selection procedures serves to allow an artisan
to ascertain that all or substantially all copies of the
chloroplast genome following such selection include the exogenous
nucleic acid. Further details relating to this technique are found
in U.S. Pat. Nos. 4,945,050 and 5,693,507, which are incorporated
herein by reference. A polypeptide can thus be produced by the
protein expression system of the chloroplast and become integrated
into the chloroplast's inner membrane.
[0121] A number of approaches are known in the art to minimize gene
flow among crops and weeds. Following is a non-limiting description
of such approaches [see also U.S. Pat. Appl. Nos. 20040098760,
20040172678 and Daniell (2002) Nat. Biotech. 20:581]. Other
approaches include male and/or seed sterility (which prevent
outcrossing, volunteer seed dispersal), cleistogamy (in which
pollination occurs prior to flower opening to thereby prevent
outcrossing) and apomixis (seed is from vegetative origin and not
from sexual cross, which controls outcrossing and volunteer seed
dispersal. See U.S. Pat. No. 6,825,397).
Maternal Inheritance
[0122] Maternal inheritance of cytoplasmic organelles is shared by
plant (chloroplasts) and animal (mitochondria) systems. Several
explanations have been offered to explain this phenomenon. It
promotes the invasion of a population by selfish cytoplasmic
factors that are overrepresented within an individual'. In
addition, maternal inheritance of cytoplasmic factors is an
evolutionary mechanism to prevent sexual transmission of disorders
or pathogens associated with males; only the nucleus (not
cytoplasm) is allowed to penetrate the ovule during fertilization
[Gressel J. Molecular Biology in Weed Control (Taylor and Francis,
London, 2002)]. It may also be an extension of the general
suppression of male nuclear genes that takes place in plants after
fertilization [Avni Mol. Gen. Genet. 225, 273-277 (1991)].
[0123] The use of chloroplast genetic engineering to promote
maternal inheritance of transgenes is highly desirable in those
instances involving a potential for outcross among genetically
modified crops or between genetically modified crops and weeds. The
prevalent pattern of plastid inheritance found in the majority of
angiosperms is uniparental-maternal and chloroplast genomes are
maternally inherited in most crops.
[0124] Maternal inheritance of the chloroplast genome is achieved
in plants during the development of the generative cells that form
sperm cells, which then fuse with the female gametes during
fertilization. The generative cells are the result of unequal
divisions during pollen formation and do not receive any
chloroplasts [Hagemann Protoplasma 152, 57-64 (1989)].
[0125] Maternal inheritance of transgenes and prevention of gene
flow through pollen in chloroplast transgenic plants have been
successfully demonstrated in several plant species, including
tobacco and tomato [Daniell Nat. Biotechnol. 16, 345-348; Ruf Nat.
Biotechnol. 19, 870-875 (2001)]. Although chloroplast genomes of
several other plant species, including potato, have been
transformed, maternal inheritance has not been demonstrated in
these studies. However, more than 30 transgenes have been stably
integrated into chloroplast genomes to confer desired plant traits
or for the use of transgenic chloroplasts as biofactories to
produce functional biopharmaceuticals or edible vaccines or
biopolymers [Daniell Trends Plant Sci. 7, 84-91 (2001); Daniell
Curr. Opin. Biotechnol. 13, 136-141].
[0126] Unlike many other containment strategies, the maternal
inheritance approach has already been tested in the field. Scott
and Wilkinson [Nat. Biotechnol. 17, 390-392 (1999)] studied plastid
inheritance in natural hybrids collected from two wild populations
growing next to oilseed rape along 34 km of the Thames River in the
United Kingdom and assessed the persistence of 18 feral oilseed
rape populations over a period of three years. They analyzed
several variables that would influence the movement of chloroplast
genes from crops to wild relatives, including the mode of
inheritance of plastids and incidence of sympatry (the occurrence
of species together in the same area), to quantify opportunities
for forming mixed populations and persistence of crops outside
agriculture limits for introgression. Despite some 0.6-0.7%
sympatry between the crop and weed species, mixed stands showed a
strong tendency toward rapid decline in plant number, seed return,
and ultimately extinction within three years. Thus, Scott and
Wilkinson concluded that gene flow should be rare if plants are
genetically engineered via the chloroplast genome.
[0127] Thus, maternal inheritance of chloroplast genomes is a
promising option for gene containment. Although plastid
transformation remains to be achieved in several major crop
species, chloroplast genetic engineering has now been shown to
confer resistance to herbicides [Daniell Nat. Biotechnol. 16,
345-348 (1998)], insects, disease [DeGray Plant Physiol. 127,
852-862 (2001)], and drought, as well as to produce antibodies
[Daniell Trends Plant Sci. 7, 84-91 (2000], biopharmaceuticals
[Daniell Trends Plant Sci. 7, 84-91 (2001)], and edible vaccines. A
recent report from the European Environment Agency (Copenhagen,
Denmark) recommends chloroplast genetic engineering as a
gene-containment approach [Eastham Genetically Modified Organisms
(GMOs): The Significance of Gene Flow Through Pollen Transfer.
Environmental Issue Report 28 (European Environmental Agency,
Copenhagen, Denmark, 2002)].
[0128] Genome Incompatability
[0129] Many cultivated crops have multiple genomes. Only one of
these crop genomes is compatible for interspecific hybridization
with weeds. For example, the D genome of wheat is compatible with
the D genome of Aegilops cylindrica (bearded goatgrass), a problem
weed in the United States; in contrast, it would be much harder to
achieve interspecific hybridization of the weed with durum wheat,
which has an AABB tetraploid B genome [Gressel. Molecular Biology
in Weed Control (Taylor and Francis, London, 2002)] provided ploidy
level is not an issue. Similarly, there is possibility for gene
transfer from the B genome of Brassica juncea (Indian or brown
mustard) to many Brassica weeds with wild species; however, thus
far most genetic engineering has been carried out Brassica napus,
which has the AACC tetraploid genome and is thus unlikely to be
compatible. The risk of transgenic traits spreading into weeds can
be reduced drastically by releasing only those transgenic lines
with incompatible genomes.
[0130] With the availability of genome information, it might become
possible to engineer crops that have a reduced likelihood of
outcrossing with weeds through incompatibility mechanisms.
[0131] Temporal and Tissue-Specific Control
[0132] Chemically inducible promoters may be used for gene
containment strategies. For example, a chemical could be used to
induce transient expression of a gene conferring herbicide
resistance before a field is sprayed with herbicide. Clearly,
genetic isolation may be possible by restricting expression of a
foreign gene to those times when the crop is not flowering. Such
promoters are currently available (see ref. WO 97/06269).
[0133] An alternative approach to switching on a foreign gene only
when a crop is not in flower would be physically to remove the gene
before flowering occurs. Keenan and Stemmer [Nat. Biotechnol. 20,
215-216 (2002)] suggest that this can be achieved by using
chemically inducible or fruit-specific promoters to activate
expression of a site-specific recombinase, such as Cre, that would
excise a foreign gene before flowering. Such systems can induce Cre
expression and result in the removal of a gene flanked by two lox
sites in either the seed (using a seed-specific promoter) or the
entire plant (using a chemically inducible promoter).
[0134] Transgenic Mitigation
[0135] Another approach for containing gene spread would be to
compromise the fitness of weeds that by introgression have acquired
positive survival traits from crop genes [Gressel Trends
Biotechnol. 17, 361-366 (1999)]. This approach, termed transgenic
mitigation (TM), is based on the premises that (1) tandem
constructs act as tightly linked genes, and their segregation from
each other is exceedingly rare; (2) TM traits are neutral or
positive for crops, but deleterious for weeds; and (3) even mildly
harmful TM traits will be eliminated from weed populations because
such plants compete strongly among themselves and have a large seed
output. Examples of processes that might be targeted by TM include
seed dormancy, seed ripening and shattering, and growth.
[0136] Weed seeds typically exhibit secondary dormancy, with those
from one harvest germinating throughout the following season and in
subsequent years, thereby maximizing fitness (and preventing all
weeds from being controlled by single treatments) while reducing
sibling competition. Abolition of secondary dormancy is neutral to
the crop, but deleterious to weeds. Steber et al. have identified
an Arabidopsis mutant that is insensitive to abscisic acid and
totally lacks secondary dormancy. Such genes associated with
dormancy (engineered or mutated) may be used for TM [Genetics 149,
509-521 (1998)].
[0137] Another characteristic of weedy plants is that they disperse
their seeds over a period of time, and most of their ripe seeds
shatter to the ground, ensuring continuity. As a result, uniformly
ripening and anti-shattering genes are harmful to weeds but neutral
for crops, whose seeds ripen uniformly and do not easily shatter;
in fact, anti-shattering genes are even advantageous for oilseed
rape, which still has shattering and volunteer weed problems. Only
weed-free "certified" seed is sown, thereby eliminating transgenic
weed seed. It is thought that the changing hormone balance in the
abscission zone of a seed influences shattering propensity.
Cytokinin overproduction may delay shattering. A SHATTERPROOF gene
has been recently isolated from Arabidopsis that prevents seed
shattering by delaying valve opening on the silique. This may be an
ideal strategy for the closely related oilseed rape.
[0138] Dwarfing has been especially valuable in generating "green
revolution" varieties of rice and wheat and brought
self-sufficiency to India and China. However, the dwarfing trait is
disadvantageous for weeds, because they can no longer compete with
the crop for light. Genetically engineered height reduction is
possible by preventing biosynthesis of gibberellins33. In addition,
a defective gibberellic acid receptor gene has been isolated that
confers gibberellin instability by competing with the native
receptor, thereby inducing dwarfing.
[0139] Promoter sequence information (e.g., SEQ ID NO: 58) allows
the generation of plants with increased expression of the
polypeptides of the present invention by modifying the promoter
sequence of the cultivated plant. Thus for instance, "knocking in"
technology or mutagenesis (e.g., chemical or radiation), can be
used to directly or indirectly generate plants with up-regulated
expression of the polypeptides of the present invention.
[0140] It will be appreciated that by localizing the cwp1 gene of
the present invention to tomato chromosome 4 of wild Lycopersicon
spp. and finer mapping to an introgression smaller than a
chromosomal fraction extending from telomeric marker TG464 to
centromeric marker CT173, it is possible to generate cultivated
tomato plants with increased cuticular water permeability using
classical breeding techniques. For example, Lycopersicon esculentum
plant may be hybridized with wild
[0141] Lycopersicon spp. plant. The fruits of the Lycopersicon
esculentum plants are then allowed to ripen and the hybrid (F1)
seeds are collected. The collected F1 seeds are then planted and F1
plants are grown and allowed to self-pollinate. Selfing may be
continued for at least one additional generation or the F1 plants
may be crossed to esculentum parental plant. Fruits from selfed or
backcrossed generations are allowed to remain on the vine past the
point of formal ripening, as determined by change of fruit color
and screened for (i) the presence of natural dehydration; and (ii)
the above described introgression. For example, minimal
introgressions containing the wild species allele can be limited to
less than 10 cM, less than 5 cM, less than 2 cM and less than 1 cM
by using the following markers, CT199, TG163, CT61, and within the
region spanning CT61 and TG464. For example markers which can be
used to generate a minimal introgression which still enable
increasing cuticular water permeability include any of the
sequences derived from the ends of the BACs shown in FIG. 3a.
[0142] Thus, the present invention also provides a cultivated
tomato plant having a genome comprising an introgression derived
from a wild Lycopersicon spp. said introgression comprising a
portion of chromosome 4 of said Lycopersicon spp. smaller than a
chromosomal fraction extending from telomeric marker TG464 to
centromeric marker CT173, said introgression being capable of
increasing cuticular water permeability of the cultivated tomato
plant.
[0143] Once cultivated and genetically modified plants of the
present invention are generated (as described above) dehydrated
fruits can be generated as follows.
[0144] Fruits are allowed to remain on the vine past normal point
of ripening. The appearance of dehydration as evidenced by
wrinkling of the fruit skin indicates reduced water content in the
fruit. Once dehydrated fruits are obtained they may be collected.
Alternatively, fruits are collected from the vine and subsequently
allowed to dehydrate (e.g., sun-drying, described in length in the
Background section.
[0145] Thus, the present invention provides polynucleotides and
polypeptides which govern cuticular water permeability in plants
expressing same and methods of using these for producing dehydrated
fruits of commercially valuable crop plants.
[0146] As used herein the term "about" refers to .+-.10%.
[0147] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0148] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0149] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan
J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn.
(1994); Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W. H. Freeman and Co., New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R.
I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Materials and Methods
[0150] Plant Material and Measurements
[0151] A set of near-isogenic introgression lines derived from a
backcross breeding program based on the inter-specific
hybridization of L. esculentum (E) and the wild species L. hirsutum
(H), distinguished by the trait of fruit dehydration was developed,
as described previously (WO 0113708) as summarized here. Plants of
E breeding line 1630 were pollinated with wild species H (LA1777).
Hybrid F.sub.1 plants were self-pollinated, generating F.sub.2
seeds. Three F.sub.2 plant were selected based on their high sugars
content when ripe. F.sub.3 seeds were sown and ten plants of each
of the F.sub.3 plants of these three F.sub.2 selections were grown,
and fruit was allowed to remain on the vine past the normal stage
of ripening and harvest. Among the F.sub.3 plants one plant
(F3-203-10) showed the characteristic of sign of fruit dehydration,
evidenced by wrinkling of fruit skin. A pedigree breeding program
was developed consisted of selfing this F.sub.3 individual until
the F.sub.4 generation followed by intense selection for fruit
dehydrating rate. Thereafter, plants were backcrossed to the E
breeding line, with the product of this cross being selfed for four
additional generations to produce a BC1F4 population. Dehydrating
individuals from this population were subjected to another
backcross to E, producing hybrid plants that were present with the
trait. Two F.sub.2 populations (2394 and 2395) were constructed
from these F.sub.1 individuals.
[0152] Initially the selection procedure was based on the phenotype
of fruit dehydration and microcracks on the fruit cuticle.
Following the development of molecular markers linked to the trait,
selection was performed according to the genotype. Cleaved
Amplified Polimorphic (CAPS) marker were used as the molecular
markers. CAPS were developed using a specific PCR product that was
cut by an endonucleases enzymes (see at "DNA Analysis" further
below).
[0153] Plants were grown in 15-1 pots in a greenhouse, according to
standard methods, as previously described (Miron and Schaffer,
1991). Fruit mean weight and dehydration rate were determined by
picking and weighing five mature red fruits from each plant,
placing them on a net-table at room temperature (about 25.degree.
C.) and weighing them every 2-3 days. The presence of microfissures
(MF) on the fruit cuticle was verified by either magnifying glass
(2.times.) or binocular microscope (10.times.).
[0154] DNA Analyses
[0155] Genomic DNA was extracted according to Fulton et al. (1995).
CAPS (Cleaved Amplified Polymorphism) markers were developed from
RFLP markers selected from high-density tomato map (Tanksley et al.
1992), as follows. BlueScript plasmid vectors (Stratagene)
containing tomato DNA inserts representing the selected RFLP
markers were kindly provided by the Tomato Genome
[0156] Center in Weizmann Institute of Science, Rehovot, Israel.
Genomic DNA insertion segments were partially sequenced at the DNA
Analysis Unit in the Hebrew University, Jerusalem, Israel, using T7
and SP6 primers (SEQ ID NO: 1 and 2, respectively). According to
these sequence analysis results, sequence-specific PCR primers were
designed using the Primer Express Program, version 1.0 (Perkin
Elmer Biosystems). A total of approximately 20 markers were
designed and these were tested to determine the existence of
polymorphisms between the L. esculentum and L. hirsutum parental
genotypes as well as between the tomato lines differing in the L.
hirsutum-derived trait.
[0157] Following are PCR primers for two markers--TG163 and TG587,
representing positions on chromosome 4.
TABLE-US-00001 TG163 F: (SEQ ID NO: 3) 5'-TGCAATCCCGAACATGAAGAC-3'
TG163 R: (SEQ ID NO: 4) 5'-CCTTCTGGTCGCATCTGTGTCT-3' TG587 F: (SEQ
ID NO: 5) 5'-TCAGGGTGAGGGGTAATAATTGAG-3' TG587 F: (SEQ ID NO: 6)
5'-GCTTAAAACTCAAGTCTCCTCGCA-3'
[0158] The amplification reactions were performed in an automated
thermocycler (Mastercycle Gradient, Eppendorf, Germany) using
thermostable Taq DNA polymerase (SuperNova Taq Polymerase, JMR
Products, Kent, UK). The reactions were carried out in 25 .mu.l
final volume that contained 10.times. reaction buffer, 0.125 mM of
each deoxynucleotide, 0.5.mu. of each primer, 2.5 Unit of Taq
polymerase and 50-100 ng of tomato genomic DNA. The conditions were
optimized for the annealing temperature for each set of primers and
the product fragment size. To identify restriction endonucleases
that would generate a polymorphism between the L. esculentum and L.
hirsutum alleles, reaction were carried out in 10 .mu.l final
volume containing 3.5 .mu.l of PCR product, 1 .mu.l of 10.times.
concentrated restriction enzyme buffer, and 1-3 unit of the
appropriate restriction endonuclease. The digestion products were
analyzed on 1% gels. DraI and HinF1 were found to be appropriate
for TG163 and TG587, respectively, and were used on the segregating
populations. A similar procedure was applied for the design of the
others CAPS markers.
[0159] All BACS (Bacterial Artificial Chromosomes) that were used
in this work were provided from Clemson University Genomic
Institute (Clemson, N.C., USA), using the Tomato Heinz 1706 BAC
Library Filters (LE_HBa). Tomato BAC library filters were screened
for a specific BAC clone by a radioactive probe, as described
below. that was labeled using the NEBlot.TM. Kit (New England
BioLabs inc. #N1500S) and according to the supplier's instructions.
Labeled BAC colonies on the filter were detected using a
phosphor-imager device (FLA-5000; FujiFilm). BAC plasmids were
purified from the matching E. coli strains using the QIAGEN.RTM.
Maxi Plasmid Purification Kit (#12263). For "Chromosome Walking"
procedure, BACs ends were sequenced using the SP6 and T7 primers
and a PCR product was developed according to the BACs end sequence.
The new purified PCR product was radioactive labeled and was used
for another round of tomato filter colonies detection.
[0160] LE_HBa 37B8 BAC clone (Clemson University Genomic Institute,
Clemson, N.C. USA) was sub-cloned into the BlueScript II ks+ vector
(Stratagene) and sequenced. The 15 kb section was completely
sequenced by developing primers and cloning by PCR and sequencing
the relevant sections, as described above. DNA sequences were
analyzed using the NCBI nucleic acid and translated protein
databases by using the BLAST software (Altschul et al., 1990).
[0161] RNA and Quantitative RT-PCR Analyses
[0162] For the preparation of cDNA, total RNA was extracted, as
previously described (Miron et al, 2002). Total RNA was used as a
template for first strand cDNA synthesis with the Super-script II
pre-amplification system reverse transcriptase kit (Gibco BRL,
LifeTechnologies, UK) at 42.degree. C. according to the supplier's
instructions.
[0163] PCR Primers
[0164] Specific primers with short amplicons for on-line
quantitative PCR were designed with the Primer Express Program,
version 1.0 (Perkin Elmer Biosystems) based on the sequences
derived from the BAC sequencing of the three ORFs: 1) ZINC gene,
forward, 5'-AATAATGCGAATCGAATCACTA-3' (SEQ ID NO: 7) and reverse,
5'-AAGGCTAAATCTCCTCCTTTCT-3' [SEQ ID NO: 8, amplicon 140 bp (SEQ ID
NO: 9)]. 2) DBP gene, forward, 5'-TGGATAAGCGGACGACTCTATTG-3' (SEQ
ID NO: 10) and reverse, 5'-CTGTTGTTTGGGAAGTGGCTTCT-3' [SEQ ID NO:
11, amplicon 116 bp (SEQ ID NO: 12)]. 3) PUT gene, forward,
5'-CTCTCCTTGGCCCAAGGCTCAA-3' (SEQ ID NO: 13) and reverse,
5'-CAGCTTTAGTGGTATCTCTCATCA-3' [SEQ ID NO: 14, amplicon 205 bp (SEQ
ID NO: 15)]. Actin was used as a reference gene, with the following
primers, based on Gene bank accession No. BF096262: forward,
5'-CACCATTGGGTCTGAGCGAT-3' (SEQ ID NO: 16) and reverse,
5'-GGGCGACAACCTTGATCTTC-3' [SEQ ID NO: 17, amplicon 251 bp (SEQ ID
NO: 18)].
[0165] The cDNA was used as template for quantitative PCR
amplification on the GeneAmp 5700 Sequence Detection System (PE
Biosystems) using SYBR Green Master Mix containing AmpliTaq Gold,
According to manufacture's instructions (PE Biosystems). The
thermocycler was programmed for 40 cycles for all reactions, with
the first step of denaturation at 95.degree. C. for 30 sec, the
annealing temperature of 62.degree. C. for 15 sec, and extension
temperature of 72.degree. C. for 30 sec. Data acquisition was done
at 77.degree. C. for 30 sec. Preliminary dissociation analyses of
the PCR products showed that product remaining above 77.degree. C.
was the specific PCR product. Standard curves containing
logarithmically increasing known cDNA levels were run with each set
of primers, in addition to the actin primers for normalization. All
real time PCR products were tested on 2% agarose gel and were sent
for sequencing for identity approval. Cloning of full-length put
gene--Full length sequence of the putative protein gene (put) was
amplified from cDNA that was extracted from HH line fruit (10 days
after anthesis), using the following primers: Put forward,
5'-GTAGTACTATATAAACCATGTGAG-3' (SEQ ID NO: 19) and reverse,
5'-CATATGTTGACATATCTAATG-3' (SEQ ID NO: 20). The full length gene
[(SEQ ID NO: 20), 930 bp) was cloned to pGEM-T easy vector
(promega) using T-A cloning procedure, and then was sub-cloned to
BlueScript II ks+ vector (Stratagene) using the EcorI (NEB #R0101)
endonuclease. The put gene (SEQ ID NO: 21) was again sub-cloned
between the cauliflower 35S promoter and the n-terminator sites of
the pBIN PLUS binary vector (Ghosh et al., 2002) using the XhoI
(NEB #R0146) and XbaI (NEB #R0145) endonucleases.
[0166] Trangenic Plants
[0167] Constructed vector comprising the put gene under the 35S
promoter was transformed into E. coli (strain DH5alpha,
Stratagene), and then were retransformed into EHA105 Agrobacterium
electro-competent cells using the method described by Walkerpeach
and Velten (1994). Plasmids were prepared using a mini-prep kit
(Qiagen #12143) and re-transformed to pBIN PLUS for sequencing to
insure the absence of deletions and other cloning inaccuracies.
[0168] Tomato transformation experiments were carried out using the
cv MicroTom as described by Meissner et al. (1997) and cv. MP1 as
described by Barg et al. (1997). Transgenic shoots were rooted on
Murashige and Skoog basal medium (Duchefa, Haarlem, The
Netherlands) supplemented with 1 mg L.sup.-1 zeatin (Duchefa
#Z0917), 100 mg L.sup.-1 kanamycin and 100 mg L.sup.-1 Chlaforan.
Standard practices of growing the transformed plants are carried
out.
Example 1
Inheritance Analysis of the Dehydration Trait
[0169] The inheritance of the trait of appearance of micro-fissures
(MF) on the fruit skin was determined in two independent
segregating F.sub.5 populations (lines 2394 and 2395) based on a
cross between a standard small fruited cultivar (line 1815) and an
advanced introgression line exhibiting the trait of dehydration
(line 1881). The distribution pattern of the appearance of
micro-fissures in the segregating populations was according to a
ratio of 3:1 for Micro-fissured: standard cuticle, with chi-square
probability values of 0.546 and 0.864 for 2394 and 2395
populations, respectively (Table 1, below).
TABLE-US-00002 TABLE 1 Segregation pattern of microfissure and
dehydration phenotypes in segregating populations 2394 and 2395.
2395 2394 Phenotype No Probability Phenotype No Probability N 16
0.272 N 15 0.234 Y 39 0.709 Y 49 0.765 Total 55 1.000 Total 64
1.000 X.sup.2 value: 0.029 X.sup.2 value: 0.424 Prob of X.sup.2:
0.864 Prob of X.sup.2: 0.546 N - non-dehydrating; Y - dehydrating;
No - number of individuals in population.
[0170] This distribution pattern is characteristic for a single
gene inheritance with dominant/recessive allelic relations.
[0171] The trait of fruit dehydration (CWP) segregated according to
a 3:1 ratio in population 2394 while in population 2395 segregation
was according to a 1:2:1 ratio with approximately half of the
population dehydrating but at an intermediate rate of dehydration.
Therefore, it is concluded that the allelic relations are either
completely dominant or semi-dominant, depending on the genetic
background of the population (FIGS. 1a-1b). From the above it can
be concluded that the trait of fruit CWP is inherited as a single
gene trait, which is termed herein as Cwp.
Example 2
Fine Mapping of Cwp Gene
[0172] Based on the high-density tomato RFLP map (Tanksley et al.
1992) a set of CAPS (Cleaved amplified polymorphism) markers were
designed. Loci representing various genomic positions, including
markers linked to QTLs for reticulated epidermis (Fulton, et al.,
2000, markers TG464, TG477, CT68 and TG68 localized on chromosomes
4, 6, 8, 12, respectively) were investigated for analysis of
linkage with the trait of micro-fissures. Each polymorphic
PCR-based molecular marker was applied to both parents and a set of
48 F.sub.2 individuals segregating for the trait.
[0173] Based on the initial set of markers the Cwp gene was mapped
to the telomeric portion of chromosome 4, linked to CT199 marker by
an estimated distance of approximately 3 cM (2 recombination events
in 96 gametes, FIG. 2a). For finer mapping of the telomeric portion
of chromosome 4 an additional group of CAPS markers were designed
for a cluster of markers located throughout this chromosomal
segment. The chromosomal introgression segment from the L. hirsutum
parent was localized between the TG163 and TG464 markers (FIG. 2b).
This introgression represents the L. hirsutum segment in the
near-isogenic line that was used as the dehydrating donor parent in
this analysis.
[0174] In order to further narrow down the introgression size a
larger F.sub.2 population (over 200 individuals) was investigated
with PCR-based markers between CT199 and TG464 markers. A closely
linked cluster (<1.5 cM) of molecular markers was defined as
flanking the Cwp gene (FIG. 2c) and based on this study the Cwp
gene was located between TG464 and CT61 (0.5 cM).
Example 3
Positional Cloning of Cwp Gene
[0175] The localization to this small introgression allowed for the
positional cloning of Cwp. For this purpose an additional 3500
segregating progeny (7000 gametes) of a heterozygous individual
derived from the near-isogenic line were subjected to CAPS marker
analysis with the marker TG464 and CT61, revealing 12 recombinants
(0.34 cM compared with 0.5 cM between the same markers in the
"first round" of fine mapping). A set of 5 contiguous BACs bridging
the linked markers TG464 and CT61 was identified and assembled
using the chromosome walking technique. In brief, this was
accomplished by sequencing the BAC end and using the BAC end as a
probe to identify a contiguous BAC.
[0176] In order to place the new BAC with respect to the
introgression, and to produce a higher resolution map polymorphic
CAPs for the two species were developed and the recombinants were
tested for these new markers.
[0177] The 5 contiguous BACs created a bridge between CT61 and
TG464 CAPS markers (FIG. 3a). For each of the 12 recombinant plants
10 selfed progenies were grown, genotyped with the appropriate
segregating markers and analysed for dehydration and the appearance
of micro-fissures. Of the 12 recombination events initially
identified, 3 were further localized between the two ends of BAC
37B8 (FIG. 3a-area restricted by two broken lines) indicating that
Cwp was located in the 37B8 BAC. To further resolve the
recombination events, BAC 37B8 was sub-cloned and the smaller
fragments were assembled in order and a segment of approximately
15,000 bp (15 kb) was identified, within which the Cwp gene was
located. (FIG. 3b, mapping and sub-cloned contigs data at a lower
resolution are not presented).
Example 4
Bioinformatical Analysis of the Candidate Genes
[0178] The segment of 15 kb in BAC 37B8 described in Example 3 was
sub-cloned into the Bluescript vectors (Stratagene), sequenced and
assembled using the SEQUENCHER software package (Gene Codes
Corporation).
[0179] A bioinformatics analysis of the 15 kb sequence after
analysis by the BLAST program (BLASTP, NCBI,
http://www(dot)ncbi(dot)nlm(dot)nih(dot)gov) revealed three
candidate open reading frames (ORFs, FIG. 4). The first ORF showed
a similarity to a protein of unknown function from Arabidopsis
thaliana (GenBank Accession No. NP 189369.1) (protein
Identity--44%, Homology--61%). This protein has two domains. The
first one is RING-finger domain (rpsBLAST--NCBI Conserved Domain
Search), a specialized type of Zn-finger of 40 to 60 residues that
binds two atoms of zinc, and is probably involved in mediating
protein-protein interactions (Borden, 1998). It was identified in
proteins with a wide range of functions, such as viral replication,
signal transduction, and development. It has two variants, the
C3HC4-type and a C3H2C3-type (RING-H2 finger), which have different
cysteine/histidine pattern. The other domain is DUF23 and it is
domain of unknown function. It is part of a family that consists of
an approximately 300 residue long region found in C. elegans and
drosophila proteins. This region contains several conserved
cysteine residues and several charged amino acids that may function
as catalytic residues. This ORF was termed "Zinc". Interestingly,
the homology of the tomato Zinc to the Arabidopsis homolog is not
at the site of the "Ring finger" but only at DUF23 one and the
"Ring finger" domain region is missing at Zinc tomato gene.
[0180] The second ORF showed similarity to a DNA-binding
bromodomain-containing protein (Arabidopsis thaliana GenBank
Accession No. NP 974153.11, protein identity--37%, Homology 56%).
This gene is a part of a DNA binding protein family that is
associated with acetylation regulation of proteins, DNA and
chromatin and are part of histone acetyltransferase regulation
(Dhalluin et al., 2000). We termed this gene "DBP" (DNA Binding
Protein).
[0181] The third ORF had similarity to a protein described merely
as an "expressed protein" (Arabidopsis thaliana At4g38260, GenBank
Accession No. NP_568038.1) (protein Identity--48%, homology--67%).
It contains a domain of unknown function (DUF833). It is part of a
family that is found in eukaryotes, prokaryotes and viruses and has
no known function. One member has been found to be expressed during
early embryogenesis in mice (Halford et al., 1993). This gene was
termed "PUT" (putative). None of these three candidate genes showed
any similarity or homology to genes that participate in known steps
of cuticle biosynthesis metabolism.
Example 5
Expression Analysis of Candidate Genes
[0182] In order to determine which of the three candidate genes is
associated with tomato fruit cuticle development, the expression
level of each of the three genes in the near-isogenic lines
differing in their Cwp allele was measured [L. hirsutum dehydrating
allele, (HH), and L. esculentum not dehydrating allele, (EE), FIGS.
5a-5b]. mRNA from ovaries and fruits of the following stages was
extracted: anthesis, 5 and 15 days after anthesis, and at immature
green, mature green and breaker developmental stages (FIGS. 5a-5b).
Fruit specimens were taken from the same segregating population
that was used for the positional cloning procedure. The expression
of each of the genes was examined by RT-PCR. DBP was expressed only
at the ovary stage and equally in both genotypes (HH and EE)
thereby indicating that the expression of this gene is not
associated with the phenotypic trait (FIG. 5b). Expression of the
Zinc gene was not observed at any fruit stage in either genotype,
similarly indicating that its expression is not associated with the
trait of dehydration (not shown).
[0183] Only PUT was expressed in the young stages of the developing
fruit and, furthermore, was expressed differentially only in fruit
of the dehydrating genotypes with the L. hirsutum allele for Cwp
(HH) (FIG. 5a). The highest expression observed in this study was
at the fruitlet stage of 15 days after anthesis.
[0184] In order to confirm the differential expression pattern of
the PUT gene, the expression of this gene in additional populations
derived from the M82 tomato industry cultivar was analyzed. One
population was an F.sub.2 population derived from a heterozygote
individual, originating from the hybridization of a dehydrating
line (line 2168) with the M82 determinate cultivar. We examined the
expression of all three segregating genotypes (HH, HE, EE), at the
stage of 5-15 days after anthesis (the stage with the highest
expression levels in the first expression analysis). As shown in
FIG. 6, a classical Mendelian expression pattern of PUT gene was
found, with the HH genotypes showing highest expression levels, the
heterozygous HE individuals showing approximately half the
expression level, and the EE genotypes lacking expression (first
three bars in FIG. 6).
[0185] In addition, the expression of the PUT gene was examined in
another NIL (near isogenic line) population the introgression line
4.4 derived from the interspecific hybridization of L. esculntum
(M82) and an additional wild species L. pennellii, containing the
analogous introgression as the L. hirsutum-derived genotypes
described here (Eshed and Zamir, 1994). This population represents
another wild allele of the PUT gene, and the fruit of IL4.4 also
show micro-fissures and dehydrate. Similar to the L. hirsutum
derived populations, the L. penellii derived introgression
containing the L. pennelii allele for Cwp (IL 4.4) showed
expression of the PUT gene in the young fruitlets, compared to M82
(FIG. 6, last two bars).
[0186] Transgenic Tomato Plants Expressing the PUT Gene
[0187] In order to show that the expression of the Put gene is
associated with the unique cuticular development trait transgenic
tomato plants were developed with the PUT gene under the control of
the 35S promoter (using the pBIN PLUS binary vector as described).
The phenotypic trait is observed in the transgenic plants,
indicating that the expression of Put is associated with the
trait.
[0188] In order to determine the gene dosage of the individual
segregating T1 plants derived from the selfing of the initial
transgenic plants 50-70 seed from each T1 plant were seeded on 1/2
MS medium containing 100 mg/ml Kanamycin. Following germination,
the percentage of seedlings with normal roots was determined. When
100% of the seedlings exhibited normal roots growth, that T1 plant
was considered homozygous for the transgene. Approximately 75% T2
seedlings with normal roots indicated that the T1 plant was
heterozygous for the transgene. Other ratios, though not observed
here, might indicate the existence of two or more unlinked copies
of the transgene. Sixteen T1 individuals from two independent T1
segregating populations were analyzed to determine their allelic
makeup. These classifications were then used to determine the
relationship between allelic dosage of the PUT gene and the
phenotypic traits.
[0189] As shown in FIGS. 7a-7b, the phenotypic trait of
microfissures (MF-) on the fruit cuticle was already observed at
the T.sub.0 generation. From 20 independent T.sub.0 transgenic
individuals 4 plants (MF1-1, MF1-4, MF1-8, MF1-12) showed varying
levels of MF on fruit skin. In addition, these transgenic plants
showed higher dehydrating rate than the wild type fruit (FIG.
7b).
[0190] Two segregating T.sub.1 populations were grown and tested
for MF presence and dehydrating rates. FIGS. 8a-8b show the effect
of the PUT transgene copy number on micro-fissures severity (scale
between 1 to 5, FIG. 8a) and weight loss percentage of the fruit
(after 14 days at room temperature, FIG. 8b). The number of PUT
gene copies were determined as in the materials and methods
section.
[0191] FIGS. 9a-9b show a comparison between transgenic tomato
individuals (T.sub.1 generation) expressing no copies, analogous to
wild type, and two copies of the PUT gene from the wild tomato
species Solanum habrochaites S. FIG. 9a-Scanning electron
micrograph presenting the intact surface of the fruit from an
individual with no copies of the PUT gene (0 copies) and the
micro-fissured fruit of an individual with two copies of the
transgene. FIG. 9b-Drying rate comparison between an individual
with no copies of the PUT gene (0 copies) and an individual with
two copies (2 copies).
[0192] These results clearly show that the expression of the PUT
gene is causal to the phenotype of microfissures and fruit
dehydration.
[0193] Phylogenetic analysis based on gene sequences indicates that
cwp is part of a gene family represented by three members in
Arabidopsis (FIGS. 10a-10b). There is an additional tomato
homologue (CWP2) showing 30% homology to the Lecwp1 gene, which is
indeed expressed in cultivated tomatoes (EST No. AW621927).
[0194] Interestingly, this homologue maps to tomato chromosome 2-1
where there is a reported QTL for tomato fruit epidermal
reticulation (Frary et al, 2004). Developing fruit of the
solanaceous cultivated pepper (Capsicum annum) also express a cwp
homologue highly similar (87%) to the Lecwp1 gene in its epidermal
tissue and pepper fruit are characterized by the horticultural
problem of post-harvest water loss, as well as by the desirable
trait of fruit dehydration in paprika cultivars. Therefore it is
likely that homologues of the CWP gene may also contribute to
cuticular modification and water permeability.
[0195] These results indicate that the expression of the cwp gene
leads to a structurally modified cuticle (based on weight and TEM)
which presumably undergoes fissuring during fruit expansion due to
reduction in elasticity. However, this phenomenon is observed only
in fruit with a highly developed fruit cuticle such as the
astomatous thick skinned cultivated tomato and is not apparent in
fruit of the wild species, with their characteristic thinner
cuticle. The deposition of cuticular components during cultivated
tomato fruit development undergoes a surge during the transition
from the immature to the mature green stage and it is reasonable
that the this coincides with the observation of the microfissure
phenotype.
[0196] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0197] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications and GenBank Accession
numbers mentioned in this specification are herein incorporated in
their entirety by reference into the specification, to the same
extent as if each individual publication, patent or patent
application or GenBank Accession number was specifically and
individually indicated to be incorporated herein by reference. In
addition, citation or identification of any reference in this
application shall not be construed as an admission that such
reference is available as prior art to the present invention.
REFERENCES
Other References are Cited in the Application
[0198] Aharoni A Dixit S Jetter R Thoenes E Arkel G Pereira A
(2004) The SHINE clade of AP2 domain transcription factors
activates wax biosynthesis, alters cuticle properties and confers
drought tolerance when overexpressed in Arabidopsis. Plant Cell (in
press). [0199] Altschul S F Gish W Miller W Myers E W Lipman D J
(1990) Basic local alignment search tool. J. Mol. Biol. 215:
403-410. [0200] Arts M G M Keijzar C J Steikema W J Pereira A
(1996) Molecular characterization of the CER1 gene of Arabidopsis
involved in epicuticular wax biosynthesis and pollen fertility.
Plant Cell 7: 2115-2127. [0201] Baker, (1982) In: The Plant
cuticle, Editors: D. F. Cutler, K. L. Alvin and C. E. Price,
London, Academic Press, [0202] Bakker J C (1988) Russeting (cuticle
cracking) in glasshouse tomatoes in relation to fruit growth. J.
Hort. Sci. 63 (3): 459-463. [0203] Barg R Pilowsky M Shabtai S
Carni N Alejandro D Szechtman BD Salts Y (1997) The TYLCV-tolerance
tomato line MP-1 is characterized by superior transformation
competence. J. Exp. Bot. 48 (316): 1919-1923. [0204] Blee E Schuber
F (1993) Biosynthesis of cutin monomers: involvement of a
lipoxygenase/peroxygenase pathway. Plant J. 4: 113-123. [0205]
Borden K L (1998) RING fingers and B-boxes: zinc-binding
protein-protein interaction domains. Biochem. Cell Biol. 76(2-3):
351-358. [0206] Chen X Goodwin S M Boroft V L Liu X Jenks M A
(2003) Cloning and characterization of the WAX2 gene of Arabidopsis
involved in cuticle membrane and wax production. Plant Cell 15:
1170-1185. [0207] Considine J Brown K (1981) Physical aspects of
fruit growth. Plant Physiol. 68: 371-376. [0208] Conter S D Burns E
E Leeper P W (1969) Pericarp anatomy of crack-resistant and
susceptible tomato fruits. J. Amer. Soc. Hort. Sci. 94: 136-137.
[0209] Dhalluin C Carlson J E Zeng L He C Aggarwal A K Zhou M M
(2000) Structure and ligand of a histone acetyltransferase
bromodomain. Nature 399(6735): 491-496. [0210] Emmons C L W Scott J
W (1997) Environmental and physiological effects on cuticle
cracking in tomato. J. Amer. Hort. Sci. 122 (6): 797-801. [0211]
Ehret D L Helmer T Hall J W (1993) Cuticle cracking in tomato
fruit. J. Hort. Scien. 68 (2) 195-201. [0212] Eshed, Y and Zamir D.
(1995) n introgression line population of Lycopersicon pennellii in
the cultivated tomato enables the identification and fine mapping
of yield associated QTL. Genetics 141:1147-1162. [0213] Flebig A
Mayfield J A Milley N L Chau S Fischer R L Prauss D (2000)
Alterations in CER6, a gene identical to CUT1, differentially
affect long-chain lipid content on the surface of pollen and stems.
Plant Cell 12: 2001-2008. [0214] Fulton T M, Chunwongse J, Tanksley
S D (1995) Microprep protocol for extraction of DNA from tomato and
other herbaceous plants. Plant Mol Biol Rep 13: 207-209. [0215]
Fulton T M Grandillo S Beck-Bunn T Fridman E Frampton A Lopez J
Petiard V Uhling J Zamir D Tanksley S D (2000) Advanced backcross
QTL analysis of a lycopersicon esculentum x lycopersicon
parviflorum cross. Theor. Appl. Genet. 100: 1025-1042. [0216] Ghosh
S B Nagi L H S Ganapathi T R Khurana P S M Bapat V A (2002) Cloning
and sequencing of potato virus Y coat protein gene from Indian
isolate and development of transgenic tobacco for PVY resistance.
Curr. Sci. 82: 855-859. [0217] Halford S Wilson D I Daw S C Roberts
C Wadey R Kamath S Wickremasinghe A Burn J Goodship J Mattei M G
(1993) Isolation of a gene expressed during early embryogenesis
from the region of 22q11 commonly deleted in DiGeorge syndrome.
Hum. Mol. Genet. 2(10):1577-1582. [0218] Holloway G J (1982)
Structure and histochemistry of plants cuticular membranes. In:
Cutler D F Cutler K L A Price C E, The Plant Cuticle. Academic
Press, London, UK, pp. 33-44. [0219] Hooker T S Millar A A Kunst L
(2002) Significance of the expression of the CER6 condensing enzyme
for cutucular wax production in Arabidopsis. Plant Physiol. 129,
1568-1580. [0220] Kolattukudy P E (1980) Biopolyester membranes of
plants: cutin and suberin. Science 208 (30): 990-999. [0221]
Koornneef M Anhart C J Theil F (1989) A genetic and phenotypic
description of eceiferum (cer) mutants of Arabidopsis thaliana. J.
Hered. 80: 118-122. [0222] Koske T J Pallas J E Jones J B (1980)
Influence of ground bed heating and cultivar on tomato fruit
cracking. Hortscience 15 760-762. [0223] Kunst L Samuels A L (2003)
Biosynthesis and secretion of plants cuticular wax. Prog. Lipid.
Res. 42(1): 51-80. [0224] Kurata T Kawabata A C Sakuradani E
Shimizu S Okada K Wada T (2003) The yore-yore gene regulates
multiple aspects of epidermal cells differentiation in Arabidopsis.
Plant J. 36: 55-66. [0225] Lownds N K Banaras M Bosland P W (1993)
Relationships between postharvest water loss and physical
properties of pepper fruit (Capsicum annuum L.). HortScience 28
(12): 1182-1184 [0226] Meissner R Jacobson Y Melamed S Levyatuv S
Shalev G Ashri A Elkind Y Levy A (1997) A new model system for
tomato genetics. Plant J 12: 14651472. [0227] Millar A A Clemens S
Zachgo S Giblin E M Taylor D C Kunst L (1997) CUT1, an Arabidopsis
gene required for cuticular wax biosynthesis and pollen fertility,
encodes a very-long-chain fatty acid condensing enzyme. Plant J.
12: 121-131. [0228] Miron D Schaffer A A (1991) Sucrose phosphate
synthase, sucrose synthase and invertase activity in developing
fruit of lycopersicon esculentum Mill. And Bonpl. Plant Physiol.
95: 623-627. [0229] Miron D Petreikov M Carmi N Shen S Levin I
Granot D Zamski E Schaffer A A (2002) Sucrose uptake, invertase
localization and gene expression in developing fruit of
lyconpersicon esculentum and the sucrose-accumulating lycopersicon
hirsutum. Physiol. Plant. 115: 35-47. [0230] Monforte A J Freidman
E Zamir D Thankslry S D (2001) Comparison of a set of allelic
QTL-NILs for chromosome 4 of tomato; Deductions about natural
variation and implications for germplasm utilization. Theor. Appl
Genet. 102:572-590. [0231] Nawrath C (2000) The biopolymers cutin
and suberin. In: Somerville C R Meyerowitz E M, The Arabidopsis
Book. Rockville, Md.: American society of Plant Biologist, Pp.
1-14. [0232] Ojimelukwe P C (1994) Effects of processing methods on
ascorbic acid retention and sensory characteristic of tomato
products. J. Food Sci. Thechnol. 31: 247-248. [0233] Peet M M
(1992) Fruit cracking in tomato. HortTechnology 2 (2): 216-223.
[0234] Peet M M Willits D H (1995) Role of excess water in tomato
fruit cracking. HortScience 30 (1): 65-68. [0235] Pruitt R E
Ville-Catzada J P Ploense S E Grossnlklaus U Lolle S J (2000)
FIDDLEHEAD, a gene required to suppress epidermal cell interaction
in Arabidopsis, encodes a putative lipid biosynthesis enzyme. Proc.
Natl. Acad. Sci. 97: 1311-1316. [0236] Reina J J Heredia A (2001)
Plant cutin biosynthesis: the involvement of a new acyltransferase.
Trends Plant Sci. 6: 296. [0237] Riederer M Schreiber L (2001)
Protecting against water loss: analysis of the barrier properties
of plant cuticles. J Exp. Bot. 52 (363): 2023-2032. [0238] Tanksley
S D Ganal M W Giovannoni J J Grandillo S Martin G B Messeguer R
Miller J C Miller L Paterson A H Pineda O Roder M S Wing R A Wu W
Young N D (1992) High density molecular linkage maps of the tomato
and potato genomes. Genetics 132: 1141-1160. [0239] Schnurr J
Shockey J Browse J (2004) The acyl-CoA synthetase encoded by LACS2
is essential for normal cuticle development in Arabidopsis. Plant
Cell 16(3): 629-642. [0240] Schonherr J (1976a) Water permeability
of isolated cuticular membranes: The effect of pH and cations on
diffusion, hydrodynamic permeability and size of polar pores in
cutin matrix. Planta 128: 113-126. [0241] Schonherr J (1976b) Water
permeability of isolated cuticular membranes: The effect of
cuticular waxes on diffusion of water. Planta 131: 159-164. [0242]
Schonherr J Schmidt H W (1979) Water permeability of plant cuticle.
Planta 144: 391-400. [0243] Todd J Post-Beittenmiller D Jaworski J
G (1999) KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase
affecting wax biosynthesis in Arabidopsis Thaliana. Plant J. 17:
119-130. [0244] Tukey L D (1959) Observations on the russeting of
apples grown in plastic bags. Proc. Am. Soc. Hortic. Sci. 74:
30-39. [0245] Vogg G Fischer S Leide J Emmanuel E Jetter R Levy A A
Riederer M (2004) Tomato fruit cuticular waxes and their effects on
transpiration barrier properties: functional characterization of a
mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA
synthase. J. Exp. Bot. 55(401): 1401-1410. [0246] Voisey P W Lyhall
L H Kloek M (1970) Tomato skin strength--its measurement and
relation to cracking. J. Amer. Soc. Hort. Sci. 95 (4): 485-488.
[0247] Walkerpeach C and Velten J (1994) Agrobacterium-mediated
gene transfer to plant cells cointegrate and binary vector system.
In: Gelvin S Schilperoort R, Plant Molecular Biology Manual. Kluwer
Academic Publishers, Belgium, Pp. 1-19. [0248] Wilson L A Sterling
C (1975) Studies on the cuticle of tomato fruit I. Fine structure
of the cuticle. Z. Pflanzenphysiol. 77: 359-371. [0249] Wellesen K
Durst F Pinot F Benveniste L Nettesheim K Wisman E Steiner-Langa S
Saedler H Yapheremov A (2001) Functional analysis of the LACERATA
gene of Arabidopsis provides evidence for different roles of fatty
acid omega-hydroxylation in development. Proc. Natl. Acad. Sci. USA
98: 9694-9699. [0250] Yahhremov A Wisman E Huijser C Wellsen K
(1999) Characterization of the FIDDLEHEAD gene of Arabidopsis
reveals a link between adhesion response and cell differentiation
in the epidermis. Plant Cell 11: 2187-2201. [0251] Young P A (1947)
Cuticle cracks in tomato fruits. Phytopathology 37: 143-145.
Sequence CWU 1
1
58122DNAArtificial sequenceSingle strand DNA oligonucleotide
1tgtaatacga ctcactatag gg 22223DNAArtificial sequenceSingle strand
DNA oligonucleotide 2aagctattta ggtgacacta tag 23321DNAArtificial
sequenceSingle strand DNA oligonucleotide 3tgcaatcccg aacatgaaga c
21422DNAArtificial sequenceSingle strand DNA oligonucleotide
4ccttctggtc gcatctgtgt ct 22524DNAArtificial sequenceSingle strand
DNA oligonucleotide 5tcagggtgag gggtaataat tgag 24624DNAArtificial
sequenceSingle strand DNA oligonucleotide 6gcttaaaact caagtctcct
cgca 24722DNAArtificial sequenceSingle strand DNA oligonucleotide
7aataatgcga atcgaatcac ta 22822DNAArtificial sequenceSingle strand
DNA oligonucleotide 8aaggctaaat ctcctccttt ct 229140DNAArtificial
sequenceTomato ZINC gene amplicon 9aataatgcga atcgaatcac tatagtttaa
acataggctt acttataata agagcggcgc 60aactacatca acttactgta aagaatcaaa
gaaaaactat ttttactatg ttgcatccag 120aaaggaggag atttagcctt
1401023DNAArtificial sequenceSingle strand DNA oligonucleotide
10tggataagcg gacgactcta ttg 231123DNAArtificial sequenceSingle
strand DNA oligonucleotide 11ctgttgtttg ggaagtggct tct
2312116DNAArtificial sequenceTomato DBP gene amplicon 12tggataagcg
gacgactcta ttggccctcc atcttctccc acccatccag gaccaaactt 60taccccggga
ggcaaaatat tttctaactt tttagaagcc acttcccaaa caacag
1161322DNAArtificial sequenceSingle strand DNA oligonucleotide
13ctctccttgg cccaaggctc aa 221424DNAArtificial sequenceSingle
strand DNA oligonucleotide 14cagctttagt ggtatctctc atca
2415205DNAArtificial sequenceTomato PUT gene amplicon 15ctctccttgg
cccaaggtaa gaattctaat gggctttttt cgatcgatat acataaatta 60tacaaatgat
atgcttttgg ttgttcattt caggctcaaa gactgaagtt aaattttaag
120aaaatgatgg atgtttacga agtgaatgac gagaaaatct gcgtcaaaga
tatgatagaa 180aaattgatga gagataccac taaag 2051620DNAArtificial
sequenceSingle strand DNA oligonucleotide 16caccattggg tctgagcgat
201720DNAArtificial sequenceSingle strand DNA oligonucleotide
17gggcgacaac cttgatcttc 2018251DNAArtificial sequenceTomato actin
amplicon 18caccattggg tctgagcgat tccgctgtcc agaagtgctg ttccaaccat
caatgatcgg 60aatggaagct gctggtattc atgaaaccac gtacaattcc atcatgaagt
gtgacgttga 120tatcaggaag gatctgtatg gaaacatcgt cctcagtggt
ggtaccacaa tgttccctgg 180tattgctgat aggatgagca aggaaattac
tgcattagct cctagtagca tgaagatcaa 240ggttgtcgcc c
2511924DNAArtificial sequenceSingle strand DNA oligonucleotide
19gtagtactat ataaaccatg tgag 242021DNAArtificial sequenceSingle
strand DNA oligonucleotide 20catatgttga catatctaat g
2121912DNALycopersicon hirsutum 21tgatcttcat cttattcttg tttttattta
tagaaacaat aaaatattta taatcaatca 60tcatgtgtat agtagtgttt atttgggaag
cagatagtag atattcatta gtgttattat 120tgaatagaga tgaatatcat
aataggccaa caaaggaagt tcattggtgg gaagatggag 180aaattgttgg
tggcaaagat gaagttggtg gtggcacttg gttggcttct tcaactaatg
240gtaaattggc ttttcttact aatgttttgg aacttcatac acttcctcat
gtcaaaacta 300gaggtgacct acctcttcga tttttacaga gcaataaaag
cccaatggag tttgcaaaag 360agttggtgaa tgaagggaat gaatacaatg
ggtttaattt aattttggca gatattgaaa 420ctaaaaaaat ggtatatgta
acaaataggc ccaaaggaga gcccataaca atacaagaag 480tccaaccagg
tattcatgtg ctgtccaatg caaaactgga ctctccttgg cccaaggctc
540aaagactgaa gttaaatttt aagaaaatgt tggatgttta cgaagtgaat
gacgagaaaa 600tctgcgtcaa agatatgata gaaaaattga tgagagatac
cactaaagct gataaaagta 660aattgccttg tatttgttct acagactggg
agttggaact tagctctatt ttcgtggaag 720ttgacactgc actggggtgt
tatggtacta gaagtacaac agcattgaca attgaagtgg 780gaggagaagt
aagcttttat gagttgtacc ttgagaacaa catgtggaaa gagcaaattg
840tcaactatcg gattgaaaaa ctccaaatgc aataaatgtt tttaatatgt
tgatatatct 900aatgttttca tg 91222270PRTLycopersicon hirsutum 22Met
Cys Ile Val Val Phe Ile Trp Glu Ala Asp Ser Arg Tyr Ser Leu 1 5 10
15 Val Leu Leu Leu Asn Arg Asp Glu Tyr His Asn Arg Pro Thr Lys Glu
20 25 30 Val His Trp Trp Glu Asp Gly Glu Ile Val Gly Gly Lys Asp
Glu Val 35 40 45 Gly Gly Gly Thr Trp Leu Ala Ser Ser Thr Asn Gly
Lys Leu Ala Phe 50 55 60 Leu Thr Asn Val Leu Glu Leu His Thr Leu
Pro His Val Lys Thr Arg 65 70 75 80 Gly Asp Leu Pro Leu Arg Phe Leu
Gln Ser Asn Lys Ser Pro Met Glu 85 90 95 Phe Ala Lys Glu Leu Val
Asn Glu Gly Asn Glu Tyr Asn Gly Phe Asn 100 105 110 Leu Ile Leu Ala
Asp Ile Glu Thr Lys Lys Met Val Tyr Val Thr Asn 115 120 125 Arg Pro
Lys Gly Glu Pro Ile Thr Ile Gln Glu Val Gln Pro Gly Ile 130 135 140
His Val Leu Ser Asn Ala Lys Leu Asp Ser Pro Trp Pro Lys Ala Gln 145
150 155 160 Arg Leu Lys Leu Asn Phe Lys Lys Met Leu Asp Val Tyr Glu
Val Asn 165 170 175 Asp Glu Lys Ile Cys Val Lys Asp Met Ile Glu Lys
Leu Met Arg Asp 180 185 190 Thr Thr Lys Ala Asp Lys Ser Lys Leu Pro
Cys Ile Cys Ser Thr Asp 195 200 205 Trp Glu Leu Glu Leu Ser Ser Ile
Phe Val Glu Val Asp Thr Ala Leu 210 215 220 Gly Cys Tyr Gly Thr Arg
Ser Thr Thr Ala Leu Thr Ile Glu Val Gly 225 230 235 240 Gly Glu Val
Ser Phe Tyr Glu Leu Tyr Leu Glu Asn Asn Met Trp Lys 245 250 255 Glu
Gln Ile Val Asn Tyr Arg Ile Glu Lys Leu Gln Met Gln 260 265 270
233155DNALycopersicon hirsutummisc_feature(1501)..(1501)n is a, c,
g, or t 23tgccgtccta ttcttagaat actcaagtaa tttaacgtag tggtgaaaat
ttgataaatt 60aattatatac taatttttca gtcttatttt atgtggtata tttaattgga
tatgtagttt 120aagaaataat aaaaacttta aaatatttat aaatttactt
ttctaaaaaa gtgaattcaa 180ttttttctct cctcataaat gtattagagt
attatcatta aaattaagtg ggactaataa 240aggtaaaaaa taaattattc
ctttaaatta tttaaccata taagaaaatg tgacattctt 300ttttagactt
gactaaaata gaaaataatg tcatatatat aaaatgagac gaaaaaagta
360aatattaatt taaaatttaa aactttaggg taatagctac tttgaattac
ctagatttca 420ataaaattca acatataata aaacatacta atttacaatt
tttaaaataa tatgactaaa 480agtcatatta ttcaaaaaac aatctatacc
gccgtcacct agttacttta atttgtgtag 540cttctagtac atacattttt
aaactttatc tgaatttaat attttaatta tattaaacat 600ttattaaaat
ttataaaatt taaattgacg taatataatg aagagagtag tactatataa
660accatgtgag tactaacatg atcttcatct tattcttgtt tttatttata
gaaacaataa 720aatagttata aaattaatca atcatgtgta tagtagtgtt
tatttgggaa gcagatagta 780gatattcatt agtgttatta ttgaatagag
atgaatatca taataggcca acaaaggaag 840ttcattggtg ggaagatgga
gaaattgttg gtggcaaaga tgaagttggt ggtggcactt 900ggttggcttc
ttcaactaat ggtaaatggc tttcttacta atgttttgga acttcataca
960cttcctcatg ccaaaactag aggtgaccta cctgttcgat ttttacaggt
acgattaaat 1020tctttatata ttatacgtta atatgtttga tctttcattt
tggttttgtt atacgaagga 1080cgagacctag aggtctttaa gacaaaacat
aaatatgcat catagtcata aactttcaat 1140aaatattcaa ttttgaatat
gcgctttcaa aggtattaca agttgagtac taaaggaatt 1200gagtttatca
agattaaatt ttgaatttga ttcttttgat catgattaat agtaatgtta
1260aatcttgtcc ttattggagt atatatatga tcaataaatc aagattttaa
attgtagtat 1320aatcttaatt ttaaagaata ttaatgttgt aaaattttag
atttaacaaa cacaaaaatc 1380atatttgtat gttataacta tagtttgtat
agttgcgctc aatatgtttg ttcgcgagct 1440gttaatatgt cactatttcg
gtttacatat acaaaagaga tcaattgcat aattttgttt 1500ngcatatacn
tnttaaacat gatacataat agaaatttca ttnattgtgt aatatatctt
1560tgtataaagc aagaaagagc gaaacacaac agaaaactgg atagggaaat
atttatattt 1620tgtatagtta taagtgtata tgacggaaat atacgtaatt
attttttata catgattttc 1680tctcgctttt atgcaaacac aaacacaatt
tatacatttg tttttgtgta aagtgagagt 1740ggcgagcgag attctataga
gagagaacca aatgaaaata tatgtattat atgcagtttt 1800ctgtagtttt
atacaaatac aaacacaatt tatacattta tttttgtgta tgagagaggc
1860gagtgagatt ctcnggggag gaaaatatat gtatatatac agttttgttt
cgctataaac 1920aaacagaaca cattttatac atttgtattt gtataaaaca
agagagacga gggagaaact 1980gctcaacgag aaattcagga agagaggtga
atgacaacta tttgttacga gttgcaagta 2040aatcaaactg cgactataac
atttagtttg aattaataat ttgttatttt aaacgatttt 2100ccgtaaaatt
taattgttaa ttgcagagca ataaaagccc aatggagttt gcaaaagagt
2160tggtgaatga agggaatgaa tacaatgggt ttaatttaat tttggcagat
attgaaacta 2220aaaaaatggt atatgtaaca aataggccca aaggagagcc
cataacaata caagaagtcc 2280aaccaggtat tcatgtgctg tccaatgcaa
aactggactc tccttggccc aaggtaagaa 2340ttctaatggg cttttttcga
tcgatataca taaattatac aaatgatatg cttttggttg 2400ttcatttcag
gctcaaagac tgaagttaaa ttttaagaaa atgttggatg tttacgaagt
2460gaatgacgag aaaatctgcg tcaaagatat gatagaaaaa ttgatgagag
ataccactaa 2520agctgataaa agtaaattgc cttgtatttg ttctacagac
tgggagttgg aacttagctc 2580tattttcgtg gaagttgaca ctgcactggg
taattcatac cgcgttataa ctaatatgtt 2640tgtttgattt taacgtactc
aaacgatgat aaaggttaaa gtagatatac aaacatttta 2700aaaataattg
aaatagttca ataatagaag tgtacatatc attaacatag tttgatgggt
2760ttttttggtg gtgtgaatat gtaggggtgt tatggtacta gaagtacaac
agcattgaca 2820attgaagtgg gaggagaagt aagcttttat gagttgtacc
ttgagaacaa catgtggaaa 2880gagcaaattg tcaactatcg gattgaaaaa
ctccaaatgc aataaatgtt tttaatatgt 2940tgatatatct aatgtttttc
atgttcatat gttgacatat ctaatgtttt catttttttt 3000ttttaattca
aataagattt tttcttcaaa aaattaaact ttttgtcttt gaatggaaat
3060tgttattcat tgtatttgta aaatgtacta cactacttgg aagacataat
gtatgtttcg 3120ggctcctttg ttttagcaac aattttagac tttca
315524750DNALycopersicon hirsutum 24atgtctatac cggtgttcat
atggaaagcg catccgttgc atcccttcct tctcttcctc 60aacagagatg aataccacaa
tcgtccaacg aaaccattgt catggtggga agatactgat 120atacttggtg
gaagggatga agttgctggt gggacttggt tggcttgtac tcgcactgga
180agacttgctt ttcttactaa tgttcgagaa atcaattcaa attcacatac
cagaagtagg 240ggagaccttc ctcttcgatt cttaaagagt gtgaagagcc
ctcgtgattt ttcagagcaa 300ctattgatag aagcaggtga atataatggg
tttaatttga tagtaactga tctttgttca 360atgactatgc tatatataac
taaccgaccg aaacacaccg gtatgtccgt cactgaggtt 420tcacccggta
ttcatgtttt atcaaatgca tcactaaact ctccatggcc taagtctcaa
480cggctggagt gcagtttcaa gcaattattg gatgaatatg gcgaatcgga
aattccaata 540gggcatgcag ctgaaagaat atgagagacg tggctcaaga
agatagtaac ccgccaggca 600tcatattctc ccgagtgtga gtaccaattg
agctccctat ttgttgacac tgaaatgtgc 660atggggcgtt tttgcccaag
aagcacttct tcactggccg tgaagaagtc ttgtgacgcc 720accttttatg
agcggttcct gagaaggttt 75025250PRTLycopersicon hirsutum 25Met Ser
Ile Pro Val Phe Ile Trp Lys Ala His Pro Leu His Pro Phe 1 5 10 15
Leu Leu Phe Leu Asn Arg Asp Glu Tyr His Asn Arg Pro Thr Lys Pro 20
25 30 Leu Ser Trp Trp Glu Asp Thr Asp Ile Leu Gly Gly Arg Asp Glu
Val 35 40 45 Ala Gly Gly Thr Trp Leu Ala Cys Thr Arg Thr Gly Arg
Leu Ala Phe 50 55 60 Leu Thr Asn Val Arg Glu Ile Asn Ser Asn Ser
His Thr Arg Ser Arg 65 70 75 80 Gly Asp Leu Pro Leu Arg Phe Leu Lys
Ser Val Lys Ser Pro Arg Asp 85 90 95 Phe Ser Glu Gln Leu Leu Ile
Glu Ala Gly Glu Tyr Asn Gly Phe Asn 100 105 110 Leu Ile Val Thr Asp
Leu Cys Ser Met Thr Met Leu Tyr Ile Thr Asn 115 120 125 Arg Pro Lys
His Thr Gly Met Ser Val Thr Glu Val Ser Pro Gly Ile 130 135 140 His
Val Leu Ser Asn Ala Ser Leu Asn Ser Pro Trp Pro Lys Ser Gln 145 150
155 160 Arg Leu Glu Cys Ser Phe Lys Gln Leu Leu Asp Glu Tyr Gly Glu
Ser 165 170 175 Glu Ile Pro Ile Gly His Ala Ala Glu Arg Ile Met Arg
Asp Val Ala 180 185 190 Gln Glu Asp Ser Asn Pro Pro Gly Ile Ile Ser
Pro Glu Cys Glu Tyr 195 200 205 Gln Leu Ser Ser Leu Phe Val Asp Thr
Glu Met Cys Met Gly Arg Phe 210 215 220 Cys Pro Arg Ser Thr Ser Ser
Leu Ala Val Lys Lys Ser Cys Asp Ala 225 230 235 240 Thr Phe Tyr Glu
Arg Phe Leu Arg Arg Phe 245 250 26762DNAArabidopsis thaliana
26atgaagatca caacagggcg acagaggcgc tgcgtttggt gggaagacgg agagacggtg
60ggaggaagag accttgttgg cggcgggacg tggctgggct gcacgaggca tggccgtctg
120gctttcctca ccaatttcaa ggaagcctcc tccttccctg ctgctaaatc
ccgtggagat 180ctgcctcttc gttacttgca gagcgaaaag agtccggccg
agtttgccga ggagatccaa 240gacgaaattt cactctacaa tggctttaac
ctggttgtcg ctcatgtctt gtccaaatcc 300atgatttaca ttaccaaccg
accaccccac ggtgacaagc tcgtgacgca agtctctccc 360gggatccatg
tcctttccaa cgccaacctc gactcccctt ggcccaagtg tctgaggctg
420agggagggtt tccaacagct tctggctgag aacgggagcg gtgaattccc
ggtgaagacc 480atggtggagg aggtgatgac caatactgtc aaggacgaag
aaaccgagct acctcacgtt 540ttcacaccag agacggaata ccatctcagc
tccatcttcg tcgacatgca gagaccaact 600gggcgttatg ggaccagaag
catctctgcg atcatcgtca agtcccatgg agatggtggt 660ggtgatggtg
agatttgctt ctacgagagg catcttgaag aaggcgattc atggaaggaa
720cacactcaac agtttgtaat aatacaaaac caaagcattt ga
76227253PRTArabidopsis thaliana 27Met Lys Ile Thr Thr Gly Arg Gln
Arg Arg Cys Val Trp Trp Glu Asp 1 5 10 15 Gly Glu Thr Val Gly Gly
Arg Asp Leu Val Gly Gly Gly Thr Trp Leu 20 25 30 Gly Cys Thr Arg
His Gly Arg Leu Ala Phe Leu Thr Asn Phe Lys Glu 35 40 45 Ala Ser
Ser Phe Pro Ala Ala Lys Ser Arg Gly Asp Leu Pro Leu Arg 50 55 60
Tyr Leu Gln Ser Glu Lys Ser Pro Ala Glu Phe Ala Glu Glu Ile Gln 65
70 75 80 Asp Glu Ile Ser Leu Tyr Asn Gly Phe Asn Leu Val Val Ala
His Val 85 90 95 Leu Ser Lys Ser Met Ile Tyr Ile Thr Asn Arg Pro
Pro His Gly Asp 100 105 110 Lys Leu Val Thr Gln Val Ser Pro Gly Ile
His Val Leu Ser Asn Ala 115 120 125 Asn Leu Asp Ser Pro Trp Pro Lys
Cys Leu Arg Leu Arg Glu Gly Phe 130 135 140 Gln Gln Leu Leu Ala Glu
Asn Gly Ser Gly Glu Phe Pro Val Lys Thr 145 150 155 160 Met Val Glu
Glu Val Met Thr Asn Thr Val Lys Asp Glu Glu Thr Glu 165 170 175 Leu
Pro His Val Phe Thr Pro Glu Thr Glu Tyr His Leu Ser Ser Ile 180 185
190 Phe Val Asp Met Gln Arg Pro Thr Gly Arg Tyr Gly Thr Arg Ser Ile
195 200 205 Ser Ala Ile Ile Val Lys Ser His Gly Asp Gly Gly Gly Asp
Gly Glu 210 215 220 Ile Cys Phe Tyr Glu Arg His Leu Glu Glu Gly Asp
Ser Trp Lys Glu 225 230 235 240 His Thr Gln Gln Phe Val Ile Ile Gln
Asn Gln Ser Ile 245 250 28801DNAArabidopsis thaliana 28atggggagag
ggagaaaaca cactgacgct gctgcagaac agagagaact ggcaattaag 60gcaaacattg
ttgatgaacc tttttctgta tcggcgattg ataggtcaat aagaaaggcg
120gaatgggtta aaactgaaac tgaccagata ttaagtggtc gttgcccaga
gaccgatggg 180acgtggttag gtatttctac tcgaggccga gtcgcttttc
ttgtggaggc agggactatt 240aacagagaca gattcaacgg cgccgagagt
cgtactcttg agttcttaga gagcaacgag 300agtccagagg actttgcaaa
gtcatcggct gcagattaca tacgtaacaa gaacacagcc 360gcctttcatc
taattgtggc cgacatagct tcaaactcaa tgctttatat ctccaaaccg
420cgtttctctg actatggcat tgtctataca gagcctgttg gtcctggtgt
tcacacacta 480tcttcagctg gactcgattc cgacgttgga tacagggact
tacgtatgag acactctttt 540tgtgagatga ttaacagaga acgactacca
ccaataaggg acattgctga gattatgtat 600gatccagtca aagcttacga
aagcgtgcta ctgagctcta tttttttcgt cgacatgaag 660attggatacg
aacactatgg aacaagaatt acgacagcat tggttgtgaa acgcaccaag
720gaagtgttgt tctttgagag gtacagggag atatttaatg atgattggga
cgaccacgac 780ttcgcgttca ccatcatcta g 80129266PRTArabidopsis
thaliana 29Met Gly Arg Gly Arg Lys His Thr Asp Ala Ala Ala Glu Gln
Arg Glu 1 5 10 15 Leu Ala Ile Lys Ala Asn
Ile Val Asp Glu Pro Phe Ser Val Ser Ala 20 25 30 Ile Asp Arg Ser
Ile Arg Lys Ala Glu Trp Val Lys Thr Glu Thr Asp 35 40 45 Gln Ile
Leu Ser Gly Arg Cys Pro Glu Thr Asp Gly Thr Trp Leu Gly 50 55 60
Ile Ser Thr Arg Gly Arg Val Ala Phe Leu Val Glu Ala Gly Thr Ile 65
70 75 80 Asn Arg Asp Arg Phe Asn Gly Ala Glu Ser Arg Thr Leu Glu
Phe Leu 85 90 95 Glu Ser Asn Glu Ser Pro Glu Asp Phe Ala Lys Ser
Ser Ala Ala Asp 100 105 110 Tyr Ile Arg Asn Lys Asn Thr Ala Ala Phe
His Leu Ile Val Ala Asp 115 120 125 Ile Ala Ser Asn Ser Met Leu Tyr
Ile Ser Lys Pro Arg Phe Ser Asp 130 135 140 Tyr Gly Ile Val Tyr Thr
Glu Pro Val Gly Pro Gly Val His Thr Leu 145 150 155 160 Ser Ser Ala
Gly Leu Asp Ser Asp Val Gly Tyr Arg Asp Leu Arg Met 165 170 175 Arg
His Ser Phe Cys Glu Met Ile Asn Arg Glu Arg Leu Pro Pro Ile 180 185
190 Arg Asp Ile Ala Glu Ile Met Tyr Asp Pro Val Lys Ala Tyr Glu Ser
195 200 205 Val Leu Leu Ser Ser Ile Phe Phe Val Asp Met Lys Ile Gly
Tyr Glu 210 215 220 His Tyr Gly Thr Arg Ile Thr Thr Ala Leu Val Val
Lys Arg Thr Lys 225 230 235 240 Glu Val Leu Phe Phe Glu Arg Tyr Arg
Glu Ile Phe Asn Asp Asp Trp 245 250 255 Asp Asp His Asp Phe Ala Phe
Thr Ile Ile 260 265 301122DNAArabidopsis thaliana 30atgcctggag
aatcgaatat catcgagtgg ccagcaagta gagtcagggt cataagtggg 60gcatcttgga
gtcgaaacgg tcagattttg agtggtcggt gcaaagctaa caacggaacc
120tggtttggta ttactaaagg tggccgagtc gcttttctcg tgaatacatc
gttgttgttg 180gaccgtgtta agtcatacag cggctcggag ttgtatcccg
ttcgtttctt ggagggcaac 240atgagtccag agcagtttgc caacgaagtg
aaagtgcatg aaaaggagac taatgaaagg 300catgcctata gtcttgtcgt
tgcagacatg acttcgagtt caatggttca tatcctgaaa 360ccctcggata
ctaagtctga tgtcgtgata gagactgttc cgtttggtgt gcatacactt
420tcttcttacg aaggtctcga ttctacagat tctgccaggg atttactcct
gagacgcttg 480tttacccaga tggttggtaa tttgggaaac gttcaacaac
gacagatgga ggagattgct 540gggaggttta tgtatgatgc tcaagcagga
agagacgcgg tgttttacca tagtagagat 600gagcatccta atggaaaact
tggaacgcaa cgctttggaa caacaagtac gacagcattg 660gttgtgaaac
gcactagaga agtgatgctc tttgagaagt acatggagca gaatggtgca
720tggaacacga acaacttcgc tttcaacatc caaaaacagc aaaagctata
tccaaatttg 780gataaagaag ctcttaagcg cgttggggta tttgcgttgg
aagaagttaa caaccatgag 840catgatattc accctgacct gatgcccagt
ttcttcgagg atgatatgct gaaagtaaaa 900tttaatgaga tgattgctag
acatgcaaaa ctgccgccaa ttaagaacat tgttgaggat 960cttatgatga
agtctccatt ttttatcgac agtgtcgatg gtgctggcaa gaaggtgagg
1020tatcgaacag tacgtacatt gggaatggac ataaaagcca acagaccaca
agggcggttc 1080tatgagaggc atttgaatga taatggtgaa tgggtaggct ag
112231373PRTArabidopsis thaliana 31Met Pro Gly Glu Ser Asn Ile Ile
Glu Trp Pro Ala Ser Arg Val Arg 1 5 10 15 Val Ile Ser Gly Ala Ser
Trp Ser Arg Asn Gly Gln Ile Leu Ser Gly 20 25 30 Arg Cys Lys Ala
Asn Asn Gly Thr Trp Phe Gly Ile Thr Lys Gly Gly 35 40 45 Arg Val
Ala Phe Leu Val Asn Thr Ser Leu Leu Leu Asp Arg Val Lys 50 55 60
Ser Tyr Ser Gly Ser Glu Leu Tyr Pro Val Arg Phe Leu Glu Gly Asn 65
70 75 80 Met Ser Pro Glu Gln Phe Ala Asn Glu Val Lys Val His Glu
Lys Glu 85 90 95 Thr Asn Glu Arg His Ala Tyr Ser Leu Val Val Ala
Asp Met Thr Ser 100 105 110 Ser Ser Met Val His Ile Leu Lys Pro Ser
Asp Thr Lys Ser Asp Val 115 120 125 Val Ile Glu Thr Val Pro Phe Gly
Val His Thr Leu Ser Ser Tyr Glu 130 135 140 Gly Leu Asp Ser Thr Asp
Ser Ala Arg Asp Leu Leu Leu Arg Arg Leu 145 150 155 160 Phe Thr Gln
Met Val Gly Asn Leu Gly Asn Val Gln Gln Arg Gln Met 165 170 175 Glu
Glu Ile Ala Gly Arg Phe Met Tyr Asp Ala Gln Ala Gly Arg Asp 180 185
190 Ala Val Phe Tyr His Ser Arg Asp Glu His Pro Asn Gly Lys Leu Gly
195 200 205 Thr Gln Arg Phe Gly Thr Thr Ser Thr Thr Ala Leu Val Val
Lys Arg 210 215 220 Thr Arg Glu Val Met Leu Phe Glu Lys Tyr Met Glu
Gln Asn Gly Ala 225 230 235 240 Trp Asn Thr Asn Asn Phe Ala Phe Asn
Ile Gln Lys Gln Gln Lys Leu 245 250 255 Tyr Pro Asn Leu Asp Lys Glu
Ala Leu Lys Arg Val Gly Val Phe Ala 260 265 270 Leu Glu Glu Val Asn
Asn His Glu His Asp Ile His Pro Asp Leu Met 275 280 285 Pro Ser Phe
Phe Glu Asp Asp Met Leu Lys Val Lys Phe Asn Glu Met 290 295 300 Ile
Ala Arg His Ala Lys Leu Pro Pro Ile Lys Asn Ile Val Glu Asp 305 310
315 320 Leu Met Met Lys Ser Pro Phe Phe Ile Asp Ser Val Asp Gly Ala
Gly 325 330 335 Lys Lys Val Arg Tyr Arg Thr Val Arg Thr Leu Gly Met
Asp Ile Lys 340 345 350 Ala Asn Arg Pro Gln Gly Arg Phe Tyr Glu Arg
His Leu Asn Asp Asn 355 360 365 Gly Glu Trp Val Gly 370
32713DNAVitis vinifera 32ggtcgcactg tatatattca acggagagga
gcagtgacgg cgtttggagg ccgagaaagt 60aagagatttc agtttctgag gcgggaaagt
acggaagcat gtgtatagca gtattcttat 120ggcaagctca cccgatttat
cctttccttc tgttgctcaa cagagacgaa tatcataatc 180ggcctactga
ggctctggca tggtggcaag gtggggagat actgggcggg cgagatgggc
240tcgccggtgg gacatggttg gcttgtagca gagatgggag gttggctttt
cttacaaatg 300tgcgagaagt tcacccaatc cccgaagcca agagcagagg
agacctaatt gttcggttct 360tggagagcaa gaagaatccc atggaatttg
cagaggaagt tgtgaaggag gcagataagt 420ataatgggtt taacttgata
atggctgatc tttgttccaa aactatgatc tatataacca 480acagaccaag
agaagctaat gtttctgttg tcgaggtttc acctggtatt catgtgctgt
540caaatgcaag tttggactca ccttggccta aggtacgaag actaggtcat
aatttcaaag 600agctcttgga taaatatggt gaaggtgaga tccccacaga
ggagatggtt gagaaattaa 660tgaagaaaca caatcaaaga cgatgaaatc
gtgctgcctc gcatctatcc tcc 71333207PRTVitis vinifera 33Glu Ile Ser
Val Ser Glu Ala Gly Lys Tyr Gly Ser Met Cys Ile Ala 1 5 10 15 Val
Phe Leu Trp Gln Ala His Pro Ile Tyr Pro Phe Leu Leu Leu Leu 20 25
30 Asn Arg Asp Glu Tyr His Asn Arg Pro Thr Glu Ala Leu Ala Trp Trp
35 40 45 Gln Gly Gly Glu Ile Leu Gly Gly Arg Asp Gly Leu Ala Gly
Gly Thr 50 55 60 Trp Leu Ala Cys Ser Arg Asp Gly Arg Leu Ala Phe
Leu Thr Asn Val 65 70 75 80 Arg Glu Val His Pro Ile Pro Glu Ala Lys
Ser Arg Gly Asp Leu Ile 85 90 95 Val Arg Phe Leu Glu Ser Lys Lys
Asn Pro Met Glu Phe Ala Glu Glu 100 105 110 Val Val Lys Glu Ala Asp
Lys Tyr Asn Gly Phe Asn Leu Ile Met Ala 115 120 125 Asp Leu Cys Ser
Lys Thr Met Ile Tyr Ile Thr Asn Arg Pro Arg Glu 130 135 140 Ala Asn
Val Ser Val Val Glu Val Ser Pro Gly Ile His Val Leu Ser 145 150 155
160 Asn Ala Ser Leu Asp Ser Pro Trp Pro Lys Val Arg Arg Leu Gly His
165 170 175 Asn Phe Lys Glu Leu Leu Asp Lys Tyr Gly Glu Gly Glu Ile
Pro Thr 180 185 190 Glu Glu Met Val Glu Lys Leu Met Lys Lys His Asn
Gln Arg Arg 195 200 205 341068DNAZea mays 34gatcagctaa gatagctgca
aaacaagcga gttacttaca accaaacaga agggtagaaa 60ccacctgaag ccatgtgcat
tgctgcatgg atttggcagg ctcaccctgt gcaccaactc 120ctcctgcttc
tcaacagaga tgagttccac agcaggccta caaaagcagt aggatggtgg
180ggtgaaggct caaagaagat ccttggtggc agggatgtgc ttggtggagg
aacatggatg 240gggtgcacca aggatggaag gcttgccttc ctgaccaatg
tgcttgaacc agatgccatg 300cccggtgcac ggactagggg agatctgcct
ctcaaattcc tgcagagcaa caagagccca 360ctcgaagttg caactgaagt
ggcagaagaa gctgatgaat acaatggctt caacctcata 420ctagctgatc
taacaacaaa tatcatggtt tatgtgtcaa accggcctaa gggtcagcct
480gcaacaattc aactcgtgtc accaggactc catgtgctgt ccaatgcaag
gctagatagc 540ccttggcaga aggcaattct cctcggtaaa aacttcaggg
agcttcttag ggagcatggt 600gctgatgagg ttgaagtgaa ggatatagtt
gagaggctaa tgactgacac cacaaaggct 660gacaaagata gactgccaaa
cactggttgt gatcccaact gggagcatgg tctgagctcc 720atcttcattg
aggtgcaaac tgaccaaggg ccctatggga cacggagcac agccgtttta
780tcagtgaact atgatggcga agctagcttg tacgagaagt atcttgagag
tggtatatgg 840aaggatcaca cagtgagtta ccagatagag tagtaggcat
tgcacaggaa aagttggcga 900cctcaaataa atagaaatat gaagcagaca
caattgtgaa tttcattatt tccctgatct 960ctagtcatct tcgtgattat
ctaagatcct accataatgc caattacatt attcactgta 1020agcagatttt
tcacttgacg ataaaatgtc aaccaaaact ttggtttt 106835271PRTZea mays
35Lys Pro Pro Glu Ala Met Cys Ile Ala Ala Trp Ile Trp Gln Ala His 1
5 10 15 Pro Val His Gln Leu Leu Leu Leu Leu Asn Arg Asp Glu Phe His
Ser 20 25 30 Arg Pro Thr Lys Ala Val Gly Trp Trp Gly Glu Gly Ser
Lys Lys Ile 35 40 45 Leu Gly Gly Arg Asp Val Leu Gly Gly Gly Thr
Trp Met Gly Cys Thr 50 55 60 Lys Asp Gly Arg Leu Ala Phe Leu Thr
Asn Val Leu Glu Pro Asp Ala 65 70 75 80 Met Pro Gly Ala Arg Thr Arg
Gly Asp Leu Pro Leu Lys Phe Leu Gln 85 90 95 Ser Asn Lys Ser Pro
Leu Glu Val Ala Thr Glu Val Ala Glu Glu Ala 100 105 110 Asp Glu Tyr
Asn Gly Phe Asn Leu Ile Leu Ala Asp Leu Thr Thr Asn 115 120 125 Ile
Met Val Tyr Val Ser Asn Arg Pro Lys Gly Gln Pro Ala Thr Ile 130 135
140 Gln Leu Val Ser Pro Gly Leu His Val Leu Ser Asn Ala Arg Leu Asp
145 150 155 160 Ser Pro Trp Gln Lys Ala Ile Leu Leu Gly Lys Asn Phe
Arg Glu Leu 165 170 175 Leu Arg Glu His Gly Ala Asp Glu Val Glu Val
Lys Asp Ile Val Glu 180 185 190 Arg Leu Met Thr Asp Thr Thr Lys Ala
Asp Lys Asp Arg Leu Pro Asn 195 200 205 Thr Gly Cys Asp Pro Asn Trp
Glu His Gly Leu Ser Ser Ile Phe Ile 210 215 220 Glu Val Gln Thr Asp
Gln Gly Pro Tyr Gly Thr Arg Ser Thr Ala Val 225 230 235 240 Leu Ser
Val Asn Tyr Asp Gly Glu Ala Ser Leu Tyr Glu Lys Tyr Leu 245 250 255
Glu Ser Gly Ile Trp Lys Asp His Thr Val Ser Tyr Gln Ile Glu 260 265
270 361152DNAHordeum vulgare 36cacacacaca caaggcgcac ggttgcaaaa
caagggagtt atttttagaa gcagggagta 60aggaaccacc tgaagccatg tgtatcgctg
catggcattt ggcaggctca cccacagcat 120cagctcctgc tgctgctcaa
cagagatgag ttccatagca ggcctacaaa ggcagtagga 180tggtggggcg
agggctcaat gaagattctt ggtggcaggg atgtactcgg tggaggaaca
240tggatgggga gcaccaaaga tggcaggctt gccttcctga ccaatgtgct
cgagcctgat 300gcaatgcccg gcgcacgcac taggggagac ctgcccctca
ggttcctgca gggaaacaag 360agcccactgg aggttgcgac tgaagtggca
aaagaagctg atgagtacaa tggcttcaac 420cttatactag ctgatctaac
caggaatgtc atggtctacg tgtcaaaccg gccaaagggg 480cagcctgcga
caattcagct cgtctcacca ggactccatg tgttgtccaa tgcaaggctt
540gatagccctt ggcagaaggc aattcgcctt ggtaaaaact tcagggagtt
tataaggaag 600catggtgatg atgaagttga agcgaaggat atagctgaca
gactaatgac tgacacgacg 660agggctgata aagataggct gccaaacacc
ggttgtgatc ccacctggga gcacggtctg 720agctccatct tcatcgaggt
gcaaactgac gaagggctct atgggacaag gagcacagca 780gttctttcag
tgaactatga tggagaagct agcttatatg aaaagtacct cgagagtggt
840atatggaaga accacacagt gcattaccag atagagtagc caatgcggac
ctaaaggcgg 900gagcccaaaa taggaagaaa gaatgaatag ctacaattgt
gcatgctgtt atttccacag 960ttgcgcttta agatcatata atgatctcta
gttatggcga ttaaattatt tactgtatgc 1020agatttatca attcagagag
agatcattca aattgttgaa tatatataca taataataat 1080aatatgatat
gatatgtata tttacagact tcatgttgcc acctttgtct atgaacatac
1140atgctttact ac 115237291PRTHordeum vulgare 37Lys Gln Gly Val Arg
Asn His Leu Lys Pro Cys Val Ser Leu His Gly 1 5 10 15 Ile Trp Gln
Ala His Pro Gln His Gln Leu Leu Leu Leu Leu Asn Arg 20 25 30 Asp
Glu Phe His Ser Arg Pro Thr Lys Ala Val Gly Trp Trp Gly Glu 35 40
45 Gly Ser Met Lys Ile Leu Gly Gly Arg Asp Val Leu Gly Gly Gly Thr
50 55 60 Trp Met Gly Ser Thr Lys Asp Gly Arg Leu Ala Phe Leu Thr
Asn Val 65 70 75 80 Leu Glu Pro Asp Ala Met Pro Gly Ala Arg Thr Arg
Gly Asp Leu Pro 85 90 95 Leu Arg Phe Leu Gln Gly Asn Lys Ser Pro
Leu Glu Val Ala Thr Glu 100 105 110 Val Ala Lys Glu Ala Asp Glu Tyr
Asn Gly Phe Asn Leu Ile Leu Ala 115 120 125 Asp Leu Thr Arg Asn Val
Met Val Tyr Val Ser Asn Arg Pro Lys Gly 130 135 140 Gln Pro Ala Thr
Ile Gln Leu Val Ser Pro Gly Leu His Val Leu Ser 145 150 155 160 Asn
Ala Arg Leu Asp Ser Pro Trp Gln Lys Ala Ile Arg Leu Gly Lys 165 170
175 Asn Phe Arg Glu Phe Ile Arg Lys His Gly Asp Asp Glu Val Glu Ala
180 185 190 Lys Asp Ile Ala Asp Arg Leu Met Thr Asp Thr Thr Arg Ala
Asp Lys 195 200 205 Asp Arg Leu Pro Asn Thr Gly Cys Asp Pro Thr Trp
Glu His Gly Leu 210 215 220 Ser Ser Ile Phe Ile Glu Val Gln Thr Asp
Glu Gly Leu Tyr Gly Thr 225 230 235 240 Arg Ser Thr Ala Val Leu Ser
Val Asn Tyr Asp Gly Glu Ala Ser Leu 245 250 255 Tyr Glu Lys Tyr Leu
Glu Ser Gly Ile Trp Lys Asn His Thr Val His 260 265 270 Tyr Gln Ile
Glu Pro Met Arg Thr Arg Arg Glu Pro Lys Ile Gly Arg 275 280 285 Lys
Asn Glu 290 38801DNAOryza sativa 38atgtgtatag ctgcatgggt ttggcaagct
cacccacagc accagctcct cctgctgctc 60aaccgggatg agttccatag caggccaacc
aaggcagtag gatggtgggg ggagggctcg 120aagaagattc ttggtggtag
agatgttctt ggtggaggga catggatggg ttgcacaaag 180gatggcaggc
tcgccttcct caccaatgtg ctcgagccgg acgccatgcc gggggcgcgc
240acaaggggag atctccccct caggttcctg cagagcaaca agagcccact
tgaagttgca 300actgaggtgg caaaagaagc tgacgagtac aacggcttca
accttgtact ggctgatctg 360accacaaacg tcatggttta tgtgtcaaat
cggccaaagg ggcagcctgc aacgatccaa 420cttgtctcac cagggctcca
tgtgttgtcc aatgcaaggc tagacagccc ttggcagaag 480gcgattcgcc
tcggtaagaa cttcagggag catcttagga agcatggtga tgatgaggtt
540gaagccaagg acatagttga gaggctaatg actgacacca caaaggctga
caaagatagg 600ctgccaaaca ctggctgtga tccaaactgg gagcacggcc
tgagctccat tttcattgag 660gtgcagactg accagggact ctacgggaca
cggagcacgg ccgttctatc agtgaactac 720gacggtgaag ctagcttgta
cgagaaatac ctggagagtg gtatatggaa ggatcacacg 780gtgcattacc
agatagagta g 80139266PRTOryza sativa 39Met Cys Ile Ala Ala Trp Val
Trp Gln Ala His Pro Gln His Gln Leu 1 5 10 15 Leu Leu Leu Leu Asn
Arg Asp Glu Phe His Ser Arg Pro Thr Lys Ala 20 25 30 Val Gly Trp
Trp Gly Glu Gly Ser Lys Lys Ile Leu Gly Gly Arg Asp 35 40 45 Val
Leu Gly Gly Gly Thr Trp Met Gly Cys Thr Lys Asp Gly Arg Leu 50 55
60 Ala Phe Leu Thr Asn Val Leu Glu Pro Asp Ala Met Pro Gly Ala Arg
65 70 75 80 Thr Arg Gly Asp Leu Pro Leu Arg Phe Leu Gln Ser Asn Lys
Ser Pro 85 90 95 Leu Glu Val Ala Thr Glu Val Ala Lys Glu Ala Asp
Glu Tyr Asn Gly 100 105 110 Phe Asn Leu Val Leu Ala Asp Leu Thr
Thr Asn Val Met Val Tyr Val 115 120 125 Ser Asn Arg Pro Lys Gly Gln
Pro Ala Thr Ile Gln Leu Val Ser Pro 130 135 140 Gly Leu His Val Leu
Ser Asn Ala Arg Leu Asp Ser Pro Trp Gln Lys 145 150 155 160 Ala Ile
Arg Leu Gly Lys Asn Phe Arg Glu His Leu Arg Lys His Gly 165 170 175
Asp Asp Glu Val Glu Ala Lys Asp Ile Val Glu Arg Leu Met Thr Asp 180
185 190 Thr Thr Lys Ala Asp Lys Asp Arg Leu Pro Asn Thr Gly Cys Asp
Pro 195 200 205 Asn Trp Glu His Gly Leu Ser Ser Ile Phe Ile Glu Val
Gln Thr Asp 210 215 220 Gln Gly Leu Tyr Gly Thr Arg Ser Thr Ala Val
Leu Ser Val Asn Tyr 225 230 235 240 Asp Gly Glu Ala Ser Leu Tyr Glu
Lys Tyr Leu Glu Ser Gly Ile Trp 245 250 255 Lys Asp His Thr Val His
Tyr Gln Ile Glu 260 265 40707DNAMedicago
truncatulamisc_feature(689)..(689)n is a, c, g, or t 40ttgtacttag
ttatagtatt attgaagcta gccaactcaa aatttgtgaa caatgtgtat 60agctttgttt
ctttggcaat ctcatccacc tttatccttt tcttcttttg aataatagag
120atgaatatca caataggcct acaaagaaag tgtcatggtg ggaagaatgt
gatatagtgg 180gaggaaggga tgaaatagga ggagggacat ggttggcttg
ttcttcacaa ggaaaagtgg 240cttttcttac caatgttttg gagcttcata
cttgccctga ggccaaaact cgtggagacc 300tacccctcat gtttctcaag
agcagcaaga atcccaaaga atttgcagaa agcttaaaaa 360gagaagctca
atattacaat ggattcaatt tagtcattgc tgatattaat tccaaatcca
420tggtatacat atcaaataga cccaagggac agccaattac tgtccaagag
gttcctcctg 480gtctacatgt actttcaaat gctaagttaa attcaccatg
gcataaggct cagcgccttc 540aatttagatt caaagagcat cttgctaaaa
atggggaagg tgagatacat gtaaaggaag 600taattaaaaa gctaatgaag
gacaaaatta aagcagacaa aagcatgcta cctaatatat 660gctcacttga
ttggggaatt caatcttanc tncatttttg ttgaaga 70741213PRTMedicago
truncatulamisc_feature(209)..(210)Xaa can be any naturally
occurring amino acid 41Leu Cys Phe Phe Gly Asn Leu Ile His Leu Tyr
Pro Phe Leu Leu Leu 1 5 10 15 Asn Asn Arg Asp Glu Tyr His Asn Arg
Pro Thr Lys Lys Val Ser Trp 20 25 30 Trp Glu Glu Cys Asp Ile Val
Gly Gly Arg Asp Glu Ile Gly Gly Gly 35 40 45 Thr Trp Leu Ala Cys
Ser Ser Gln Gly Lys Val Ala Phe Leu Thr Asn 50 55 60 Val Leu Glu
Leu His Thr Cys Pro Glu Ala Lys Thr Arg Gly Asp Leu 65 70 75 80 Pro
Leu Met Phe Leu Lys Ser Ser Lys Asn Pro Lys Glu Phe Ala Glu 85 90
95 Ser Leu Lys Arg Glu Ala Gln Tyr Tyr Asn Gly Phe Asn Leu Val Ile
100 105 110 Ala Asp Ile Asn Ser Lys Ser Met Val Tyr Ile Ser Asn Arg
Pro Lys 115 120 125 Gly Gln Pro Ile Thr Val Gln Glu Val Pro Pro Gly
Leu His Val Leu 130 135 140 Ser Asn Ala Lys Leu Asn Ser Pro Trp His
Lys Ala Gln Arg Leu Gln 145 150 155 160 Phe Arg Phe Lys Glu His Leu
Ala Lys Asn Gly Glu Gly Glu Ile His 165 170 175 Val Lys Glu Val Ile
Lys Lys Leu Met Lys Asp Lys Ile Lys Ala Asp 180 185 190 Lys Ser Met
Leu Pro Asn Ile Cys Ser Leu Asp Trp Gly Ile Gln Ser 195 200 205 Xaa
Xaa His Phe Cys 210 42627DNASolanum tuberosum 42gatatggaaa
gcgcatccgc tgtatccctt cctcctattc ctcaacagag atgaatacca 60caatcgtgat
atacttggtg gaagggatga agttgctggt gggacttggt tggcttgtac
120tcgcaccgga agacttgctt tccttactaa tgttcgagaa atcaattcaa
attcacatac 180caaaagtagg ggagaccttc ctcttcgatt cttgaagagt
gtaaagagcc ctcatgattt 240ttcagagcaa cttttgaaag aagcaggcga
atataatggg tttaacttga tagtagctga 300tctttgttca atgactatgc
ttgatataac caaccgacca aaacacaccg gtatgtccgg 360cactgaagtt
tcacccggta ttcacgtttt atcaaatgca acactagact ctccatggcc
420taagtctcaa cggctggagt acagtttcaa gcaattattg gatgaatatg
gcgaatctga 480aattccaata gggcagacag ctgaaagaat aatgagagac
ttggctaaag aagatagcaa 540cctgccaggc atctattccc ctgagtgtga
gtaccagttg agctccatat tcgttgacac 600tgaaatgtcc atggggcgtt ttggcac
62743208PRTSolanum tuberosum 43Ile Trp Lys Ala His Pro Leu Tyr Pro
Phe Leu Leu Phe Leu Asn Arg 1 5 10 15 Asp Glu Tyr His Asn Arg Asp
Ile Leu Gly Gly Arg Asp Glu Val Ala 20 25 30 Gly Gly Thr Trp Leu
Ala Cys Thr Arg Thr Gly Arg Leu Ala Phe Leu 35 40 45 Thr Asn Val
Arg Glu Ile Asn Ser Asn Ser His Thr Lys Ser Arg Gly 50 55 60 Asp
Leu Pro Leu Arg Phe Leu Lys Ser Val Lys Ser Pro His Asp Phe 65 70
75 80 Ser Glu Gln Leu Leu Lys Glu Ala Gly Glu Tyr Asn Gly Phe Asn
Leu 85 90 95 Ile Val Ala Asp Leu Cys Ser Met Thr Met Leu Asp Ile
Thr Asn Arg 100 105 110 Pro Lys His Thr Gly Met Ser Gly Thr Glu Val
Ser Pro Gly Ile His 115 120 125 Val Leu Ser Asn Ala Thr Leu Asp Ser
Pro Trp Pro Lys Ser Gln Arg 130 135 140 Leu Glu Tyr Ser Phe Lys Gln
Leu Leu Asp Glu Tyr Gly Glu Ser Glu 145 150 155 160 Ile Pro Ile Gly
Gln Thr Ala Glu Arg Ile Met Arg Asp Leu Ala Lys 165 170 175 Glu Asp
Ser Asn Leu Pro Gly Ile Tyr Ser Pro Glu Cys Glu Tyr Gln 180 185 190
Leu Ser Ser Ile Phe Val Asp Thr Glu Met Ser Met Gly Arg Phe Gly 195
200 205 44423DNAGlycine max 44gtgctcccca gattgttcta ttttggcttt
tataaagaat tgtttagatc ctttgaattg 60aagaatgtgt atagctttgt ttctttggca
agcccaccca ctctaccctt tccttctttt 120gaacaacaga gatgaatatc
acaacaggcc tacgaagcca gtgtcatggt gggaagatat 180tgatatagtt
ggaggaagag atgagattgc tggaggaaca tggttggctt gttcaagaga
240aggaagagtt gcttttctga ccaatgtttt ggagcttcgt tcccttcctg
aggctaaaag 300cagaggagac ctacctgtct catttcttaa gagtggaaag
catccgaaag aatttgcaga 360aagtctaaaa atggaagctc attattacaa
tgggttcaac ttgattgtgg ccgatattcc 420gtc 42345119PRTGlycine max
45Met Cys Ile Ala Leu Phe Leu Trp Gln Ala His Pro Leu Tyr Pro Phe 1
5 10 15 Leu Leu Leu Asn Asn Arg Asp Glu Tyr His Asn Arg Pro Thr Lys
Pro 20 25 30 Val Ser Trp Trp Glu Asp Ile Asp Ile Val Gly Gly Arg
Asp Glu Ile 35 40 45 Ala Gly Gly Thr Trp Leu Ala Cys Ser Arg Glu
Gly Arg Val Ala Phe 50 55 60 Leu Thr Asn Val Leu Glu Leu Arg Ser
Leu Pro Glu Ala Lys Ser Arg 65 70 75 80 Gly Asp Leu Pro Val Ser Phe
Leu Lys Ser Gly Lys His Pro Lys Glu 85 90 95 Phe Ala Glu Ser Leu
Lys Met Glu Ala His Tyr Tyr Asn Gly Phe Asn 100 105 110 Leu Ile Val
Ala Asp Ile Pro 115 46542DNABeta vulgaris 46cccacgcgtc cgcccacgcg
tccgcccacg cgtccgcgga cgcgtgggtc gacccacgcg 60tccgtttgaa ccacttttca
attttcgagc tgaaacatga aagtgcatta attcacaccc 120aaacctgcaa
cacatctttc tgaatagctc aaaattcgaa attccactca tgcaagagca
180agaatttagc atgaacatga aatgacaaat ttgaaatttc caccactaat
catgaaaaac 240ccatgaaaaa gaaacgtgat gtgcatcgca atatttcaat
ggcaatccca cccactttac 300ccatttcttc tactcctcaa ccgcgacgaa
tatcataccc ggccaacaaa tccagcaggg 360tggtgggaag gtgaagaaat
tgttggtggg aaagatgaag ttggtggtgg gacatggttg 420gcttgttcca
aaggtggaag aattgctttt cttaccaatt ttagagagag agaatcaatt
480cctcatgcta aaagtagagg agatttgcct gttcgttttc ttaagtgtaa
gaaagatccg 540gc 5424794PRTBeta vulgaris 47Met Cys Ile Ala Ile Phe
Gln Trp Gln Ser His Pro Leu Tyr Pro Phe 1 5 10 15 Leu Leu Leu Leu
Asn Arg Asp Glu Tyr His Thr Arg Pro Thr Asn Pro 20 25 30 Ala Gly
Trp Trp Glu Gly Glu Glu Ile Val Gly Gly Lys Asp Glu Val 35 40 45
Gly Gly Gly Thr Trp Leu Ala Cys Ser Lys Gly Gly Arg Ile Ala Phe 50
55 60 Leu Thr Asn Phe Arg Glu Arg Glu Ser Ile Pro His Ala Lys Ser
Arg 65 70 75 80 Gly Asp Leu Pro Val Arg Phe Leu Lys Cys Lys Lys Asp
Pro 85 90 48190DNACapsicum annuum 48agaatgtgca tagcagtgtt
tatttggcaa gcagacagta gatattcatt agtgttgttg 60ttgaacagag atgaatatca
caataggcca acaaaggcag ttcattggtg ggaaggtgga 120gatcaaatag
ttggtggtaa agatgacgtt ggtggtggta cttggttacc ttcttcaaca
180aatggtaaat 1904962PRTCapsicum annuum 49Met Cys Ile Ala Val Phe
Ile Trp Gln Ala Asp Ser Arg Tyr Ser Leu 1 5 10 15 Val Leu Leu Leu
Asn Arg Asp Glu Tyr His Asn Arg Pro Thr Lys Ala 20 25 30 Val His
Trp Trp Glu Gly Gly Asp Gln Ile Val Gly Gly Lys Asp Asp 35 40 45
Val Gly Gly Gly Thr Trp Leu Pro Ser Ser Thr Asn Gly Lys 50 55 60
50719DNALactuca sativamisc_feature(687)..(687)n is a, c, g, or t
50gggaggaaga gtgtcattcc ttactaacgt cttggagctt cacactctcc cggaagccaa
60aactagagga gaccttccac ttcgtttctt ggagagcaat aagagtccag aggaatttgc
120aaaggaattg gtgaaggagg ttcatgagta caatgggttc aacctcataa
cccttgacat 180ttcttcaaaa acgatgtttt atatatcaaa tagaccaaaa
agtgaacctc caactgttca 240acaggttcaa ccaggcatcc atgtcctctc
caatgccaag ctcgactccc cttggccaaa 300ggctcaacgt ttgaagttta
attttaaaaa gttgcttagc gcatatgata aagacgaaga 360tatacccatg
aaggatatga tggacaaact aatgagagac accatgaaag cagaaaagag
420tcaacttcct aatatttgtt ccattgattg ggagcataat ctaagctcga
tatttgttga 480agtagacacc ccgttgggtc gttatgggac gagaagcatg
attgcactaa gtatcaaaga 540taccgaagaa gcaagttttc atgagaccta
cattgaaaga ggattttggt gggagaaaac 600cgtcgattat tatgttactc
cacaagttaa aataaaagat atcgtcttct aagactaaat 660atacgttaca
aatatttaaa atacagnctt tctctctata tatatcttat atataaaaa
71951216PRTLactuca sativa 51Gly Gly Arg Val Ser Phe Leu Thr Asn Val
Leu Glu Leu His Thr Leu 1 5 10 15 Pro Glu Ala Lys Thr Arg Gly Asp
Leu Pro Leu Arg Phe Leu Glu Ser 20 25 30 Asn Lys Ser Pro Glu Glu
Phe Ala Lys Glu Leu Val Lys Glu Val His 35 40 45 Glu Tyr Asn Gly
Phe Asn Leu Ile Thr Leu Asp Ile Ser Ser Lys Thr 50 55 60 Met Phe
Tyr Ile Ser Asn Arg Pro Lys Ser Glu Pro Pro Thr Val Gln 65 70 75 80
Gln Val Gln Pro Gly Ile His Val Leu Ser Asn Ala Lys Leu Asp Ser 85
90 95 Pro Trp Pro Lys Ala Gln Arg Leu Lys Phe Asn Phe Lys Lys Leu
Leu 100 105 110 Ser Ala Tyr Asp Lys Asp Glu Asp Ile Pro Met Lys Asp
Met Met Asp 115 120 125 Lys Leu Met Arg Asp Thr Met Lys Ala Glu Lys
Ser Gln Leu Pro Asn 130 135 140 Ile Cys Ser Ile Asp Trp Glu His Asn
Leu Ser Ser Ile Phe Val Glu 145 150 155 160 Val Asp Thr Pro Leu Gly
Arg Tyr Gly Thr Arg Ser Met Ile Ala Leu 165 170 175 Ser Ile Lys Asp
Thr Glu Glu Ala Ser Phe His Glu Thr Tyr Ile Glu 180 185 190 Arg Gly
Phe Trp Trp Glu Lys Thr Val Asp Tyr Tyr Val Thr Pro Gln 195 200 205
Val Lys Ile Lys Asp Ile Val Phe 210 215 52522DNASorghum bicolor
52agctaagata gttgcaaaca agcgagttac ttacaaccaa ccaaaggagt agaaaccacc
60tgaagccatg tgcattgctg catggatttg gcaggctcac cctgtgcacc aactcctcct
120gcttctcaac agagatgagt tccacagcag gcctacaaaa gcagtaggat
ggtggggaga 180aggctcaaag aagattcttg gtggcaggga tgtgcttggt
ggaggaacat ggatggggtg 240caccaaggat ggaaggcttg ccttcctgac
caatgtgctt gaaccagatg ccatgcccgg 300tgcacggact aggggagatc
tgcctctcag gttcctgcag agcaacaaga gcccactcga 360agttgcaact
gaagtggcag aagaagctca taaatacaat ggcttcaacc tcatactagc
420tgatctaaca acaaatatca tggtctatgt gtcaaaccgg cctaaggggc
agcctgcaac 480aattcaactc gtctcaccag gactccatgt gctgtccaat gc
52253151PRTSorghum bicolor 53Met Cys Ile Ala Ala Trp Ile Trp Gln
Ala His Pro Val His Gln Leu 1 5 10 15 Leu Leu Leu Leu Asn Arg Asp
Glu Phe His Ser Arg Pro Thr Lys Ala 20 25 30 Val Gly Trp Trp Gly
Glu Gly Ser Lys Lys Ile Leu Gly Gly Arg Asp 35 40 45 Val Leu Gly
Gly Gly Thr Trp Met Gly Cys Thr Lys Asp Gly Arg Leu 50 55 60 Ala
Phe Leu Thr Asn Val Leu Glu Pro Asp Ala Met Pro Gly Ala Arg 65 70
75 80 Thr Arg Gly Asp Leu Pro Leu Arg Phe Leu Gln Ser Asn Lys Ser
Pro 85 90 95 Leu Glu Val Ala Thr Glu Val Ala Glu Glu Ala His Lys
Tyr Asn Gly 100 105 110 Phe Asn Leu Ile Leu Ala Asp Leu Thr Thr Asn
Ile Met Val Tyr Val 115 120 125 Ser Asn Arg Pro Lys Gly Gln Pro Ala
Thr Ile Gln Leu Val Ser Pro 130 135 140 Gly Leu His Val Leu Ser Asn
145 150 541132DNATriticum aestivummisc_feature(17)..(17)n is a, c,
g, or t 54aatcctcagg gttacgncga cccacgcgtc cgcaaacaca caaggcgcac
ggttgcgaaa 60caagggaatt atttagaagc aggaaggaac acctgaagcc atgtgtatcg
ctgcatggat 120ttggcaggct cacccacagc atcagctcct gcttctgctc
aacagagatg agttccatag 180caggcctaca aaggcagtag gatggtgggg
ggagggctca atgaagattc ttggcggcag 240ggatgtactt ggtggaggaa
catggatggg gagcaccaaa gatggcagac ttgccttcct 300gaccaatgtg
ctcgagcctg atgcgatgcc tggcgcacgc actaggggag acctgcccct
360caggttcctg cagggcaaca agagcccact ggaggttgca actgaagtcg
caaaagaagc 420tgatgagtac aatggcttca accttatact agctgatcta
accaggaatg tcatggttta 480tgtgtcaaac cggccaaagg ggcagcctgc
gacgattcag ctcgtctcac caggactcca 540tgtgttgtcc aatgcaaggc
tagacagccc ttggcagaag gcaattcgcc ttggtaaaaa 600cttcagggag
tttataagga agcatggtga tgatgaagtt gaagcgaagg atatagctga
660tagactaatg actgacacca cgagggctga taaagatagg ctgccaaaca
ccggttgtga 720tcccaactgg gagcacggtc tgagctccat cttcatcgag
gtgcaaactg acgaagggct 780ctatgggaca aggagcacag cagttctttc
agtgaactat gatggagaag ctagcttata 840tgagaagtac ctcgagagtg
gtatatggaa gaaccacaca gtgcattacc agatagaatt 900gccaatgcgc
acctaaaggc aggagcctca aataggaaga aagaatgaat agctaccatt
960gtgcatgctg ttatttccac agttgcgctt taagatcaca taatgatctc
taattatggc 1020aattaaatta tttactgtat gcggatctat aaattcagag
acagatcaag tcaaattgtt 1080gaatatatat acataataat aatatgatat
agtatgtgta tttacagact tc 113255271PRTTriticum aestivum 55Met Cys
Ile Ala Ala Trp Ile Trp Gln Ala His Pro Gln His Gln Leu 1 5 10 15
Leu Leu Leu Leu Asn Arg Asp Glu Phe His Ser Arg Pro Thr Lys Ala 20
25 30 Val Gly Trp Trp Gly Glu Gly Ser Met Lys Ile Leu Gly Gly Arg
Asp 35 40 45 Val Leu Gly Gly Gly Thr Trp Met Gly Ser Thr Lys Asp
Gly Arg Leu 50 55 60 Ala Phe Leu Thr Asn Val Leu Glu Pro Asp Ala
Met Pro Gly Ala Arg 65 70 75 80 Thr Arg Gly Asp Leu Pro Leu Arg Phe
Leu Gln Gly Asn Lys Ser Pro 85 90 95 Leu Glu Val Ala Thr Glu Val
Ala Lys Glu Ala Asp Glu Tyr Asn Gly 100 105 110 Phe Asn Leu Ile Leu
Ala Asp Leu Thr Arg Asn Val Met Val Tyr Val 115 120 125 Ser Asn Arg
Pro Lys Gly Gln Pro Ala Thr Ile Gln Leu Val Ser Pro 130 135 140 Gly
Leu His Val Leu Ser Asn Ala Arg Leu Asp Ser Pro Trp Gln Lys 145 150
155 160 Ala Ile Arg Leu Gly Lys Asn Phe Arg Glu Phe Ile Arg Lys His
Gly 165 170 175 Asp Asp Glu Val Glu Ala Lys Asp Ile Ala Asp Arg Leu
Met Thr Asp 180 185 190 Thr Thr Arg Ala Asp Lys Asp Arg Leu Pro Asn
Thr Gly Cys Asp Pro 195 200 205 Asn Trp Glu His Gly Leu Ser Ser Ile
Phe Ile Glu Val Gln Thr Asp 210 215 220 Glu Gly Leu Tyr
Gly Thr Arg Ser Thr Ala Val Leu Ser Val Asn Tyr 225 230 235 240 Asp
Gly Glu Ala Ser Leu Tyr Glu Lys Tyr Leu Glu Ser Gly Ile Trp 245 250
255 Lys Asn His Thr Val His Tyr Gln Ile Glu Leu Pro Met Arg Thr 260
265 270 561090DNASaccharum officinarum 56agcaaaacaa gcgagttact
tacaaccaac caaggagtag aaaccacctg aagccatgtg 60cattgctgca tggatttggc
aggctcaccc tgtgcaccaa ctcctcctga ttctcaacag 120agatgagttc
cactgcaggc ctacaaaagc agtaggatgg tggggagaag gctcaaagaa
180gattcttggc ggcagggatg tgcttggtgg aggaacatgg atgggttgca
ccaaggatgg 240caggcttgcc ttcctgacca atgtgcttga accagatgcc
atgcccggtg cacggactag 300gggagatctg cctctcaggt tcctgcagag
caacaagagc ccactcgaag ttgcaactga 360agtggcagaa gaagctcatg
aatacaatgg gttcaacctc atactagctg atctaacaac 420aaatatcatg
gtctatgtgt caaatcggcc taaggggcag cctgcaacaa ttcaactcgt
480ctcaccagga ctccatgtgc tgtccaatgc aaggctagat agcccttggc
agaaggcaat 540tcgccttggt aaaaacttca aggagcttct tagggagcat
ggtgacgatg agattgaagt 600gaaggatata gttgagaggc taatgactga
caccacaaag gctgacaaag atagactgcc 660aaacactggt tgtgatccca
actgggagca tggtctgagc tccatcttca tcgaggtgca 720aactgaccaa
gggctctacg ggacacggag cacagccgtt ttatcagtga actatgatgg
780tgaagctagc ttgtacgaga agtaccttga gagtggtata tggaaggacc
acacagtgaa 840ttaccagata gagtagtagg cattgcacag gaaaagctgg
caacctcaaa taaatagaga 900tatgaagcag acacaattgt ggatttcatt
ctttccctaa tccctagtca ccttcacgac 960tatctaagat cccatcatga
tgccaattac attatttact gtaagcagat ttgtcacttg 1020acgataaaat
gtcaagcaga agtttaagtt taaatatata caccaaatat ataaatttac
1080agacttcgtg 109057266PRTSaccharum officinarum 57Met Cys Ile Ala
Ala Trp Ile Trp Gln Ala His Pro Val His Gln Leu 1 5 10 15 Leu Leu
Ile Leu Asn Arg Asp Glu Phe His Cys Arg Pro Thr Lys Ala 20 25 30
Val Gly Trp Trp Gly Glu Gly Ser Lys Lys Ile Leu Gly Gly Arg Asp 35
40 45 Val Leu Gly Gly Gly Thr Trp Met Gly Cys Thr Lys Asp Gly Arg
Leu 50 55 60 Ala Phe Leu Thr Asn Val Leu Glu Pro Asp Ala Met Pro
Gly Ala Arg 65 70 75 80 Thr Arg Gly Asp Leu Pro Leu Arg Phe Leu Gln
Ser Asn Lys Ser Pro 85 90 95 Leu Glu Val Ala Thr Glu Val Ala Glu
Glu Ala His Glu Tyr Asn Gly 100 105 110 Phe Asn Leu Ile Leu Ala Asp
Leu Thr Thr Asn Ile Met Val Tyr Val 115 120 125 Ser Asn Arg Pro Lys
Gly Gln Pro Ala Thr Ile Gln Leu Val Ser Pro 130 135 140 Gly Leu His
Val Leu Ser Asn Ala Arg Leu Asp Ser Pro Trp Gln Lys 145 150 155 160
Ala Ile Arg Leu Gly Lys Asn Phe Lys Glu Leu Leu Arg Glu His Gly 165
170 175 Asp Asp Glu Ile Glu Val Lys Asp Ile Val Glu Arg Leu Met Thr
Asp 180 185 190 Thr Thr Lys Ala Asp Lys Asp Arg Leu Pro Asn Thr Gly
Cys Asp Pro 195 200 205 Asn Trp Glu His Gly Leu Ser Ser Ile Phe Ile
Glu Val Gln Thr Asp 210 215 220 Gln Gly Leu Tyr Gly Thr Arg Ser Thr
Ala Val Leu Ser Val Asn Tyr 225 230 235 240 Asp Gly Glu Ala Ser Leu
Tyr Glu Lys Tyr Leu Glu Ser Gly Ile Trp 245 250 255 Lys Asp His Thr
Val Asn Tyr Gln Ile Glu 260 265 58743DNAArtificial
sequencefunctional fragments of the cwp1 gene promoter 58tgccgtccta
ttcttagaat actcaagtaa tttaacgtag tggtgaaaat ttgataaatt 60aattatatac
taatttttca gtcttatttt atgtggtata tttaattgga tatgtagttt
120aagaaataat aaaaacttta aaatatttat aaatttactt ttctaaaaaa
gtgaattcaa 180ttttttctct cctcataaat gtattagagt attatcatta
aaattaagtg ggactaataa 240aggtaaaaaa taaattattc ctttaaatta
tttaaccata taagaaaatg tgacattctt 300ttttagactt gactaaaata
gaaaataatg tcatatatat aaaatgagac gaaaaaagta 360aatattaatt
taaaatttaa aactttaggg taatagctac tttgaattac ctagatttca
420ataaaattca acatataata aaacatacta atttacaatt tttaaaataa
tatgactaaa 480agtcatatta ttcaaaaaac aatctatacc gccgtcacct
agttacttta atttgtgtag 540cttctagtac atacattttt aaactttatc
tgaatttaat attttaatta tattaaacat 600ttattaaaat ttataaaatt
taaattgacg taatataatg aagagagtag tactatataa 660accatgtgag
tactaacatg atcttcatct tattcttgtt tttatttata gaaacaataa
720aatagttata aaattaatca atc 743
* * * * *
References