U.S. patent application number 12/660501 was filed with the patent office on 2011-02-03 for transgenic plants with enhanced growth characteristics.
This patent application is currently assigned to Los Alamos National Security, LLC. Invention is credited to Penelope S. Anderson, Thomas J. Knight, Pat J. Unkefer.
Application Number | 20110030089 12/660501 |
Document ID | / |
Family ID | 43528271 |
Filed Date | 2011-02-03 |
United States Patent
Application |
20110030089 |
Kind Code |
A1 |
Unkefer; Pat J. ; et
al. |
February 3, 2011 |
Transgenic plants with enhanced growth characteristics
Abstract
The invention relates to transgenic plants exhibiting
dramatically enhanced growth rates, greater seed and fruit/pod
yields, earlier and more productive flowering, more efficient
nitrogen utilization, increased tolerance to high salt conditions,
and increased biomass yields. In one embodiment, transgenic plants
engineered to over-express both glutamine phenylpyruvate
transaminase (GPT) and glutamine synthetase (GS) are provided. The
GPT+GS double-transgenic plants of the invention consistently
exhibit enhanced growth characteristics, with T0 generation lines
showing an increase in biomass over wild type counterparts of
between 50% and 300%. Generations that result from sexual crosses
and/or selfing typically perform even better, with some of the
double-transgenic plants achieving an astounding four-fold biomass
increase over wild type plants.
Inventors: |
Unkefer; Pat J.; (Los
Alamos, NM) ; Anderson; Penelope S.; (Los Alamos,
NM) ; Knight; Thomas J.; (Raymond, ME) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
425 MARKET STREET
SAN FRANCISCO
CA
94105-2482
US
|
Assignee: |
Los Alamos National Security,
LLC
Los Alamos
NM
University of Maine System Board of Trustees
Bangor
ME
|
Family ID: |
43528271 |
Appl. No.: |
12/660501 |
Filed: |
February 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12551271 |
Aug 31, 2009 |
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12660501 |
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61190520 |
Aug 29, 2008 |
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Current U.S.
Class: |
800/278 ;
435/419; 800/298; 800/305; 800/306; 800/307; 800/309; 800/310;
800/313; 800/314; 800/315; 800/316; 800/317; 800/320; 800/321;
800/323; 800/323.1; 800/323.2; 800/323.3 |
Current CPC
Class: |
C12Y 206/01064 20130101;
C12N 9/93 20130101; C12N 15/8267 20130101; C12N 15/8261 20130101;
C12N 15/827 20130101; C12N 9/1096 20130101; Y02A 40/146 20180101;
C12Y 603/01002 20130101; C12N 15/8273 20130101; C12N 15/8222
20130101 |
Class at
Publication: |
800/278 ;
435/419; 800/298; 800/305; 800/306; 800/307; 800/309; 800/310;
800/313; 800/314; 800/315; 800/316; 800/317; 800/320; 800/321;
800/323; 800/323.1; 800/323.2; 800/323.3 |
International
Class: |
A01H 1/06 20060101
A01H001/06; C12N 5/10 20060101 C12N005/10; A01H 5/00 20060101
A01H005/00 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
Contract No. W-7405-ENG-36 awarded by the United States Department
of Energy to The Regents of The University of California, and
Contract No. DE-AC52-06NA25396, awarded by the United States
Department of Energy to Los Alamos National Security, LLC. The
government has certain rights in this invention.
Claims
1. A transgenic plant comprising a GPT transgene and a GS
transgene, wherein each of said GPT transgene and said GS transgene
is operably linked to a plant promoter.
2. (canceled)
3. The transgenic plant of claim 1, wherein each plant promoter is
heterologous to the plant.
4. The transgenic plant of claim 1, wherein the plant promoter
operably linked to the GPT transgene is heterologous to the species
from which the GPT transgene is derived, and wherein the plant
promoter operably linked to the GS transgene is heterologous to the
species from which the GS transgene is derived.
5. The transgenic plant of claim 4, wherein the GPT transgene
encodes a polypeptide having an amino acid sequence selected from
the group consisting of (a) SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO:
15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ
ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:
35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49, and (b) an
amino acid sequence that is at least 75% identical to any one of
SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID
NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,
SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID
NO: 46 and SEQ ID NO: 49 and has GPT activity.
6. The transgenic plant of claim 4, wherein the GS transgene
encodes a polypeptide having an amino acid sequence selected form
the group consisting of (a) SEQ ID NO: 4, SEQ ID NO: 7 from residue
11, and SEQ ID NO: 41, and (b) an amino acid sequence that is at
least 75% identical to SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO:
41.
7. The transgenic plant according to claim 1, wherein the GPT and
GS transgenes are incorporated into the genome of the plant.
8. The transgenic plant according to claim 1, wherein the GPT
and/or the GS transgenes are incorporated into the plastome of the
plant.
9-15. (canceled)
16. The transgenic plant according to claim 1, which is a plant of
the phylum Tracheophyta.
17. The transgenic plant of claim 16, wherein the transgenic plant
is selected from the group consisting of fruits including apples,
avocado, banana, blackberry, blueberry, cherries, cranberries,
cantaloupes, grapefruit, lemons, limes, nectarines, oranges,
peaches, pineapples, pears, plums, tangelos, tangerines, papaya,
mango, strawberry, raspberry, grape, kiwi fruit, olives, and
coconuts; vegetables including brussel sprouts, radish, mung beans,
carrots, sugarbeet, yams, horseradish, flax, sesame, turnip, sweet
potato, cabbage, canola, cucumbers, eggplant, lettuce, onion, okra,
parsnips, pumpkins and spinach; flowering plants including lily,
carnation, chrysanthemum, petunia, geranium, violet, gladioli,
orchid, and lilac; trees including oil palm; ornamental plants;
canola; lupins; and cotton.
18. The transgenic plant of claim 16, wherein the transgenic plant
is selected from the group consisting of plants of the families
Poaceae, Gossypium, Fabaceae, Brassicaceae, Rutaceae, Rubiaceae,
Cucurbitaceae, Rosaceae, Asteraceae, Amaranthaceae and
Brassicaceae.
19. The transgenic plant of claim 16, wherein the transgenic plant
is selected from the group consisting of plants of the genera
Avena, Hordeum, Oryza, Panicum, Phleum, Saccharum, Secale, Sorghum,
Triticum, Zea, Pennisetum, Lycopersicon, Capiscum, Fagopyrum,
Triticosecale, Chenopodium, Digitaria, Manihot, Ipomoea, Olea,
Daucus, Pastinaca, Raphanus, Dioscorea, Armoracia, Elaeis, Linum,
Carthamus, Sesamum, Vitis, and Solanum.
20-22. (canceled)
23. A method of producing a plant having increased nitrogen use
efficiency relative to an analogous wild type or untransformed
plant, comprising: (a) introducing a GPT transgene into the plant;
(b) introducing a GS transgene into the plant or a progeny of the
plant; (c) expressing the GPT transgene and the GS transgene in the
plant or the progeny of the plant; and, (d) selecting a plant
having an increased nitrogen use efficiency relative to a plant of
the same species that does not comprise a GPT transgene or a GS
transgene.
24-29. (canceled)
30. A transplastomic plant or cell line carrying a GPT transgene
and a GS transgene expression cassette, wherein said expression
cassette is flanked by sequences from the plant or plant cell's
plastome.
31-35. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 12/551,271, filed Aug. 31, 2009, which
application claims priority to U.S. Provisional Application No.
61/190,520 filed Aug. 29, 2008.
BACKGROUND OF THE INVENTION
[0003] As the human population increases worldwide, and available
farmland continues to be destroyed or otherwise compromised, the
need for more effective and sustainable agriculture systems is of
paramount interest to the human race. Improving crop yields,
protein content, and plant growth rates represent major objectives
in the development of agriculture systems that can more effectively
respond to the challenges presented.
[0004] In recent years, the importance of improved crop production
technologies has only increased as yields for many well-developed
crops have tended to plateau. Many agricultural activities are time
sensitive, with costs and returns being dependent upon rapid
turnover of crops or upon time to market. Therefore, rapid plant
growth is an economically important goal for many agricultural
businesses that involve high-value crops such as grains,
vegetables, berries and other fruits.
[0005] Genetic engineering has and continues to play an
increasingly important yet controversial role in the development of
sustainable agriculture technologies. A large number of genetically
modified plants and related technologies have been developed in
recent years, many of which are in widespread use today (Factsheet:
Genetically Modified Crops in the United States, Pew Initiative on
Food and Biotechnology, August 2004,
http://pewagbiotech.org/resources/factsheets). The adoption of
transgenic plant varieties is now very substantial and is on the
rise, with approximately 250 million acres planted with transgenic
plants in 2006.
[0006] While acceptance of transgenic plant technologies may be
gradually increasing, particularly in the United States, Canada and
Australia, many regions of the World remain slow to adopt
genetically modified plants in agriculture, notably Europe.
Therefore, consonant with pursuing the objectives of responsible
and sustainable agriculture, there is a strong interest in the
development of genetically engineered plants that do not introduce
toxins or other potentially problematic substances into plants
and/or the environment. There is also a strong interest in
minimizing the cost of achieving objectives such as improving
herbicide tolerance, pest and disease resistance, and overall crop
yields. Accordingly, there remains a need for transgenic plants
that can meet these objectives.
[0007] The goal of rapid plant growth has been pursued through
numerous studies of various plant regulatory systems, many of which
remain incompletely understood. In particular, the plant regulatory
mechanisms that coordinate carbon and nitrogen metabolism are not
fully elucidated. These regulatory mechanisms are presumed to have
a fundamental impact on plant growth and development.
[0008] The metabolism of carbon and nitrogen in photosynthetic
organisms must be regulated in a coordinated manner to assure
efficient use of plant resources and energy. Current understanding
of carbon and nitrogen metabolism includes details of certain steps
and metabolic pathways which are subsystems of larger systems. In
photosynthetic organisms, carbon metabolism begins with CO.sub.2
fixation, which proceeds via two major processes, termed C-3 and
C-4 metabolism. In plants with C-3 metabolism, the enzyme ribulose
bisphosphate carboxylase (RuBisCo) catalyzes the combination of
CO.sub.2 with ribulose bisphosphate to produce 3-phosphoglycerate,
a three carbon compound (C-3) that the plant uses to synthesize
carbon-containing compounds. In plants with C-4 metabolism,
CO.sub.2 is combined with phosphoenol pyruvate to form acids
containing four carbons (C-4), in a reaction catalyzed by the
enzyme phosphoenol pyruvate carboxylase. The acids are transferred
to bundle sheath cells, where they are decarboxylated to release
CO.sub.2, which is then combined with ribulose bisphosphate in the
same reaction employed by C-3 plants.
[0009] Numerous studies have found that various metabolites are
important in plant regulation of nitrogen metabolism. These
compounds include the organic acid malate and the amino acids
glutamate and glutamine. Nitrogen is assimilated by photosynthetic
organisms via the action of the enzyme glutamine synthetase (GS)
which catalyzes the combination of ammonia with glutamate to form
glutamine. GS plays a key role in the assimilation of nitrogen in
plants by catalyzing the addition of ammonium to glutamate to form
glutamine in an ATP-dependent reaction (Miflin and Habash, 2002,
Journal of Experimental Botany, Vol. 53, No. 370, pp. 979-987). GS
also reassimilates ammonia released as a result of photorespiration
and the breakdown of proteins and nitrogen transport compounds. GS
enzymes may be divided into two general classes, one representing
the cytoplasmic form (GS1) and the other representing the plastidic
(i.e., chloroplastic) form (GS2).
[0010] Previous work has demonstrated that increased expression
levels of GS1 result in increased levels of GS activity and plant
growth, although reports are inconsistent. For example, Fuentes et
al. reported that CaMV S35 promoter-driven overexpression of
Alfalfa GS1 (cytoplasmic form) in tobacco resulted in increased
levels of GS expression and GS activity in leaf tissue, increased
growth under nitrogen starvation, but no effect on growth under
optimal nitrogen fertilization conditions (Fuentes et al., 2001, J.
Exp. Botany 52: 1071-81). Temple et al. reported that transgenic
tobacco plants overexpressing the full length Alfalfa GS1 coding
sequence contained greatly elevated levels of GS transcript, and GS
polypeptide which assembled into active enzyme, but did not report
phenotypic effects on growth (Temple et al., 1993, Molecular and
General Genetics 236: 315-325). Corruzi et al. have reported that
transgenic tobacco overexpressing a pea cytosolic GS1 transgene
under the control of the CaMV S35 promoter show increased GS
activity, increased cytosolic GS protein, and improved growth
characteristics (U.S. Pat. No. 6,107,547). Unkefer et al. have more
recently reported that transgenic tobacco plants overexpressing the
Alfalfa GS1 in foliar tissues, which had been screened for
increased leaf-to-root GS activity following genetic segregation by
selfing to achieve increased GS1 transgene copy number, were found
to produce increased 2-hydroxy-5-oxoproline levels in their foliar
portions, which was found to lead to markedly increased growth
rates over wildtype tobacco plants (see, U.S. Pat. Nos. 6,555,500;
6,593,275; and 6,831,040).
[0011] Unkefer et al. have further described the use of
2-hydroxy-5-oxoproline (also known as 2-oxoglutaramate) to improve
plant growth (U.S. Pat. Nos. 6,555,500; 6,593,275; 6,831,040). In
particular, Unkefer et al. disclose that increased concentrations
of 2-hydroxy-5-oxoproline in foliar tissues (relative to root
tissues) triggers a cascade of events that result in increased
plant growth characteristics. Unkefer et al. describe methods by
which the foliar concentration of 2-hydroxy-5-oxoproline may be
increased in order to trigger increased plant growth
characteristics, specifically, by applying a solution of
2-hydroxy-5-oxoproline directly to the foliar portions of the plant
and over-expressing glutamine synthetase preferentially in leaf
tissues.
[0012] A number of transaminase and hydrolyase enzymes known to be
involved in the synthesis of 2-hydroxy-5-oxoproline in animals have
been identified in animal liver and kidney tissues (Cooper and
Meister, 1977, CRC Critical Reviews in Biochemistry, pages 281-303;
Meister, 1952, J. Biochem. 197: 304). In plants, the biochemical
synthesis of 2-hydroxy-5-oxoproline has been known but has been
poorly characterized. Moreover, the function of
2-hydroxy-5-oxoproline in plants and the significance of its pool
size (tissue concentration) are unknown. Finally, the art provides
no specific guidance as to precisely what transaminase(s) or
hydrolase(s) may exist and/or be active in catalyzing the synthesis
of 2-hydroxy-5-oxoproline in plants, and no such plant
transaminases have been reported, isolated or characterized.
SUMMARY OF THE INVENTION
[0013] The invention relates to transgenic plants exhibiting
dramatically enhanced growth rates, greater seed and fruit/pod
yields, earlier and more productive flowering, more efficient
nitrogen utilization, increased tolerance to high salt conditions,
and increased biomass yields. In one embodiment, transgenic plants
engineered to over-express both glutamine phenylpyruvate
transaminase (GPT) and glutamine synthetase (GS) are provided. The
GPT+GS double-transgenic plants of the invention consistently
exhibit enhanced growth characteristics, with T0 generation lines
showing an increase in biomass over wild type counterparts of
between 50% and 300%. Generations that result from sexual crosses
and/or selfing typically Perform even better, with some of the
double-transgenic plants achieving an astounding four-fold biomass
increase over wild type plants. Similarly, flower and fruit or pod
yields are also tremendously improved, with T0 generation lines
typically showing 50% to 70% increases over their wild type
counterparts, and in some cases showing a 100% increase. Transgenic
plants exhibiting such enhanced growth phenotypic characteristics
have been successfully generated across a spectrum of individual
plant species, using various transformation methodologies,
different expression vectors and promoters, and heterologous and
homologous transgene sequences from a variety of species, as
exemplified by the numerous working examples provided herein. This
invention, therefore, provides a fundamental break-though
technology that has the potential to transform virtually all areas
of agriculture.
[0014] Applicants have identified the enzyme glutamine
phenylpyruvate transaminase (GPT) as a catalyst of
2-hydroxy-5-oxoproline (2-oxoglutaramate) synthesis in plants.
2-oxoglutaramate is a powerful signal metabolite which regulates
the function of a large number of genes involved in the
photosynthesis apparatus, carbon fixation and nitrogen metabolism.
The invention provides isolated nucleic acid molecules encoding
GPT, and discloses the novel finding that the encoded enzyme is
directly involved in the synthesis of 2-hydroxy-5-oxoproline. This
aspect of the invention is exemplified herein by the disclosure of
GPT polynucleotides encoding GPTs from several species, including
Arabidopsis, Grape, Rice, Soybean, Barley, Bamboo and a non-plant
homolog from Zebra fish, most of which have been expressed as
recombinant GPTs and confirmed as having GPT activity.
[0015] The invention further provides transgenic plants which
express both a GPT transgene and a GS transgene. The expression of
these two transgenes in such "double-transgene" plants results in a
substantially increased rate of carbon dioxide fixation and an
extremely potent growth enhancing effect, as these plants exhibit
very significantly and sometimes tremendously enhanced growth rates
and flower/fruit/pod/seed yields. Methods for the generation of
such growth-enhanced transgenic plants are provided.
[0016] By preferentially increasing the concentration of the signal
metabolite 2-oxoglutaramate (i.e., in foliar tissues), the
transgenic plants of the invention are capable of producing higher
overall yields over shorter periods of time, and therefore may
provide agricultural industries with enhanced productivity across a
wide range of crops. Importantly, unlike many transgenic plants
described to date, the invention utilizes natural plant genes
encoding a natural plant enzyme. The enhanced growth
characteristics of the transgenic plants of the invention is
achieved essentially by introducing additional GPT and GS capacity
into the plant. Thus, the transgenic plants of the invention do not
express any toxic substances, growth hormones, viral or bacterial
gene products, and are therefore free of many of the concerns that
have heretofore impeded the adoption of transgenic plants in
certain parts of the World.
[0017] In one embodiment, the invention provides a transgenic plant
comprising a GPT transgene and a GS transgene, wherein said GPT
transgene and said GS transgene are operably linked to a plant
promoter. In a specific embodiment, the GS transgene is a GS1
transgene. In another specific embodiment, the GPT transgene
encodes a polypeptide having an amino acid sequence selected from
the group consisting of (a) SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO:
15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ
ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:
35, SEQ ID NO: 36, SEQ ID NO: 46 and SEQ ID NO: 49, and (b) an
amino acid sequence that is at least 75% identical to any one of
SEQ ID NO: 2; SEQ ID NO: 9; SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID
NO: 21, SEQ ID NO 24, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32,
SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID
NO: 46 and SEQ ID NO: 49 and has GPT activity. In yet another
specific embodiment, the GS transgene encodes a polypeptide having
an amino acid sequence selected from the group consisting of (a)
SEQ ID NO: 4, SEQ ID NO: 7 from residue 11 and SEQ ID NO: 41, and
(b) an amino acid sequence that is at least 75% identical to SEQ ID
NO: 4, SEQ ID NO: 7 or SEQ ID NO: 41. In some embodiments, the GPT
and GS transgenes are incorporated into the genome of the
plant.
[0018] In a particular aspect of the invention, the GPT transgene
and a GS transgene construct is incorporated into the genome of a
plant selected from the group consisting of: maize, rice, sugar
cane, wheat, oats, sorghum, switch grass, soya bean, tubers (such
as potatoes), canola, lupins or cotton.
[0019] The invention also provides progeny of any generation of the
transgenic plants of the invention, wherein said progeny comprises
a GPT transgene and a GS transgene, as well as a seed of any
generation of the transgenic plants of the invention, wherein said
seed comprises said GPT transgene and said GS transgene. The
transgenic plants of the invention may display one or more enhanced
growth characteristics rate when compared to an analogous wild-type
or untransformed plant, including without limitation increased
growth rate, biomass yield, seed yield, flower or flower bud yield,
fruit or pod yield, larger leaves, and may also display increased
levels of GPT and/or GS activity, and/or increased levels of
2-oxoglutaramate. In some embodiments, the transgenic plants of the
invention display increased nitrogen use efficiency or increased
tolerance to salt or saline conditions.
[0020] In a further aspect of the invention there is provided a
transplastomic plant or cell line carrying a GPT transgene and a GS
transgene expression cassette, said expression cassette being
flanked by sequences from the plant or plant cell's plastome.
[0021] Further still, the invention provides a method for preparing
a transplastomic plant or cell line carrying a GPT transgene and a
GS transgene construct, said method comprising the steps of: (a)
inserting into at least one expression cassette at least a GPT
transgene and a GS transgene, wherein said expression cassette is
flanked by sequences from the plant or plant cell's plastome.
[0022] Methods for producing the transgenic plants of the invention
and seeds thereof are also provided, including methods for
producing a plant having enhanced growth properties, increased
nitrogen use efficiency and increased tolerance to germination or
growth in salt or saline conditions, relative to an analogous wild
type or untransformed plant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] 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.
[0024] FIG. 1. Nitrogen assimilation and 2-oxoglutaramate
biosynthesis: schematic of metabolic pathway.
[0025] FIG. 2. Photograph showing comparison of transgenic tobacco
plants over-expressing either GS1 or GPT, compared to wild type
tobacco plant. From left to right: wild type plant, Alfalfa GS1
transgene, Arabidopsis GPT transgene. See Examples 3 and 5,
infra.
[0026] FIG. 3. Photograph showing comparison of transgenic
Micro-Tom tomato plants over-expressing either GS1 or GPT, compared
to wild type tomato plant. From left to right: wild type plant,
Alfalfa GS1 transgene, Arabidopsis GPT transgene. See Examples 4
and 6, infra.
[0027] FIG. 4. Photographs showing comparisons of leaf sizes
between wild type and GS1 or GPT transgenic tobacco plants. A:
Comparison between leaves from GS1 transgenic tobacco (bottom leaf)
and wild type (top leaf). B: Comparison between leaves from GPT
transgenic tobacco (bottom leaf) and wild type (top leaf).
[0028] FIG. 5. Photographs showing comparisons of transgenic
tobacco plants generated from various crosses between GS1 and GPT
transgenic tobacco lines with wild type and single transgene
plants. A-C: Cross 2, 3 and 7, respectively. See Example 7,
infra.
[0029] FIG. 6. Photographs showing comparisons of leaf sizes
between wild type and crosses between GS1 and GPT transgenic
tobacco plants. A: Comparison between leaves from GSXGPT Cross 3
(bottom leaf) and wild type (top leaf). B: Comparison between
leaves from GSXGPT Cross 7 (bottom leaf) and wild type (top leaf).
See Example 7, infra.
[0030] FIG. 7. Photograph of transgenic pepper plant (right) and
wild type control pepper plant (left), showing larger pepper fruit
yield in the transgenic plant relative to the wild type control
plant. See Example 8, infra.
[0031] FIG. 8. Transgenic bean plants compared to wild type control
bean plants (several transgenic lines expressing Arabidopsis GPT
and GS transgenes). Upper Left: plant heights on various days;
Upper right: flower bud numbers; Lower left: flower numbers; Lower
right: bean pod numbers. Wildtype is the control, and lines 2A, 4A
and 5B are all transgenic plant lines. See Example 9, infra.
[0032] FIG. 9. Photograph of transgenic bean plant (right) and wild
type control bean plant (left), showing increased growth in the
transgenic plant relative to the wild type control plant.
Transgenic line expressing Arabidopsis GPT and GS transgenes. See
Example 9, infra.
[0033] FIG. 10. Transgenic bean plants pods, flowers and flower
buds compared to wild type control bean plants (transgenic line
expressing grape GPT and Arabidopsis GS transgenes). See Example
10, infra.
[0034] FIG. 11. Photograph of transgenic bean plant (right) and
wild type control bean plant (left), showing increased growth in
the transgenic plant relative to the wild type control plant.
Transgenic line expressing Grape GPT and Arabidopsis GS transgenes.
See Example 10, infra.
[0035] FIG. 12. Transgenic Cowpea Line A plants compared to wild
type control Cowpea plants (transgenic line expressing Arabidopsis
GPT and GS transgenes), showing that the transgenic plants grow
faster and flower and set pods sooner than wild type control
plants. (A) Relative height and longest leaf measurements as of May
21, (B) Relative trifolate leafs and flower buds as of June 18, (C)
Relative numbers of flowers, flower buds and pea pods as of June
22. See Example 11, infra.
[0036] FIG. 13. Photograph of transgenic Cowpea Line A plant
(right) and wild type control Cowpea plant (left), showing
increased growth in the transgenic plant relative to the wild type
control plant. Transgenic line expressing Arabidopsis GPT and GS
transgenes. See Example 11, infra.
[0037] FIG. 14. Transgenic Cowpea Line G plants compared to wild
type control Cowpea plants (transgenic line expressing Grape GPT
and Arabidopsis GS transgenes), showing that the transgenic plants
grow faster and flower and set pods sooner than wild type control
plants. (A) plant heights, (B) flowers and pea pod numbers, (C)
leaf bud and trifolate numbers. See Example 12, infra.
[0038] FIG. 15. Photograph of transgenic Cowpea Line G plant
(right) and wild type control Cowpea plant (left), showing
increased growth in the transgenic plant relative to the wild type
control plant. Transgenic line expressing Grape GPT and Arabidopsis
GS transgenes. See Example 12, infra.
[0039] FIG. 16. Photograph of transgenic Cantaloupe plant (right)
and wild type control Cantaloupe plant (left), showing increased
growth in the transgenic plant relative to the wild type control
plant. Transgenic line expressing Arabidopsis GPT and GS
transgenes. See Example 14, infra.
[0040] FIG. 17. Photograph of transgenic Pumpkin plants (right) and
wild type control Pumpkin plants (left), showing increased growth
in the transgenic plants relative to the wild type control plants.
Transgenic lines expressing Arabidopsis GPT and GS transgenes. See
Example 15, infra.
[0041] FIG. 18. Photograph of transgenic Arabidopsis plants (right)
and wild type control Arabidopsis plants (left), showing increased
growth in the transgenic plants relative to the wild type control
plants. Transgenic lines expressing Arabidopsis GPT and GS
transgenes. See Example 16, infra.
[0042] FIG. 19. Transgenic tomato plants expressing Arabidopsis GPT
and GS transgenes compared to control tomato plants. (A) Photograph
of transgenic tomato plant leaves (right) vs. wild type control
leaves (left) showing larger leaves in the transgenic plant. (B)
Photograph of transgenic tomato plants (right) and wild type
control plants (left), showing increased growth in the transgenic
plants relative to the wild type control plants. See Example 17,
infra.
[0043] FIG. 20. Photograph of transgenic Camelina plant (right) and
wild type control Camelina plant (left), showing increased growth
in the transgenic plant relative to the wild type control plant.
Transgenic line expressing Arabidopsis GPT and GS transgenes. See
Example 18, infra.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0044] Unless otherwise defined, all terms of art, notations and
other scientific terminology used herein are intended to have the
meanings commonly understood by those of skill in the art to which
this invention pertains. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready
reference, and the inclusion of such definitions herein should not
necessarily be construed to represent a substantial difference over
what is generally understood in the art. The techniques and
procedures described or referenced herein are generally well
understood and commonly employed using conventional methodology by
those skilled in the art, such as, for example, the widely utilized
molecular cloning methodologies described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current
Protocols in Molecular Biology (Ausbel et al., eds., John Wiley
& Sons, Inc. 2001; Transgenic Plants: Methods and Protocols
(Leandro Pena, ed., Humana Press, 1.sup.st edition, 2004); and,
Agrobacterium Protocols (Wan, ed., Humana Press, 2.sup.nd edition,
2006). As appropriate, procedures involving the use of commercially
available kits and reagents are generally carried out in accordance
with manufacturer defined protocols and/or parameters unless
otherwise noted.
[0045] Each document, reference, patent application or patent cited
in this text is expressly incorporated herein in its entirety by
reference, and each should be read and considered as part of this
specification. That the document, reference, patent application or
patent cited in this specification is not repeated herein is merely
for conciseness.
[0046] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof ("polynucleotides") in either
single- or double-stranded form. Unless specifically limited, the
term "polynucleotide" encompasses nucleic acids containing known
analogues of natural nucleotides which have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.
degenerate codon substitutions) and complementary sequences and as
well as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al., 1991, Nucleic Acid Res. 19: 5081; Ohtsuka et al., 1985 J.
Biol. Chem. 260: 2605-2608; and Cassol et al., 1992; Rossolini et
al., 1994, Mol. Cell. Probes 8: 91-98). The term nucleic acid is
used interchangeably with gene, cDNA, and mRNA encoded by a
gene.
[0047] The term "promoter" refers to anucleic acid control sequence
or sequences that direct transcription of an operably linked
nucleic acid. As used herein, a "plant promoter" is a promoter that
functions in plants. Promoters include necessary nucleic acid
sequences near the start site of transcription, such as, in the
case of a polymerase II type promoter, a TATA element. A promoter
also optionally includes distal enhancer or repressor elements,
which can be located as much as several thousand base pairs from
the start site of transcription. A "constitutive" promoter is a
promoter that is active under most environmental and developmental
conditions. An "inducible" promoter is a promoter that is active
under environmental or developmental regulation. The term "operably
linked" refers to a functional linkage between a nucleic acid
expression control sequence (such as a promoter, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence directs
transcription of the nucleic acid corresponding to the second
sequence.
[0048] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers.
[0049] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0050] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0051] The term "plant" includes whole plants, plant organs (e.g.,
leaves, stems, flowers, roots, reproductive organs, embryos and
parts thereof, etc.), seedlings, seeds and plant cells and progeny
thereof. The class of plants which can be used in the method of the
invention is generally as broad as the class of higher plants
amenable to transformation techniques, including angiosperms
(monocotyledonous and dicotyledonous plants), as well as
gymnosperms. It includes plants of a variety of ploidy levels,
including polyploid, diploid, haploid and hemizygous.
[0052] The terms "GPT polynucleotide" and "GPT nucleic acid" are
used interchangeably herein, and refer to a full length or partial
length polynucleotide sequence of a gene which encodes a
polypeptide involved in catalyzing the synthesis of
2-oxoglutaramate, and includes polynucleotides containing both
translated (coding) and un-translated sequences, as well as the
complements thereof. The term "GPT coding sequence" refers to the
part of the gene which is transcribed and encodes a GPT protein.
The term "targeting sequence" refers to the amino terminal part of
a protein which directs the protein into a subcellular compartment
of a cell, such as a chloroplast in a plant cell. GPT
polynucleotides are further defined by their ability to hybridize
under defined conditions to the GPT polynucleotides specifically
disclosed herein, or to PCR products derived therefrom.
[0053] A "GPT transgene" is a nucleic acid molecule comprising a
GPT polynucleotide which is exogenous to transgenic plant, or plant
embryo, organ or seed, harboring the nucleic acid molecule, or
which is exogenous to an ancestor plant, or plant embryo, organ or
seed thereof, of a transgenic plant harboring the GPT
polynucleotide. More particularly, the exogenous GPT transgene will
be heterogeneous with any GPT polynucleotide sequence present in
wild-type plant, or plant embryo, organ or seed into which the GPT
transgene is inserted. To this extent the scope of the
heterogeneity required need only be a single nucleotide difference.
However, preferably the heterogeneity will be in the order of an
identity between sequences selected from the following identities:
0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.
[0054] The terms "GS polynucleotide" and "GS nucleic acid" are used
interchangeably herein, and refer to a full length or partial
length polynucleotide sequence of a gene which encodes a glutamine
synthetase protein, and includes polynucleotides containing both
translated (coding) and un-translated sequences, as well as the
complements thereof. The term "GS coding sequence" refers to the
part of the gene which is transcribed and encodes a GS protein. The
terms "GS1 polynucleotide" and "GS1 nucleic acid" are used
interchangeably herein, and refer to a full length or partial
length polynucleotide sequence of a gene which encodes a glutamine
synthetase isoform 1 protein, and includes polynucleotides
containing both translated (coding) and un-translated sequences, as
well as the complements thereof. The term "GS1 coding sequence"
refers to the part of the gene which is transcribed and encodes a
GS1 protein.
[0055] A "GS transgene" is a nucleic acid molecule comprising a GS
polynucleotide which is exogenous to transgenic plant, or plant
embryo, organ or seed, harboring the nucleic acid molecule, or
which is exogenous to an ancestor plant, or plant embryo, organ or
seed thereof, of a transgenic plant harboring the GS
polynucleotide. A "GS1 transgene" is a nucleic acid molecule
comprising a GS1 polynucleotide which is exogenous to transgenic
plant, or plant embryo, organ or seed, harboring the nucleic acid
molecule, or which is exogenous to an ancestor plant, or plant
embryo, organ or seed thereof, of a transgenic plant harboring the
GS1 polynucleotide. More particularly, the exogenous GS or GS1
transgene will be heterogeneous with any GS or GS1 polynucleotide
sequence present in wild-type plant, or plant embryo, organ or seed
into which the GS or GS1 transgene is inserted. To this extent the
scope of the heterogeneity required need only be a single
nucleotide difference. However, preferably the heterogeneity will
be in the order of an identity between sequences selected from the
following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%,
and 20%.
[0056] Exemplary GPT polynucleotides of the invention are presented
herein, and include GPT coding sequences for Arabidopsis, Rice,
Barley, Bamboo, Soybean, Grape, Clementine orange and Zebra Fish
GPTs.
[0057] Partial length GPT polynucleotides include polynucleotide
sequences encoding N- or C-terminal truncations of GPT, mature GPT
(without targeting sequence) as well as sequences encoding domains
of GPT. Exemplary GPT polynucleotides encoding N-terminal
truncations of GPT include Arabidopsis-30, -45 and -56 constructs,
in which coding sequences for the first 30, 45, and 56,
respectively, amino acids of the full length GPT structure of SEQ
ID NO: 2 are eliminated.
[0058] In employing the GPT polynucleotides of the invention in the
generation of transformed cells and transgenic plants, one of skill
will recognize that the inserted polynucleotide sequence need not
be identical, but may be only "substantially identical" to a
sequence of the gene from which it was derived, as further defined
below. The term "GPT polynucleotide" specifically encompasses such
substantially identical variants. Similarly, one of skill will
recognize that because of codon degeneracy, a number of
polynucleotide sequences will encode the same polypeptide, and all
such polynucleotide sequences are meant to be included in the term
GPT polynucleotide. In addition, the term specifically includes
those sequences substantially identical (determined as described
below) with an GPT polynucleotide sequence disclosed herein and
that encode polypeptides that are either mutants of wild type GPT
polypeptides or retain the function of the GPT polypeptide (e.g.,
resulting from conservative substitutions of amino acids in a GPT
polypeptide). The term "GPT polynucleotide" therefore also includes
such substantially identical variants.
[0059] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refers to
those nucleic acids which encode identical or essentially identical
amino acid sequences, or where the nucleic acid does not encode an
amino acid sequence, to essentially identical sequences. Because of
the degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
[0060] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0061] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0062] Macromolecular structures such as polypeptide structures can
be described in terms of various levels of organization. For a
general discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell (3.sup.rd ed., 1994) and Cantor and
Schimmel, Biophysical Chemistry Part I: The Conformation of
Biological Macromolecules (1980). "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains.
Domains are portions of a polypeptide that form a compact unit of
the polypeptide and are typically 25 to approximately 500 amino
acids long. Typical domains are made up of sections of lesser
organization such as stretches of .beta.-sheet and .alpha.-helices.
"Tertiary structure" refers to the complete three dimensional
structure of a polypeptide monomer. "Quaternary structure" refers
to the three dimensional structure formed by the noncovalent
association of independent tertiary units. Anisotropic terms are
also known as energy terms.
[0063] The term "isolated" refers to material which is
substantially or essentially free from components which normally
accompany the material as it is found in its native or natural
state. However, the term "isolated" is not intended refer to the
components present in an electrophoretic gel or other separation
medium. An isolated component is free from such separation media
and in a form ready for use in another application or already in
use in the new application/milieu. An "isolated" antibody is one
that has been identified and separated and/or recovered from a
component of its natural environment. Contaminant components of its
natural environment are materials that would interfere with
diagnostic or therapeutic uses for the antibody, and may include
enzymes, hormones, and other proteinaceous or non-proteinaceous
solutes. In preferred embodiments, the antibody will be purified
(1) to greater than 95% by weight of antibody as determined by the
Lowry method, and most preferably more than 99% by weight, (2) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (3) to homogeneity by SDS-PAGE under reducing or nonreducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes the antibody in situ within recombinant
cells since at least one component of the antibody's natural
environment will not be present. Ordinarily, however, isolated
antibody will be prepared by at least one purification step.
[0064] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, a nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
nucleic acid encoding a protein from one source and a nucleic acid
encoding a peptide sequence from another source. Similarly, a
heterologous protein indicates that the protein comprises two or
more subsequences that are not found in the same relationship to
each other in nature (e.g., a fusion protein).
[0065] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%,
or 95% identity over a specified region, when compared and aligned
for maximum correspondence over a comparison window, or designated
region as measured using a sequence comparison algorithms, or by
manual alignment and visual inspection. This definition also refers
to the complement of a test sequence, which has substantial
sequence or subsequence complementarity when the test sequence has
substantial identity to a reference sequence. This definition also
refers to the complement of a test sequence, which has substantial
sequence or subsequence complementarity when the test sequence has
substantial identity to a reference sequence.
[0066] When percentage of sequence identity is used in reference to
polypeptides, it is recognized that residue positions that are not
identical often differ by conservative amino acid substitutions,
where amino acids residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the polypeptide. Where sequences differ in
conservative substitutions, the percent sequence identity may be
adjusted upwards to correct for the conservative nature of the
substitution.
[0067] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0068] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment
algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443, by
the search for similarity method of Pearson & Lipman, 1988,
Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0069] A preferred example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., 1977, Nuc. Acids Res. 25:3389-3402 and Altschul et al.,
1990, J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0
are used, typically with the default parameters described herein,
to determine percent sequence identity for the nucleic acids and
proteins of the invention. Software for performing BLAST analyses
is publicly available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a word length (W) of 11, an expectation
(E) of 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a word length
of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix
(see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands.
[0070] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0071] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, highly
stringent conditions are selected to be about 5-10.degree. C. lower
than the thermal melting point (Tm) for the specific sequence at a
defined ionic strength pH. Low stringency conditions are generally
selected to be about 15-30.degree. C. below the Tm. Tm is the
temperature (under defined ionic strength, pH, and nucleic
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). Stringent conditions will be those in
which the salt concentration is less than about 1.0M sodium ion,
typically about 0.01 to 1.0M sodium ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g., greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. For selective
or specific hybridization, a positive signal is at least two times
background, preferably 10 times background hybridization.
[0072] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cased, the nucleic acids typically hybridize under moderately
stringent hybridization conditions.
[0073] Genomic DNA or cDNA comprising GPT polynucleotides may be
identified in standard Southern blots under stringent conditions
using the GPT polynucleotide sequences disclosed here. For this
purpose, suitable stringent conditions for such hybridizations are
those which include a hybridization in a buffer of 40% formamide,
1M NaCl, 1% SDS at 37.degree. C., and at least one wash in
0.2.times.SSC at a temperature of at least about 50.degree. C.,
usually about 55.degree. C. to about 60.degree. C., for 20 minutes,
or equivalent conditions. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions may be utilized
to provide conditions of similar stringency.
[0074] A further indication that two polynucleotides are
substantially identical is if the reference sequence, amplified by
a pair of oligonucleotide primers, can then be used as a probe
under stringent hybridization conditions to isolate the test
sequence from a cDNA or genomic library, or to identify the test
sequence in, e.g., a northern or Southern blot.
Transgenic Plants:
[0075] The invention provides novel transgenic plants exhibiting
substantially enhanced agronomic characteristics, including faster
growth, greater mature plant fresh weight and total biomass,
earlier and more abundant flowering, and greater fruit, pod and
seed yields. The transgenic plants of the invention are generated
by introducing into a plant one or more expressible genetic
constructs capable of driving the expression of one or more
polynucleotides encoding glutamine synthetase (GS) and glutamine
phenylpyruvate transaminase (GPT). In an exemplary embodiment,
single-transgene parental lines carrying either a GPT or GS1
transgene coding sequence are generated, preferably selfed until
homozygous for the transgene, then crossed to generate progeny
plants containing both transgenes.
[0076] The transgenic plants of the invention may be any vascular
plant of the phylum Tracheophyta, including angiosperms and
gymnosperms. Angiosperms may be a monocotyledonous (monocot) or a
dicotyledonous (dicot) plant. Important monocots include those of
the grass families, such as the family Poaceae and Gramineae,
including plants of the genus Avena (Avena sativa, oats), genus
Hordeum (i.e., Hordeum vulgare, Barley), genus Oryza (i.e., Oryza
sativa, rice, cultivated rice varieties), genus Panicum (Panicum
spp., Panicum virgatum, Switchgrass), genus Phleum (Phleum
pratense, Timothy-grass), genus Saccharum (i.e., Saccharum
officinarum, Saccharum spontaneum, hybrids thereof, Sugarcane),
genus Secale (i.e., Secale cereale, Rye), genus Sorghum (Sorghum
vulgare, Sorghum), genus Triticum (wheat, various classes,
including T. aestivum and T. durum), genus Fagopyrum (buckwheat,
including F. esculentum), genus Triticosecale (Triticale, various
hybrids of wheat and rye), genus Chenopodium (quinoa, including C.
quinoa), genus Zea (i.e., Zea mays, numerous varieties) as well as
millets (i.e., Pennisetum glaucum) including the genus Digitaria
(D. exilis).
[0077] Important dicots include those of the family Solanaceae,
such as plants of the genus Lycopersicon (Lycopersicon esculentum,
tomato), genus Capiscum (Capsicum annuum, peppers), genus Solanum
(Solanum tuberosum, potato, S. lycopersicum, tomato); genus Manihot
(cassaya, M. esculenta), genus Ipomoea (sweet potato, I. batatas),
genus Olea (olives, including O. europaea); plants of the Gossypium
family (i.e., Gossypium spp., G. hirsutum, G. herbaceum, cotton);
the Legumes (family Fabaceae), such as peas (Pisum spp, P.
sativum), beans (Glycine spp., Glycine max(soybean); Phaseolus
vulgaris, common beans, Vigna radiata, mung bean), chickpeas (Cicer
arietinum)), lentils (Lens culinaris), peanuts (Arachis hypogaea);
coconuts (Cocos nucifera) as well as various other important crops
such as camelina (Camelina sativa, family Brassicaceae), citrus
(Citrus spp, family Rutaceae), coffee (Coffea spp, family
Rubiaceae), melon (Cucumis spp, family Cucurbitaceae), squash
(Cucurbita spp, family Cucurbitaceae), roses (Rosa spp, family
Rosaceae), sunflower (Helianthus annuus, family Asteraceae), sugar
beets (Beta spp, family Amaranthaceae), including sugarbeet, B.
vulgaris), genus Daucus (carrots, including D. carota), genus
Pastinaca (parsnip, including P. sativa), genus Raphanus (radish,
including R. sativus), genus Dioscorea (yams, including D.
rotundata and D. cayenensis), genus Armoracia (horseradish,
including A. rusticana), genus Elaeis (Oil palm, including E.
guineensis), genus Linum (flax, including L. usitatissimum), genus
Carthamus (safflower, including C. tinctorius L.), genus Sesamum
(sesame, including S. indicum), genus Vitis (grape, including Vitis
vinifera), and plants of the genus Brassica (family Brassicaceae,
i.e., broccoli, brussel sprouts, cabbage, swede, turnip, rapeseed
B. napus, and cauliflower).
[0078] Other specific plants which may be transformed to generate
the transgenic plants of the invention include various other fruits
and vegetables, such as apples, asparagus, avocado, banana,
blackberry, blueberry, brussel sprout, cabbage, cotton, canola,
carrots, radish, cucumbers, cherries, cranberries, cantaloupes,
eggplant, grapefruit, lemons, limes, nectarines, oranges, peaches,
pineapples, pears, plums, tangelos, tangerines, papaya, mango,
strawberry, raspberry, lettuce, onion, grape, kiwi fruit, okra,
parsnips, pumpkins, and spinach. In addition various flowering
plants, trees and ornamental plants may be used to generate
transgenic varietals, including without limitation lily, carnation,
chrysanthemum, petunia, geranium, violet, gladioli, lupine, orchid
and lilac.
[0079] In stable transformation embodiments of the invention, one
or more copies of the expressible genetic construct become
integrated into the host plant genome, thereby providing increased
GS and GPT enzyme capacity into the plant, which serves to mediate
increased synthesis of 2-oxoglutaramate, which in turn signals
metabolic gene expression, resulting in increased plant growth and
the enhancement other agronomic characteristics. 2-oxoglutaramate
is a metabolite which is an extremely potent effector of gene
expression, metabolism and plant growth (U.S. Pat. No. 6,555,500),
and which may play a pivotal role in the coordination of the carbon
and nitrogen metabolism systems (Lancien et al., 2000, Enzyme
Redundancy and the Importance of 2-Oxoglutarate in Higher Plants
Ammonium Assimilation, Plant Physiol. 123: 817-824). See, also, the
schematic of the 2-oxoglutaramate pathway shown in FIG. 1.
[0080] In one aspect of the invention, applicants have isolated a
nucleic acid molecule encoding the Arabidopsis glutamine
phenylpyruvate transaminase (GPT) enzyme (see Example 1, infra),
and have demonstrated for the first time that the expressed
recombinant enzyme is active and capable of catalyzing the
synthesis of the signal metabolite, 2-oxoglutaramate (Example 2,
infra). Further, applicants have demonstrated for the first time
that over-expression of the Arabidopsis glutamine transaminase gene
in a transformed heterologous plant results in enhanced CO.sub.2
fixation rates and increased growth characteristics (Example 3,
infra).
[0081] Applicants' previous work demonstrated that over-expression
of Alfalfa GS1 gene under the control of a strong constitutive
promoter results in transgenic tobacco plants with higher levels of
GS activity in the leaves. These plants outgrow their wild-type
counterparts, fix CO.sub.2 faster, contain increased concentrations
of total protein, as well as increased concentrations of glutamine
and 2-oxoglutaramate, and show increased rates of uptake of nitrate
through their roots.
[0082] As disclosed herein (see Example 3, infra), over-expression
of a transgene comprising the full-length Arabidopsis GPT coding
sequence in transgenic tobacco plants also results in faster
CO.sub.2 fixation, and increased levels of total protein, glutamine
and 2-oxoglutaramate. These transgenic plants also grow faster than
wild-type plants (FIG. 2). Similarly, in preliminary studies
conducted with tomato plants (see Example 4, infra), tomato plants
transformed with the Arabidopsis GPT transgene showed significant
enhancement of growth rate, flowering, and seed yield in relation
to wild type control plants (FIG. 3 and Example 4, infra).
[0083] In one particular embodiment, exemplified herein by way of
Examples 3, 5 and 7, infra, a first set of parental
single-transgene tobacco plant lines carrying the Alfalfa GS1 gene,
including 5' and 3' untranslated regions, were generated using
Agrobacterium mediated gene transformation, under selective
pressure, together with screening for the fastest growing
phenotype, and selfing to transgene/phenotype homozygosity (see
Example 5, infra). A second set of parental single-transgene
tobacco plant lines carrying the full length coding sequence of
Arabidopsis GPT were generated in the same manner (Example 3,
infra). High growth rate performing plants from each of the
parental lines were then sexually crossed to yield progeny lines
(Example 7, infra).
[0084] The resulting progeny from multiple crosses of Arabidopsis
GS1 and GPT transgenic tobacco plants produce far better and quite
surprising increases in growth rates over the single-transgene
parental lines as well as wildtype plants. FIG. 5 shows photographs
of double-transgene progeny from single-transgene GS1.times.GPT
plant crosses, relative to wild type and single-transgene parental
plants. FIG. 6 shows photographs comparing leaf sizes of
double-transgene progeny and wild type plants. Experimentally
observed growth rates in these double transgenic plants ranged
between 200% and 300% over wild-type plants (Example 7, infra).
Moreover, total biomass levels increased substantially in the
double-transgene plants, with whole plant fresh weights typically
being about two to three times the wild-type plant weights.
Similarly, seed yields showed similar increases in the
double-transgene plants, with seed pod production typically two to
three times the wild type average, and overall seed yields
exceeding wild-type plant yields by 300-400%.
[0085] In addition to the transgenic tobacco plants referenced
above, various other species of transgenic plants comprising GPT
and GS transgenes are specifically exemplified herein. As
exemplified herein, transgenic plants showing enhanced growth
characteristics have been generated in two species of Tomato (see
Examples 4 and 17), Pepper (Example 8), Beans (Examples 9 and 10),
Cowpea (Examples 11 and 12), Alfalfa (Example 13), Cantaloupe
(Example 14), Pumpkin (Example 15), Arabidopsis (Example 16) and
Camilena (Example 18). These transgenic plants of the invention
were generated using a variety of transformation methodologies,
including Agrobacterium-mediated callus, floral dip, seed
inoculation, pod inoculation, and direct flower inoculation, as
well as combinations thereof, and via sexual crosses of single
transgene plants, as exemplified herein. Different GPT and GS
transgenes were successfully employed in generating the transgenic
plants of the invention, as exemplified herein.
[0086] The invention also provides methods of generating a
transgenic plant having enhanced growth and other agronomic
characteristics. In one embodiment, a method of generating a
transgenic plant having enhanced growth and other agronomic
characteristics comprises introducing into a plant cell an
expression cassette comprising a nucleic acid molecule encoding a
GPT transgene, under the control of a suitable promoter capable of
driving the expression of the transgene, so as to yield a
transformed plant cell, and obtaining a transgenic plant which
expresses the encoded GPT. In another embodiment, a method of
generating a transgenic plant having enhanced growth and other
agronomic characteristics comprises introducing into a plant cell
one or more nucleic acid constructs or expression cassettes
comprising nucleic acid molecules encoding a GPT transgene and an
GS transgene, under the control of one or more suitable promoters
(and, optionally, other regulatory elements) capable of driving the
expression of the transgenes, so as to yield a plant cell
transformed thereby, and obtaining a transgenic plant which
expresses the GPT and GS transgenes.
[0087] Based on the results disclosed herein, it is clear that any
number of GPT and GS polynucleotides may be used to generate the
transgenic plants of the invention. Both GS1 and GPT proteins are
highly conserved among various plant species, and it is evident
from the experimental data disclosed herein that closely-related
non-plant GPTs may be used as well (e.g., Danio rerio GPT). With
respect to GPT, numerous GPT polynucleotides derived from different
species have been shown to be active and useful as GPT transgenes.
Similarly, different GS polynucleotides may be used, including
without limitation any plant GS1 encoding polynucleotide that
generates GS activity in a host cell transformed with an
expressible GS1 construct.
[0088] In a specific embodiment, the GPT transgene is a GPT
polynucleotide encoding an Arabidopsis derived GPT, such as the GPT
of SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 30, and the GS
transgene is a GS polynucleotide encoding an Alfalfa derived GS1
(i.e., SEQ ID NO: 4) or an Arabidopsis derived GS1 (SEQ ID NO: 7).
The GPT transgene may be encoded by the nucleotide sequence of SEQ
ID NO: 1; a nucleotide sequence having at least 75% and more
preferably at least 80% identity to SEQ ID NO: 1, and encoding a
polypeptide having GPT activity; a nucleotide sequence encoding the
polypeptide of SEQ ID NO: 2, or a polypeptide having at least 75%
and more preferably at least 80% sequence identity thereto which
has GPT activity; and a nucleotide sequence encoding the
polypeptide of SEQ ID NO: 2 truncated at its amino terminus by
between 30 to 56 amino acid residues, or a polypeptide having at
least 75% and more preferably at least 80% sequence identity
thereto which has GPT activity. The GS1 transgene may be encoded by
the polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 6 or a nucleotide
sequence having at least 75% and more preferably at least 80%
identity to SEQ ID NO: 3 or SEQ ID NO: 6, and encoding a
polypeptide having GPT activity; and a nucleotide sequence encoding
the polypeptide of SEQ ID NO: 4 or 7, or a polypeptide having at
least 75% and more preferably at least 80% sequence identity
thereto which has GS activity.
[0089] In another specific embodiment, the GPT transgene is a GPT
polynucleotide encoding a Grape derived GPT, such as the Grape GPTs
of SEQ ID NO: 9 and SEQ ID NO: 31, and the GS transgene is a GS1
polynucleotide. The GPT transgene may be encoded by the nucleotide
sequence of SEQ ID NO: 8; a nucleotide sequence having at least 75%
and more preferably at least 80% identity to SEQ ID NO: 8, and
encoding a polypeptide having GPT activity; a nucleotide sequence
encoding the polypeptide of SEQ ID NO: 9 or SEQ ID NO: 31, or a
polypeptide having at least 75% and more preferably at least 80%
sequence identity thereto which has GPT activity.
[0090] In yet another specific embodiment, the GPT transgene is a
GPT polynucleotide encoding a Rice derived GPT, such as the Rice
GPTs of SEQ ID NO: 11 and SEQ ID NO: 32, and the GS transgene is a
GS1 polynucleotide. The GPT transgene may be encoded by the
nucleotide sequence of SEQ ID NO: 10; a nucleotide sequence having
at least 75% and more preferably at least 80% identity to SEQ ID
NO: 10, and encoding a polypeptide having GPT activity; a
nucleotide sequence encoding the polypeptide of SEQ ID NO: 11 or
SEQ ID NO: 32, or a polypeptide having at least 75% and more
preferably at least 80% sequence identity thereto which has GPT
activity.
[0091] In yet another specific embodiment, the GPT transgene is a
GPT polynucleotide encoding a Soybean derived GPT, such as the
Soybean GPTs of SEQ ID NO: 13, SEQ ID NO: 33 or SEQ ID NO: 33 with
a further Isoleucine at the N-terminus of the sequence, and the GS
transgene is a GS1 polynucleotide. The GPT transgene may be encoded
by the nucleotide sequence of SEQ ID NO: 12; a nucleotide sequence
having at least 75% and more preferably at least 80% identity to
SEQ ID NO: 12, and encoding a polypeptide having GPT activity; a
nucleotide sequence encoding the polypeptide of SEQ ID NO: 13 or
SEQ ID NO: 33 or SEQ ID NO: 33 with a further Isoleucine at the
N-terminus of the sequence, or a polypeptide having at least 75%
and more preferably at least 80% sequence identity thereto which
has GPT activity.
[0092] In yet another specific embodiment, the GPT transgene is a
GPT polynucleotide encoding a Barley derived GPT, such as the
Barley GPTs of SEQ ID NO: 15 and SEQ ID NO: 34, and the GS
transgene is a GS1 polynucleotide. The GPT transgene may be encoded
by the nucleotide sequence of SEQ ID NO: 14; a nucleotide sequence
having at least 75% and more preferably at least 80% identity to
SEQ ID NO: 10, and encoding a polypeptide having GPT activity; a
nucleotide sequence encoding the polypeptide of SEQ ID NO: 15 or
SEQ ID NO: 34, or a polypeptide having at least 75% and more
preferably at least 80% sequence identity thereto which has GPT
activity.
[0093] In yet another specific embodiment, the GPT transgene is a
GPT polynucleotide encoding a Zebra fish derived GPT, such as the
Zebra fish GPTs of SEQ ID NO: 17 and SEQ ID NO: 35, and the GS
transgene is a GS1 polynucleotide. The GPT transgene may be encoded
by the nucleotide sequence of SEQ ID NO: 16; a nucleotide sequence
having at least 75% and more preferably at least 80% identity to
SEQ ID NO: 16, and encoding a polypeptide having GPT activity; a
nucleotide sequence encoding the polypeptide of SEQ ID NO: 17 or
SEQ ID NO: 35, or a polypeptide having at least 75% and more
preferably at least 80% sequence identity thereto which has GPT
activity.
[0094] In yet another specific embodiment, the GPT transgene is a
GPT polynucleotide encoding a Bamboo derived GPT, such as the
Bamboo GPT of SEQ ID NO: 36, and the GS transgene is a GS1
polynucleotide. The GPT transgene may be encoded by a nucleotide
sequence encoding the polypeptide of SEQ ID NO: 36, or a
polypeptide having at least 75% and more preferably at least 80%
sequence identity thereto which has GPT activity.
[0095] Other GPT polynucleotides suitable for use as GPT transgenes
in the practice of the invention may be obtained by various means,
as will be appreciated by one skilled in the art, tested for the
ability to direct the expression of a GPT with GPT activity in a
recombinant expression system (i.e., E. coli (see Examples 20-23),
in a transient in planta expression system (see Example 19), or in
a transgenic plant (see Examples 1-18).
Transgene Constructs/Expression Vectors
[0096] In order to generate the transgenic plants of the invention,
the gene coding sequence for the desired transgene(s) must be
incorporated into a nucleic acid construct (also interchangeably
referred to herein as a/an (transgene) expression vector,
expression cassette, expression construct or expressible genetic
construct), which can direct the expression of the transgene
sequence in transformed plant cells. Such nucleic acid constructs
carrying the transgene(s) of interest may be introduced into a
plant cell or cells using a number of methods known in the art,
including but not limited to electroporation, DNA bombardment or
biolistic approaches, microinjection, and via the use of various
DNA-based vectors such as Agrobacterium tumefaciens and
Agrobacterium rhizogenes vectors. Once introduced into the
transformed plant cell, the nucleic acid construct may direct the
expression of the incorporated transgene(s) (i.e., GPT), either in
a transient or stable fashion. Stable expression is preferred, and
is achieved by utilizing plant transformation vectors which are
able to direct the chromosomal integration of the transgene
construct. Once a plant cell has been successfully transformed, it
may be cultivated to regenerate a transgenic plant.
[0097] A large number of expression vectors suitable for driving
the constitutive or induced expression of inserted genes in
transformed plants are known. In addition, various transient
expression vectors and systems are known. To a large extent,
appropriate expression vectors are selected for use in a particular
method of gene transformation (see, infra). Broadly speaking, a
typical plant expression vector for generating transgenic plants
will comprise the transgene of interest under the expression
regulatory control of a promoter, a selectable marker for assisting
in the selection of transformants, and a transcriptional terminator
sequence.
[0098] More specifically, the basic elements of a nucleic acid
construct for use in generating the transgenic plants of the
invention are: a suitable promoter capable of directing the
functional expression of the transgene(s) in a transformed plant
cell, the transgene (s) (i.e., GPT coding sequence) operably linked
to the promoter, preferably a suitable transcription termination
sequence (i.e., nopaline synthetic enzyme gene terminator) operably
linked to the transgene, and sometimes other elements useful for
controlling the expression of the transgene, as well as one or more
selectable marker genes suitable for selecting the desired
transgenic product (i.e., antibiotic resistance genes).
[0099] As Agrobacterium tumefaciens is the primary transformation
system used to generate transgenic plants, there are numerous
vectors designed for Agrobacterium transformation. For stable
transformation, Agrobacterium systems utilize "binary" vectors that
permit plasmid manipulation in both E. coli and Agrobacterium, and
typically contain one or more selectable markers to recover
transformed plants (Hellens et al., 2000, Technical focus: A guide
to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446-451).
Binary vectors for use in Agrobacterium transformation systems
typically comprise the borders of T-DNA, multiple cloning sites,
replication functions for Escherichia coli and A. tumefaciens, and
selectable marker and reporter genes.
[0100] So-called "super-binary" vectors provide higher
transformation efficiencies, and generally comprise additional
virulence genes from a Ti (Komari et al., 2006, Methods Mol. Biol.
343: 15-41). Super binary vectors are typically used in plants
which exhibit lower transformation efficiencies, such as cereals.
Such additional virulence genes include without limitation virB,
virE, and virG (Vain et al., 2004, The effect of additional
virulence genes on transformation efficiency, transgene integration
and expression in rice plants using the pGreen/pSoup dual binary
vector system. Transgenic Res. 13: 593-603; Srivatanakul et al.,
2000, Additional virulence genes influence transgene expression:
transgene copy number, integration pattern and expression. J. Plant
Physiol. 157, 685-690; Park et al., 2000, Shorter T-DNA or
additional virulence genes improve Agrobacterium-mediated
transformation. Theor. Appl. Genet. 101, 1015-1020; Jin et al.,
1987, Genes responsible for the supervirulence phenotype of
Agrobacterium tumefaciens A281. J. Bacteriol. 169: 4417-4425).
[0101] In the embodiments exemplified herein (see Examples, infra),
expression vectors which place the inserted transgene(s) under the
control of the constitutive CaMV 35S promoter and the RuBisCo
promoter are employed. A number of expression vectors which utilize
the CaMV 35S and RuBsCo promoter are known and/or commercially
available and/or derivable using ordinary skill in the art.
Additionally, numerous promoters suitable for directing the
expression of the transgene are known and may be used in the
practice of the invention, as further described, infra.
Plant Promoters
[0102] A large number of promoters which are functional in plants
are known in the art. In constructing GPT and GS transgene
constructs, the selected promoter(s) may be constitutive,
non-specific promoters such as the Cauliflower Mosaic Virus 35S
ribosomal promoter (CaMV 35S promoter), which is widely employed
for the expression of transgenes in plants. Examples of other
strong constitutive promoters include without limitation the rice
actin 1 promoter, the CaMV 19S promoter, the Ti plasmid nopaline
synthase promoter, the alcohol dehydrogenase promoter and the
sucrose synthase promoter.
[0103] Alternatively, in some embodiments, it may be desirable to
select a promoter based upon the desired plant cells to be
transformed by the transgene construct, the desired expression
level of the transgene, the desired tissue or subcellular
compartment for transgene expression, the developmental stage
targeted, and the like.
[0104] For example, when expression in photosynthetic tissues and
compartments is desired, a promoter of the ribulose bisphosphate
carboxylase (RuBisCo) gene may be employed. In the Examples which
follow, expressible nucleic acid constructs comprising GPT and GS1
transgenes under the control of a tomato RuBisCo promoter were
prepared and used in the generation of transgenic plants or to
assay for GPT activity in planta or in E. coli.
[0105] When the expression in seeds is desired, promoters of
various seed storage protein genes may be employed. For expression
in fruits, a fruit-specific promoter such as tomato 2A11 may be
used. Examples of other tissue specific promoters include the
promoters encoding lectin (Vodkin et al., 1983, Cell 34:1023-31;
Lindstrom et al., 1990, Developmental Genetics 11:160-167), corn
alcohol dehydrogenase 1 (Vogel et al, 1989, J. Cell. Biochem.
(Suppl. 0) 13:Part D; Dennis et al., 1984, Nucl. Acids Res., 12(9):
3983-4000), corn light harvesting complex (Simpson, 1986, Science,
233: 34-38; Bansal et al., 1992, Proc. Natl. Acad. Sci. USA, 89:
3654-3658), corn heat shock protein (Odell et al., 1985, Nature,
313: 810-812; Rochester et al., 1986, EMBO J., 5: 451-458), pea
small subunit RuBP carboxylase (Poulsen et al., 1986, Mol. Gen.
Genet., 205(2): 193-200; Cashmore et al., 1983, Gen. Eng. Plants,
Plenum Press, New York, pp 29-38), Ti plasmid mannopine synthase
and Ti plasmid nopaline synthase (Langridge et al., 1989, Proc.
Natl. Acad. Sci. USA, 86: 3219-3223), petunia chalcone isomerase
(Van Tunen et al., 1988, EMBO J. 7(5): 1257-1263), bean glycine
rich protein 1 (Keller et al., 1989, EMBO J. 8(5): 1309-1314),
truncated CaMV 35s (Odell et al., 1985, supra), potato patatin
(Wenzler et al., 1989, Plant Mol. Biol. 12: 41-50), root cell
(Conkling et al., 1990, Plant Physiol. 93: 1203-1211), maize zein
(Reina et al., 1990, Nucl. Acids Res. 18(21): 6426; Kriz et al.,
1987, Mol. Gen. Genet. 207(1): 90-98; Wandelt and Feix, 1989, Nuc.
Acids Res. 17(6): 2354; Langridge and Feix, 1983, Cell 34:
1015-1022; Reina et al., 1990, Nucl. Acids Res. 18(21): 6426),
globulin-1 (Belanger and Kriz, 1991, Genetics 129: 863-872),
.alpha.-tubulin (Carpenter et al., 1992, Plant Cell 4(5): 557-571;
Uribe et al., 1998, Plant Mol. Biol. 37(6): 1069-1078), cab
(Sullivan, et al., 1989, Mol. Gen. Genet. 215(3): 431-440), PEPCase
(Hudspeth and Grula, 1989, Plant Mol. Biol. 12: 579-589), R gene
complex (Chandler et al., 1989, The Plant Cell 1: 1175-1183),
chalcone synthase (Franken et al., 1991, EMBO J. 10(9): 2605-2612)
and glutamine synthetase promoters (U.S. Pat. No. 5,391,725;
Edwards et al., 1990, Proc. Natl. Acad. Sci. USA 87: 3459-3463;
Brears et al., 1991, Plant J. 1(2): 235-244).
[0106] In addition to constitutive promoters, various inducible
promoter sequences may be employed in cases where it is desirable
to regulate transgene expression as the transgenic plant
regenerates, matures, flowers, etc. Examples of such inducible
promoters include promoters of heat shock genes, protection
responding genes (i.e., phenylalanine ammonia lyase; see, for
example Bevan et al., 1989, EMBO J. 8(7): 899-906), wound
responding genes (i.e., cell wall protein genes), chemically
inducible genes (i.e., nitrate reductase, chitinase) and dark
inducible genes (i.e., asparagine synthetase; see, for example U.S.
Pat. No. 5,256,558). Also, a number of plant nuclear genes are
activated by light, including gene families encoding the major
chlorophyll a/b binding proteins (cab) as well as the small subunit
of ribulose-1,5-bisphosphate carboxylase (rbcS) (see, for example,
Tobin and Silverthorne, 1985, Annu. Rev. Plant Physiol. 36:
569-593; Dean et al., 1989, Annu. Rev. Plant Physiol. 40:
415-439.).
[0107] Other inducible promoters include ABA- and turgor-inducible
promoters, the auxin-binding protein gene promoter (Schwob et al.,
1993, Plant J. 4(3): 423-432), the UDP glucose flavonoid
glycosyl-transferase gene promoter (Ralston et al., 1988, Genetics
119(1): 185-197); the MPI proteinase inhibitor promoter (Cordero et
al., 1994, Plant J. 6(2): 141-150), the glyceraldehyde-3-phosphate
dehydrogenase gene promoter (Kohler et al., 1995, Plant Mol. Biol.
29(6): 1293-1298; Quigley et al., 1989, J. Mol. Evol. 29(5):
412-421; Martinez et al., 1989, J. Mol. Biol. 208(4): 551-565) and
light inducible plastid glutamine synthetase gene from pea (U.S.
Pat. No. 5,391,725; Edwards et al., 1990, supra).
[0108] For a review of plant promoters used in plant transgenic
plant technology, see Potenza et al., 2004, In Vitro Cell. Devel.
Biol-Plant, 40(1): 1-22. For a review of synthetic plant promoter
engineering, see, for example, Venter, M., 2007, Trends Plant Sci.,
12(3): 118-124.
Glutamine Phenylpyruvate Transaminase (GPT) Transgene
[0109] The present invention discloses for the first time that
plants contain a glutamine phenylpyruvate transaminase (GPT) enzyme
which is directly functional in the synthesis of the signal
metabolite 2-hydroxy-5-oxoproline. Until now, no plant glutamine
phenylpyruvate transaminase with a defined function has been
described. Applicants have isolated and tested GPT polynucleotide
coding sequences derived from several plant and animal species, and
have successfully incorporated the gene into heterologous
transgenic host plants which exhibit markedly improved growth
characteristics, including faster growth, higher foliar protein
content, increased glutamine synthetase activity in foliar tissue,
and faster CO.sub.2 fixation rates.
[0110] In the practice of the invention, the GPT gene functions as
one of at least two transgenes incorporated into the transgenic
plants of the invention, the other being the glutamine sythetase
gene (see infra).
[0111] It is expected that all plant species contain a GPT which
functions in the same metabolic pathway, involving the biosynthesis
of the signal metabolite 2-hydroxy-5-oxoproline. Thus, in the
practice of the invention, any plant gene encoding a GPT homolog or
functional variants thereof may be useful in the generation of
transgenic plants of this invention. Moreover, given the structural
similarity between various plant GPT protein structures and the
putative (and biologically active) GPT homolog from Danio rerio
(Zebra fish) (see Example 22), other non-plant GPT homologs may be
used in preparing GPT transgenes for use in generating the
transgenic plants of the invention.
[0112] When individually compared (by BLAST alignment) to the
Arabidopsis mature protein sequence provided in SEQ ID NO: 30, the
following sequence identities and homologies (BLAST "positives",
including similar amino acids) were obtained for the following
mature GPT protein sequences:
TABLE-US-00001 [SEQ ID] or FIG. NO. ORIGIN % IDENTITY % POSITIVE
[31] Grape 84 93 [32] Rice 83 91 [33] Soybean 83 93 [34] Barley 82
91 [35] Zebra fish 83 92 [36] Bamboo 81 90 FIG. 2 Corn 79 90 FIG. 2
Castor 84 93 FIG. 2 Poplar 85 93
[0113] Underscoring the conserved nature of the structure of the
GPT protein across most plant species, the conservation seen within
the above plant species extends to the non-human putative GPTs from
Zebra fish and Chlamydomonas. In the case of Zebra fish, the extent
of identity is very high (83% amino acid sequence identity with the
mature Arabidopsis GPT of SEQ ID NO: 30, and 92% homologous taking
similar amino acid residues into account). The Zebra fish mature
GPT was confirmed by expressing it in E. coli and demonstrating
biological activity (synthesis of 2-oxoglutaramate).
[0114] In order to determine whether putative GPT homologs would be
suitable for generating the growth-enhanced transgenic plants of
the invention, one need initially express the coding sequence
thereof in E. coli or another suitable host and determine whether
the 2-oxoglutaramate signal metabolite is synthesized at increased
levels (see Examples 19-23). Where such an increase is
demonstrated, the coding sequence may then be introduced into both
homologous plant hosts and heterologous plant hosts, and growth
characteristics evaluated. Any assay that is capable of detecting
2-oxoglutaramate with specificity may be used for this purpose,
including without limitation the NMR and HPLC assays described in
Example 2, infra. In addition, assays which measure GPT activity
directly may be employed, such as the GPT activity assay described
in Example 7.
[0115] Any plant GPT with 2-oxoglutaramate synthesis activity may
be used to transform plant cells in order to generate transgenic
plants of the invention. There appears to be a high level of
structural homology among plant species, which appears to extend
beyond plants, as evidenced by the close homology between various
plant GPT proteins and the putative Zebra fish GPT homolog.
Therefore, various plant GPT genes may be used to generate
growth-enhanced transgenic plants in a variety of heterologous
plant species. In addition, GPT transgenes expressed in a
homologous plant would be expected to result in the desired
enhanced-growth characteristics as well (i.e., rice glutamine
transaminase over-expressed in transgenic rice plants), although it
is possible that regulation within a homologous cell may attenuate
the expression of the transgene in some fashion that may not be
operable in a heterologous cell.
Glutamine Synthetase (GS) Transgene:
[0116] In the practice of the invention, the glutamine synthetase
(GS) gene functions as one of at least two transgenes incorporated
into the transgenic plants of the invention (GPT being the other of
the two).
[0117] Glutamine synthetase plays a key role in nitrogen metabolism
in plants, as well as in animals and bacteria. The GS enzyme
catalyzes the addition of ammonium to glutamate to synthesize
glutamine in an ATP-dependent reaction. GS enzymes from assorted
species show highly conserved amino acid residues considered to be
important for active site function, indicating that GS enzymes
function similarly (for review, see Eisenberg et al., Biochimica et
Biophysica Acta, 1477:122 145, 2000).
[0118] GS is distributed in different subcellular locations
(chloroplast and cytoplasm) and is found in various plant tissues,
including leaf, root, shoot, seeds and fruits. There are two major
isoforms of plant GS: the cystolic isoform (GS1) and the plastidic
(chloroplastic) isoform (GS2). GS2 is principally found in leaf
tissue and functions in the assimilation of ammonia produced by
photorespiration or by nitrate reduction. GS1 is mainly found in
leaf and root tissue, typically exists in a number of different
isoforms in higher plants, and functions to assimilate ammonia
produced by all other physiological processes (Coruzzi, 1991, Plant
Science 74: 145-155; McGrath and Coruzzi, 1991, Plant J. 1(3):
275-280; Lam et al., 1996, Ann. Rev. Plant Physiol. Plant Mol.
Biol. 47: 569-593; Stitt, 1999, Curr. Op. Plant Biol. 2: 178-186;
Oliveira et al., 2001, Brazilian J. Med. Biol. Res. 34: 567-575).
Multiple GS genes are associated with a complex promoter repertoire
which enable the expression of GS in an organ and tissue specific
manner, as well as in an environmental factor-dependent manner.
[0119] Plant glutamine synthetase consists of eight subunits, and
the native enzyme in plants has a molecular mass ranging from 320
to 380 kD, each subunit having a molecular mass of between 38 and
45 kD. The GS1 genes of several plants, especially legumes, have
been cloned and sequenced (Tischer et al., 1986, Mol Gen Genet.
203: 221-229; Gebhardt et al., 1986, EMBO J. 5: 1429-1435; Tingey
et al., 1987, EMBO J. 6: 1-9; Tingey et al., 1988, J Biol. Chem.
263: 9651-9657; Bennett et al., 1989, Plant Mol. Biol. 12: 553-565;
Boron and Legocki, 1993, Gene 136: 95-102; Roche et al., 1993,
Plant Mol. Biol. 22: 971-983; Marsolier et al., 1995, Plant Mol.
Biol. 27: 1-15; Temple et al., 1995, Mol Plant-Microbe Interact. 8:
218-227). All have been found to be encoded by nuclear genes (for
review, see, Morey et al., 2002, Plant Physiol. 128(1):
182-193).
[0120] Chloroplastic GS2 appears to be encoded by a single gene,
while various cystoloic GS1 isoforms are encoded within multigene
families (Tingey et al., 1987, supra; Sakamoto et al., 1989, Plant
Mol. Biol. 13: 611-614; Brears et al, 1991, supra; Li et al., 1993,
Plant Mol. Biol., 23:401-407; Dubois et al., 1996, Plant Mol.
Biol., 31:803-817; Lam et al., 1996, supra). GS1 multigene families
appear to encode different subunits which may combine to form homo-
or hetero-octamers, and the different members show a unique
expression pattern suggesting that the gene members are
differentially regulated, which may relate to the various
functional roles of glutamine synthetase plays in overall nitrogen
metabolism (Gebhardt et al., 1986, supra; Tingey et al., 1987,
supra; Bennett et al., 1989, supra; Walker and Coruzzi, 1989,
supra; Peterman and Goodman, 1991, Mol Gen Genet. 1991;
330:145-154; Marsolier et al., 1995, supra; Temple et al., 1995,
supra; Dubois et al., 1996, supra).
[0121] In one embodiment, a GS1 gene coding sequence is employed to
generate GS transgene constructs. In particular embodiments,
further described in the Examples, infra, the Alfalfa or
Arabidopsis GS1 gene coding sequence is used to generate a
transgene construct that may be used to generate a transgenic plant
expressing the GS1 transgene. As an example, such a construct may
be used to transform Agrobacteria. The transformed Agrobacteria are
then used to generate T.sub.0 transgenic plants. Example 5
demonstrates the generation of T.sub.0 GS1 transgenic tobacco
plants using this approach. Similarly, Examples 6 and 17
demonstrates the generation of T.sub.0 GS1 transgenic tomato
plants, Example 8 demonstrates the generation of T.sub.0 GS1
transgenic pepper plants, Examples 9 and 10 demonstrate the
generation of T.sub.0 GS1 transgenic bean plants, Examples 11 and
12 demonstrate the generation of T.sub.0 GS1 transgenic cowpea
plants, Example 13 demonstrates the generation of T.sub.0 GS1
transgenic alfalfa plants, Example 14 demonstrates the generation
of T.sub.0 GS1 transgenic cantaloupe plants, Example 15
demonstrates the generation of T.sub.0 GS1 transgenic pumpkin
plants, Example 16 demonstrates the generation of T.sub.0 GS1
transgenic Arabidopsis plants, and Example 18 demonstrates the
generation of T.sub.0 GS1 transgenic Cantaloupe plants.
Transcription Terminators:
[0122] In preferred embodiments, a 3' transcription termination
sequence is incorporated downstream of the transgene in order to
direct the termination of transcription and permit correct
polyadenylation of the mRNA transcript. Suitable transcription
terminators are those which are known to function in plants,
including without limitation, the nopaline synthase (NOS) and
octopine synthase (OCS) genes of Agrobacterium tumefaciens, the T7
transcript from the octopine synthase gene, the 3' end of the
protease inhibitor I or II genes from potato or tomato, the CaMV
35S terminator, the tml terminator and the pea rbcS E9 terminator.
In addition, a gene's native transcription terminator may be used.
In specific embodiments, described by way of the Examples, infra,
the nopaline synthase transcription terminator is employed.
Selectable Markers:
[0123] Selectable markers are typically included in transgene
expression vectors in order to provide a means for selecting
transformants. While various types of markers are available,
various negative selection markers are typically utilized,
including those which confer resistance to a selection agent that
inhibits or kills untransformed cells, such as genes which impart
resistance to an antibiotic (such as kanamycin, gentamycin,
anamycin, hygromycin and hygromycinB) or resistance to a herbicide
(such as sulfonylurea, gulfosinate, phosphinothricin and
glyphosate). Screenable markers include, for example, genes
encoding .beta.-glucuronidase (Jefferson, 1987, Plant Mol. Biol.
Rep 5: 387-405), genes encoding luciferase (Ow et al., 1986,
Science 234: 856-859) and various genes encoding proteins involved
in the production or control of anthocyanin pigments (See, for
example, U.S. Pat. No. 6,573,432). The E. coli glucuronidase gene
(gus, gusA or uidA) has become a widely used selection marker in
plant transgenics, largely because of the glucuronidase enzyme's
stability, high sensitivity and ease of detection (e.g.,
fluorometric, spectrophotometric, various histochemical methods).
Moreover, there is essentially no detectable glucuronidase in most
higher plant species.
Transformation Methodologies and Systems:
[0124] Various methods for introducing the transgene expression
vector constructs of the invention into a plant or plant cell are
well known to those skilled in the art, and any capable of
transforming the target plant or plant cell may be utilized.
[0125] Agrobacterium-mediated transformation is perhaps the most
common method utilized in plant transgenics, and protocols for
Agrobacterium-mediated transformation of a large number of plants
are extensively described in the literature (see, for example,
Agrobacterium Protocols, Wan, ed., Humana Press, 2.sup.nd edition,
2006). Agrobacterium tumefaciens is a Gram negative soil bacteria
that causes tumors (Crown Gall disease) in a great many dicot
species, via the insertion of a small segment of tumor-inducing DNA
("T-DNA", `transfer DNA`) into the plant cell, which is
incorporated at a semi-random location into the plant genome, and
which eventually may become stably incorporated there. Directly
repeated DNA sequences, called T-DNA borders, define the left and
the right ends of the T-DNA. The T-DNA can be physically separated
from the remainder of the Ti-plasmid, creating a `binary vector`
system.
[0126] Agrobacterium transformation may be used for stably
transforming dicots, monocots, and cells thereof (Rogers et al.,
1986, Methods Enzymol., 118: 627-641; Hernalsteen et al., 1984,
EMBO J., 3: 3039-3041; Hoykass-Van Slogteren et al., 1984, Nature,
311: 763-764; Grimsley et al., 1987, Nature 325: 167-1679; Boulton
et al., 1989, Plant Mol. Biol. 12: 31-40; Gould et al., 1991, Plant
Physiol. 95: 426-434). Various methods for introducing DNA into
Agrobacteria are known, including electroporation, freeze/thaw
methods, and triparental mating. The most efficient method of
placing foreign DNA into Agrobacterium is via electroporation (Wise
et al., 2006, Three Methods for the Introduction of Foreign DNA
into Agrobacterium, Methods in Molecular Biology, vol. 343:
Agrobacterium Protocols, 2/e, volume 1; Ed., Wang, Humana Press
Inc., Totowa, N.J., pp. 43-53). In addition, given that a large
percentage of T-DNAs do not integrate, Agrobacterium-mediated
transformation may be used to obtain transient expression of a
transgene via the transcriptional competency of unincorporated
transgene construct molecules (Helens et al., 2005, Plant Methods
1:13).
[0127] A large number of Agrobacterium transformation vectors and
methods have been described (Karimi et al., 2002, Trends Plant Sci.
7(5): 193-5), and many such vectors may be obtained commercially
(for example, Invitrogen, Carlsbad, Calif.). In addition, a growing
number of "open-source" Agrobacterium transformation vectors are
available (for example, pCambia vectors; Cambia, Canberra,
Australia). See, also, subsection herein on TRANSGENE CONSTRUCTS,
supra. In a specific embodiment described further in the Examples,
a pMON316-based vector was used in the leaf disc transformation
system of Horsch et. al. (Horsch et al., 1995, Science
227:1229-1231) to generate growth enhanced transgenic tobacco and
tomato plants.
[0128] Other commonly used transformation methods that may be
employed in generating the transgenic plants of the invention
include, without limitation microprojectile bombardment, or
biolistic transformation methods, protoplast transformation of
naked DNA by calcium, polyethylene glycol (PEG) or electroporation
(Paszkowski et al., 1984, EMBO J. 3: 2727-2722; Potrykus et al.,
1985, Mol. Gen. Genet. 199: 169-177; Fromm et al., 1985, Proc. Nat.
Acad. Sci. USA 82: 5824-5828; Shimamoto et al., 1989, Nature, 338:
274-276.
[0129] Biolistic transformation involves injecting millions of
DNA-coated metal particles into target cells or tissues using a
biolistic device (or "gene gun"), several kinds of which are
available commercially. Once inside the cell, the DNA elutes off
the particles and a portion may be stably incorporated into one or
more of the cell's chromosomes (for review, see Kikkert et al.,
2005, Stable Transformation of Plant Cells by Particle
Bombardment/Biolistics, in: Methods in Molecular Biology, vol. 286:
Transgenic Plants: Methods and Protocols, Ed. L. Pena, Humana Press
Inc., Totowa, N.J.).
[0130] Electroporation is a technique that utilizes short,
high-intensity electric fields to permeabilize reversibly the lipid
bilayers of cell membranes (see, for example, Fisk and Dandekar,
2005, Introduction and Expression of Transgenes in Plant
Protoplasts, in: Methods in Molecular Biology, vol. 286: Transgenic
Plants: Methods and Protocols, Ed. L. Pena, Humana Press Inc.,
Totowa, N.J., pp. 79-90; Fromm et al., 1987, Electroporation of DNA
and RNA into plant protoplasts, in Methods in Enzymology, Vol. 153,
Wu and Grossman, eds., Academic Press, London, UK, pp. 351-366;
Joersbo and Brunstedt, 1991, Electroporation: mechanism and
transient expression, stable transformation and biological effects
in plant protoplasts. Physiol. Plant. 81, 256-264; Bates, 1994,
Genetic transformation of plants by protoplast electroporation.
Mol. Biotech. 2: 135-145; Dillen et al., 1998,
Electroporation-mediated DNA transfer to plant protoplasts and
intact plant tissues for transient gene expression assays, in Cell
Biology, Vol. 4, ed., Celis, Academic Press, London, UK, pp.
92-99). The technique operates by creating aqueous pores in the
cell membrane, which are of sufficiently large size to allow DNA
molecules (and other macromolecules) to enter the cell, where the
transgene expression construct (as T-DNA) may be stably
incorporated into plant genomic DNA, leading to the generation of
transformed cells that can subsequently be regenerated into
transgenic plants.
[0131] Newer transformation methods include so-called "floral dip"
methods, which offer the promise of simplicity, without requiring
plant tissue culture, as is the case with all other commonly used
transformation methodologies (Bent et al., 2006, Arabidopsis
thaliana Floral Dip Transformation Method, Methods Mol Biol, vol.
343: Agrobacterium Protocols, 2/e, volume 1; Ed., Wang, Humana
Press Inc., Totowa, N.J., pp. 87-103; Clough and Bent, 1998, Floral
dip: a simplified method for Agrobacterium-mediated transformation
of Arabidopsis thaliana, Plant J. 16: 735-743). However, with the
exception of Arabidopsis, these methods have not been widely used
across a broad spectrum of different plant species. Briefly, floral
dip transformation is accomplished by dipping or spraying flowering
plants in with an appropriate strain of Agrobacterium tumefaciens.
Seeds collected from these T.sub.0 plants are then germinated under
selection to identify transgenic T.sub.1 individuals. Example 16
demonstrated floral dip inoculation of Arabidopsis to generate
transgenic Arabidopsis plants.
[0132] Other transformation methods include those in which the
developing seeds or seedlings of plants are transformed using
vectors such as Agrobacterium vectors. For example, as exemplified
in Example 8, such vectors may be used to transform developing
seeds by injecting a suspension or mixture of the vector (i.e.,
Agrobacteria) directly into the seed cavity of developing pods
(i.e., pepper pods, bean pods, pea pods and the like). Seedlings
may be transformed as described for Alfalfa in Example 13.
Germinating seeds may be transformed as described for Camelina in
Example 18. Intra-fruit methods, in which the vector is injected
into fruit or developing fruit, may be used as described for
Cantaloupe melons in Example 14 and pumpkins in Example 15.
[0133] Still other transformation methods include those in which
the flower structure is targeted for vector inoculation, such as
the flower inoculation methods described for beans in Examples 9
and 10, peas in Examples 11 and 12 and tomatoes in Example 17.
[0134] In addition, although transgenes are most commonly inserted
into the nuclear DNA of plant cells, trangenes may also be inserted
into plastidic DNA (i.e., into the plastome of the chloroplast). In
most flowering plants, plastids do not occur in the pollen cells,
and therefore transgenic DNA incorporated within a plastome will
not be passed on through propagation, thereby restricting the trait
from migrating to wild type plants. Plastid transformation,
however, is more complex than cell nucleus transformation, due to
the presence of many thousands of plastomes per cell (as high as
10,000).
[0135] Transplastomic lines are genetically stable only if all
plastid copies are modified in the same way, i.e. uniformly. This
is typically achieved through repeated regeneration under certain
selection conditions to eliminate untransformed plastids, by
segregating transplastomic and untransformed plastids, resulting in
the selection of homoplasmic cells carrying the transgene construct
and the selectable marker stably integrated therein. Plastid
transformation has been successfully performed in various plant
species, including tobacco, potatoes, oilseed rape, rice and
Arabidopsis.
[0136] To generate transplastomic lines carrying GPT and GS
transgenes, the transgene expression cassette is inserted into
flanking sequences from the plastome. Using homologous
recombination, the transgene expression cassette becomes integrated
into the plastome via a natural recombination process. The basic
DNA delivery techniques for plastid transformation include particle
bombardment of leaves or polyethylene glycol-mediated DNA
transformation of protoplasts. Transplastomic plants carrying
transgenes in the plastome may be expressed at very high levels,
due to the fact that many plastids (i.e., chloroplasts) per cell,
each carrying many copies of the plastome. This is particularly the
case in foliar tissue, where a single mature leaf cell may contain
over 10,000 copies of the plastome. Following a successful
transformation of the plastome, the transplastomic events carry the
transgene on every copy of the plastid genetic material. This can
result in the transgene expression levels representing as much as
half of the total protein produced in the cell.
[0137] Plastid transformation methods and vector systems are
described, for example, in recent U.S. Pat. Nos. 7,528,292;
7,371,923; 7,235,711; and, 7,193,131. See also U.S. Pat. Nos.
6,680,426 and 6,642,053.
[0138] The foregoing plant transformation methodologies may be used
to introduce transgenes into a number of different plant cells and
tissues, including without limitation, whole plants, tissue and
organ explants including chloroplasts, flowering tissues and cells,
protoplasts, meristem cells, callus, immature embryos and gametic
cells such as microspores, pollen, sperm and egg cells, tissue
cultured cells of any of the foregoing, any other cells from which
a fertile regenerated transgenic plant may be generated. Callus is
initiated from tissue sources including, but not limited to,
immature embryos, seedling apical meristems, microspores and the
like. Cells capable of proliferating as callus are also recipient
cells for genetic transformation.
[0139] Methods of regenerating individual plants from transformed
plant cells, tissues or organs are known and are described for
numerous plant species.
[0140] As an illustration, transformed plantlets (derived from
transformed cells or tissues) are cultured in a root-permissive
growth medium supplemented with the selective agent used in the
transformation strategy (i.e., and antibiotic such as kanamycin).
Once rooted, transformed plantlets are then transferred to soil and
allowed to grow to maturity. Upon flowering, the mature plants are
preferably selfed (self-fertilized), and the resultant seeds
harvested and used to grow subsequent generations. Examples 3-6
describe the regeneration of transgenic tobacco and tomato
plants.
[0141] T.sub.0 transgenic plants may be used to generate subsequent
generations (e.g., T.sub.1, T.sub.2, etc.) by selfing of primary or
secondary transformants, or by sexual crossing of primary or
secondary transformants with other plants (transformed or
untransformed). For example, as described in Example 7, infra,
individual plants over expressing the Alfalfa GS1 gene and
outperforming wildtype plants were crossed with individual plants
over-expressing the Arabidopsis GPT gene and outperforming wildtype
plants, by simple sexual crossing using manual pollen transfer.
Reciprocal crosses were made such that each plant served as the
male in a set of crosses and each plant served as the female in a
second set of crosses. During the mature plant growth stage, the
plants are typically examined for growth phenotype, CO.sub.2
fixation rate, etc. (see following subsection).
Selection of Growth-Enhanced Transgenic Plants:
[0142] Transgenic plants may be selected, screened and
characterized using standard methodologies. The preferred
transgenic plants of the invention will exhibit one or more
phenotypic characteristics indicative of enhanced growth and/or
other desirable agronomic properties. Transgenic plants are
typically regenerated under selective pressure in order to select
transformants prior to creating subsequent transgenic plant
generations. In addition, the selective pressure used may be
employed beyond T.sub.0 generations in order to ensure the presence
of the desired transgene expression construct or cassette.
[0143] T.sub.0 transformed plant cells, calli, tissues or plants
may be identified and isolated by selecting or screening for the
genetic composition of and/or the phenotypic characteristics
encoded by marker genes contained in the transgene expression
construct used for the transformation. For example, selection may
be conducted by growing potentially-transformed plants, tissues or
cells in a growth medium containing a repressive amount of
antibiotic or herbicide to which the transforming genetic construct
can impart resistance. Further, the transformed plant cells,
tissues and plants can be identified by screening for the activity
of marker genes (i.e., .beta.-glucuronidase) which may be present
in the transgene expression construct.
[0144] Various physical and biochemical methods may be employed for
identifying plants containing the desired transgene expression
construct, as is well known. Examples of such methods include
Southern blot analysis or various nucleic acid amplification
methods (i.e., PCR) for identifying the transgene, transgene
expression construct or elements thereof, Northern blotting, S1
RNase protection, reverse transcriptase PCR (RT-PCR) amplification
for detecting and determining the RNA transcription products; and
protein gel electrophoresis, Western blotting, immunoprecipitation,
enzyme immunoassay, and the like may be used for identifying the
protein encoded and expressed by the transgene.
[0145] In another approach, expression levels of genes, proteins
and/or metabolic compounds that are know to be modulated by
transgene expression in the target plant may be used to identify
transformants. In one embodiment of the present invention,
increased levels of the signal metabolite 2-oxoglutaramate may be
used to screen for desirable transformants, as exemplified in the
Examples. Similarly, increased levels of GPT and/or GS activity may
be assayed, as exemplified in the Examples.
[0146] Ultimately, the transformed plants of the invention may be
screened for enhanced growth and/or other desirable agronomic
characteristics. Indeed, some degree of phenotypic screening is
generally desirable in order to identify transformed lines with the
fastest growth rates, the highest seed yields, etc., particularly
when identifying plants for subsequent selfing, cross-breeding and
back-crossing. Various parameters may be used for this purpose,
including without limitation, growth rates, total fresh weights,
dry weights, seed and fruit yields (number, weight), seed and/or
seed pod sizes, seed pod yields (e.g., number, weight), leaf sizes,
plant sizes, increased flowering, time to flowering, overall
protein content (in seeds, fruits, plant tissues), specific protein
content (i.e., GS), nitrogen content, free amino acid, and specific
metabolic compound levels (i.e., 2-oxoglutaramate). Generally,
these phenotypic measurements are compared with those obtained from
a parental identical or analogous plant line, an untransformed
identical or analogous plant, or an identical or analogous
wild-type plant (i.e., a normal or parental plant). Preferably, and
at least initially, the measurement of the chosen phenotypic
characteristic(s) in the target transgenic plant is done in
parallel with measurement of the same characteristic(s) in a normal
or parental plant. Typically, multiple plants are used to establish
the phenotypic desirability and/or superiority of the transgenic
plant in respect of any particular phenotypic characteristic.
[0147] Preferably, initial transformants are selected and then used
to generate T.sub.1 and subsequent generations by selfing
(self-fertilization), until the transgene genotype breeds true
(i.e., the plant is homozygous for the transgene). In practice,
this is accomplished by screening at each generation for the
desired traits and selfing those individuals, often repeatedly
(i.e., 3 or 4 generations). As exemplified herein, transgenic plant
lines propagated through at least one sexual generation (Tobacco,
Arabidopsis, Tomato) demonstrated higher transgene product
activities compared to lines that did not have the benefit of
sexual reproduction and the concomitant increase in transgene copy
number.
[0148] Stable transgenic lines may be crossed and back-crossed to
create varieties with any number of desired traits, including those
with stacked transgenes, multiple copies of a transgene, etc.
Various common breeding methods are well know to those skilled in
the art (see, e.g., Breeding Methods for Cultivar Development,
Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987)).
Additionally, stable transgenic plants may be further modified
genetically, by transforming such plants with further transgenes or
additional copies of the parental transgene. Also contemplated are
transgenic plants created by single transformation events which
introduce multiple copies of a given transgene or multiple
transgenes.
[0149] In another aspect, the invention provides transgenic plants
characterized by increased nitrogen use efficiency. Nitrogen use
efficiency may be expressed as plant yield per given amount of
nitrogen. In the Examples provided herein, the transgene and
control plants all received the same nutrient solutions in the same
amounts. The transgenic plants were consistently characterized by
higher yields, and thus have higher nitrogen use efficiencies.
[0150] In yet another aspect, the invention provides transgenic
plants and seeds thereof with increased tolerance to high salt
growth conditions. This aspect of the invention is exemplified by
Example 24, which describes the germination of transgenic tobacco
plant seeds in very high salt conditions (200 mM NaCl). While
counterpart wild type tobacco seeds germinated at a rate of only
about 10%, on average, the transgenic tobacco seeds achieved nearly
the same rate of germination obtained under no salt conditions for
both transgenic and wild type seeds, or about 92%.
EXAMPLES
[0151] Various aspects of the invention are further described and
illustrated by way of the several examples which follow, none of
which are intended to limit the scope of the invention.
Example 1
Isolation of Arabidopsis Gluamine Phenylpyruvate Transaminase (GPT)
Gene
[0152] In an attempt to locate a plant enzyme that is directly
involved in the synthesis of the signal metabolite
2-oxoglutaramate, applicants hypothesized that the putative plant
enzyme might bear some degree of structural relationship to a human
protein that had been characterized as being involved in the
synthesis of 2-oxoglutaramate. The human protein, glutamine
transaminase K (E.C. 2.6.1.64) (also referred in the literature as
cysteine conjugate .beta.-lyase, kyneurenine aminotransferase,
glutamine phenylpyruvate transaminase, and other names), had been
shown to be involved in processing of cysteine conjugates of
halogenated xenobiotics (Perry et al., 1995, FEBS Letters
360:277-280). Rather than having an activity involved in nitrogen
assimilation, however, human cysteine conjugate .beta.-lyase has a
detoxifying activity in humans, and in animals (Cooper and Meister,
1977, supra). Nevertheless, the potential involvement of this
protein in the synthesis of 2-oxoglutaramate was of interest.
[0153] Using the protein sequence of human cysteine conjugate
.beta.-lyase, a search against the TIGR Arabidopsis plant database
of protein sequences identified one potentially related sequence, a
polypeptide encoded by a partial sequence at the Arabidopsis gene
locus at At1q77670, sharing approximately 36% sequence
homology/identity across aligned regions.
[0154] The full coding region of the gene was then amplified from
an Arabidopsis cDNA library (Stratagene) with the following primer
pair:
TABLE-US-00002 [SEQ ID NO: 37] 5'-CCCATCGATGTACC
TGGACATAAATGGTGTGATG-3' [SEQ ID NO: 38]
5'-GATGGTACCTCAGACTTTTCTCTTAAGCTTCTGCTTC-3'
[0155] These primers were designed to incorporate Cla I (ATCGAT)
and Kpn I (GGTACC) restriction sites to facilitate subsequent
subcloning into expression vectors for generating transgenic
plants. Takara ExTaq DNA polymerase enzyme was used for high
fidelity PCR using the following conditions: initial denaturing
94.degree. C. for 4 minutes, 30 cycles of 94.degree. C. 30 second,
annealing at 55.degree. C. for 30 seconds, extension at 72.degree.
C. for 90 seconds, with a final extension of 72.degree. C. for 7
minutes. The amplification product was digested with Cla I and Kpn
I restriction enzymes, isolated from an agarose gel electrophoresis
and ligated into vector pMon316 (Rogers, et. al. 1987 Methods in
Enzymology 153:253-277) which contains the cauliflower mosaic virus
(CaMV, also CMV) 35S constitutive promoter and the nopaline
synthase (NOS) 3' terminator. The ligation product was transformed
into DH5a cells and transformants sequenced to verify the
insert.
[0156] A 1.3 kb cDNA was isolated and sequenced, and found to
encode a full length protein of 440 amino acids in length,
including a putative chloroplast signal sequence.
Example 2
Production of Biologically Active Recombinant Arabidopsis Glutamine
Phenylpyruvate Transaminase (GPT)
[0157] To test whether the protein encoded by the cDNA isolated as
described in Example 1, supra, is capable of catalyzing the
synthesis of 2-oxoglutaramate, the cDNA was expressed in E. coli,
purified, and assayed for its ability to synthesize
2-oxoglutaramate using a standard method.
NMR Assay for 2-Oxoglutaramate:
[0158] Briefly, the resulting purified protein was added to a
reaction mixture containing 150 mM Tris-HCl, pH 8.5, 1 mM beta
mercaptoethanol, 200 mM glutamine, 100 mM glyoxylate and 200 .mu.M
pyridoxal 5'-phosphate. The reaction mixture without added test
protein was used as a control. Test and control reaction mixtures
were incubated at 37.degree. C. for 20 hours, and then clarified by
centrifugation to remove precipitated material. Supernatants were
tested for the presence and amount of 2-oxoglutaramate using
.sup.13C NMR with authentic chemically synthesized 2-oxoglutaramate
as a reference. The products of the reaction are 2-oxoglutaramate
and glycine, while the substrates (glutamine and glyoxylate)
diminish in abundance. The cyclic 2-oxoglutaramate gives rise to a
distinctive signal allowing it to be readily distinguished from the
open chain glutamine precursor.
HPLC Assay for 2-Oxoglutaramate:
[0159] An alternative assay for GPT activity uses HPLC to determine
2-oxoglutaramate production, following a modification of Calderon
et al., 1985, J Bacteriol 161(2): 807-809. Briefly, a modified
extraction buffer consisting of 25 mM Tris-HCl pH 8.5, 1 mM EDTA,
20 .mu.M FAD, 10 mM Cysteine, and .about.1.5% (v/v)
Mercaptoethanol. Tissue samples from the test material (i.e., plant
tissue) are added to the extraction buffer at approximately a 1/3
ratio (w/v), incubated for 30 minutes at 37.degree. C., and stopped
with 200 .mu.l of 20% TCA. After about 5 minutes, the assay mixture
is centrifuged and the supernatant used to quantify
2-oxoglutaramate by HPLC, using an ION-300 7.8 mm ID.times.30 cm L
column, with a mobile phase in 0.01N H.sub.2SO.sub.4, a flow rate
of approximately 0.2 ml/min, at 40.degree. C. Injection volume is
approximately 20 and retention time between about 38 and 39
minutes. Detection is achieved with 210 nm UV light.
Results Using NMR Assay:
[0160] This experiment revealed that the test protein was able to
catalyze the synthesis of 2-oxoglutaramate. Therefore, these data
indicate that the isolated cDNA encodes a glutamine phenylpyruvate
transaminase that is directly involved in the synthesis of
2-oxoglutaramate in plants. Accordingly, the test protein was
designated Arabidopsis glutamine phenylpyruvate transaminase, or
"GPT".
[0161] The nucleotide sequence of the Arabidopsis GPT coding
sequence is shown in the Table of Sequences, SEQ ID NO. 1. The
translated amino acid sequence of the GPT protein is shown in SEQ
ID NO. 2.
Example 3
Creation of Transgenic Tobacco Plants Over-Expressing Arabidopsis
GPT
[0162] Generation of Plant Expression Vector pMON-PJU:
[0163] Briefly, the plant expression vector pMon316-PJU was
constructed as follows. The isolated cDNA encoding Arabidopsis GPT
(Example 1) was cloned into the ClaI-KpnI polylinker site of the
pMON316 vector, which places the GPT gene under the control of the
constitutive cauliflower mosaic virus (CaMV) 35S promoter and the
nopaline synthase (NOS) transcriptional terminator. A kanamycin
resistance gene was included to provide a selectable marker.
Agrobacterium-Mediated Plant Transformations:
[0164] pMON-PJU and a control vector pMon316 (without inserted DNA)
were transferred to Agrobacterium tumefaciens strain pTiTT37ASE
using a standard electroporation method (McCormac et al., 1998,
Molecular Biotechnology 9:155-159), followed by plating on LB
plates containing the antibiotics spectinomycin (100 micro gm/ml)
and kanamycin (50 micro gm/ml). Antibiotic resistant colonies of
Agrobacterium were examined by PCR to assure that they contained
plasmid.
[0165] Nicotiana tabacum cv. Xanthi plants were transformed with
pMON-PJU transformed Agrobacteria using the leaf disc
transformation system of Horsch et. al. (Horsch et al., 1995,
Science 227:1229-1231). Briefly, sterile leaf disks were inoculated
and cultured for 2 days, then transferred to selective MS media
containing 100 .mu.g/ml kanamycin and 500 .mu.g/ml clafaran.
Transformants were confirmed by their ability to form roots in the
selective media.
Generation of GPT Transgenic Tobacco Plants:
[0166] Sterile leaf segments were allowed to develop callus on
Murashige & Skoog (M&S) media from which the transformant
plantlets emerged. These plantlets were then transferred to the
rooting-permissive selection medium (M&S medium with kanamycin
as the selection agent). The healthy, and now rooted, transformed
tobacco plantlets were then transferred to soil and allowed to grow
to maturity and upon flowering the plants were selfed and the
resultant seeds were harvested. During the growth stage the plants
had been examined for growth phenotype and the CO.sub.2 fixation
rate was measured for many of the young transgenic plants.
Production of T1 and T2 Generation GPT Transgenic Plants:
[0167] Seeds harvested form the T.sub.0 generation of the
transgenic tobacco plants were germinated on M&S media
containing kanamycin (100 mg/L) to enrich for the transgene. At
least one fourth of the seeds did not germinate on this media
(kanamycin is expected to inhibit germination of the seeds without
resistance that would have been produced as a result of normal
genetic segregation of the gene) and more than half of the
remaining seeds were removed because of demonstrated sensitivity
(even mild) to the kanamycin.
[0168] The surviving plants (T.sub.1 generation) were thriving and
these plants were then selfed to produce seeds for the T.sub.2
generation. Seeds from the T.sub.1 generation were germinated on MS
media supplemented for the transformant lines with kanamycin (10
mg/liter). After 14 days they were transferred to sand and provided
quarter strength Hoagland's nutrient solution supplemented with 25
mM potassium nitrate. They were allowed to grow at 24.degree. C.
with a photoperiod of 16 h light and 8 hr dark with a light
intensity of 900 micromoles per meter squared per second. They were
harvested 14 days after being transferred to the sand culture.
Characterization of GPT Transgenic Plants:
[0169] Harvested transgenic plants (both GPT transgenes and vector
control transgenes) were analyzed for glutamine sythetase activity
in root and leaf, whole plant fresh weight, total protein in root
and leaf, and CO.sub.2 fixation rate (Knight et al., 1988, Plant
Physiol. 88: 333). Non-transformed, wild-type A. tumefaciens plants
were also analyzed across the same parameters in order to establish
a baseline control.
[0170] Growth characteristic results are tabulated below in Table
I. Additionally, a photograph of the GPT transgenic plant compared
to a wild type control plant is shown in FIG. 2 (together with GS1
transgenic tobacco plant, see Example 5). Across all parameters
evaluated, the GPT transgenic tobacco plants showed enhanced growth
characteristics. In particular, the GPT transgenic plants exhibited
a greater than 50% increase in the rate of CO.sub.2 fixation, and a
greater than two-fold increase in glutamine synthetase activity in
leaf tissue, relative to wild type control plants. In addition, the
leaf-to-root GS ratio increased by almost three-fold in the
transaminase transgenic plants relative to wild type control. Fresh
weight and total protein quantity also increased in the transgenic
plants, by about 50% and 80% (leaf), respectively, relative to the
wild type control. These data demonstrate that tobacco plants
overexpressing the Arabidopsis GPT transgene achieve significantly
enhanced growth and CO.sub.2 fixation rates.
TABLE-US-00003 TABLE I Leaf Root Protein mg/gram fresh weight Wild
type - control 8.3 2.3 Line PN1-8 a second control 8.9 2.98 Line
PN9-9 13.7 3.2 Glutamine Synthetase activity, micromoles/min/mg
protein Wild type (Ratio of leaf:root = 4.1:1) 4.3 1.1 PN1-8 (Ratio
of leaf:root = 4.2:1) 5.2 1.3 PN9-9 (Ratio of leaf:root = 10.9:1)
10.5 0.97 Whole Plant Fresh Weight, g Wild type 21.7 PN1-8 26.1
PN9-9 33.1 CO.sub.2 Fixation Rate, umole/m2/sec Wild type 8.4 PN1-8
8.9 PN9-9 12.9 Data = average of three plants Wild type - Control
plants; not regenerated or transformed. PN1 lines were produced by
regeneration after transformation using a construct without
inserted gene. A control against the processes of regeneration and
transformation. PN 9 lines were produced by regeneration after
transformation using a construct with the Arabidopsis GPT gene.
Example 4
Generation of Transgenic Tomato Plants Carrying Arabidopsis GPT
Transgene
[0171] Transgenic Lycopersicon esculentum (Micro-Tom Tomato) plants
carrying the Arabidopsis GPT transgene were generated using the
vectors and methods described in Example 3. T.sub.0 transgenic
tomato plants were generated and grown to maturity. Initial growth
characteristic data of the GPT transgenic tomato plants is
presented in Table II. The transgenic plants showed significant
enhancement of growth rate, flowering, and seed yield in relation
to wild type control plants. In addition, the transgenic plants
developed multiple main stems, whereas wild type plants developed
with a single main stem. A photograph of a GPT transgenic tomato
plant compared to a wild type plant is presented in FIG. 3
(together with GS1 transgenic tomato plants, see Example 6).
TABLE-US-00004 TABLE II Growth Wildtype GPT Transgenic
Characteristics Tomato Tomato Stem height, cm 6.5 18, 12, 11 major
stems Stems 1 3 major, 0 other Buds 2 16 Flowers 8 12 Fruit 0 3
Example 5
Generation of Transgenic Tobacco Plants Overexpressing Alfalfa
Gs1
[0172] Generation of Plant Expression Vector pGS111:
[0173] Transgenic tobacco plants overexpressing the Alfalfa GS1
gene were generated as previously described (Temple et al., 1993,
Mol. Gen. Genetics 236: 315-325). Briefly, the plant expression
vector pGS111 was constructed by inserting the entire coding
sequence together with extensive regions of both the 5' and 3'
untranslated regions of the Alfalfa GS1 gene [SEQ ID NO: 3]
(DasSarma at al., 1986, Science, Vol 232, Issue 4755, 1242-1244)
into pMON316 (Rogers et al., 1987, supra), placing the transgene
under the control of the constitutive cauliflower mosaic virus
(CaMV) 35S promoter and the nopaline synthase (NOS) transcriptional
terminator. A kanamycin resistance gene was included to provide a
selectable marker.
Generation of GS1 Transformants:
[0174] pGS111 was transferred to Agrobacterium tumefaciens strain
pTiTT37ASE using triparental mating as described (Rogers et al.,
1987, supra; Unkefer et al., U.S. Pat. No. 6,555,500). Nicotiana
tabacum cv. Xanthi plants were transformed with pGS111 transformed
Agrobacteria using the leaf disc transformation system of Horsch
et. al. (Horsch et al., 1995, Science 227:1229-1231). Transformants
were selected and regenerated on MS medium containing 100 .mu.g/ml
kanamycin. Shoots were rooted on the same medium (with kanamycin,
absent hormones) and transferred to potting
soil:perlite:vermiculite (3:1:1), grown to maturity, and allowed to
self. Seeds were harvested from this T.sub.0 generation, and
subsequent generations produced by selfing and continuing selection
with kanamycin. The best growth performers were used to yield a T3
progeny for crossing with the best performing GPT over-expressing
lines identified as described in Example 3. A photograph of the GS1
transgenic plant compared to a wild type control plant is shown in
FIG. 2 (together with GPT transgenic tobacco plant, see Example
3)
Example 6
Generation of Transgenic Tomato Plants Carrying Alfalfa GS1
Transgene
[0175] Transgenic Lycopersicon esculentum (Micro-Tom Tomato) plants
carrying the Alfalfa GS1 transgene were generated using the vector
described in Example 5 and a transformation protocol essentially as
described (Sun et al., 2006. Plant Cell Physiol. 46(3) 426-31).
T.sub.0 transgenic tomato plants were generated and grown to
maturity. Initial growth characteristic data of the GPT transgenic
tomato plants is presented in Table III. The transgenic plants
showed significant enhancement of growth rate, flowering, and seed
yield in relation to wild type control plants. In addition, the
transgenic plants developed multiple main stems, whereas wild type
plants developed with a single main stem. A photograph of a GS1
transgenic tomato plant compared to a wild type plant is presented
in FIG. 3 (together with GPT transgenic tomato plant, see Example
4).
TABLE-US-00005 TABLE III Growth Wildtype GS1 Transgenic
Characteristics Tomato Tomato Stem height, cm 6.5 16, 7, 5 major
stems Stems 1 3 major, 3 med, 1 sm Buds 2 2 Flowers 8 13 Fruit 0
4
Example 7
Generation of Double Transgenic Tobacco Plants Carrying GS1 and GPT
Transgenes
[0176] In an effort to determine whether the combination of GS1 and
GPT transgenes in a single transgenic plant might improve the
extent to which growth and other agronomic characteristics may be
enhanced, a number of sexual crosses between high producing lines
of the single transgene (GS1 or GPT) transgenic plants were carried
out. The results obtained are dramatic, as these crosses repeatedly
generated progeny plants having surprising and heretofore unknown
increases in growth rates, biomass yield, and seed production.
Materials and Methods:
[0177] Single-transgene, transgenic tobacco plants overexpressing
GPT or GS1 were generated as described in Examples 3 and 4,
respectively. Several of fastest growing T.sub.2 generation GPT
transgenic plant lines were crossed with the fastest growing T3
generation GS1 transgenic plant lines using reciprocal crosses. The
progeny were then selected on kanamycin containing M&S media as
described in Example 3, and their growth, flowering and seed yields
examined.
[0178] Tissue extractions for GPT and GS activities: GPT activity
was extracted from fresh plant tissue after grinding in cold 100 mM
Tris-HCl, pH 7.6, containing 1 mm ethylenediaminetetraacetic, 200
mM pyridoxal phosphate and 6 mM mercaptoethanol in a ratio of 3 ml
per gram of tissue. The extract was clarified by centrifugation and
used in the assay. GS activity was extracted from fresh plant
tissue after grinding in cold 50 mM Imidazole, pH 7.5 containing 10
mM MgCl2, and 12.5 mM mercaptoethanol in a ratio of 3 ml per gram
of tissue. The extract was clarified by centrifugation and used in
the assay. GPT activity was assayed as described in Calderon and
Mora, 1985, Journal Bacteriology 161:807-809. GS activity was
measured as described in Shapiro and Stadtmann, 1970, Methods in
Enzymology 17A: 910-922. Both assays involve an incubation with
substrates and cofactor at the proper pH. Detection was by
HPLC.
Results:
[0179] The results are presented in two ways. First, specific
growth characteristics are tabulated in Tables IV.A and IV.B
(biomass, seed yields, growth rate, GS activity, GPT activity,
2-oxoglutaramate activity, etc). Second, photographs of progeny
plants and their leaves are shown in comparison to single-transgene
and wild type plants and leaves are presented in FIG. 5 and FIG. 6,
which show much larger whole plants, larger leaves, and earlier
and/or more abundant flowering in comparison to the parental
single-transgene plants and wild type control plants.
[0180] Referring to Table IV.A, double-transgene progeny plants
form these crosses showed tremendous increases total biomass (fresh
weight), with fresh weights ranging from 45-89 grams per individual
progeny plant, compared to a range of only 19-24 grams per
individual wild type plant, representing on average, about a two-
to three-fold increase over wild type plants, and representing at
the high end, an astounding four-fold increase in biomass over wild
type plants. Taking the 24 individual double-transgene progeny
plants evaluated, the average individual plant biomass was about
2.75 times that of the average wild type control plant. Four of the
progeny lines showed approximately 2.5 fold greater average per
plant fresh weights, while two lines showed over three-fold greater
fresh weights in comparison to wild type plants.
[0181] In comparison to the single-transgene parental lines, the
double-transgene progeny plants also showed far more than an
additive growth enhancement. Whereas GPT single-transgene lines
show as much as about a 50% increase over wild type biomass, and
GS1 single-transgene lines as much as a 66% increase, progeny
plants averaged almost a 200% increase over wild type plants.
[0182] Similarly, the double transgene progeny plants flowered
earlier and more prolifically than either the wild type or single
transgene parental lines, and produced a far greater number of seed
pods as well as total number of seeds per plant. Referring again to
Table IV.A, on average, the double-transgene progeny produced over
twice the number of seed pods produced by wild type plants, with
two of the high producer plants generating over three times the
number of seed pods compared to wild type. Total seed yield in
progeny plants, measured on a per plant weight basis, ranged from
about double to nearly quadruple the number produced in wild type
plants.
TABLE-US-00006 TABLE IV.A FRESH WEIGHT SEED PODS SEED YIELD GS
ACTIVITY PLANT LINE g/whole plant #pods/plant g/plant LEAF ROOT L/R
RATIO Wild Type Tobacco Wild type 1 18.73 26 0.967 Wild type 2
24.33 24 1.07 Wild type 3 23.6 32 0.9 Wild type 4 18.95 32 1.125 WT
Average 21.4025 28.5 1.0155 7.75 1.45 5.34 Cross 1 X1L1a .times.
PA9-9ff 1 59.21 62 2.7811 2 65.71 56 3 55.36 72 4 46.8 56 Cross 1
Average 56.77 61.5 +274% 14.98 1.05 14.27 Compared to WT +265%
+216% +193% -28% +267% Cross 2 PA9-2 .times. L9 1 70.83 61 1.76 2
49.17 58 3.12 3 50.23 90 NA 4 45.77 Cross 2 Average 54 58.3 2.44
16.32 1.81 9.02 Compared to WT +252% +205% +240% +211% +125% +169%
Cross 3 PA9-9ff .times. L1a 1 89.1 77 3.687 2 78.18 3 58.34 4 61.79
Cross 3 Average 71.85 77 (one plant) 3.678 (one plant) 15.92 1.38
11.54 Compared to WT +336% +270% +362% +205% -5% +216% Cross 5
PA9-10aa .times. L1a 1 65.34 45 2.947 2 53.28 64 3.3314 3 49.85 42
1.5667 4 44.63 42 2.5013 Cross 5 Average 53.275 48.25 2.86928 13.03
1.8 7.24 Compared to WT +244% +169% +283% +168% Cross 6 PA9-17b
.times. L1a 1 56.7 64 2.492 2 55.05 66 2.162 3 51.51 59 1.8572 4
45.38 72 4.742 Cross 6 Average 52.16 65.25 2.8133 14.114.752
1.1.1124 13.29 Compared to WT +244% +229% +277% Cross 7 PA9-20aa
.times. L1b 1 76.26 67 2.0535 2 66.27 42 1.505 3 72.26 72 2.3914 4
63.91 91 2.87 Cross 7 Average 69.675 68 2.204975 14.12 1.24 11.39
Compared to WT +326% +239% +217% Control PA9-9ff 1 32.18 N/A 2
32.64 N/A 3 34.67 N/A 4 25.18 N/A Average 31.17 N/A 11.57 1.14
10.15 Compared to WT +148% Control GS L1a 1 41.74 N/A 2 36.24 N/A 3
33.8 N/A 4 30.48 N/A Average 35.57 N/A 13.15 1.23 10.69 Compared to
WT +166%
[0183] Table IV.B shows growth rate, biomass and yield, and
biochemical characteristics of Line XX (Line 3 further selfed)
compared to the single transgene line expressing GS1 and wild type
control tobacco. All parameters are greatly increased in the double
transgenic plant (Line XX). Notably, 2-oxoglutaramate activity was
almost 17-fold higher, and seed yield and foliar biomass was
three-fold higher, in Line XX plants versus control plants.
TABLE-US-00007 TABLE IV.B Specific Foliar Fruit/ GS Activity GPT
Activity 2-oxoglu- Trans Plant Growth Rate Biomass Flowers/ Seed
umol/min/ nmol/h/ taramate Gene Type mg/g/d FWt, g Buds Yield g
gFWt gFWt nmol/gFWt Assay Wildtype, 228 21.40 28.5 1.02 7.75 16.9
68.9 No avg Line 1 GS 269 35.57 NM NM 11.6 NM 414 Yes Line XX 339
59.71 62.9 2.94 16.3 243.9 1,153.6 Yes NM Not Measured
Example 8
Generation of Double Transgenic Pepper Plants Carrying GS1 and GPT
Transgenes
[0184] In this example, Big Jim chili pepper plants (New Mexico
varietal) were transformed with the Arabidopsis GPT full length
coding sequence of SEQ ID NO: 1 under the control of the CMV 35S
promoter, and the Arabidopsis GS1 coding sequence included in SEQ
ID NO: 6 under the control of the RuBisCo promoter, using
Agrobacterium-mediated transfer to seed pods. After 3 days, seeds
were harvested and used to generate T0 plants and screened for
transformants. The resulting double-transgenic plants showed higher
pod yields, faster growth rates, and greater biomass yields in
comparison to the control plants.
Materials and Methods:
[0185] Solanaceae Capisicum Pepper plants ("Big Jim" varietal) were
transformed with the Arabidopsis GPT full length coding sequence of
SEQ ID NO: 1 under the control of the CMV 35S promoter within the
expression vector pMON (see Example 3), and the Arabidopsis GS1
coding sequence included in SEQ ID NO: 6 under the control of the
RuBisCo promoter within the expression vector pCambia 1201 (Tomato
rubisco rbcS3C promoter: Kyozulka et al., 1993, Plant Physiol. 103:
991-1000; SEQ ID NO: 22; vector construct of SEQ ID NO: 6), using
Agrobacterium-mediated transfer to seed pods.
[0186] For this and all subsequent examples, the Cambia 1201 or
1305.1 vectors were constructed according to standard cloning
methods (Sambrook et al., 1989, supra, Saiki et al., 1988, Science
239: 487-491). The vector is supplied with a 35S CaMV promoter;
that promoter was replaced with RcbS-3C promoter from tomato to
control the expression of the target gene. The Cambia 1201 vectors
contain bacterial chlorophenicol and plant hygromycin resistance
selectable marker genes. The Cambia 1305.1 vectors contain
bacterial chlorophenicol and hygromycin resistance selectable
marker genes.
[0187] The transgene expression vectors pMON (GPT transgene) and
pCambia 1201 (GS transgene) were transferred to separate
Agrobacterium tumefaciens strain LBA4404 cultures using a standard
electroporation method (McCormac et al., 1998, Molecular
Biotechnology 9:155-159). Transformed Agrobacterium were selected
on media containing 50 .mu.g/ml of either streptamycin for pMON
constructs or chloroamphenicol for the Cambia constructs.
Transformed Agrobacterium cells were grown in LB culture media
containing 25 .mu.g/ml of antibiotic for 36 hours. At the end of
the 36 hr growth period cells were collected by centrifugation and
cells from each transformation were resuspended in 100 ml LB broth
without antibiotic.
[0188] Pepper plants were then transformed with a mixture of the
resulting Agrobacterium cell suspensions using a transformation
protocol in which the Agrobacterium is injected directly into the
seed cavity of developing pods. Briefly, developing pods were
injected with the 200 ml mixture in order to inoculate immature
seeds with the Agrobacteria essentially as described (Wang and
Waterhouse, 1997, Plant Mol. Biol. Reporter 15: 209-215). In order
to induce Agrobacteria virulence and improve transformation
efficiencies, 10 .mu.g/ml acetosyringonone was added to the
Agrobacteria cultures prior to pod inoculations (see, Sheikholeslam
and Weeks, 1986, Plant Mol. Biol. 8: 291-298).
[0189] Using a syringe, pods were injected with a liberal quantity
of the Agrobacterium vector mixture, and left to incubate for about
3 days. Seeds were then harvested and germinated, and developing
plants observed for phenotypic characteristics including growth and
antibiotic resistance. Plants carrying the transgenes were green,
whereas untransformed plants showed signs of chlorosis in leaf
tips. Vigorous growing transformants were further cultivated and
compared to wild type pepper plants grown under identical
conditions.
Results:
[0190] The results are presented in FIG. 7 and Table V. FIG. 7
shows a photograph of a GPT+GS double transgenic pepper plant
compared to a control plant grown for the same time under identical
conditions. This photograph shows tremendous pepper yield in the
transgenic line compared to the control plant.
[0191] Table V presents biomass yield and GS activity, as well as
transgene genotyping, in the transgenic lines compared to the wild
type control. Referring to Table V, double-transgene progeny plants
showed tremendous increases total biomass (fresh weight), with
fresh weights, ranging from 393-662 grams per individual transgenic
plant, compared to an average of 328 grams per wild type plant.
Transgenic line A5 produced more than twice the total biomass of
the controls. Moreover, pepper yields in the transgenic lines were
greatly improved over wild type plants, and were 50% greater than
control plants (on average). Notably, one of the transgene lines
produced twice as many peppers as the control plant average.
TABLE-US-00008 TABLE V TRANSGENIC PEPPER GROWTH/BIOMASS AND
REPRODUCTION Biomass, Yield GS activity Transgene Foliar Fresh
Peppers, g Umoles/min/ Presence Plant type Wt, g DWt gFWt Assay
Wildtype, avg 328.2 83.7 1.09 Negative Line A2 457.3 184.2 1.57 GPT
- Yes Line A5 661.7 148.1 1.8 GPT - Yes Line B1 493.4 141.0 1.3 GPT
- Yes Line B4 393.1 136.0 1.6 GPT - Yes Line C1 509.4 152.9 1.55
GPT - Yes FWt Fresh Weight; DWt Dry Weight
Example 9
Generation of Double Transgenic Bean Plants Carrying Arabidopsis
GS1 and GPT Transgenes
[0192] In this example, yellow wax bean plants (Phaseolus vulgaris)
were transformed with the Arabidopsis GPT full length coding
sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter
within the expression vector pCambia 1201, and the Arabidopsis GS1
coding sequence included in SEQ ID NO: 6 under the control of the
RuBisCo promoter within the expression vector pCambia 1201, using
Agrobacterium-mediated transfer into flowers.
Materials and Methods:
[0193] The transgene expression vectors pCambia 1201-GPT (including
construct of SEQ ID NO: 27) and pCambia 1201-GS (including
construct of SEQ ID NO: 6) were transferred to separate
Agrobacterium tumefaciens strain LBA4404 cultures using a standard
electroporation method (McCormac et al., 1998, Molecular
Biotechnology 9:155-159). Transformed Agrobacterium were selected
on media containing 50 .mu.g/ml of chloroamphenicol. Transformed
Agrobacterium cells were grown in LB culture media containing 25
.mu.g/ml of antibiotic for 36 hours. At the end of the 36 hr growth
period cells were collected by centrifugation and cells from each
transformation were resuspended in 100 ml LB broth without
antibiotic.
[0194] Bean plants were then transformed with a mixture of the
resulting Agrobacterium cell suspensions using a transformation
protocol in which the Agrobacteria is injected directly into the
flower structure (Yasseem, 2009, Plant Mol. Biol. Reporter 27:
20-28). In order to induce Agrobacteria virulence and improve
transformation efficiencies, 10 .mu.g/ml acetosyringonone was added
to the Agrobacteria cultures prior to flower inoculation. Briefly,
once flowers bloomed, the outer structure encapsulating the
reproductive organs was gently opened with forceps in order to
permit the introduction of the Agrobacteria mixture, which was
added to the flower structure sufficient to flood the anthers.
[0195] Plants were grown until bean pods developed, and seeds were
harvested and used to generate transgenic plants. Transgenic plants
were then grown together with control bean plants under identical
conditions, photographed and phenotypically characterized. Growth
rates were measured for both transgenic and control plants. In this
and all examples, Glutamine synthetase (GS) activity was assayed
according to the methods in Shapiro and Stadtmann, 1970, Methods in
Enzymology 17A: 910-922; and, Glutamine phenylpyruvate transaminase
(GPT) activity was assayed according to the methods in Calderon et
al., 1985, J. Bacteriol. 161: 807-809. See details in Example 7,
Methods, supra.
Results:
[0196] The results are presented in FIG. 8, FIG. 9 and Table
VI.
[0197] FIG. 8 shows GPT+GS transgenic bean line A growth rate data
relative to control plants, including plant heights on various days
into cultivation, as well as numbers of flower buds, flowers, and
bean pods. These data show that the GPT+GS double transgenic bean
plants outgrew their counterpart control plants. The transgenic
plants grew taller, flowered earlier and produced more flower buds
and flowers, and developed bean pods and produced more bean pods
that the wild type control plants.
TABLE-US-00009 TABLE VI TRANSGENIC BEANS LINE A GS Activity Bean
Pod GPT Activity umoles/min/ Antibiotic Plant Type Yield FWt, g
nmoles/h/gFWt gFWt Resistance Wildtype, avg 126.6 101.9 25.2
Negative 2A 211.5 NM NM + 4A 207.7 NM NM + 5B 205.7 984.7 101.3 +
WT Wildtype; FWt Fresh Weight; NM Not Measured
[0198] Table VI presents bean pod yield, GPT and GS activity, as
well as antibiotic resistance status, in the transgenic lines
compared to the wild type control (average of several robust
control plants; control plants that did not grow well were excluded
from the analyses). Referring to Table VI, double-transgene progeny
plants showed substantial bean pod biomass increases (fresh pod
weight) in comparison to the control plants, with bean pod biomass
yields consistently above 200 grams per individual transgenic
plant, compared to an average of 127 grams per wild type plant,
representing an over 60% increase in pod yield in the double
transgene lines relative to control plant(s).
[0199] Lastly, FIG. 9 shows a photograph of a GPT+GS double
transgenic bean plant compared to a control plant grown for the
same time under identical conditions, showing increased growth in
the transgenic plant.
Example 10
Generation of Double Transgenic Bean Plants Carrying Arabidopsis
GS1 and Grape GPT Transgenes
[0200] In this example, yellow wax bean plants (Phaseolus vulgaris)
were transformed with the Grape GPT full length coding sequence
included in SEQ ID NO: 8 under the control of the RuBisCo promoter
within the expression vector pCambia 1305.1, and the Arabidopsis
GS1 coding sequence included in SEQ ID NO: 6 under the control of
the RuBisCo promoter within the expression vector pCambia 1201,
using Agrobacterium-mediated transfer into developing pods.
Materials and Methods:
[0201] The transgene expression vectors pCambia 1201-GPT(grape)
(including construct of SEQ ID NO: 8) and pCambia 1201-GS
(including construct of SEQ ID NO: 6) were transferred to separate
Agrobacterium tumefaciens strain LBA4404 cultures using a standard
electroporation method (McCormac et al., 1998, Molecular
Biotechnology 9:155-159). Transformed Agrobacterium were selected
on media containing 50 .mu.g/ml of chloroamphenicol. Transformed
Agrobacterium cells were grown in LB culture media containing 25
.mu.g/ml of antibiotic for 36 hours. At the end of the 36 hr growth
period cells were collected by centrifugation and cells from each
transformation were resuspended in 100 ml LB broth without
antibiotic.
[0202] Bean plants were then transformed with a mixture of the
resulting Agrobacterium cell suspensions using a transformation
protocol in which the Agrobacteria is injected directly into the
flower structure. In order to induce Agrobacteria virulence and
improve transformation efficiencies, 10 .mu.g/ml acetosyringonone
was added to the Agrobacteria cultures prior to flower inoculation.
Briefly, once flowers bloomed, the outer structure encapsulating
the reproductive organs was gently opened with forceps in order to
permit the introduction of the Agrobacteria mixture, which was
added to the flower structure sufficient to flood the anthers.
[0203] Plants were grown until bean pods developed, and seeds were
harvested and used to generate transgenic plants. Transgenic plants
were then grown together with control bean plants under identical
conditions, photographed and phenotypically characterized. Growth
rates were measured for both transgenic and control plants.
Results:
[0204] The results are presented in FIG. 10, FIG. 11 and Table
VII.
[0205] FIG. 10 shows GPT+GS transgenic bean line G growth rate data
relative to control plants, specifically including numbers of
flower buds, flowers, and bean pods. These data show that the
GPT+GS double transgenic bean plants outgrew their counterpart
control plants. Notably, the transgenic plants produced
substantially more bean pods that the wild type control plants.
TABLE-US-00010 TABLE VII TRANSGENIC BEANS LINE G: POD YIELDS Plant
Type Bean Pod Yield FWt, g Antibiotic Resistance Wild type, avg
157.9 Negative G1 200.5 + G2 178.3 + WT Wildtype; FWt Fresh Weight;
NM Not Measured
[0206] Table VII presents bean pod yield and antibiotic resistance
status, in the transgenic lines compared to the wild type control
(average of several robust control plants; control plants that did
not grow well were excluded from the analyses). Referring to Table
VII, double-transgene progeny plants showed substantial bean pod
biomass increases (fresh pod weight) in comparison to the control
plants, with bean pod biomass yields of 200.5 (line G1) and 178
grams (line G2) per individual transgenic plant, compared to an
average of 158 grams per individual wild type plant, representing
approximately a 27% increase in pod yield in the double transgene
lines relative to control plants.
[0207] Lastly, FIG. 11 shows a photograph of a GPT+GS double
transgenic bean plant compared to a control plant grown for the
same time under identical conditions. The transgenic plant shows
substantially increased size and biomass, larger leaves and a more
mature flowering compared to the control plant.
Example 11
Generation of Double Transgenic Cowpea Plants Carrying Arabidopsis
GS1 and GPT Transgenes
[0208] In this example, common Cowpea plants were transformed with
the Arabidopsis GPT full length coding sequence of SEQ ID NO: 1
under the control of the CMV 35S promoter within the expression
vector pMON, and the Arabidopsis GS1 coding sequence included in
SEQ ID NO: 6 under the control of the RuBisCo promoter within the
expression vector pCambia 1201, using Agrobacterium-mediated
transfer into flowers. Materials and methods were as in Example 9,
supra.
Results:
[0209] The results are presented in FIGS. 12 and 13, and Table VI.
FIG. 12 shows relative growth rates for the GPT+GS transgenic
Cowpea line A and wild type control Cowpea at several intervals
during cultivation, including (FIG. 12A) height and longest leaf
measurements, (FIG. 12B) trifolate leafs and flower buds, and (FIG.
12C) flowers, flower buds and pea pods. These data show that the
GPT+GS double transgenic Cowpea plants outgrew their counterpart
control plants. The transgenic plants grew faster and taller, had
longer leaves, and set flowers and pods sooner than wild type
control plants.
TABLE-US-00011 TABLE VIII TRANSGENIC COWPEA LINE A Pea Pod Yield,
GPT Activity GS Activity Antibiotic Plant Type FWt, g nmoles/h/gFWt
umol/min/gFWt Resistance Wildtype, avg 74.7 44.4 28.3 Negative 4A
112.8 NM 41.3 + 8B 113.8 736.2 54.9 + WT Wildtype; FWt Fresh
Weight; NM Not Measured
[0210] Table VIII presents pea pod yield, GPT and GS activity, as
well as antibiotic resistance status, in the transgenic lines
compared to the wild type control (average of several robust
control plants; control plants that did not grow well were excluded
from the analyses). Referring to Table VIII, double-transgene
progeny plants showed substantial pea pod biomass increases (fresh
pod weight) in comparison to the control plants, with average
transgenic plant pea pod biomass yields nearly 52% greater than the
yields measured in control plant(s).
[0211] Lastly, FIG. 13 shows a photograph of a GPT+GS double
transgenic bean plant compared to a control plant grown for the
same time under identical conditions, showing increased biomass and
pod yield in the transgenic plant relative to the wild type control
plant.
Example 12
Generation of Double Transgenic Cowpea Plants Carrying Arabidopsis
GS1 and Grape GPT Transgenes
[0212] In this example, common Cowpea plants were transformed with
the Grape GPT full length coding sequence included in SEQ ID NO: 8
under the control of the RuBisCo promoter within the expression
vector pCambia 1305.1 (including construct of SEQ ID NO: 8), and
the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under
the control of the RuBisCo promoter within the expression vector
pCambia 1201 (including construct of SEQ ID NO: 6), using
Agrobacterium-mediated transfer into flowers. Materials and methods
were as in Example 11, supra.
Results:
[0213] The results are presented in FIGS. 14 and 15, and Table
IX.
[0214] FIG. 14 shows relative growth rates for the GPT+GS
transgenic Cowpea line G and wild type control Cowpea. These data
show that the transgenic plants are consistently higher (FIG. 14A),
produce substantially more flowers, flower buds and pea pods (FIG.
14B), and develop trifolates and leaf buds faster (FIG. 14C).
TABLE-US-00012 TABLE IX TRANSGENIC COWPEA LINE G Pod Yield, GPT
Activity GS Activity Antibiotic Plant Type FWt, g nmoles/h/gFWT
umol/min/gFWt Resistance Wildtype, 59.7 44.4 26.7 Negative avg G9
102.0 555.6 34.5 + WT Wildtype; FWt Fresh Weight; NM Not
Measured
[0215] Table IX presents pea pod yield, GPT and GS activity, as
well as antibiotic resistance status, in the transgenic lines
compared to the wild type control (average of several robust
control plants; control plants that did not grow well were excluded
from the analyses). Referring to Table IX, double-transgene progeny
plants showed substantial pea pod biomass increases (fresh pod
weight) in comparison to the control plants, with average pea pod
biomass yields 70% greater in the transgenic plants compared to
control plant(s).
[0216] Lastly, FIG. 15 shows a photograph of a GPT+GS double
transgenic pea plant compared to a control plant grown for the same
time under identical conditions, showing increased height, biomass
and leaf size in the transgenic plant relative to the wild type
control plant.
Example 13
Generation of Double Transgenic Alfalfa Plants Carrying Arabidopsis
GS1 and GPT Transgenes
[0217] In this example, Alfalfa plants (Medicago sativa, var Ladak)
were transformed with the Arabidopsis GPT full length coding
sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter
within the expression vector pMON316 (see Example 3, supra), and
the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under
the control of the RuBisCo promoter within the expression vector
pCambia 1201 (including construct of SEQ ID NO: 6), using
Agrobacterium-mediated transfer into seedling plants. Agrobacterium
vectors and mixtures were prepared for seedling inoculations as
described in Example 11, supra.
Seedling Inoculations:
[0218] When Alfalfa seedlings were still less than about 1/2 inch
tall, they were soaked in paper toweling that had been flooded with
the Agrobacteria mixture containing both transgene constructs. The
seedlings were left in the paper toweling for two to three days,
removed and then planted in potting soil. Resulting T0 and control
plants were then grown for the first 30 days in a growth chamber,
thereafter cultivated in a greenhouse, and then harvested 42 days
after sprouting. At this point, only the transgenic Alfalfa line
displayed flowers, as the wild type plants only displayed immature
flower buds. The plants were characterized as to flowering status
and total biomass.
Results:
[0219] The results are presented in Table X. The data shows that
the transgenic Alfalfa plants grew faster, flowered sooner, and
yielded on average about a 62% biomass increase relative to the
control plants.
TABLE-US-00013 TABLE X TRANSGENIC ALFALFA VS. CONTROL Plant Type
Biomass at Sacrifice, g Flowering Stage Wildtype, avg 6.03 Small
defined buds No buds swelling. No flowers Transgene #5 10.38 4 Open
flowers Transgene # 11 9.03 Flower buds swelling Transgene #13 9.95
Flower buds swelling
Example 14
Generation of Double Transgenic Cantaloupe Plants Carrying
Arabidopsis GS1 and GPT Transgenes
[0220] In this example, Cantaloupe plants (Cucumis melo var common)
were transformed with the Arabidopsis GPT full length coding
sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter
within the expression vector pMON316 (see Example 3, supra), and
the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under
the control of the RuBisCo promoter within the expression vector
pCambia 1201 (including construct of SEQ ID NO: 6), using
Agrobacterium-mediated transfer via injection into developing
melons. Agrobacterium vectors and mixtures were prepared for
intra-melon inoculations as described in Example 8, supra.
Inoculations into developing melons were carried out essentially as
described in
Example 8
[0221] The plants were characterized as to flowering status and
total biomass relative to control melon plants grown under
identical conditions.
[0222] The results are presented in FIG. 16 and Table XI. Referring
to Table XI, the transgenic plants showed substantial foliar plant
biomass increases in comparison to the control plants, with an
average increase in biomass of 63%. Moreover, a tremendous increase
in flower and flower bud yields was observed in all five transgenic
lines. Control plants displayed no flowers and only 5 buds at
sacrifice, on average. In sharp contrast, the transgenic plants
displayed between 2 and 5 flowers per plant, and between 21 and 30
flower buds, per plant, indicating a substantially higher growth
rate and flower yield. Increased flower yield would be expected to
translate into correspondingly higher melon yields in the
transgenic plants. Referring to FIG. 16 (a photograph comparing
transgenic Cantaloupe plants to control Cantaloupe plants), the
transgenic Cantaloupe plants show dramatically increased height,
overall biomass and flowering status relative to the control
plants.
TABLE-US-00014 TABLE XI TRANGENIC CANTALOUPE VERSUS CONTROL Biomass
Flowers/Flower Antibiotic Plant Type Foliar FWt, g Buds at
Sacrifice Resistance Wildtype, avg 22.8 0/5 Negative Line 1 37.0
3/21 + Line 2 35.0 2/30 + Line 3 37.1 3/27 + Line 4 40.6 5/26 +
Line 5 35.7 4/30 + FWt Fresh Weight
Example 15
Generation of Double Transgenic Pumpkin Plants Carrying Arabidopsis
GS1 and GPT Transgenes
[0223] In this example, common Pumpkin plants (Cucurbita maxima)
were transformed with the Arabidopsis GPT full length coding
sequence of SEQ ID NO: 1 under the control of the CMV 35S promoter
within the expression vector pMON316 (see Example 3, supra), and
the Arabidopsis GS1 coding sequence included in SEQ ID NO: 6 under
the control of the RuBisCo promoter within the expression vector
pCambia 1201 (including construct of SEQ ID NO: 6), using
Agrobacterium-mediated transfer via injection into developing
pumpkins, essentially as described in Example 14, supra. The
transgenic and control pumpkin plants were grown under identical
conditions until the emergence of flower buds in the control
plants, then all plants were characterized as to flowering status
and total biomass.
[0224] The results are presented in FIG. 17 and Table XII.
Referring to Table XII, the transgenic plants showed substantial
foliar plant biomass increases in comparison to the control plants,
with an increase in average biomass yield of 67% over control
plants. Moreover, an increase in flower bud yields was observed in
four of the five transgenic lines in comparison to control. Control
plants displayed only 4 buds at sacrifice (average). In contrast,
four transgenic plant lines displayed between 8 and 15 flowers buds
per plant, representing a two- to nearly four-fold yield
increase.
TABLE-US-00015 TABLE XII TRANGENIC PUMPKIN VERSUS CONTROL Biomass
Flower Buds at Antibiotic Plant Type Foliar FWt, g Sacrifice
Resistance Wildtype, avg 47.7 4.2 Negative Line 1 (Photo) 82.3 8
Line 2 74.3 8 + Line 3 80.3 9 + Line 4 (Photo) 77.8 4 + Line 5 84.5
15 + FWt Fresh Weight;
[0225] Referring to FIG. 17 (a photograph comparing transgenic
pumpkin plants to control plants), the transgenic pumpkin plants
show substantially increased plant size, overall biomass and leaf
sizes and numbers relative to the control plants.
Example 16
Generation of Double Transgenic Arabidopsis Plants Carrying
Arabidopsis GS1 and GPT Transgenes
[0226] In this example, Arabidopsis thaliana plants were
transformed with the truncated Arabidopsis GPT coding sequence of
SEQ ID NO: 18 under the control of the CMV 35S promoter within the
expression vector pMON316 (see Example 3, supra), and transgenic
plants thereafter transformed with the Arabidopsis GS1 coding
sequence included in SEQ ID NO: 6 under the control of the RuBisCo
promoter within the expression vector pCambia 1201 (including
construct of SEQ ID NO: 6), using Agrobacterium-mediated "floral
dip" transfer as described (Harrison et al., 2006, Plant Methods
2:19-23; Clough and Bent, 1998, Plant J. 16:735-743). Agrobacterium
vectors pMON316 carrying GPT and pCambia 1201 carrying GS1 were
prepared as described in Examples 3 and 11, respectively.
[0227] Transformation of two different cultures of Agrobacterium
with either a pMon 316+ Arabidopsis GTP construct or with a Cambia
1201+ Arabidopsis GS construct was done by electroporation using
the method of Weigel and Glazebrook 2002. The transformed
Agrobacterium were then grown under antibiotic selection, collected
by centrifugation resuspended in LB broth with antibiotic and used
in the floral dip of Arabidopsis inflorescence. Floral dipped
Arabidopsis plants were taken to maturity and self-fertilized and
seeds were collected. Seeds from twice dipped plants were first
geminated on a media containing 20 ug/ml of kanamycin and by
following regular selection procedures surviving seedlings were
transferred to media containing 20 ug of hygromycin. Plants (3)
surviving the selection process on both antibiotics were
self-fertilized and seeds were collected. Seeds from the T1
generation were germinated on MS media containing 20 ug/ml of
hygromycin and surviving seedlings were taken to maturity,
self-fertilized and seeds collected. This seed population the T2
generation was then used for subsequent growth studies.
[0228] The results are presented in FIG. 18 and Table XIII.
Referring to Table XIII, which shows data from 6 wild type and 6
transgenic Arabidopsis plants (averaged), the transgenic plants
displayed increased levels of both GPT and GS activity. GPT
activity was over twenty-fold higher than the control plants.
Moreover, the transgenic plant fresh foliar weight average was well
over four-fold that of the wild type control plant average. A
photograph of young transgene Arabidopsis plants in comparison to
wild type control Arabidopsis plants grown under identical
conditions is shown in FIG. 18, and reveals a consistent and very
significant growth/biomass increase in transgenic plants relative
to the control plants.
TABLE-US-00016 TABLE XIII TRANSGENIC ARABIDOPSIS VERSUS CONTROL
Biomass, g GS Activity Fresh foliar GPT Activity umol/min/gF
Antibiotic Plant type wt nmol/h/gFWt Wt Resistance Wildtype, avg
0.246 18.4 7.0 Negative Transgene 1.106 395.6 18.2 Positive
Example 17
Generation of Transgenic Tomato Plants Carrying Arabidopsis GPT and
GS1 Transgenes
[0229] In this example, tomato plants (Solanum lycopersicon, "Money
Maker" variety) were transformed with the Arabidopsis GPT full
length coding sequence of SEQ ID NO: 1 under the control of the CMV
35S promoter within the expression vector pMON316 (see Example 3,
supra), and the Arabidopsis GS1 coding sequence included in SEQ ID
NO: 6 under the control of the RuBisCo promoter within the
expression vector pCambia 1201 (including construct of SEQ ID NO:
6). Single transgene (GPT) transgenic tomato plants were generated
and grown to flowering essentially as described in Example 4. The
Arabidopsis GS1 transgene was then introduced into the
single-transgene T0 plants using Agrobacterium-mediated transfer
via injection directly into flowers (as described in Example 8).
The transgenic and control tomato plants were grown under identical
conditions and characterized as to growth phenotype
characteristics. Resulting T0 double-transgene plants were then
grown to maturity, photographed along with control tomato plants,
and phenotypically characterized.
[0230] The results are presented in FIG. 19 and in Table X.
Referring to Table XIX, double-transgene tomato plants showed
substantial foliar plant biomass increases in comparison to the
control plants, with an increase in average biomass yield of 45%
over control. Moreover, as much as a 70% increase in tomato fruit
yield was observed in the transgenic lines compared to control
plants (e.g., 51 tomatoes harvested from Line 4C, versus and
average of approximately 30 tomatoes from control plants). A much
higher level of GPT activity was observed in the transgenic plants
(e.g., line 4C displaying an approximately 32-fold higher GPT
activity in comparison to the average GPT activity measured in
control plants). GS activity was also higher in the transgenic
plants relative to control plants (almost double in Line 4C).
[0231] With respect to growth phenotype, and referring to FIG. 19,
the transgenic tomato plants displayed substantially larger leaves
compared to control plants (FIG. 19A). In addition, it can be seen
that the transgenic tomato plants were substantially larger, taller
and of a greater overall biomass (see FIG. 19B).
TABLE-US-00017 TABLE XIX TRANSGENIC TOMATO GROWTH AND REPRODUCTION
Total Tomatoes GPT GS Biomass Harvested Activity Activity Transgene
Foliar until nmoles/ umoles/ Presence Plant Type FWt, g Sacrifice
h/gFWt min/gFWt Assay Wildtype, 891 30.2 287 14.27 Negative avg
Line 6C 1288 43 9181 18.3 + Line 4C 1146 51 1718 26.4 +
Example 18
Generation of Transgenic Camilena Plants Carrying Arabidopsis GPT
and GS1 Transgenes
[0232] In this example, Camelina plants (Camelina sativa, Var MT
303) were transformed with the Arabidopsis GPT full length coding
sequence of SEQ ID NO: 1 under the control of the RuBisCo promoter
within the expression vector pCambia 1201, and the Arabidopsis GS1
coding sequence included in SEQ ID NO: 6 under the control of the
RuBisCo promoter within the expression vector pCambia 1201, using
Agrobacterium-mediated transfer into germinating seeds according to
the method described in Chee et al., 1989, Plant Physiol. 91:
1212-1218. Agrobacterium vectors and mixtures were prepared for
seed inoculations as described in Example 11, supra.
[0233] Transgenic and control Camelina plants were grown under
identical conditions (30 days in a growth chamber and then moved to
greenhouse cultivation) for 39 days, and characterized as to
biomass, growth characteristics and flowering stage.
[0234] The results are presented in Table XX and FIG. 20. Referring
to Table XX, it can be seen that total biomass in the transgenic
plants was, on average, almost double control plant biomass. Canopy
diameter was also significantly improved in the transgenic plants.
FIG. 20 shows a photograph of transgenic Camelina compared to
control. The transgenic plant is noticeably larger and displays
more advanced flowering.
TABLE-US-00018 TABLE XX TRANSGENIC CAMELINA VERSUS CONTROL
Height/Canopy Plant Type Diameter, inches Biomass G Flowering Stage
Wildtype, avg 14/4 8.35 Partial flowering Transgene C-1 15.5/5
16.54 Full flowering Transgene C-3 14/7 14.80 Initial flowering
Example 19
Activity of Barley GPT Transgene in Planta
[0235] In this example, the putative coding sequence for Barley GPT
was isolated and expressed from a transgene construct using an in
planta transient expression assay. Biologically active recombinant
Barley GPT was produced, and catalyzed the increased synthesis of
2-oxoglutaramate, as confirmed by HPLC.
[0236] The Barley (Hordeum vulgare) GPT coding sequence was
determined and synthesized. The DNA sequence of the Barley GPT
coding sequence used in this example is provided in SEQ ID NO: 14,
and the encoded GPT protein amino acid sequence is presented in SEQ
ID NO: 15.
[0237] The coding sequence for Barley GPT was inserted into the
1305.1 cambia vector, and transferred to Agrobacterium tumefaciens
strain LBA404 using a standard electroporation method (McCormac et
al., 1998, Molecular Biotechnology 9:155-159), followed by plating
on LB plates containing hygromycin (50 micro gm/ml). Antibiotic
resistant colonies of Agrobacterium were selected for analysis.
[0238] The transient tobacco leaf expression assay consisted of
injecting a suspension of transformed Agrobacterium (1.5-2.0 OD
650) into rapidly growing tobacco leaves. Intradermal injections
were made in a grid across the leaf surface to assure that a
significant amount of the leaf surface would be exposed to the
Agrobacterium. The plant was then allowed to grow for 3-5 days when
the tissue was extracted as described for all other tissue
extractions and the GPT activity measured.
[0239] GPT activity in the inoculated leaf tissue (1217
nanomoles/gFWt/h) was three-fold the level measured in the control
plant leaf tissue (407 nanomoles/gFWt/h), indicating that the
Hordeum GPT construct directed the expression of biologically
active GPT in a transgenic plant.
Example 20
Isolation and Expression of Recombinant Rice GPT Gene Coding
Sequence and Analysis of Biological Activity
[0240] In this example, the putative coding sequence for rice GPT
was isolated and expressed in E. coli. Biologically active
recombinant rice GPT was produced, and catalyzed the increased
synthesis of 2-oxoglutaramate, as confirmed by HPLC.
Materials and Methods:
[0241] Rice GPT Coding Sequence and Expression in E. coli:
[0242] The rice (Oryza sativa) GPT coding sequence was determined
and synthesized, inserted into a PET28 vector, and expressed in E.
coli. Briefly, E. coli cells were transformed with the expression
vector and transformants grown overnight in LB broth diluted and
grown to OD 0.4, expression induced with
isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and
harvested. A total of 25.times.106 cells were then assayed for
biological activity using the NMR assay, below. Untransformed, wild
type E. coli cells were assayed as a control. An additional control
used E. coli cells transformed with an empty vector.
[0243] The DNA sequence of the rice GPT coding sequence used in
this example is provided in SEQ ID NO: 10, and the encoded GPT
protein amino acid sequence is presented in SEQ ID NO: 11.
HPLC Assay for 2-Oxoglutaramate:
[0244] HPLC was used to determine 2-oxoglutaramate production in
GPT-overexpressing E. coli cells, following a modification of
Calderon et al., 1985, J Bacteriol 161(2): 807-809. Briefly, a
modified extraction buffer consisting of 25 mM Tris-HCl pH 8.5, 1
mM EDTA, 20 .mu.M Pyridoxal phosphate, 10 mM Cysteine, and
.about.1.5% (v/v) Mercaptoethanol was used. Samples (lysate from E.
coli cells, 25.times.106 cells) were added to the extraction buffer
at approximately a 1/3 ratio (w/v), incubated for 30 minutes at
37.degree. C., and stopped with 200 .mu.l of 20% TCA. After about 5
minutes, the assay mixture is centrifuged and the supernatant used
to quantify 2-oxoglutaramate by HPLC, using an ION-300 7.8 mm
ID.times.30 cm L column, with a mobile phase in 0.01N
H.sub.2SO.sub.4, a flow rate of approximately 0.2 ml/min, at
40.degree. C. Injection volume is approximately 20 and retention
time between about 38 and 39 minutes. Detection is achieved with
210 nm UV light.
[0245] NMR analysis comparison with authentic 2-oxoglutaramate was
used to establish that the Arabidopisis full length sequence
expresses a GPT with 2-oxoglutaramate synthesis activity. Briefly,
authentic 2-oxoglutarmate (structure confirmed with NMR) made by
chemical synthesis to validate the HPLC assay, above, by confirming
that the product of the assay (molecule synthesized in response to
the expressed GPT) and the authentic 2-oxoglutaramate elute at the
same retention time. In addition, when mixed together the assay
product and the authentic compound elute as a single peak.
Furthermore, the validation of the HPLC assay also included
monitoring the disappearance of the substrate glutamine and showing
that there was a 1:1 molar stoechiometry between glutamine consumed
to 2-oxoglutaramte produced. The assay procedure always included
two controls, one without the enzyme added and one without the
glutamine added. The first shows that the production of the
2-oxoglutaramate was dependent upon having the enzyme present, and
the second shows that the production of the 2-oxoglutaramate was
dependent upon the substrate glutamine.
Results:
[0246] Expression of the rice GPT coding sequence of SEQ ID NO: 10
resulted in the over-expression of recombinant GPT protein having
2-oxoglutaramate synthesis-catalyzing bioactivity. Specifically,
1.72 nanomoles of 2-oxoglutaramate activity was observed in the E.
coli cells overexpressing the recombinant rice GPT, compared to
only 0.02 nanomoles of 2-oxoglutaramate activity in control E. coli
cells, an 86-fold activity level increase over control.
Example 21
Isolation and Expression of Recombinant Soybean GPT Gene Coding
Sequence and Analysis of Biological Activity
[0247] In this example, the putative coding sequence for soybean
GPT was isolated and expressed in E. coli. Biologically active
recombinant soybean GPT was produced, and catalyzed the increased
synthesis of 2-oxoglutaramate, as confirmed by HPLC.
Materials and Methods:
[0248] Soybean GPT Coding Sequence and Expression in E. coli:
[0249] The soybean (Glycine max) GPT coding sequence was determined
and synthesized, inserted into a PET28 vector, and expressed in E.
coli. Briefly, E. coli cells were transformed with the expression
vector and transformants grown overnight in LB broth diluted and
grown to OD 0.4, expression induced with
isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and
harvested. A total of 25.times.106 cells were then assayed for
biological activity using the HPLC assay, below. Untransformed,
wild type E. coli cells were assayed as a control. An additional
control used E. coli cells transformed with an empty vector.
[0250] The DNA sequence of the soybean GPT coding sequence used in
this example is provided in SEQ ID NO: 12, and the encoded GPT
protein amino acid sequence is presented in SEQ ID NO: 13.
HPLC Assay for 2-Oxoglutaramate:
[0251] HPLC was used to determine 2-oxoglutaramate production in
GPT-overexpressing E. coli cells, as described in Example 20,
supra.
Results:
[0252] Expression of the soybean GPT coding sequence of SEQ ID NO:
12 resulted in the over-expression of recombinant GPT protein
having 2-oxoglutaramate synthesis-catalyzing bioactivity.
Specifically, 31.9 nanomoles of 2-oxoglutaramate activity was
observed in the E. coli cells overexpressing the recombinant
soybean GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate
activity in control E. coli cells, a nearly 1.600-fold activity
level increase over control.
Example 22
Isolation and Expression of Recombinant Zebra Fish GPT Gene Coding
Sequence and Analysis of Biological Activity
[0253] In this example, the putative coding sequence for Zebra fish
GPT was isolated and expressed in E. coli. Biologically active
recombinant Zebra fish GPT was produced, and catalyzed the
increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.
Materials and Methods:
[0254] Zebra Fish GPT Coding Sequence and Expression in E.
coli:
[0255] The Zebra fish (Danio rerio) GPT coding sequence was
determined and synthesized, inserted into a PET28 vector, and
expressed in E. coli. Briefly, E. coli cells were transformed with
the expression vector and transformants grown overnight in LB broth
diluted and grown to OD 0.4, expression induced with
isopropyl-B-D-thiogalactoside (0.4 micromolar), grown for 3 hr and
harvested. A total of 25.times.106 cells were then assayed for
biological activity using the HPLC assay, below. Untransformed,
wild type E. coli cells were assayed as a control. An additional
control used E. coli cells transformed with an empty vector.
[0256] The DNA sequence of the Zebra fish GPT coding sequence used
in this example is provided in SEQ ID NO: 16, and the encoded GPT
protein amino acid sequence is presented in SEQ ID NO: 17.
HPLC Assay for 2-Oxoglutaramate:
[0257] HPLC was used to determine 2-oxoglutaramate production in
GPT-overexpressing E. coli cells, as described in Example 20,
supra.
Results:
[0258] Expression of the Zebra fish GPT coding sequence of SEQ ID
NO: 16 resulted in the over-expression of recombinant GPT protein
having 2-oxoglutaramate synthesis-catalyzing bioactivity.
Specifically, 28.6 nanomoles of 2-oxoglutaramate activity was
observed in the E. coli cells overexpressing the recombinant Zebra
fish GPT, compared to only 0.02 nanomoles of 2-oxoglutaramate
activity in control E. coli cells, a more than 1.400-fold activity
level increase over control.
Example 23
Generation and Expression of Recombinant Truncated Arabidopsis GPT
Gene Coding Sequences and Analysis of Biological Activity
[0259] In this example, two different truncations of the
Arabidopsis GPT coding sequence were designed and expressed in E.
coli, in order to evaluate the activity of GPT proteins in which
the putative chloroplast signal peptide is absent or truncated.
Recombinant truncated GPT proteins corresponding to the full length
Arabidopsis GPT amino acid sequence of SEQ ID NO: 2, truncated to
delete either the first 30 amino-terminal amino acid residues, or
the first 45 amino-terminal amino acid residues, were successfully
expressed and showed biological activity in catalyzing the
increased synthesis of 2-oxoglutaramate, as confirmed by HPLC.
Materials and Methods:
[0260] Truncated Arabidopsis GPT Coding Sequences and Expression in
E. coli:
[0261] The DNA coding sequence of a truncation of the Arabidopsis
thaliana GPT coding sequence of SEQ ID NO: 1 was designed,
synthesized, inserted into a PET28 vector, and expressed in E.
coli. The DNA sequence of the truncated Arabidopsis GPT coding
sequence used in this example is provided in SEQ ID NO: 20 (-45 AA
construct), and the corresponding truncated GPT protein amino acid
sequence is provided in SEQ ID NO: 21. Briefly, E. coli cells were
transformed with the expression vector and transformants grown
overnight in LB broth diluted and grown to OD 0.4, expression
induced with isopropyl-B-D-thiogalactoside (0.4 micromolar), grown
for 3 hr and harvested. A total of 25.times.10.sup.6 cells were
then assayed for biological activity using HPLC as described in
Example 20. Untransformed, wild type E. coli cells were assayed as
a control. An additional control used E. coli cells transformed
with an empty vector.
[0262] Expression of the truncated-45 Arabidopsis GPT coding
sequence of SEQ ID NO: 20 resulted in the over-expression of
biologically active recombinant GPT protein (2-oxoglutaramate
synthesis-catalyzing bioactivity). Specifically, 16.1 nanomoles of
2-oxoglutaramate activity was observed in the E. coli cells
overexpressing the truncated-45 GPT, compared to only 0.02
nanomoles of 2-oxoglutaramate activity in control E. coli cells, a
more than 800-fold activity level increase over control. For
comparison, the full length Arabidopsis gene coding sequence
expressed in the same E. coli assay generated 2.8 nanomoles of
2-oxoglutaramate activity, or roughly less than one-fifth the
activity observed from the truncated recombinant GPT protein.
Example 24
GPT+GS Transgenic Tobacco Seed Germination Tolerates High Salt
Concentrations
[0263] In this example, seeds form the double transgene tobacco
line XX-3 (Cross 3 in Table 4, see Example 7) were tested in a seed
germination assay designed to evaluate tolerance to high salt
concentrations.
Materials and Methods:
[0264] Tobacco seeds from the wild type and XX-3 populations were
surfaced sterilized (5% bleach solution for 5 minutes followed by a
10% ethanol wash for 3 minutes) and rinsed with sterile distilled
water. The surface sterilized seeds were then spread on Murashige
and Skoog media (10% agarose) without sucrose and containing either
0 or 200 mM NaCl. The seeds were allowed to germinate in darkness
for 2 days followed by 6 days under a 16:8 photoperiod at
24.degree. C. On day eight the rate of germination was determined
by measuring the percentage of seeds from the control or transgene
plants that had germinated.
Results:
[0265] The results are tabulated in Table XXI below. The rate of
germination of the transgenic plant line seeds under zero salt
conditions was the same as observed with wild type control plant
seeds. In stark contrast, the germination rate of the transgenic
plant line seeds under very high salt conditions far exceeded the
rate seen in wild type control seeds. Whereas over 81% of the
transgenic plant seeds had germinated under the high salt
conditions, only about 9% of the wild type control plant seeds had
germinated by the same time point. These data indicate that the
transgenic seeds are capable of germinating very well under high
salt concentrations, an important trait for plant growth in areas
of increasingly high water and/or soil salinity.
TABLE-US-00019 TABLE XXI TRANSGENIC TOBACCO PLANTS GERMINATE AND
TOLERATE HIGH SALT Control (0 mM NaCl) Test (200 mM NaCl)a Plant
type % Germination % Germination Wild type 92, 87, 94 9, 11, 8
Transgene line XX-3 92, 91, 94 84, 82, 78
Example 25
Method for Generating Transgenic Maize Plants Carrying Hordeum GPT
and GS1 Transgenes
[0266] This example provides a method for generating transgenic
maize plants expressing GPT and GS1 transgenes. Maize (Zea mays,
hybrid line Hi-II) type II callus is biolistically transformed with
an expression cassette comprising the hordeum glutamine synthetase
(GS1) coding sequence of SEQ ID NO: 40 under the control of the
rice RuBisCo small subunit promoter of SEQ ID NO: 39 (expression
casette of SEQ ID NO: 42), and the hordeum GPT coding sequence of
SEQ ID NO: 45 under the control of the corn ubiquitin (Ubil)
promoter of SEQ ID NO: 44. Transformation of maize callus is
achieved by particle bombardment.
Vector Constructs:
[0267] An expression cassette comprising the hordeum GS1 and GPT
genes, under the control of the rice RuBisCo small subunit and corn
ubiquitin promoters, respectively, is cloned into the plasmid
pAHC25 (Christensen and Quail, 1996, Transgenic Research 5:213-218)
modified to include a bar gene conferring resistance to bialophos,
or a similar vector, in order to generate the transgene expression
vector.
Transformation and Regeneration:
[0268] The transgene expression vector is introduced into immature
zygotic embryo source callus of parent maize hybrid line Hi-II
(A188xB73 origin) (Armstrong et al., 1991, Maize Genetics Coop
Newsletter 65:92-93) using particle bombardment, essentially as
described (Frame et al., 2000, In Vitro Cell. Dev. Biol-Plant
36:21-29; this method was developed by and is routinely used at the
Iowa State University Center for Plant Transformation).
[0269] More specifically, immature zygotic embryo source callus is
prepared for transformation by serial culturing on a
callus-initiating medium (N6E, Songstad et al., 1996, In vitro Cell
Dev. Biol.-Plant 32:179-183). Washed gold particles are coated with
the plasmid construct and used to bombard the callus with a PDS
1000/He biolistic gun as described (Sanford et al., 1993, Methods
in Enzymology 217: 483-509). After 7-10 days on initiation medium,
the callus is then transferred to selection medium containing
bialophos (N6S, Songstad et al., 1996, supra) and allowed to grow.
Following the development of bialophos resistant clones, callus
pieces are transferred to a regeneration medium (Armstrong and
Green, 1985, Planta 164:207-214) containing bialophos and allowed
to grow for several weeks. Thereafter, the resulting plantlets are
transferred to regeneration medium without the selection agent, and
cultivated.
[0270] Transgenic corn plants may be grown and evaluated through
maturity, and seeds harvested for use in generating subsequent
generations of an event. Various phenotypic characteristics may be
observed in T.sub.0 events, as well as in T.sub.1 and subsequent
generations, and used to select seed sources for the development of
subsequent generations. High performing lines may be selfed to
achieve trait homozygosity and/or crossed.
Example 26
Method for Generating Transgenic Rice Plants Carrying Hordeum GPT
and GS1 Transgenes
[0271] This example provides a method for generating transgenic
rice plants expressing GPT and GS1 transgenes. Rice (Oryza sativa,
Japonica cultivar Nipponbare) type II calus is transformed with the
hordeum glutamine synthetase (GS1) coding sequence of SEQ ID NO: 40
under the control of the rice RuBisCo small subunit promoter of SEQ
ID NO: 39 (expression cassette of SEQ ID NO: 42), and the hordeum
GPT coding sequence of SEQ ID NO: 45 under the control of the corn
ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation is
achieved by Agrobacterium-mediated transformation.
Vector Constructs:
[0272] An expression cassette comprising the hordeum GS1 and GPT
genes, under the control of the rice RuBisCo small subunit and corn
ubiquitin promoters, respectively, is cloned into base vector
pTF101.1, using standard molecular cloning methodologies, to
generate the transgene expression vector. Base vector pTF101.1 is a
derivative of the pPZP binary vector (Hajdukiewicz et al 1994,
Plant Mol. Biol. 25:989-994), which includes the right and left
T-DNA border fragments from a nopaline strain of A. tumefaciens, a
broad host origin of replication (pVS1) and a
spectinomycin-resistant marker gene (aadA) for bacterial selection.
The plant selectable marker gene cassette includes the
phosphinothricin acetyl transferase (bar) gene from Streptomyces
hygroscopicus that confers resistance to the herbicides glufosinate
and bialophos. The soybean vegetative storage protein terminator
(Mason et al., 1993) follows the 3' end of the bar gene.
Media:
[0273] YEP Medium: 5 g/L yeast extract, 10 g/L peptone, 5 g/L
NaCl.sub.2, 15 g/L Bacto-agar. pH to 6.8 with NaOH. After
autoclaving, the appropriate antibiotics are added to the medium
when it has cooled to 50.degree. C.
[0274] Infection Medium: N6 salts and vitamins (Chu et al., 1975,
Sci. Sinica 18: 659-668), 1.5 mg/L 2,4-dichlorophenoxyacetic acid
(2,4-D), 0.7 g/L L-proline, 68.4 g/L sucrose, and 36 g/L glucose
(pH 5.2). This medium is filter-sterilized and stored at 4.degree.
C. Acetosyringone (AS, 100 .mu.M) is added just prior to use
(prepared from 100 .mu.M stocks of filter-sterilized AS, dissolved
in DMSO to 200 mM then diluted 1:1 with water).
[0275] Callus Induction Medium: N6 salts and vitamins, 300 mg/L
casamino acids, 2.8 g/L L-proline, 30 g/L sucrose, and 4 g/L
gelrite (pH 5.8). Filter sterilized N6 Vitamins and 2 mg/L 2,4-D,
are added to this medium after autoclaving.
[0276] Co-cultivation Medium (make fresh): N6 salts and vitamins,
300 mg/L casamino acids, 30 g/L sucrose, 10 g/L glucose, and 4 g/L
gelrite (pH 5.8). Filter sterilized N6 vitamins, acetosyringone
(AS) 100 .mu.M and 2 mg/L 2,4-D are added to this medium after
autoclaving.
[0277] Selection Medium: N6 salts and vitamins, 300 mg/L casamino
acids, 2.8 g/L L-proline, 30 g/L sucrose, and 4 g/L gelrite (pH
5.8). Filter sterilized N6 vitamins, 2 mg/L 2,4-D, 2 mg/L Bialaphos
(Shinyo Sangyo, Japan) and 500 mg/L carbenicillin are added to this
medium after autoclaving.
[0278] Regeneration Medium I: MS salts and vitamins (Murashige and
Skoog, 1962), 2 g/L casamino acids, 30 g/L sucrose, 30 g/L
sorbitol, and 4 g/L gelrite (pH 5.8). Filter sterilized MS
vitamins, 100 mg/L cefotaxime, 100 mg/L vancomycin, 0.02 mg/L NAA
(naphthaleneacetic acid), 2 mg/L kinetin (Toki, 1997, supra) and 2
mg/L Bialaphos are added to this medium after autoclaving.
[0279] Regeneration Medium II: MS Salts and vitamins, 100 mg/L
myo-inositol, 30 g/L sucrose, 3 g/L gelrite, (pH 5.8).
Transformation and Regeneration:
[0280] Japonica rice cultivar Nipponbare is transformed with
Agrobacterium tumefaciens strain EHA101 (Hood et al., 1986, J.
Bacteriol. 168:1291-1301), transformed with the pTF101.1 transgene
expression vector carrying the hordeum GS1+GPT expression cassette.
The vector system pTF101.1 in EHA101 is maintained on YEP medium
(An et al., 1988) containing 100 mg/L spectinomycin (for pTF101.1)
and 50 mg/L kanamycin (for EHA101).
[0281] Briefly, callus tissue derived from the mature rice embryo
is used as the starting material for transformation. Callus
induction, co-cultivation, selection and regeneration I media are
based on those of Hiei et al., 1994, The Plant Journal 6
(2):271-282.
[0282] More specifically, calli are induced as follows. First,
15-20 rice seeds are dehusked and rinsed in 10 ml of 70% Ethanol
(50 ml conical tube) by vigorously shaking the tube for one minute,
followed by rinsing once with sterile water. Then, 10 ml of 50%
commercial bleach (5.25% hypochlorite) is added and placed on a
shaker for 30 minutes (low setting). The bleach solution is then
poured-off and the seeds rinsed five times with .about.10 ml of
sterilized water each time. With a small portion of the final
rinse, the seeds are poured onto sterilized filter paper (in a
sterile petri plate) and then allowed to dry. Using sterile
forceps, several (i.e., 5) seeds are transferred to the surface of
individual sterile petri plates containing callus induction medium.
The plates are wrapped with vent tape and incubated in the light
(16:8 photoperiod) at 29.degree. C. Seeds are observed every few
days and those showing signs of contamination are discarded.
[0283] After two to three weeks, developing callus is visible on
the scutellum of the mature seed. Calli are then subcultured to
fresh induction medium and allowed to proliferate. Four days prior
to infection, the callus tissue is cut into 2-4 mm pieces and
transferred to fresh induction medium.
[0284] The selection medium uses modifications from Toki (Toki,
1997, Plant Molecular Biology Reporter 15:16-21) whereby bialophos
(2 mg/L) is employed for plant selection and carbenicillin (500
mg/L) for counter selection against Agrobacterium. Regeneration II
medium is as described (Armstrong and Green, 1985, Planta
164:207-214).
[0285] Agrobacterium culture is grown (i.e., for 3 days at
19.degree. C., or 2 days at 28.degree. C.) on YEP medium amended
with spectinomycin (100 mg/L) and kanamycin (50 mg/L). An aliquot
of the culture is then suspended in .about.15 ml of liquid
infection medium supplemented with 100 .mu.M AS in a 50 ml conical
tube (no pre-induction). The optical density is adjusted to <0.1
(OD.sub.550=0.06-0.08) before use.
[0286] For infection, rice calli are first placed into
bacteria-free infection medium+AS (50 ml conical). This pre-wash is
removed and replaced with 10 ml of the prepared Agrobacterium
suspension (OD.sub.550<0.1). Then, the conical is fastened onto
a vortex shaker (low setting) for two minutes. After infection,
calli are poured out of the conical onto a stack of sterile filter
paper in a 100.times.15 petri dish to blot dry. Then, they are
transferred off the filter paper and onto the surface of
co-cultivation medium with sterile forceps. Co-cultivation plates
are wrapped with vent tape and incubated in the dark at 25.degree.
C. for three days. After three days of co-cultivation, the calli
are washed five times with 5 ml of the liquid infection medium (no
AS) supplemented with carbenicillin (500 mg/L) and vancomycin (100
mg/L). Calli are blotted dry on sterile filter paper as before.
Individual callus pieces are transferred off the paper and onto
selection medium containing 2 mg/L bialaphos. Selection plates are
wrapped with parafilm and placed in the light at 29.degree. C.
[0287] For selection of stable transformation events, plant tissue
is cultured onto fresh selection medium every two weeks. This
should be done with the aid of a microscope to look for any
evidence of Agrobacterium overgrowth. If overgrowth is noted, the
affected calli should be avoided (contaminated calli should not be
transferred). The remaining tissue is then carefully transferred,
preferably using newly sterilized forceps for each calli. Putative
clones begin to appear after six to eight weeks on selection. A
clone is recognized as white, actively growing callus and is
distinguishable from the brown, unhealthy non-transformed tissue.
Individual transgenic events are identified and the white, actively
growing tissue is transferred to individual plates in order to
produce enough tissue to take to regeneration. Regeneration of
transgenic plants is accomplished by selecting new lobes of growth
from the callus tissue and transferring them onto Regeneration
Medium I (light, 25.degree. C.). After two to three weeks, the
maturing tissue is transferred to Regeneration Medium II for
germination (light, 25.degree. C.). When the leaves are
approximately 4-6 cm long and have developed good-sized roots, the
plantlets may be transferred (on an individual basis, typically
7-14 days after germination begins) to soilless mix using sterile
conditions.
[0288] Transgenic rice plants may be grown and evaluated through
maturity, and seeds harvested for use in generating subsequent
generations of an event. Various phenotypic characteristics may be
observed in T.sub.0 events, as well as in T.sub.1 and subsequent
generations, and used to select seed sources for the development of
subsequent generations. High performing lines may be selfed to
achieve trait homozygosity and/or crossed.
Example 27
Method for Generating Transgenic Sugarcane Plants Carrying Hordeum
GPT and GS1 Transgenes
[0289] This example provides a method for generating transgenic
sugarcane plants expressing GPT and GS1 transgenes. Sugarcane
(Saccharum spp L) is biolistically transformed with an expression
cassette comprising the hordeum glutamine synthetase (GS1) coding
sequence of SEQ ID NO: 40 under the control of the rice RuBisCo
small subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ
ID NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45
under the control of the corn ubiquitin (Ubil) promoter of SEQ ID
NO: 44. Transformation of sugarcane callus is achieved by particle
bombardment.
Vector Constructs:
[0290] An expression cassette comprising the hordeum GS1 and GPT
genes, under the control of the rice RuBisCo small subunit and corn
ubiquitin promoters, respectively, are cloned into a small plasmid
well established for sugarcane expression, such as pAHC20 (Thomson
et al., 1987, EMBO J. 6:2519-2523), using standard molecular
cloning methodologies, to generate the transgene expression vector.
The plasmid used contains a selectable marker against either the
phospinothricin family of herbicides or the antibiotics geneticin
or kanamycin, each of which have been shown effective (Ingelbrecht
et al., 1999, Plant Physiology 119:1187-1197; Gallo-Maegher &
Irvine, 1996, Crop Science 36:1367-1374).
Transformation and Regeneration:
[0291] The plasmid containing the expression cassette encoding the
hordeum GS1 and GPT coding sequences is introduced into embryogenic
callus prepared for transformation by the basic method of
Gallo-Maegher and Irvine (Gallo-Maegher and Irvine, 1996, supra)
and Ingelbrecht et al. (Ingelbrecht et al., 1999, supra) with the
improved stimulation of shoot regeneration with thidiazuron
(Gallo-Maegher et al., 2000, In vitro Cell Dev. Biol.-Plant
36:37-40). This particle bombardment method is effective in
transforming sugarcane (see, for example, Gilbert et al., 2005,
Crop Science 45:2060-2067; and see the foregoing references).
Regenerable sugarcane varieties, such as the commercial varieties
CP65-357 and CP72-1210, may be used to generate transgene
events.
[0292] Briefly, 7- to 40-week old calli are bombarded with
plasmid-coated tungsten or gold particles. Two days after
bombardment the calli are transferred to selection medium. Four
weeks later the resistant calli are transferred to shoot-induction
medium containing the selection agent and sub-cultured every two
weeks for approximately 12 weeks, at which time the shoots are
transferred to Magenta boxes containing rooting medium with
selection agent. The shoots are maintained on this medium for
approximately 8 weeks, at which time those with good root
development are transferred to potting mix and the adapted to
atmospheric growth.
[0293] Transgenic sugarcane plants may be grown and evaluated
through maturity, and seeds harvested for use in generating
subsequent generations of an event. Various phenotypic
characteristics may be observed in T.sub.0 events, as well as in
T.sub.1 and subsequent generations, and used to select seed sources
for the development of subsequent generations. High performing
lines may be selfed to achieve trait homozygosity and/or
crossed.
Example 28
Method for Generating Transgenic Wheat Plants Carrying Hordeum GPT
And GS1 Transgenes
[0294] This example provides a method for generating transgenic
wheat plants expressing
[0295] GPT and GS1 transgenes. Wheat (Triticum spp.) is
biolistically transformed with an expression cassette comprising
the hordeum glutamine synthetase (GS1) coding sequence of SEQ ID
NO: 40 under the control of the rice RuBisCo small subunit promoter
of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42), and the
hordeum GPT coding sequence of SEQ ID NO: 45 under the control of
the corn ubiquitin (Ubil) promoter of SEQ ID NO: 44. Transformation
of wheat callus is achieved by particle bombardment.
Vector Constructs:
[0296] An expression cassette comprising the hordeum GS1 and GPT
genes, under the control of the rice RuBisCo small subunit and corn
(maize) ubiquitin promoters, respectively, are cloned into a
plasmid such as pAHC17, which contains the bar gene to provide the
desired resistance to the phosphinothricin-class of herbicides for
selection of transformants, using standard molecular cloning
methodologies, to generate the transgene expression vector.
Transformation and Regeneration:
[0297] Wheat is transformed biolistically, and transgenic events
regenerated, essentially as described (Weeks et al., 1993, Plant
Physiology. 102:1077-1084; Blechl and Anderson, 1996, Nat. Biotech.
14:875-879; Okubara et. al., 2002, Theoretical and Applied
Genetics. 106:74-83). These methods were developed and are
routinely practiced at the US Department of Agriculture,
Agricultural Research Service, Western Regional Research Center
(Albany Calif.). The highly regenerable hexaploid spring wheat
cultivar `Bobwhite` is used as the source of immature embryos for
bombardment with plasmid-coated particles.
[0298] Bombarded embryos are cultured without selection for 1-3
weeks in the dark on MS media before transferring them to shoot
induction medium (MS media plus hormones and selection agent
bialophos (1, 1.5, 2, 3 mg/L) for 2-8 weeks with subculturing
weekly (Blechl et al., 2007, J Cereal Science 45:172-183). Shoots
that formed are transferred to rooting medium also containing the
selection agent (bialophos 3 mg/L) (Weeks et al., 1993, supra).
Well-rooted plantlets are transferred to potting media and adapted
to atmospheric growth conditions.
[0299] Transgenic wheat plants may be grown and evaluated through
maturity, and seeds harvested for use in generating subsequent
generations of an event. Various phenotypic characteristics may be
observed in T.sub.0 events, as well as in T.sub.1 and subsequent
generations, and used to select seed sources for the development of
subsequent generations. High performing lines may be selfed to
achieve trait homozygosity and/or crossed.
Example 29
Method for Generating Transgenic Sorghum Plants Carrying Hordeum
GPT and GS1 Transgenes
[0300] This example provides a method for generating transgenic
sorghum plants expressing GPT and GS1 transgenes. Sorghum (Sorghum
spp L) is transformed with Agrobacterium carrying an expression
cassette encoding the hordeum glutamine synthetase (GS1) coding
sequence of SEQ ID NO: 40 under the control of the rice RuBisCo
subunit promoter of SEQ ID NO: 39 (expression cassette of SEQ ID
NO: 42), and the hordeum GPT coding sequence of SEQ ID NO: 45 under
the control of the corn ubiquitin (Ubil) promoter of SE ID NO:
44.
Vector Constructs:
[0301] An expression cassette comprising the hordeum GS1 and GPT
genes, under the control of the rice RuBisCo small subunit and corn
ubiquitin promoters, respectively, is cloned into a stable binary
vector such as pZY101 (Vega et al 2008, Plant Cell Rep.
27:297-305), using standard molecular cloning methodologies, to
generate the transgene expression vector.
Transformation and Regeneration:
[0302] Agrobacterium-mediated transformation and recovery Of
transgenic sorghum plants is as described (Lu et al., 2009, Plant
Cell Tissue Organ Culture 99:97-108). These methods are routinely
used by the University of Missouri Plant Transformation Core
Facility. The public sorghum line, P898012, is grown as described
(Lu et al., 2009, supra) and transformed with Agrobacterium
tumefaciens strain EHA101 (Hood et al., 1986, supra) transformed
with the transgene expression vector.
[0303] More specifically, Agrobacterium (0.3-0.4 OD) harboring the
transgene expression vector is used to inoculate immature sorghum
embryos for 5 minutes. The embryos are then transferred onto filter
paper on top of their co-cultivation medium, containing
acetosyringone to enhance the effectiveness of the infection.
Embryos are incubated for 3-5 days and then transferred for another
4 days on resting medium (containing carbenicillin) and then
transferred onto callus induction medium (with selection agent PPT)
with weekly transfers. Once somatic embyrogenic cells develop they
are transferred onto shooting medium (with carbenicillin and PPT)
until shoots (2-5 cm long) develop. Shoots are transferred to
Magenta boxes with rooting medium (with PPT) and maintained in 16 h
light and 8 h darkness until 8-20 cm tall well-rooted plantlets are
produced. They are then transferred to potting mix and adapted to
atmospheric conditions.
[0304] Transgenic sorghum plants may be grown and evaluated through
maturity, and seeds harvested for use in generating subsequent
generations of an event. Various phenotypic characteristics may be
observed in T.sub.0 events, as well as in T.sub.1 and subsequent
generations, and used to select seed sources for the development of
subsequent generations. High performing lines may be selfed to
achieve trait homozygosity and/or crossed.
Example 30
Method for Generating Transgenic Switchgrass Plants Carrying
Hordeum GPT and GS1 Transgenes
[0305] This example provides a method for generating transgenic
switchgrass plants expressing GPT and GS1 transgenes. Switchgrass
(Panicum virgatum) is transformed with Agrobacterium carrying a
transgene expression vector including an expression cassette
encoding the hordeum glutamine synthetase (GS1) coding sequence of
SEQ ID NO: 40 under the control of the rice RuBisCo small subunit
promoter of SEQ ID NO: 39 (expression cassette of SEQ ID NO: 42),
and the hordeum GPT coding sequence of SEQ ID NO: 45 under the
control of the corn ubiquitin (Ubil) promoter of SE ID NO: 44.
Vector Constructs:
[0306] An expression cassette comprising the hordeum GS1 and GPT
genes, under the control of the rice RuBisCo small subunit and corn
(maize) ubiquitin promoters, respectively, is cloned into a Cambia
vector thirteen hundred series (i.e., 1305.1) containing the HPT
gene which provides hygromycin resistance for selection of the
Switchgrass events, using standard molecular cloning methodologies,
to generate the transgene expression vector.
Transformation and Regeneration:
[0307] Agrobacterium-mediated transformation and recovery of
transgenic switchgrass plants is essentially as described (Somleva
et al., 2002, Crop Science 42:2080-2087; Somleva 2006, Switchgrass
(Panicum virgatum L.) In Methods in Molecular Biology Vol 344.
Agrobacterium Protocols 2/e, Volume 2. Ed K. Wang Humana Press
Inc., Totowa, N.J.; Xi et al 2009, Bioengineering Research
2:275-283). These methods are routinely used by the Plant
Biotechnology Resource and Outreach Center at Michigan State
University.
[0308] Briefly, explants of embryonic callus from the mature
caryopses of the public Switchgrass cv. Alamo are transformed with
Agrobacterium tumefaciens strain EHA105 (Hood et al., 1986, supra)
carrying the transgene expression vector. Agrobacterium (0.8-1.0
OD) harboring the transgene expression vector and pretreated with
acetosynringone is used to inoculate the switchgrass callus for 10
minutes and then co-cultivated for 4-6 days in the dark. The
explants are then washed free of the agrobacterium and placed on
selection medium containing the antibiotic timentin and hygromycin;
selection requires 2-6 months. Subculturing is carried out at
4-week intervals. Regeneration is accomplished in 4-8 weeks on
media containing GA3, timentin and hygromycin under a photoperiod
of 16 h light and 8 dark. The plantlets are then transferred to
Magenta boxes with regeneration medium containing GA3, timentin and
hygromycin for another 4 weeks as before. The plants are then
transferred to soil and adapted to atmospheric growth.
[0309] Transgenic switchgrass plants may be grown and evaluated
through maturity, and seeds harvested for use in generating
subsequent generations of an event. Various phenotypic
characteristics may be observed in T.sub.0 events, as well as in
T.sub.1 and subsequent generations, and used to select seed sources
for the development of subsequent generations. High performing
lines may be selfed to achieve trait homozygosity and/or
crossed.
Example 31
Method for Generating Transgenic Soybean Plants Carrying
Arabidopsis GPT and GS1 Transgenes
[0310] This example provides a method for generating transgenic
soybean plants expressing GPT and GS1 transgenes. Soybean (Glycine
max) is transformed with Agrobacterium carrying a transgene
expression vector including an expression cassette encoding the
Arabidopsis glutamine synthetase (GS1) coding sequence of SEQ ID
NO: 7 under the control of the tomato RuBisCo small subunit
promoter of SEQ ID NO: 22 (expression cassette of SEQ ID NO: 47),
and the Arabidopsis GPT coding sequence of SEQ ID NO: 1 under the
control of the 35S cauliflower mosaic virus (CMV) promoter
(expression cassette of SEQ ID NO: 27).
Vector Constructs:
[0311] An expression cassette comprising the hordeum GS1 and GPT
genes, under the control of the tomato RuBisCo small subunit and
35S CMV promoters, respectively, is cloned into pTF101.1, using
standard molecular cloning methodologies, to generate the transgene
expression vector. pTF101.1 is a derivative of the pPZP binary
vector (Hajdukiewicz et al 1994, Plant Mol. Biol. 25:989-994),
which includes the right and left T-DNA border fragments from a
nopaline strain of A. tumefaciens, a broad host origin of
replication (pVS1) and a spectinomycin-resistant marker gene (aadA)
for bacterial selection. The plant selectable marker gene cassette
includes the phosphinothricin acetyl transferase (bar) gene from
Streptomyces hygroscopicus that confers resistance to the
herbicides glufosinate and bialophos. The soybean vegetative
storage protein terminator (Mason et al., 1993) follows the 3' end
of the bar gene.
Media:
[0312] YEP Solid Medium: 5 g/L Yeast extract, 10 g/L Peptone, 5 g/L
NaCl.sub.2, 12 g/L Bacto-agar. pH to 7.0 with NaOH. Appropriate
antibiotics should be added to the medium after autoclaving. Pour
into sterile 100.times.15 plates (.about.25 ml per plate).
[0313] YEP Liquid Medium: 5 g/L Yeast extract, 10 g/L Peptone, 5
g/L NaCl.sub.2. pH to 7.0 with NaOH. Appropriate antibiotics should
be added to the medium prior to inoculation.
[0314] Co-cultivation Medium: 1/10.times. B5 major salts,
1/10.times. B5 minor salts, 2.8 mg/L Ferrous, 3.8 mg/L NaEDTA, 30
g/L Sucrose, 3.9 g/L MES, and 4.25 g/L Noble agar (pH 5.4). Filter
sterilized 1.times. B5 vitamins, GA3 (0.25 mg/L), BAP (1.67 mg/L),
Cysteine (400 mg/L), Dithiothrietol (154.2 mg/L), and 40 mg/L
acetosyringone are added to this medium after autoclaving. Pour
into sterile 100.times.15 mm plates (.about.88 plates/L). When
solidified, overlay the co-cultivation medium with sterile filter
paper to reduce bacterial overgrowth during co-cultivation (Whatman
#1, 70 mm).
[0315] Infection Medium: 1/10.times. B5 major salts, 1/10.times. B5
minor salts, 2.8 mg/L Ferrous, 3.8 mg/L NaEDTA, 30 g/L Sucrose, 3.9
g/L MES (pH 5.4). Filter sterilized 1.times. B5 vitamins, GA3 (0.25
mg/L), BAP (1.67 mg/L), and 40 mg/L acetosyringone are added to
this medium after autoclaving.
[0316] Shoot Induction Washing Medium: 1.times. B5 major salts,
1.times. B5 minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 30 g/L
Sucrose, and 0.59 g/L MES (pH 5.7). Filter sterilized 1.times. B5
vitamins, BAP (1.11 mg/L), Timentin (100 mg/L), Cefotaxime (200
mg/L), and Vancomycin (50 mg/L) are added to this medium after
autoclaving.
[0317] Shoot Induction Medium I: 1.times. B5 major salts, 1.times.
B5 minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 30 g/L Sucrose,
0.59 g/L MES, and 7 g/L Noble agar (pH 5.7). Filter sterilized
1.times. B5 vitamins, BAP (1.11 mg/L), Timentin (50 mg/L),
Cefotaxime (200 mg/L), and Vancomycin (50 mg/L) are added to this
medium after autoclaving. Pour into sterile 100.times.20 mm plates
(26 plates/L).
[0318] Shoot Induction Medium II: 1.times. B5 major salts, 1.times.
B5 minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 30 g/L Sucrose,
0.59 g/L MES, and 7 g/L Noble agar (pH 5.7). Filter sterilized
1.times. B5 vitamins, BAP (1.11 mg/L), Timentin (50 mg/L),
Cefotaxime (200 mg/L), Vancomycin (50 mg/L) and Glufosinate (6
mg/L) are added to this medium after autoclaving. Pour into sterile
100.times.20 mm plates (26 plates/L).
[0319] Shoot Elongation Medium: 1.times.MS major salts, 1.times.MS
minor salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 30 g/L Sucrose, 0.59
g/L MES, and 7 g/L Noble agar (pH 5.7). Filter sterilized 1.times.
B5 vitamins, Asparagine (50 mg/L), L-Pyroglutamic Acid (100 mg/L),
IAA (0.1 mg/L), GA3 (0.5 mg/L), Zeatin-R (1 mg/L), Timentin (50
mg/L), Cefotaxime (200 mg/L), Vancomycin (50 mg/L), and Glufosinate
(6 mg/L) are added to this medium after autoclaving. Pour into
sterile 100.times.25 mm plates (22 plates/L).
[0320] Rooting Medium: 1.times. MS major salts, 1.times.MS minor
salts, 28 mg/L Ferrous, 38 mg/L NaEDTA, 20 g/L Sucrose, 0.59 g/L
MES, and 7 g/L Noble agar (pH 5.6). Filter sterilized 1.times. B5
vitamins, Asparagine (50 mg/L), and L-Pyroglutamic Acid (100 mg/L)
are added to this medium after autoclaving. Pour into sterile
150.times.25 mm vial (10 ml/vial).
Transformation and Regeneration:
[0321] Agrobacterium cultures are prepared for infecting seed
explants as follows. The vector system, pTF102 in EHA101, is
cultured on YEP medium (An et al., 1988) containing 100 mg/L
spectinomycin (for pTF102), 50 mg/L kanamycin (for EHA101), and 25
mg/L chloramphenicol (for EHA101). 24 hours prior to infection a 2
ml culture of Agrobacterium is started by inoculating a loop of
bacteria from the fresh YEP plate in YEP liquid medium amended with
antibiotics. This culture is allowed to grow to saturation (8-10
hours) at 28.degree. C. in a shaker incubator (-250 rpm). Then 0.2
ml of starter culture is transferred to a 1 L flask containing 250
ml of YEP medium amended with antibiotics. The culture is allowed
to grow overnight at 28.degree. C., 250 rpm to log phase
(OD650=0.3-0.6 for EHA105) or late log phase (OD.sub.650=1.0-1.2
for EHA101). The Agrobacterium culture is then pelleted at 3,500
rpm for 10 minutes at 20.degree. C., and the pellet resuspended in
infection medium by pipetting through the pellet. Bacterial cell
densities are adjusted to a final OD650=0.6 (for EHA105) or
OD650=0.6 to 1.0 (for EHA101). Agrobacteria-containing infection
medium is shaken at 60 rpm for at least 30 minutes before use.
[0322] Explants are prepared for inoculation as follows. Seeds are
sterilized, ideally with a combination of bleach solution and
exposure to chlorine gas. Prior to infection, (-20 hours), sees are
imbibed with deionized sterile water in the dark. Imbibed soybean
seeds are transferred to a sterile 100.times.15 petri plate for
dissection. Using a scalpel (i.e., #15 blade), longitudinal cuts
are made along the hilum to separate the cotyledons and remove the
seed coat. The embryonic axis found at the nodal end of the
cotyledons is excised, and any remaining axial shoots/buds attached
to the cotyledonary node are also removed.
[0323] Agrobacterium-mediated transformation is conducted as
follows. Half-seed explants are dissected into a 100.times.25 mm
petri plate and 30 ml Agrobacterium-containing infection media
added thereto, such that the explants are completely covered by the
infection media. Explants are allowed to incubate at room
temperature for a short period of time (i.e., 30 minutes),
preferably with occasional gentle agitation.
[0324] After infection, the explants are transferred to
co-cultivation medium, preferably so that the flat, axial side is
touching the filter paper. These plates are typically wrapped in
parafilm, and cultivated for 5 days at 24.degree. C. under an 18:6
photoperiod. Following this co-cultivation, shoot growth is induced
by first washing the explants in shoot induction washing medium at
room temperature, followed by placing the explants in shoot
induction medium I, such that the explants are oriented with the
nodal end of the cotyledon imbedded in the medium and the
regeneration region flush to the surface with flat side up
(preferably at a 30-45.degree. angle). Explants are incubated at
24.degree. C., 18:6 photoperiod, for 14 days. Explants are
thereafter transferred to shoot induction medium II and maintained
under the same conditions for another 14 days.
[0325] Following shoot induction, explants are transferred to shoot
elongation medium, as follows. First, cotyledons are removed from
the explants. A fresh cut at the base of the shoot pad flush to the
medium is made, and the explants transferred to shoot elongation
medium (containing glufosinate) and incubated at 24.degree. C.,
18:6 photoperiod, for 2-8 weeks. Preferably, explant tissue is
transferred to fresh shoot elongation medium every 2 weeks, and at
transfer, a fresh horizontal slice at the base of the shoot pad is
made.
[0326] When shoots surviving the glufosinate selection have reached
.about.3 cm length, they are excised from the shoot pad, briefly
dipped in indole-3-butyric acid (1 mg/ml, 1-2 minutes), then
transferred to rooting medium for acclimatization (i.e., in
150.times.25 mm glass vials with the stems of the shoots embedded
approximately 1/2 cm into the media). When well rooted, the shoots
are transferred to soil and plantlets grown at 24.degree. C., 18:6
photoperiod, for at least one week, watering as needed. When the
plantlets have at least two healthy trifoliates, an herbicide paint
assay may be applied to confirm resistance to glufosinate. Briefly,
using a cotton swab, Liberty herbicide (150 mg l-1) is applied to
the upper leaf surface along the midrib of two leaves on two
different trifoliates. Painted plants are transferred to the
greenhouse and covered with a humidome. Plantlets are scored 3-5
days after painting. Resistant plantlets may be transplanted
immediately to larger pots (i.e., 2 gal).
Example 32
Method for Generating Transgenic Potato Plants Carrying Arabidopsis
GPT and GS1 Transgenes
[0327] This example provides a method for generating transgenic
potato plants expressing GPT and GS1 transgenes. Potato (Solanum
tuberosum, cultivar Desiree) is transformed with Agrobacterium
carrying a transgene expression vector including an expression
cassette encoding the Arabidopsis glutamine synthetase (GS1) coding
sequence of SEQ ID NO: 7 under the control of the tomato RuBisCo
small subunit promoter of SEQ ID NO: 22 (expression cassette of SEQ
ID NO: 47), and the Arabidopsis GPT coding sequence of SEQ ID NO: 1
under the control of the 35S cauliflower mosaic virus (CMV)
promoter (expression cassette of SEQ ID NO: 27).
Vector Constructs:
[0328] An expression cassette comprising the hordeum GS1 and GPT
genes, under the control of the tomato RuBisCo small subunit and
35S CMV promoters, respectively, is cloned into the Cambia 2201
vector which provides kanamycin resistance.
Transformation and Regeneration:
[0329] A suitable Agrobacterium tumefaciens strain such as
UC-Riverside Agro-1 strain is employed and used for infecting
potato explant tissue (see, Narvaez-Vasquez et al., 1992, Plant Mo.
Biol. 20:1149-1157). Cultures are maintained at 28.degree. C. in
liquid medium containing 10 g/L Yeast extract, 10 g/L Peptone, 5
g/L NaCl.sub.2, 10 mg/L kanamycin, 30 mg/L tetracycline, and 9.81
g/L Acetosyringone (50 mM). Overnight cultures are diluted with
liquid MS medium (4.3 g/L MS salts, 20 g/L sucrose, 1 mg/L
thiamine, 100 mg/L inositol and 7 g/L phytoagar, pH to 5.8.) to
10.sup.8 Agrobacterium cells/ml for the infection of plant tissues
(co-cultivation).
[0330] Potato leaf discs or tuber discs may be used as the explants
to be inoculated. Discs are pre-conditioned by incubation on feeder
plates for two to three days at 25.degree. C. under dark
conditions. Pre-conditioned explants are infected with
Agrobacterium by soaking in 20 ml of sterile liquid MS medium
(supra), containing 10.sup.8 Agrobacterium cells/ml for about 20
minutes. Before or during the co-cultivation, the explants are
carefully punched with a syringe needle, or scalpel blade. Then,
the explants are blotted dry with sterile filter paper, and
incubated again in feeder plates for another two days. Explants are
then transferred to liquid medium with transgene-transformed
Agrobacterium, and incubated for three days at 28.degree. C. under
dark conditions for calli and shoot development (development (2-4
cm) in the presence of kanamycin (100 mg/L).
[0331] Following co-cultivation, supra, the explants are washed
three times with sterile liquid medium and finally rinsed with the
same medium containing 500 mg/l of cefotaxime. The explants are
blotted dry with sterile filter paper and placed on shoot induction
medium (4.3 g/L MS salts, 10 mg/L thiamine, 1 mg/L nicotinic acid,
1 mg/L pyridxine, 100 mg/L inositol, 30 g/L sucrose, 1 mg/L zeatin,
0.5 mg/L IAA, 7 g/L phytoagar, 250 mg/L Cefotaxime, 500 mg/L
Carbenicillin, 100 mg/L Kanamycin) for 4-6 weeks. Thereafter,
plantlets are transferred to rooting medium (4.3 g/L MS salts, 10
mg/L thiamine, 1 mg/L nicotinic acid, 1 mg/L pyridxine, 100 mg/L
inositol, 20 g/L sucrose, 50 .mu.g/L IAA, 7 g/L phytoagar, 50 mg/L
Kanamycin and 500 mg/L Vancomycin) for 3-4 weeks.
[0332] All publications, patents, and patent applications cited in
this specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0333] The present invention is not to be limited in scope by the
embodiments disclosed herein, which are intended as single
illustrations of individual aspects of the invention, and any which
are functionally equivalent are within the scope of the invention.
Various modifications to the models and methods of the invention,
in addition to those described herein, will become apparent to
those skilled in the art from the foregoing description and
teachings, and are similarly intended to fall within the scope of
the invention. Such modifications or other embodiments can be
practiced without departing from the true scope and spirit of the
invention.
TABLE-US-00020 SEQ ID NO: 1 Arabidopsis glutamine phenylpyruvate
transaminase DNA coding sequence:
ATGTACCTGGACATAAATGGTGTGATGATCAAACAGTTTAGCTTCAAAGCCTCTC
TTCTCCCATTCTCTTCTAATTTCCGACAAAGCTCCGCCAAAATCCATCGTCCTAT
CGGAGCCACCATGACCACAGTTTCGACTCAGAACGAGTCTACTCAAAAACCCGT
CCAGGTGGCGAAGAGATTAGAGAAGTTCAAGACTACTATTTTCACTCAAATGAG
CATATTGGCAGTTAAACATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTC
GACGGTCCTGATTTTGTTAAAGAAGCTGCGATCCAAGCTATTAAAGATGGTAAAA
ACCAGTATGCTCGTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGCGC
GGTTTCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGAAAGAAGTTACTGTTAC
ATCTGGTTGCACAGAAGCCATAGCTGCAGCTATGTTGGGTTTAATAAACCCTGG
TGATGAAGTCATTCTCTTTGCACCGTTTTATGATTCCTATGAAGCAACACTCTCTA
TGGCTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCACCGGACTTCTCCATCC
CTTTGGAAGAGCTTAAAGCTGCGGTAACTAACAAGACTCGAGCCATCCTTATGA
ACACTCCGCACAACCCGACCGGGAAGATGTTCACTAGGGAGGAGCTTGAAACC
ATTGCATCTCTCTGCATTGAAAACGATGTGCTTGTGTTCTCGGATGAAGTATACG
ATAAGCTTGCGTTTGAAATGGATCACATTTCTATAGCTTCTCTTCCCGGTATGTA
TGAAAGAACTGTGACCATGAATTCCCTGGGAAAGACTTTCTCTTTAACCGGATG
GAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGAGTTCGACAAG
CACACTCTTACCTCACATTCGCCACATCAACACCAGCACAATGGGCAGCCGTTG
CAGCTCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGAAAAGAGATTACAATG
TGAAAAAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCGGATTTACAGTGTTCC
CATCGAGCGGGACTTACTTTGTGGTTGCTGATCACACTCCATTTGGAATGGAGA
ACGATGTTGCTTTCTGTGAGTATCTTATTGAAGAAGTTGGGGTCGTTGCGATCC
CAACGAGCGTCTTTTATCTGAATCCAGAAGAAGGGAAGAATTTGGTTAGGTTTG
CGTTCTGTAAAGACGAAGAGACGTTGCGTGGTGCAATTGAGAGGATGAAGCAG
AAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 2 Arabidopsis GPT amino acid
sequence MYLDINGVMIKQFSFKASLLPFSSNFRQSSAKIHRPIGATMTTVSTQNESTQKPVQV
AKRLEKFKTTIFTQMSILAVKHGAINLGQGFPNFDGPDFVKEAAIQAIKDGKNQYARG
YGIPQLNSAIAARFREDTGLVVDPEKEVTVTSGCTEAIAAAMLGLINPGDEVILFAPFY
DSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVTNKTRAILMNTPHNPTGKMFTRE
ELETIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYERTVTMNSLGKTFSLTG
WKIGWAIAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKAPESYFKELKRDYNVK
KETLVKGLKEVGFTVFPSSGTYFVVADHTPFGMENDVAFCEYLIEEVGVVAIPTSVF
YLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLKRKV SEQ ID NO: 3 Alfalfa GS1 DNA
coding sequence (upper case) with 5' and 3' untranslated sequences
(indicated in lower case).
atttccgttttcgttttcatttgattcattgaatcaaatcgaatcgaatctttaggattcaatacag
attccttagattttactaagtttgaaaccaaaaccaaaacATGTCTCTCCTTTCAGAT
CTTATCAACCTTGACCTCTCCGAAACCACCGAGAAAATCATCGCCG
AATACATATGGATTGGTGGATCTGGTTTGGACTTGAGGAGCAAAGC
AAGGACTCTACCAGGACCAGTTACTGACCCTTCACAGCTTCCCAAG
TGGAACTATGATGGTTCCAGCACAGGTCAAGCTCCTGGAGAAGAT
AGTGAAGTTATTATCTACCCACAAGCCATTTTCAAGGACCCATTTA
GAAGGGGTAACAATATCTTGGTTATGTGTGATGCATACACTCCAGC
TGGAGAGCCCATTCCCACCAACAAGAGACATGCAGCTGCCAAGAT
TTTCAGCCATCCTGATGTTGTTGCTGAAGTACCATGGTATGGTATT
GAGCAAGAATACACCTTGTTGCAGAAAGACATCAATTGGCCTCTTG
GTTGGCCAGTTGGTGGTTTTCCTGGACCTCAGGGACCATACTATTG
TGGAGCTGGTGCTGACAAGGCATTTGGCCGTGACATTGTTGACTC
ACATTACAAAGCCTGTCTTTATGCCGGCATCAACATCAGTGGAATC
AATGGTGAAGTGATGCCTGGTCAATGGGAATTCCAAGTTGGTCCCT
CAGTTGGTATCTCTGCTGGTGATGAGATATGGGTTGCTCGTTACAT
TTTGGAGAGGATCACTGAGGTTGCTGGTGTGGTGCTTTCCTTTGAC
CCAAAACCAATTAAGGGTGATTGGAATGGTGCTGGTGCTCACACAA
ATTACAGCACCAAGTCTATGAGAGAAGATGGTGGCTATGAAGTCAT
CTTGAAAGCAATTGAGAAGCTTGGGAAGAAGCACAAGGAGCACAT
TGCTGCTTATGGAGAAGGCAACGAGCGTAGATTGACAGGGCGACA
TGAGACAGCTGACATTAACACCTTCTTATGGGGTGTTGCAAACCGT
GGTGCGTCGATTAGAGTTGGAAGGGACACAGAGAAAGCAGGGAAA
GGTTATTTCGAGGATAGGAGGCCATCATCTAACATGGATCCATATG
TTGTTACTTCCATGATTGCAGACACCACCATTCTCTGGAAACCATA
Agccaccacacacacatgcattgaagtatttgaaagtcattgttgattccgcattagaatttgg
tcattgttttttctaggatttggatttgtgttattgttatggttcacactttgtttgtttgaatttgaggc
cttgttataggtttcatatttctttctcttgttctaagtaaatgtcagaataataatgtaat SEQ
ID NO: 4 Alfalfa GS1 amino acid sequence
MSLLSDLINLDLSETTEKIIAEYIWIGGSGLDLRSKARTLPGPVTDPSQLPKWNYDGS
STGQAPGEDSEVIIYPQAIFKDPFRRGNNILVMCDAYTPAGEPIPTNKRHAAAKIFSH
PDVVAEVPWYGIEQEYTLLQKDINWPLGWPVGGFPGPQGPYYCGAGADKAFGRDI
VDSHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISAGDEIWVARYILERITEVA
GVVLSFDPKPIKGDWNGAGAHTNYSTKSMREDGGYEVILKAIEKLGKKHKEHIAAYG
EGNERRLTGRHETADINTFLWGVANRGASIRVGRDTEKAGKGYFEDRRPSSNMDP
YVVTSMIADTTILWKP SEQ ID NO: 5 Alfalfa GS1 DNA coding sequence
(upper case) with 5' and 3' untranslated sequences (indicated in
lower case) and vector sequences from ClaI to SmaI/SspI and
SspI/SmaI to SalI/XhoI (lower case, underlined).
atcgatgaattcgagctcggtacccatttccgttttcgttttcatttgattcattgaatcaaatcga
atcgaatctttaggattcaatacagattccttagattttactaagtttgaaaccaaaaccaaaa
cATGTCTCTCCTTTCAGATCTTATCAACCTTGACCTCTCCGAAACCA
CCGAGAAAATCATCGCCGAATACATATGGATTGGTGGATCTGGTTT
GGACTTGAGGAGCAAAGCAAGGACTCTACCAGGACCAGTTACTGA
CCCTTCACAGCTTCCCAAGTGGAACTATGATGGTTCCAGCACAGGT
CAAGCTCCTGGAGAAGATAGTGAAGTTATTATCTACCCACAAGCCA
TTTTCAAGGACCCATTTAGAAGGGGTAACAATATCTTGGTTATGTG
TGATGCATACACTCCAGCTGGAGAGCCCATTCCCACCAACAAGAG
ACATGCAGCTGCCAAGATTTTCAGCCATCCTGATGTTGTTGCTGAA
GTACCATGGTATGGTATTGAGCAAGAATACACCTTGTTGCAGAAAG
ACATCAATTGGCCTCTTGGTTGGCCAGTTGGTGGTTTTCCTGGACC
TCAGGGACCATACTATTGTGGAGCTGGTGCTGACAAGGCATTTGG
CCGTGACATTGTTGACTCACATTACAAAGCCTGTCTTTATGCCGGC
ATCAACATCAGTGGAATCAATGGTGAAGTGATGCCTGGTCAATGGG
AATTCCAAGTTGGTCCCTCAGTTGGTATCTCTGCTGGTGATGAGAT
ATGGGTTGCTCGTTACATTTTGGAGAGGATCACTGAGGTTGCTGGT
GTGGTGCTTTCCTTTGACCCAAAACCAATTAAGGGTGATTGGAATG
GTGCTGGTGCTCACACAAATTACAGCACCAAGTCTATGAGAGAAGA
TGGTGGCTATGAAGTCATCTTGAAAGCAATTGAGAAGCTTGGGAAG
AAGCACAAGGAGCACATTGCTGCTTATGGAGAAGGCAACGAGCGT
AGATTGACAGGGCGACATGAGACAGCTGACATTAACACCTTCTTAT
GGGGTGTTGCAAACCGTGGTGCGTCGATTAGAGTTGGAAGGGACA
CAGAGAAAGCAGGGAAAGGTTATTTCGAGGATAGGAGGCCATCAT
CTAACATGGATCCATATGTTGTTACTTCCATGATTGCAGACACCAC
CATTCTCTGGAAACCATAAgccaccacacacacatgcattgaagtatttgaaagtc
attgttgattccgcattagaatttggtcattgttttttctaggatttggatttgtgttattgttatggttc
acactttgtttgtttgaatttgaggccttgttataggtttcatatttctttctcttgttctaagtaaatg
tcagaataataatgtaatggggatcctctagagtcgag SEQ ID NO: 6 Arabidopsis GS1
coding sequence Cambia 1201 vector + rbcS3C + arabidopsis GS1Bold
ATG is the start site,
AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG
ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC
AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG
TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA
ACCAATTATTTCAGCA TCTCTGCTCTCAGATCTCGTTAACCTCAACCTCA
CCGATGCCACCGGGAAAATCATCGCCGAATACATATGGATCGGTGGATCTGGA
ATGGATATCAGAAGCAAAGCCAGGACACTACCAGGACCAGTGACTGATCCATCA
AAGCTTCCCAAGTGGAACTACGACGGATCCAGCACCGGTCAGGCTGCTGGAGA
AGACAGTGAAGTCATTCTATACCCTCAGGCAATATTCAAGGATCCCTTCAGGAAA
GGCAACAACATCCTGGTGATGTGTGATGCTTACACACCAGCTGGTGATCCTATT
CCAACCAACAAGAGGCACAACGCTGCTAAGATCTTCAGCCACCCCGACGTTGC
CAAGGAGGAGCCTTGGTATGGGATTGAGCAAGAATACACTTTGATGCAAAAGGA
TGTGAACTGGCCAATTGGTTGGCCTGTTGGTGGCTACCCTGGCCCTCAGGGAC
CTTACTACTGTGGTGTGGGAGCTGACAAAGCCATTGGTCGTGACATTGTGGATG
CTCACTACAAGGCCTGTCTTTACGCCGGTATTGGTATTTCTGGTATCAATGGAGA
AGTCATGCCAGGCCAGTGGGAGTTCCAAGTCGGCCCTGTTGAGGGTATTAGTT
CTGGTGATCAAGTCTGGGTTGCTGGATACCTTCTCGAGAGGATCACTGAGATCT
CTGGTGTAATTGTCAGCTTCGACCCGAAACCAGTCCCGGGTGACTGGAATGGA
GCTGGAGCTCACTGCAACTACAGCACTAAGACAATGAGAAACGATGGAGGATTA
GAAGTGATCAAGAAAGCGATAGGGAAGCTTCAGCTGAAACACAAAGAACACATT
GCTGCTTACGGTGAAGGAAACGAGCGTCGTCTCACTGGAAAGCACGAAACCGC
AGACATCAACACATTCTCTTGGGGAGTCGCGAACCGTGGAGCGTCAGTGAGAG
TGGGACGTGACACAGAGAAGGAAGGTAAAGGGTACTTCGAAGACAGAAGGCCA
GCTTCTAACATGGATCCTTACGTTGTCACCTCCATGATCGCTGAGACGACCATA CTCGGTTGA
SEQ ID NO: 7 Arabidopsis GS1 amino acid sequence Vector sequences
at N-terminus in italics
MVDLRNRRTSMSLLSDLVNLNLTDATGKIIAEYIWIGGSGMDIRSKARTLPGPVTDPS
KLPKWNYDGSSTGQAAGEDSEVILYPQAIFKDPFRKGNNILVMCDAYTPAGDPIPTN
KRHNAAKIFSHPDVAKEEPWYGIEQEYTLMQKDVNWPIGWPVGGYPGPQGPYYC
GVGADKAIGRDIVDAHYKACLYAGIGISGINGEVMPGQWEFQVGPVEGISSGDQVW
VARYLLERITEISGVIVSFDPKPVPGDWNGAGAHCNYSTKTMRNDGGLEVIKKAIGK
LQLKHKEHIAAYGEGNERRLTGKHETADINTFSWGVANRGASVRVGRDTEKEGKG
YFEDRRPASNMDPYVVTSMIAETTILG SEQ ID NO: 8 Grape GPT coding DNA
sequence Showing Cambia 1305.1 with (3' end of) rbcS3C + Vitis
vinifera GPT (Grape). Bold ATG is the start site, parentheses are
the catI intron and the underlined actagt is the speI cloning site
used to splice in the GPT gene.
AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG
ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC
AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG
TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA
ACCAATTATTTCAGCA TAGATCTGAGG(GTAAATTTCTAGTTTTTCTCCT
TCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCT
TTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAA
CTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAG)A ACCGACGA
ATGCAGCTCTCTCAATGTACCTGGACATTCCCAGAGTTGC
TTAAAAGACCAGCCTTTTTAAGGAGGAGTATTGATAGTATTTCGAGTAGAAGTAG
GTCCAGCTCCAAGTATCCATCTTTCATGGCGTCCGCATCAACGGTCTCCGCTCC
AAATACGGAGGCTGAGCAGACCCATAACCCCCCTCAACCTCTACAGGTTGCAAA
GCGCTTGGAGAAATTCAAAACAACAATCTTTACTCAAATGAGCATGCTTGCCATC
AAACATGGAGCAATAAACCTTGGCCAAGGGTTTCCCAACTTTGATGGTCCTGAG
TTTGTCAAAGAAGCAGCAATTCAAGCCATTAAGGATGGGAAAAACCAATATGCTC
GTGGATATGGAGTTCCTGATCTCAACTCTGCTGTTGCTGATAGATTCAAGAAGG
ATACAGGACTCGTGGTGGACCCCGAGAAGGAAGTTACTGTTACTTCTGGATGTA
CAGAAGCAATTGCTGCTACTATGCTAGGCTTGATAAATCCTGGTGATGAGGTGA
TCCTCTTTGCTCCATTTTATGATTCCTATGAAGCCACTCTATCCATGGCTGGTGC
CCAAATAAAATCCATCACTTTACGTCCTCCGGATTTTGCTGTGCCCATGGATGAG
CTCAAGTCTGCAATCTCAAAGAATACCCGTGCAATCCTTATAAACACTCCCCATA
ACCCCACAGGAAAGATGTTCACAAGGGAGGAACTGAATGTGATTGCATCCCTCT
GCATTGAGAATGATGTGTTGGTGTTTACTGATGAAGTTTACGACAAGTTGGCTTT
CGAAATGGATCACATTTCCATGGCTTCTCTTCCTGGGATGTACGAGAGGACCGT
GACTATGAATTCCTTAGGGAAAACTTTCTCCCTGACTGGATGGAAGATTGGTTG
GACAGTAGCTCCCCCACACCTGACATGGGGAGTGAGGCAAGCCCACTCATTCC
TCACGTTTGCTACCTGCACCCCAATGCAATGGGCAGCTGCAACAGCCCTCCGG
GCCCCAGACTCTTACTATGAAGAGCTAAAGAGAGATTACAGTGCAAAGAAGGCA
ATCCTGGTGGAGGGATTGAAGGCTGTCGGTTTCAGGGTATACCCATCAAGTGG
GACCTATTTTGTGGTGGTGGATCACACCCCATTTGGGTTGAAAGACGATATTGC
GTTTTGTGAGTATCTGATCAAGGAAGTTGGGGTGGTAGCAATTCCGACAAGCGT
TTTCTACTTACACCCAGAAGATGGAAAGAACCTTGTGAGGTTTACCTTCTGTAAA
GACGAGGGAACTCTGAGAGCTGCAGTTGAAAGGATGAAGGAGAAACTGAAGCC
TAAACAATAGGGGCACGTGA SEQ ID NO: 9 Grape GPT amino acid sequence
MVDLRNRRTSMQLSQCTWTFPELLKRPAFLRRSIDSISSRSRSSSKYPSFMASAST
VSAPNTEAEQTHNPPQPLQVAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGP
EFVKEAAIQAIKDGKNQYARGYGVPDLNSAVADRFKKDTGLVVDPEKEVTVTSGCT
EAIAATMLGLINPGDEVILFAPFYDSYEATLSMAGAQIKSITLRPPDFAVPMDELKSAI
SKNTRAILINTPHNPTGKMFTREELNVIASLCIENDVLVFTDEVYDKLAFEMDHISMAS
LPGMYERTVTMNSLGKTFSLTGWKIGWTVAPPHLTWGVRQAHSFLTFATCTPMQW
AAATALRAPDSYYEELKRDYSAKKAILVEGLKAVGFRVYPSSGTYFVVVDHTPFGLK
DDIAFCEYLIKEVGVVAIPTSVFYLHPEDGKNLVRFTFCKDEGTLRAAVERMKEKLKP KQ SEQ
ID NO: 10 Rice GPT DNA coding sequence Rice GPT codon optimized for
E. coli expression; untranslated sequences shown in lower case
atgtggATGAACCTGGCAGGCTTTCTGGCAACCCCGGCAACCGCAACCGCAACCC
GTCATGAAATGCCGCTGAACCCGAGCAGCAGCGCGAGCTTTCTGCTGAGCAGC
CTGCGTCGTAGCCTGGTGGCGAGCCTGCGTAAAGCGAGCCCGGCAGCAGCAG
CAGCACTGAGCCCGATGGCAAGCGCAAGCACCGTGGCAGCAGAAAACGGTGC
AGCAAAAGCAGCAGCAGAAAAACAGCAGCAGCAGCCGGTGCAGGTGGCGAAA
CGTCTGGAAAAATTTAAAACCACCATTTTTACCCAGATGAGCATGCTGGCGATTA
AACATGGCGCGATTAACCTGGGCCAGGGCTTTCC
GAACTTTGATGGCCCGGATTTTGTGAAAGAAGCGGCGATTCAGGCGATTAACGC
GGGCAAAAACCAGTATGCGCGTGGCTATGGCGTGCCGGAACTGAACAGCGCGA
TTGCGGAACGTTTTCTGAAAGATAGCGGCCTGCAGGTGGATCCGGAAAAAGAA
GTGACCGTGACCAGCGGCTGCACCGAAGCGATTGCGGCGACCATTCTGGGCCT
GATTAACCCGGGCGATGAAGTGATTCTGTTTGCGCCGTTTTATGATAGCTATGA
AGCGACCCTGAGCATGGCGGGCGCGAACGTGAAAGCGATTACCCTGCGTCCG
CCGGATTTTAGCGTGCCGCTGGAAGAACTGAAAGCGGCCGTGAGCAAAAACAC
CCGTGCGATTATGATTAACACCCCGCATAACCCGACCGGCAAAATGTTTACCCG
TGAAGAACTGGAATTTATTGCGACCCTGTGCAAAGAAAACGATGTGCTGCTGTT
TGCGGATGAAGTGTATGATAAACTGGCGTTTGAAGCGGATCATATTAGCATGGC
GAGCATTCCGGGCATGTATGAACGTACCGTGACCATGAACAGCCTGGGCAAAA
CCTTTAGCCTGACCGGCTGGAAAATTGGCTGGGCGATTGCGCCGCCGCATCTG
ACCTGGGGCGTGCGTCAGGCACATAGCTTTCTGACCTTTGCAACCTGCACCCC
GATGCAGGCAGCCGCCGCAGCAGCACTGCGTGCACCGGATAGCTATTATGAAG
AACTGCGTCGTGATTATGGCGCGAAAAAAGCGCTGCTGGTGAACGGCCTGAAA
GATGCGGGCTTTATTGTGTATCCGAGCAGCGGCACCTATTTTGTGATGGTGGAT
CATACCCCGTTTGGCTTTGATAACGATATTGAATTTTGCGAATATCTGATTCGTG
AAGTGGGCGTGGTGGCGATTCCGCCGAGCGTGTTTTATCTGAACCCGGAAGAT
GGCAAAAACCTGGTGCGTTTTACCTTTTGCAAAGATGATGAAACCCTGCGTGCG
GCGGTGGAACGTATGAAAACCAAACTGCGTAAAAAAAAGCTTgcggccgcactcgagc
accaccaccaccaccactga SEQ ID NO: 11 Rice GPT amino acid sequence
Includes amino terminal amino acids MW for cloning and His tag
sequences from pet28 vector in italics.
MWMNLAGFLATPATATATRHEMPLNPSSSASFLLSSLRRSLVASLRKASPAAAAAL
SPMASASTVAAENGAAKAAAEKQQQQPVQVAKRLEKFKTTIFTQMSMLAIKHGAINL
GQGFPNFDGPDFVKEAAIQAINAGKNQYARGYGVPELNSAIAERFLKDSGLQVDPE
KEVTVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAGANVKAITLRPPDFS
VPLEELKAAVSKNTRAIMINTPHNPTGKMFTREELEFIATLCKENDVLLFADEVYDKL
AFEADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSFL
TFATCTPMQAAAAAALRAPDSYYEELRRDYGAKKALLVNGLKDAGFIVYPSSGTYF
VMVDHTPFGFDNDIEFCEYLIREVGVVAIPPSVFYLNPEDGKNLVRFTFCKDDETLR
AAVERMKTKLRKKKLAAALEHHHHHH SEQ ID NO: 12 Soybean GPT DNA coding
sequence TOPO 151D WITH SOYBEAN for E. coli expression From
starting codon. Vector sequences are italicized
ATGCATCATCACCATCACCATGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTC
GATTCTACGGAAAACCTGTATTTTCAGGGAATTGATCCCTTCACCGCGAAACGT
CTGGAAAAATTTCAGACCACCATTTTTACCCAGATGAGCCTGCTGGCGATTAAAC
ATGGCGCGATTAACCTGGGCCAGGGCTTTCCGAACTTTGATGGCCCGGAATTT
GTGAAAGAAGCGGCGATTCAGGCGATTCGTGATGGCAAAAACCAGTATGCGCG
TGGCTATGGCGTGCCGGATCTGAACATTGCGATTGCGGAACGTTTTAAAAAAGA
TACCGGCCTGGTGGTGGATCCGGAAAAAGAAATTACCGTGACCAGCGGCTGCA
CCGAAGCGATTGCGGCGACCATGATTGGCCTGATTAACCCGGGCGATGAAGTG
ATTATGTTTGCGCCGTTTTATGATAGCTATGAAGCGACCCTGAGCATGGCGGGC
GCGAAAGTGAAAGGCATTACCCTGCGTCCGCCGGATTTTGCGGTGCCGCTGGA
AGAACTGAAAAGCACCATTAGCAAAAACACCCGTGCGATTCTGATTAACACCCC
GCATAACCCGACCGGCAAAATGTTTACCCGTGAAGAACTGAACTGCATTGCGAG
CCTGTGCATTGAAAACGATGTGCTGGTGTTTACCGATGAAGTGTATGATAAACT
GGCGTTTGATATGGAACATATTAGCATGGCGAGCCTGCCGGGCATGTTTGAACG
TACCGTGACCCTGAACAGCCTGGGCAAAACCTTTAGCCTGACCGGCTGGAAAAT
TGGCTGGGCGATTGCGCCGCCGCATCTGAGCTGGGGCGTGCGTCAGGCGCAT
GCGTTTCTGACCTTTGCAACCGCACATCCGTTTCAGTGCGCAGCAGCAGCAGCA
CTGCGTGCACCGGATAGCTATTATGTGGAACTGAAACGTGATTATATGGCGAAA
CGTGCGATTCTGATTGAAGGCCTGAAAGCGGTGGGCTTTAAAGTGTTTCCGAGC
AGCGGCACCTATTTTGTGGTGGTGGATCATACCCCGTTTGGCCTGGAAAACGAT
GTGGCGTTTTGCGAATATCTGGTGAAAGAAGTGGGCGTGGTGGCGATTCCGAC
CAGCGTGTTTTATCTGAACCCGGAAGAAGGCAAAAACCTGGTGCGTTTTACCTT
TTGCAAAGATGAAGAAACCATTCGTAGCGCGGTGGAACGTATGAAAGCGAAACT
GCGTAAAGTCGACTAA SEQ ID NO: 13 Soybean GPT amino acid sequence
Translated protein product, vector sequences italicized
MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTAKRLEKFQTTIFTQMSLLAIKHGAI
NLGQGFPNFDGPEFVKEAAIQAIRDGKNQYARGYGVPDLNIAIAERFKKDTGLVVDP
EKEITVTSGCTEAIAATMIGLINPGDEVIMFAPFYDSYEATLSMAGAKVKGITLRPPDF
AVPLEELKSTISKNTRAILINTPHNPTGKMFTREELNCIASLCIENDVLVFTDEVYDKL
AFDMEHISMASLPGMFERTVTLNSLGKTFSLTGWKIGWAIAPPHLSWGVRQAHAFL
TFATAHPFQCAAAAALRAPDSYYVELKRDYMAKRAILIEGLKAVGFKVFPSSGTYFV
VVDHTPFGLENDVAFCEYLVKEVGVVAIPTSVFYLNPEEGKNLVRFTFCKDEETIRS
AVERMKAKLRKVD SEQ ID NO: 14 Barley GPT DNA coding sequence Coding
sequence from start with intron removed TAGATCTGAGGAACCGACGA
ATGGCATCCGCCCCCGCCTCCGC
CTCCGCGGCCCTCTCCACCGCCGCCCCCGCCGACAACGGGGCCGCCAAGCCC
ACGGAGCAGCGGCCGGTACAGGTGGCTAAGCGATTGGAGAAGTTCAAAACAAC
AATTTTCACACAGATGAGCATGCTCGCAGTGAAGCATGGAGCAATAAACCTTGG
ACAGGGGTTTCCCAATTTTGATGGCCCTGACTTTGTCAAAGATGCTGCTATTGA
GGCTATCAAAGCTGGAAAGAATCAGTATGCAAGAGGATATGGTGTGCCTGAATT
GAACTCAGCTGTTGCTGAGAGATTTCTCAAGGACAGTGGATTGCACATCGATCC
TGATAAGGAAGTTACTGTTACATCTGGGTGCACAGAAGCAATAGCTGCAACGAT
ATTGGGTCTGATCAACCCTGGGGATGAAGTCATACTGTTTGCTCCATTCTATGAT
TCTTATGAGGCTACACTGTCCATGGCTGGTGCGAATGTCAAAGCCATTACACTC
CGCCCTCCGGACTTTGCAGTCCCTCTTGAAGAGCTAAAGGCTGCAGTCTCGAA
GAATACCAGAGCAATAATGATTAATACACCTCACAACCCTACCGGGAAAATGTTC
ACAAGGGAGGAACTTGAGTTCATTGCTGATCTCTGCAAGGAAAATGACGTGTTG
CTCTTTGCCGATGAGGTCTACGACAAGCTGGCGTTTGAGGCGGATCACATATCA
ATGGCTTCTATTCCTGGCATGTATGAGAGGACCGTCACTATGAACTCCCTGGGG
AAGACGTTCTCCTTGACCGGATGGAAGATCGGCTGGGCGATAGCACCACCGCA
CCTGACATGGGGCGTAAGGCAGGCACACTCCTTCCTCACATTCGCCACCTCCA
CGCCGATGCAATCAGCAGCGGCGGCGGCCCTGAGAGCACCGGACAGCTACTT
TGAGGAGCTGAAGAGGGACTACGGCGCAAAGAAAGCGCTGCTGGTGGACGGG
CTCAAGGCGGCGGGCTTCATCGTCTACCCTTCGAGCGGAACCTACTTCATCATG
GTCGACCACACCCCGTTCGGGTTCGACAACGACGTCGAGTTCTGCGAGTACTT
GATCCGCGAGGTCGGCGTCGTGGCCATCCCGCCAAGCGTGTTCTACCTGAACC
CGGAGGACGGGAAGAACCTGGTGAGGTTCACCTTCTGCAAGGACGACGACACG
CTAAGGGCGGCGGTGGACAGGATGAAGGCCAAGCTCAGGAAGAAATGA SEQ ID NO: 15
Barley GPT amino acid sequence Translated sequence from start site
(intron removed)
MVDLRNRRTSMASAPASASAALSTAAPADNGAAKPTEQRPVQVAKRLEKFKTTIFT
QMSMLAVKHGAINLGQGFPNFDGPDFVKDAAIEAIKAGKNQYARGYGVPELNSAVA
ERFLKDSGLHIDPDKEVTVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAG
ANVKAITLRPPDFAVPLEELKAAVSKNTRAIMINTPHNPTGKMFTREELEFIADLCKE
NDVLLFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPP
HLTWGVRQAHSFLTFATSTPMQSAAAAALRAPDSYFEELKRDYGAKKALLVDGLKA
AGFIVYPSSGTYFIMVDHTPFGFDNDVEFCEYLIREVGVVAIPPSVFYLNPEDGKNLV
RFTFCKDDDTLRAAVDRMKAKLRKK SEQ ID NO: 16 Zebra fish GPT DNA coding
sequence Danio rerio sequence designed for expression in E coli.
Bold, italicized nucleotides added for cloning or from pET28b
vector. GTGGCGAAACGTCTGGAAAAATTTAAAACCACCATTTTTACCCAGATGA
GCATGCTGGCGATTAAACATGGCGCGATTAACCTGGGCCAGGGCTTTCCGAAC
TTTGATGGCCCGGATTTTGTGAAAGAAGCGGCGATTCAGGCGATTCGTGATGGC
AACAACCAGTATGCGCGTGGCTATGGCGTGCCGGATCTGAACATTGCGATTAG
CGAACGTTATAAAAAAGATACCGGCCTGGCGGTGGATCCGGAAAAAGAAATTAC
CGTGACCAGCGGCTGCACCGAAGCGATTGCGGCGACCGTGCTGGGCCTGATT
AACCCGGGCGATGAAGTGATTGTGTTTGCGCCGTTTTATGATAGCTATGAAGCG
ACCCTGAGCATGGCGGGCGCGAAAGTGAAAGGCATTACCCTGCGTCCGCCGG
ATTTTGCGCTGCCGATTGAAGAACTGAAAAGCACCATTAGCAAAAACACCCGTG
CGATTCTGCTGAACACCCCGCATAACCCGACCGGCAAAATGTTTACCCCGGAAG
AACTGAACACCATTGCGAGCCTGTGCATTGAAAACGATGTGCTGGTGTTTAGCG
ATGAAGTGTATGATAAACTGGCGTTTGATATGGAACATATTAGCATTGCGAGCCT
GCCGGGCATGTTTGAACGTACCGTGACCATGAACAGCCTGGGCAAAACCTTTA
GCCTGACCGGCTGGAAAATTGGCTGGGCGATTGCGCCGCCGCATCTGACCTGG
GGCGTGCGTCAGGCGCATGCGTTTCTGACCTTTGCAACCAGCAACCCGATGCA
GTGGGCAGCAGCAGTGGCACTGCGTGCACCGGATAGCTATTATACCGAACTGA
AACGTGATTATATGGCGAAACGTAGCATTCTGGTGGAAGGCCTGAAAGCGGTG
GGCTTTAAAGTGTTTCCGAGCAGCGGCACCTATTTTGTGGTGGTGGATCATACC
CCGTTTGGCCATGAAAACGATATTGCGTTTTGCGAATATCTGGTGAAAGAAGTG
GGCGTGGTGGCGATTCCGACCAGCGTGTTTTATCTGAACCCGGAAGAAGGCAA
AAACCTGGTGCGTTTTACCTTTTGCAAAGATGAAGGCACCCTGCGTGCGGCGGT
GGATCGTATGAAAGAAAAACTGCGTAAA SEQ ID NO: 17 Zebra fish GPR amino
acid sequence Amino acid sequence of Danio rerio cloned and
expressed in E. coli (bold, italicized amino acids are added from
vector/cloning and His tag on C-terminus)
VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAIRDGNNQ
YARGYGVPDLNIAISERYKKDTGLAVDPEKEITVTSGCTEAIAATVLGLINPGDEVIVF
APFYDSYEATLSMAGAKVKGITLRPPDFALPIEELKSTISKNTRAILLNTPHNPTGKMF
TPEELNTIASLCIENDVLVFSDEVYDKLAFDMEHISIASLPGMFERTVTMNSLGKTFSL
TGWKIGWAIAPPHLTWGVRQAHAFLTFATSNPMQWAAAVALRAPDSYYTELKRDY
MAKRSILVEGLKAVGFKVFPSSGTYFVVVDHTPFGHENDIAFCEYLVKEVGVVAIPT
SVFYLNPEEGKNLVRFTFCKDEGTLRAAVDRMKEKLRK SEQ ID NO: 18 Arabidopsis
truncated GPT-30 construct DNA sequence Arabidopsis GPT coding
sequence with 30 amino acids removed from the targeting sequence.
ATGGCCAAAATCCATCGTCCTATCGGAGCCACCATGACCACAGTTTCGACTCAG
AACGAGTCTACTCAAAAACCCGTCCAGGTGGCGAAGAGATTAGAGAAGTTCAAG
ACTACTATTTTCACTCAAATGAGCATATTGGCAGTTAAACATGGAGCGATCAATT
TAGGCCAAGGCTTTCCCAATTTCGACGGTCCTGATTTTGTTAAAGAAGCTGCGA
TCCAAGCTATTAAAGATGGTAAAAACCAGTATGCTCGTGGATACGGCATTCCTCA
GCTCAACTCTGCTATAGCTGCGCGGTTTCGTGAAGATACGGGTCTTGTTGTTGA
TCCTGAGAAAGAAGTTACTGTTACATCTGGTTGCACAGAAGCCATAGCTGCAGC
TATGTTGGGTTTAATAAACCCTGGTGATGAAGTCATTCTCTTTGCACCGTTTTAT
GATTCCTATGAAGCAACACTCTCTATGGCTGGTGCTAAAGTAAAAGGAATCACTT
TACGTCCACCGGACTTCTCCATCCCTTTGGAAGAGCTTAAAGCTGCGGTAACTA
ACAAGACTCGAGCCATCCTTATGAACACTCCGCACAACCCGACCGGGAAGATGT
TCACTAGGGAGGAGCTTGAAACCATTGCATCTCTCTGCATTGAAAACGATGTGC
TTGTGTTCTCGGATGAAGTATACGATAAGCTTGCGTTTGAAATGGATCACATTTC
TATAGCTTCTCTTCCCGGTATGTATGAAAGAACTGTGACCATGAATTCCCTGGGA
AAGACTTTCTCTTTAACCGGATGGAAGATCGGCTGGGCGATTGCGCCGCCTCAT
CTGACTTGGGGAGTTCGACAAGCACACTCTTACCTCACATTCGCCACATCAACA
CCAGCACAATGGGCAGCCGTTGCAGCTCTCAAGGCACCAGAGTCTTACTTCAAA
GAGCTGAAAAGAGATTACAATGTGAAAAAGGAGACTCTGGTTAAGGGTTTGAAG
GAAGTCGGATTTACAGTGTTCCCATCGAGCGGGACTTACTTTGTGGTTGCTGAT
CACACTCCATTTGGAATGGAGAACGATGTTGCTTTCTGTGAGTATCTTATTGAAG
AAGTTGGGGTCGTTGCGATCCCAACGAGCGTCTTTTATCTGAATCCAGAAGAAG
GGAAGAATTTGGTTAGGTTTGCGTTCTGTAAAGACGAAGAGACGTTGC
GTGGTGCAATTGAGAGGATGAAGCAGAAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 19
Arabidopsis truncated GPT-30 construct amino acid sequence
MAKIHRPIGATMTTVSTQNESTQKPVQVAKRLEKFKTTIFTQMSILAVKHGAINLGQG
FPNFDGPDFVKEAAIQAIKDGKNQYARGYGIPQLNSAIAARFREDTGLVVDPEKEVT
VTSGCTEAIAAAMLGLINPGDEVILFAPFYDSYEATLSMAGAKVKGITLRPPDFSIPLE
ELKAAVTNKTRAILMNTPHNPTGKMFTREELETIASLCIENDVLVFSDEVYDKLAFEM
DHISIASLPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSYLTFATS
TPAQWAAVAALKAPESYFKELKRDYNVKKETLVKGLKEVGFTVFPSSGTYFVVADH
TPFGMENDVAFCEYLIEEVGVVAIPTSVFYLNPEEGKNLVRFAFCKDEETLRGAIER MKQKLKRKV
SEQ ID NO: 20: Arabidopsis truncated GPT-45 construct DNA sequence
Arabidopsis GPT coding sequence with 45 residues in the targeting
sequence removed
ATGGCGACTCAGAACGAGTCTACTCAAAAACCCGTCCAGGTGGCGAAGAGATTA
GAGAAGTTCAAGACTACTATTTTCACTCAAATGAGCATATTGGCAGTTAAACATG
GAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTCGACGGTCCTGATTTTGTTAA
AGAAGCTGCGATCCAAGCTATTAAAGATGGTAAAAACCAGTATGCTCGTGGATA
CGGCATTCCTCAGCTCAACTCTGCTATAGCTGCGCGGTTTCGTGAAGATACGGG
TCTTGTTGTTGATCCTGAGAAAGAAGTTACTGTTACATCTGGTTGCACAGAAGCC
ATAGCTGCAGCTATGTTGGGTTTAATAAACCCTGGTGATGAAGTCATTCTCTTTG
CACCGTTTTATGATTCCTATGAAGCAACACTCTCTATGGCTGGTGCTAAAGTAAA
AGGAATCACTTTACGTCCACCGGACTTCTCCATCCCTTTGGAAGAGCTTAAAGC
TGCGGTAACTAACAAGACTCGAGCCATCCTTATGAACACTCCGCACAACCCGAC
CGGGAAGATGTTCACTAGGGAGGAGCTTGAAACCATTGCATCTCTCTGCATTGA
AAACGATGTGCTTGTGTTCTCGGATGAAGTATACGATAAGCTTGCGTTTGAAATG
GATCACATTTCTATAGCTTCTCTTCCCGGTATGTATGAAAGAACTGTGACCATGA
ATTCCCTGGGAAAGACTTTCTCTTTAACCGGATGGAAGATCGGCTGGGCGATTG
CGCCGCCTCATCTGACTTGGGGAGTTCGACAAGCACACTCTTACCTCACATTCG
CCACATCAACACCAGCACAATGGGCAGCCGTTGCAGCTCTCAAGGCACCAGAG
TCTTACTTCAAAGAGCTGAAAAGAGATTACAATGTGAAAAAGGAGACTCTGGTTA
AGGGTTTGAAGGAAGTCGGATTTACAGTGTTCCCATCGAGCGGGACTTACTTTG
TGGTTGCTGATCACACTCCATTTGGAATGGAGAACGATGTTGCTTTCTGTGAGTA
TCTTATTGAAGAAGTTGGGGTCGTTGCGATCCCAACGAGCGTCTTTTATCTGAAT
CCAGAAGAAGGGAAGAATTTGGTTAGGTTTGCGTTCTGTAAAGACGAAGAGACG
TTGCGTGGTGCAATTGAGAGGATGAAGCAGAAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 21:
Arabidopsis truncated GPT-45 construct amino acid sequence
MATQNESTQKPVQVAKRLEKFKTTIFTQMSILAVKHGAINLGQGFPNFDGPDFVKEA
AIQAIKDGKNQYARGYGIPQLNSAIAARFREDTGLVVDPEKEVTVTSGCTEAIAAAML
GLINPGDEVILFAPFYDSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVTNKTRAIL
MNTPHNPTGKMFTREELETIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYER
TVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKA
PESYFKELKRDYNVKKETLVKGLKEVGFTVFPSSGTYFVVADHTPFGMENDVAFCE
YLIEEVGVVAIPTSVFYLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLKRKV SEQ ID NO:
22: Tomato Rubisco promoter TOMATO RuBisCo rbcS3C promoter sequence
from KpnI to NcoI
GGTACCGTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGTAATTTTAC
TTTGTTGTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTTTTAATACTAATCTGC
TTCTGAAACTTGTAATGTATGTATATCAGTTTCTTATAATTTATCCAAGTAATATCT
TCCATTCTCTATGCAATTGCCTGCATAAGCTCGACAAAAGAGTACATCAACCCCT
CCTCCTCTGGACTACTCTAGCTAAACTTGAATTTCCCCTTAAGATTATGAAATTG
ATATATCCTTAACAAACGACTCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTC
TTTTGGGTATATATAGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTG
AAATGGATACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAG
GAGTAACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCTGTCTT
AGACAGATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTTCGCACTGAG
GGTATACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAAAGTTCCCATCGAG
CTCTAAAATCACATCTTTGGAATGAAATTCAATTAGAGATAAGTTGCTTCATAGCA
TAGGTAAAATGGAAGATGTGAAGTAACCTGCAATAATCAGTGAAATGACATTAAT
ACACTAAATACTTCATATGTAATTATCCTTTCCAGGTTAACAATACTCTATAAAGT
AAGAATTATCAGAAATGGGCTCATCAAACTTTTGTACTATGTATTTCATATAAGGA
AGTATAACTATACATAAGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAG
AGACTGTTTTCGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAA
TTATCTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGTTTG
TACCATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCATAAACAGTTT
TCCCCAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTAACAGTTCAAATATAT
GGCGCAGAAATTACTCTATGCCCAAAACCAAACGAGAAAGAAACAAAATACAGG
GGTTGCAGACTTTATTTTCGTGTTAGGGTGTGTTTTTTCATGTAATTAATCAAAAA
ATATTATGACAAAAACATTTATACATATTTTTACTCAACACTCTGGGTATCAGGGT
GGGTTGTGTTCGACAATCAATATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTA
ATATTTTCAGTTATACCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTC
GTCAGAAAGAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGA
AAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGAGTGA
GGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCACAAAATCCAA
TGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCGTTAGATAGG
AAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTAACCAATTATT
TCAGCACCATGG SEQ ID NO: 23: Bamboo GPT DNA coding sequence
ATGGCCTCCGCGGCCGTCTCCACCGTCGCCACCGCCGCCGACGGCGTCGCGA
AGCCGACGGAGAAGCAGCCGGTACAGGTCGCAAAGCGTTTGGAAAAGTTTAAG
ACAACAATTTTCACACAGATGAGCATGCTTGCCATCAAGCATGGAGCAATAAAC
CTCGGCCAGGGCTTTCCGAATTTTGATGGCCCTGACTTTGTGAAAGAAGCTGCT
ATTCAAGCTATCAATGCTGGGAAGAATCAGTATGCAAGAGGATATGGTGTGCCT
GAACTGAACTCGGCTGTTGCTGAAAGGTTCCTGAAGGACAGTGGCTTGCAAGTC
GATCCCGAGAAGGAAGTTACTGTCACATCTGGGTGCACGGAAGCGATAGCTGC
AACGATATTGGGTCTTATCAACCCTGGCGATGAAGTGATCTTGTTTGCTCCATTC
TATGATTCATACGAGGCTACGCTGTCGATGGCTGGTGCCAATGTAAAAGCCATT
ACTCTCCGTCCTCCAGATTTTGCAGTCCCTCTTGAGGAGCTAAAGGCCACAGTC
TCTAAGAACACCAGAGCGATAATGATAAACACACCACACAATCCTACTGGGAAA
ATGTTTTCTAGGGAAGAACTTGAATTCATTGCTACTCTCTGCAAGAAAAATGATG
TGTTGCTTTTTGCTGATGAGGTCTATGACAAGTTGGCATTTGAGGCAGATCATAT
ATCAATGGCTTCTATTCCTGGCATGTATGAGAGGACTGTGACTATGAACTCTCTG
GGGAAGACATTCTCTCTAACAGGATGGAAGATCGGTTGGGCAATAGCACCACCA
CACCTGACATGGGGTGTAAGGCAGGCACACTCATTCCTCACATTTGCCACCTGC
ACACCAATGCAATCGGCGGCGGCGGCGGCTCTTAGAGCACCAGATAGCTACTA
TGGGGAGCTGAAGAGGGATTACGGTGCAAAGAAAGCGATACTAGTCGACGGAC
TCAAGGCTGCAGGTTTTATTGTTTACCCTTCAAGTGGAACATACTTTGTCATGGT
CGATCACACCCCGTTTGGTTTCGACAATGATATTGAGTTCTGCGAGTATTTGATC
CGCGAAGTCGGTGTTGTCGCCATACCACCAAGCGTATTTTATCTCAACCCTGAG
GATGGGAAGAACTTGGTGAGGTTCACCTTCTGCAAGGATGATGATACGCTGAGA
GCCGCAGTTGAGAGGATGAAGACAAAGCTCAGGAAAAAATGA SEQ ID NO: 24: Bamboo
GPT amino acid sequence
MASAAVSTVATAADGVAKPTEKQPVQVAKRLEKFKTTIFTQMSMLAIKHGAINLGQG
FPNFDGPDFVKEAAIQAINAGKNQYARGYGVPELNSAVAERFLKDSGLQVDPEKEV
TVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAGANVKAITLRPPDFAVPL
EELKATVSKNTRAIMINTPHNPTGKMFSREELEFIATLCKKNDVLLFADEVYDKLAFE
ADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSFLTFA
TCTPMQSAAAAALRAPDSYYGELKRDYGAKKAILVDGLKAAGFIVYPSSGTYFVMV
DHTPFGFDNDIEFCEYLIREVGVVAIPPSVFYLNPEDGKNLVRFTFCKDDDTLRAAVE
RMKTKLRKK SEQ ID NO: 25: 1305.1 + rbcS3C promoter + catI intron
with rice GPT gene. Cambia1305.1 with (3' end of) rbcS3C + rice GPT
coding sequence. Underlined ATG is start site, parentheses are the
catI intron and the underlined actagt is the speI cloning site used
to splice in the rice gene.
AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG
ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC
AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG
TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA
ACCAATTATTTCAGCA TAGATCTGAGG(GTAAATTTCTAGTTTTTCTCCT
TCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCT
TTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAA
CTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAG)A ACCGACGA
ATGAATCTGGCCGGCTTTCTCGCCACGCCCGCGACCGCG
ACCGCGACGCGGCATGAGATGCCGTTAAATCCCTCCTCCTCCGCCTCCTTCCTC
CTCTCCTCGCTCCGCCGCTCGCTCGTCGCGTCGCTCCGGAAGGCCTCGCCGG
CGGCGGCCGCGGCGCTCTCCCCCATGGCCTCCGCGTCCACCGTCGCCGCCGA
GAACGGCGCCGCCAAGGCGGCGGCGGAGAAGCAGCAGCAGCAGCCTGTGCA
GGTTGCAAAGCGGTTGGAAAAGTTTAAGACGACCATTTTCACACAGATGAGTAT
GCTTGCCATCAAGCATGGAGCAATAAACCTTGGCCAGGGTTTTCCGAATTTCGA
TGGCCCTGACTTTGTAAAAGAGGCTGCTATTCAAGCTATCAATGCTGGGAAGAA
TCAGTACGCAAGAGGATATGGTGTGCCTGAACTGAACTCAGCTATTGCTGAAAG
ATTCCTGAAGGACAGCGGACTGCAAGTCGATCCGGAGAAGGAAGTTACTGTCA
CATCTGGATGCACAGAAGCTATAGCTGCAACAATTTTAGGTCTAATTAATCCAGG
CGATGAAGTGATATTGTTTGCTCCATTCTATGATTCATATGAGGCTACCCTGTCA
ATGGCTGGTGCCAACGTAAAAGCCATTACTCTCCGTCCTCCAGATTTTTCAGTC
CCTCTTGAAGAGCTAAAGGCTGCAGTCTCGAAGAACACCAGAGCTATTATGATA
AACACCCCGCACAATCCTACTGGGAAAATGTTTACAAGGGAAGAACTTGAGTTT
ATTGCCACTCTCTGCAAGGAAAATGATGTGCTGCTTTTTGCTGATGAGGTCTAC
GACAAGTTAGCTTTTGAGGCAGATCATATATCAATGGCTTCTATTCCTGGCATGT
ATGAGAGGACCGTGACCATGAACTCTCTTGGGAAGACATTCTCTCTTACAGGAT
GGAAGATCGGTTGGGCAATCGCACCGCCACACCTGACATGGGGTGTAAGGCAG
GCACACTCATTCCTCACGTTTGCGACCTGCACACCAATGCAAGCAGCTGCAGCT
GCAGCTCTGAGAGCACCAGATAGCTACTATGAGGAACTGAGGAGGGATTATGG
AGCTAAGAAGGCATTGCTAGTCAACGGACTCAAGGATGCAGGTTTCATTGTCTA
TCCTTCAAGTGGAACATACTTCGTCATGGTCGACCACACCCCATTTGGTTTCGA
CAATGATATTGAGTTCTGCGAGTATTTGATTCGCGAAGTCGGTGTTGTCGCCATA
CCACCTAGTGTATTTTATCTCAACCCTGAGGATGGGAAGAACTTGGTGAGGTTC
ACCTTTTGCAAGGATGATGAGACGCTGAGAGCCGCGGTTGAGAGGATGAAGAC
AAAGCTCAGGAAAAAATGA SEQ ID NO: 26: HORDEUM GPT SEQUENCE IN VECTOR
Cambia1305.1 with (3' end of) rbcS3C + hordeum (IDI4) coding
sequence. Underlined ATG is start site, parentheses are the catI
intron and the underlined actagt is the speI cloning site used to
splice in the hordeum gene.
AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG
ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC
AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG
TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA
ACCAATTATTTCAGCACC TAGATCTGAGG(GTAAATTTCTAGTTTTTCTCCT
TCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCT
TTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAA
CTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAG)A ACCGACGA
ATGGCATCCGCCCCCGCCTCCGCCTCCGCGGCCCTCTCC
ACCGCCGCCCCCGCCGACAACGGGGCCGCCAAGCCCACGGAGCAGCGGCCG
GTACAGGTGGCTAAGCGATTGGAGAAGTTCAAAACAACAATTTTCACACAGATG
AGCATGCTCGCAGTGAAGCATGGAGCAATAAACCTTGGACAGGGGTTTCCCAAT
TTTGATGGCCCTGACTTTGTCAAAGATGCTGCTATTGAGGCTATCAAAGCTGGA
AAGAATCAGTATGCAAGAGGATATGGTGTGCCTGAATTGAACTCAGCTGTTGCT
GAGAGATTTCTCAAGGACAGTGGATTGCACATCGATCCTGATAAGGAAGTTACT
GTTACATCTGGGTGCACAGAAGCAATAGCTGCAACGATATTGGGTCTGATCAAC
CCTGGGGATGAAGTCATACTGTTTGCTCCATTCTATGATTCTTATGAGGCTACAC
TGTCCATGGCTGGTGCGAATGTCAAAGCCATTACACTCCGCCCTCCGGACTTTG
CAGTCCCTCTTGAAGAGCTAAAGGCTGCAGTCTCGAAGAATACCAGAGCAATAA
TGATTAATACACCTCACAACCCTACCGGGAAAATGTTCACAAGGGAGGAACTTG
AGTTCATTGCTGATCTCTGCAAGGAAAATGACGTGTTGCTCTTTGCCGATGAGG
TCTACGACAAGCTGGCGTTTGAGGCGGATCACATATCAATGGCTTCTATTCCTG
GCATGTATGAGAGGACCGTCACTATGAACTCCCTGGGGAAGACGTTCTCCTTGA
CCGGATGGAAGATCGGCTGGGCGATAGCACCACCGCACCTGACATGGGGCGT
AAGGCAGGCACACTCCTTCCTCACATTCGCCACCTCCACGCCGATGCAATCAGC
AGCGGCGGCGGCCCTGAGAGCACCGGACAGCTACTTTGAGGAGCTGAAGAGG
GACTACGGCGCAAAGAAAGCGCTGCTGGTGGACGGGCTCAAGGCGGCGGGCT
TCATCGTCTACCCTTCGAGCGGAACCTACTTCATCATGGTCGACCACACCCCGT
TCGGGTTCGACAACGACGTCGAGTTCTGCGAGTACTTGATCCGCGAGGTCGGC
GTCGTGGCCATCCCGCCAAGCGTGTTCTACCTGAACCCGGAGGACGGGAAGAA
CCTGGTGAGGTTCACCTTCTGCAAGGACGACGACACGCTAAGGGCGGCGGTG
GACAGGATGAAGGCCAAGCTCAGGAAGAAATGATTGAGGGGCG SEQ ID NO: 27
Expression cassette, Arabidopsis GPT coding sequence (ATG
underlined) under control of CMV 35S promoter (italics; promoter
from Cambia 1201)
CATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGG
CGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTC
AACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCT
CAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACC
TCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAA
GGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGA
AGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCA
TCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTG
ATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACC
CTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACGGGGGACTCTTGA
CCATGTACCTGGACATAAATGGTGTGATGATCAAACAGTTTAGCTTCAAAGCCTC
TCTTCTCCCATTCTCTTCTAATTTCCGACAAAGCTCCGCCAAAATCCATCGTCCT
ATCGGAGCCACCATGACCACAGTTTCGACTCAGAACGAGTCTACTCAAAAACCC
GTCCAGGTGGCGAAGAGATTAGAGAAGTTCAAGACTACTATTTTCACTCAAATG
AGCATATTGGCAGTTAAACATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATT
TCGACGGTCCTGATTTTGTTAAAGAAGCTGCGATCCAAGCTATTAAAGATGGTAA
AAACCAGTATGCTCGTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGC
GCGGTTTCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGAAAGAAGTTACTGT
TACATCTGGTTGCACAGAAGCCATAGCTGCAGCTATGTTGGGTTTAATAAACCCT
GGTGATGAAGTCATTCTCTTTGCACCGTTTTATGATTCCTATGAAGCAACACTCT
CTATGGCTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCACCGGACTTCTCCA
TCCCTTTGGAAGAGCTTAAAGCTGCGGTAACTAACAAGACTCGAGCCATCCTTA
TGAACACTCCGCACAACCCGACCGGGAAGATGTTCACTAGGGAGGAGCTTGAA
ACCATTGCATCTCTCTGCATTGAAAACGATGTGCTTGTGTTCTCGGATGAAGTAT
ACGATAAGCTTGCGTTTGAAATGGATCACATTTCTATAGCTTCTCTTCCCGGTAT
GTATGAAAGAACTGTGACCATGAATTCCCTGGGAAAGACTTTCTCTTTAACCGGA
TGGAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGAGTTCGACA
AGCACACTCTTACCTCACATTCGCCACATCAACACCAGCACAATGGGCAGCCGT
TGCAGCTCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGAAAAGAGATTACAA
TGTGAAAAAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCGGATTTACAGTGTT
CCCATCGAGCGGGACTTACTTTGTGGTTGCTGATCACACTCCATTTGGAATGGA
GAACGATGTTGCTTTCTGTGAGTATCTTATTGAAGAAGTTGGGGTCGTTGCGAT
CCCAACGAGCGTCTTTTATCTGAATCCAGAAGAAGGGAAGAATTTGGTTAGGTT
TGCGTTCTGTAAAGACGAAGAGACGTTGCGTGGTGCAATTGAGAGGATGAAGC
AGAAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 28 Cambia p1305.1 with (3' end
of) rbcS3C + Arabidopsis GPT coding sequence. Underlined ATG is
start site, parentheses are the catI intron and the underlined
actagt is the speI cloning site used to splice in the Arabidopsis
gene. AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGG
ACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC
AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCG
TTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTA
ACCAATTATTTCAGCA TAGATCTGAGG(GTAAATTTCTAGTTTTTCTCCT
TCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCT
TTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAA
CTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACAG)A ACCGACGA
ATGTACCTGGACATAAATGGTGTGATGATCAAACAGTTTA
GCTTCAAAGCCTCTCTTCTCCCATTCTCTTCTAATTTCCGACAAAGCTCCGCCAA
AATCCATCGTCCTATCGGAGCCACCATGACCACAGTTTCGACTCAGAACGAGTC
TACTCAAAAACCCGTCCAGGTGGCGAAGAGATTAGAGAAGTTCAAGACTACTAT
TTTCACTCAAATGAGCATATTGGCAGTTAAACATGGAGCGATCAATTTAGGCCAA
GGCTTTCCCAATTTCGACGGTCCTGATTTTGTTAAAGAAGCTGCGATCCAAGCTA
TTAAAGATGGTAAAAACCAGTATGCTCGTGGATACGGCATTCCTCAGCTCAACT
CTGCTATAGCTGCGCGGTTTCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGA
AAGAAGTTACTGTTACATCTGGTTGCACAGAAGCCATAGCTGCAGCTATGTTGG
GTTTAATAAACCCTGGTGATGAAGTCATTCTCTTTGCACCGTTTTATGATTCCTAT
GAAGCAACACTCTCTATGGCTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCA
CCGGACTTCTCCATCCCTTTGGAAGAGCTTAAAGCTGCGGTAACTAACAAGACT
CGAGCCATCCTTATGAACACTCCGCACAACCCGACCGGGAAGATGTTCACTAG
GGAGGAGCTTGAAACCATTGCATCTCTCTGCATTGAAAACGATGTGCTTGTGTT
CTCGGATGAAGTATACGATAAGCTTGCGTTTGAAATGGATCACATTTCTATAGCT
TCTCTTCCCGGTATGTATGAAAGAACTGTGACCATGAATTCCCTGGGAAAGACTT
TCTCTTTAACCGGATGGAAGATCGGCTGGGCGATTGCGCCGCCTCATCTGACTT
GGGGAGTTCGACAAGCACACTCTTACCTCACATTCGCCACATCAACACCAGCAC
AATGGGCAGCCGTTGCAGCTCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGA
AAAGAGATTACAATGTGAAAAAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCG
GATTTACAGTGTTCCCATCGAGCGGGACTTACTTTGTGGTTGCTGATCACACTC
CATTTGGAATGGAGAACGATGTTGCTTTCTGTGAGTATCTTATTGAAGAAGTTGG
GGTCGTTGCGATCCCAACGAGCGTCTTTTATCTGAATCCAGAAGAAGGGAAGAA
TTTGGTTAGGTTTGCGTTCTGTAAAGACGAAGAGACGTTGCGTGGTGCAATTGA
GAGGATGAAGCAGAAGCTTAAGAGAAAAGTCTGA SEQ ID NO: 29 Arabidpsis GPT
coding sequence (mature protein, no targeting sequence)
GTGGCGAAGAGATTAGAGAAGTTCAAGACTACTATTTTCACTCAAATGAGCATAT
TGGCAGTTAAACATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTCGACG
GTCCTGATTTTGTTAAAGAAGCTGCGATCCAAGCTATTAAAGATGGTAAAAACCA
GTATGCTCGTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGCGCGGTT
TCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGAAAGAAGTTACTGTTACATCT
GGTTGCACAGAAGCCATAGCTGCAGCTATGTTGGGTTTAATAAACCCTGGTGAT
GAAGTCATTCTCTTTGCACCGTTTTATGATTCCTATGAAGCAACACTCTCTATGG
CTGGTGCTAAAGTAAAAGGAATCACTTTACGTCCACCGGACTTCTCCATCCCTTT
GGAAGAGCTTAAAGCTGCGGTAACTAACAAGACTCGAGCCATCCTTATGAACAC
TCCGCACAACCCGACCGGGAAGATGTTCACTAGGGAGGAGCTTGAAACCATTG
CATCTCTCTGCATTGAAAACGATGTGCTTGTGTTCTCGGATGAAGTATACGATAA
GCTTGCGTTTGAAATGGATCACATTTCTATAGCTTCTCTTCCCGGTATGTATGAA
AGAACTGTGACCATGAATTCCCTGGGAAAGACTTTCTCTTTAACCGGATGGAAG
ATCGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGAGTTCGACAAGCACA
CTCTTACCTCACATTCGCCACATCAACACCAGCACAATGGGCAGCCGTTGCAGC
TCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGAAAAGAGATTACAATGTGAAA
AAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCGGATTTACAGTGTTCCCATCG
AGCGGGACTTACTTTGTGGTTGCTGATCACACTCCATTTGGAATGGAGAACGAT
GTTGCTTTCTGTGAGTATCTTATTGAAGAAGTTGGGGTCGTTGCGATCCCAACG
AGCGTCTTTTATCTGAATCCAGAAGAAGGGAAGAATTTGGTTAGGTTTGCGTTCT
GTAAAGACGAAGAGACGTTGCGTGGTGCAATTGAGAGGATGAAGCAGAAGCTT
AAGAGAAAAGTCTGA SEQ ID NO: 30 Arabidpsis GPT amino acid sequence
(mature protein, no targeting sequence)
VAKRLEKFKTTIFTQMSILAVKHGAINLGQGFPNFDGPDFVKEAAIQAIKDGKNQYAR
GYGIPQLNSAIAARFREDTGLVVDPEKEVTVTSGCTEAIAAAMLGLINPGDEVILFAP
FYDSYEATLSMAGAKVKGITLRPPDFSIPLEELKAAVTNKTRAILMNTPHNPTGKMFT
REELETIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYERTVTMNSLGKTFSL
TGWKIGWAIAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKAPESYFKELKRDYN
VKKETLVKGLKEVGFTVFPSSGTYFVVADHTPFGMENDVAFCEYLIEEVGVVAIPTS
VFYLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLKRKV SEQ ID NO: 31 Grape GPT
amino acid sequence (mature protein, no targeting sequence)
VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPEFVKEAAIQAIKDGKNQYAR
GYGVPDLNSAVADRFKKDTGLVVDPEKEVTVTSGCTEAIAATMLGLINPGDEVILFA
PFYDSYEATLSMAGAQIKSITLRPPDFAVPMDELKSAISKNTRAILINTPHNPTGKMFT
REELNVIASLCIENDVLVFTDEVYDKLAFEMDHISMASLPGMYERTVTMNSLGKTFS
LTGWKIGWTVAPPHLTWGVRQAHSFLTFATCTPMQWAAATALRAPDSYYEELKRD
YSAKKAILVEGLKAVGFRVYPSSGTYFVVVDHTPFGLKDDIAFCEYLIKEVGVVAIPT
SVFYLHPEDGKNLVRFTFCKDEGTLRAAVERMKEKLKPKQ SEQ ID NO: 32 Rice GPT
amino acid sequence (mature protein, no targeting sequence)
VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAINAGKNQYAR
GYGVPELNSAIAERFLKDSGLQVDPEKEVTVTSGCTEAIAATILGLINPGDEVILFAPF
YDSYEATLSMAGANVKAITLRPPDFSVPLEELKAAVSKNTRAIMINTPHNPTGKMFT
REELEFIATLCKENDVLLFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSL
TGWKIGWAIAPPHLTWGVRQAHSFLTFATCTPMQAAAAAALRAPDSYYEELRRDY
GAKKALLVNGLKDAGFIVYPSSGTYFVMVDHTPFGFDNDIEFCEYLIREVGVVAIPPS
VFYLNPEDGKNLVRFTFCKDDETLRAAVERMKTKLRKK SEQ ID NO: 33 Soybean GPT
amino acid sequence (-1 mature protein, no targeting sequence)
AKRLEKFQTTIFTQMSLLAIKHGAINLGQGFPNFDGPEFVKEAAIQAIRDGKNQYARG
YGVPDLNIAIAERFKKDTGLVVDPEKEITVTSGCTEAIAATMIGLINPGDEVIMFAPFY
DSYEATLSMAGAKVKGITLRPPDFAVPLEELKSTISKNTRAILINTPHNPTGKMFTRE
ELNCIASLCIENDVLVFTDEVYDKLAFDMEHISMASLPGMFERTVTLNSLGKTFSLTG
WKIGWAIAPPHLSWGVRQAHAFLTFATAHPFQCAAAAALRAPDSYYVELKRDYMAK
RAILIEGLKAVGFKVFPSSGTYFVVVDHTPFGLENDVAFCEYLVKEVGVVAIPTSVFY
LNPEEGKNLVRFTFCKDEETIRSAVERMKAKLRKVD SEQ ID NO: 34 Barley GPT amino
acid sequence (mature protein, no targeting sequence)
VAKRLEKFKTTIFTQMSMLAVKHGAINLGQGFPNFDGPDFVKDAAIEAIKAGKNQYA
RGYGVPELNSAVAERFLKDSGLHIDPDKEVTVTSGCTEAIAATILGLINPGDEVILFAP
FYDSYEATLSMAGANVKAITLRPPDFAVPLEELKAAVSKNTRAIMINTPHNPTGKMFT
REELEFIADLCKENDVLLFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSL
TGWKIGWAIAPPHLTWGVRQAHSFLTFATSTPMQSAAAAALRAPDSYFEELKRDYG
AKKALLVDGLKAAGFIVYPSSGTYFIMVDHTPFGFDNDVEFCEYLIREVGVVAIPPSV
FYLNPEDGKNLVRFTFCKDDDTLRAAVDRMKAKLRKK SEQ ID NO: 35 Zebra fish GPT
amino acid sequence (mature protein, no targeting sequence)
VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAIRDGNNQYA
RGYGVPDLNIAISERYKKDTGLAVDPEKEITVTSGCTEAIAATVLGLINPGDEVIVFAP
FYDSYEATLSMAGAKVKGITLRPPDFALPIEELKSTISKNTRAILLNTPHNPTGKMFTP
EELNTIASLCIENDVLVFSDEVYDKLAFDMEHISIASLPGMFERTVTMNSLGKTFSLT
GWKIGWAIAPPHLTWGVRQAHAFLTFATSNPMQWAAAVALRAPDSYYTELKRDYM
AKRSILVEGLKAVGFKVFPSSGTYFVVVDHTPFGHENDIAFCEYLVKEVGVVAIPTSV
FYLNPEEGKNLVRFTFCKDEGTLRAAVDRMKEKLRK SEQ ID NO: 36 Bamboo GPT amino
acid sequence (mature protein, no targeting sequence)
VAKRLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVKEAAIQAINAGKNQYAR
GYGVPELNSAVAERFLKDSGLQVDPEKEVTVTSGCTEAIAATILGLINPGDEVILFAP
FYDSYEATLSMAGANVKAITLRPPDFAVPLEELKATVSKNTRAIMINTPHNPTGKMFS
REELEFIATLCKKNDVLLFADEVYDKLAFEADHISMASIPGMYERTVTMNSLGKTFSL
TGWKIGWAIAPPHLTWGVRQAHSFLTFATCTPMQSAAAAALRAPDSYYGELKRDY
GAKKAILVDGLKAAGFIVYPSSGTYFVMVDHTPFGFDNDIEFCEYLIREVGVVAIPPS
VFYLNPEDGKNLVRFTFCKDDDTLRAAVERMKTKLRKK SEQ ID NO: 39 Rice rubisco
promoter deposited in NCBI GenBank: AF143510.1 PstI cloning sites
in bold; NcoI cloning site in italics, catI intron and part of Gus
plus protein from Cambia 1305.1 vector in bold underline (sequence
removed and not translated), 3' terminal SpeI cloning site in
double underline. The construct also includes a PmlI 1305.1 cloning
site CACGTG (also cuts in rice rbsc promoter), and a ZraI cloning
site GACGTC, which can be added by PCR to clone into PmlI site of
vector). CTGCAGCAAAGAAACGTTATTAGTTGGTGCTTTTGGTGGTAGGAATGTAGTTTTC
TGACAAAGTCAATTACTGAATATAAAAAAAATCTGCACAGCTCTGCGTCAACAGT
TGTCCAAGGGATGCCTCAAAAATCTGTGCAGATTATCAGTCGTCACGCAGAAGC
AGAACATCATGGTGTGCTAGGTCAGCTTCTTGCATTGGGCCATGAATCCGGTTG
GTTGTTAATCTCTCCTCTCTTATTCTCTTATATTAAGATGCATAACTCTTTTATGTA
GTCTAAAAAAAAATCCAGTGGATCGGATAGTAGTACGTCATGGTGCCATTAGGT
ACCGTTGAACCTAACAGATATTTATGCATGTGTATATATATAGCTATATAGACAAA
ATTGATGCCGATTATAGACCCAAAAGCAATAGGTATATATAATATAATACAGACC
ACACCACCAAACTAAGAATCGATCAAATAGACAAGGCATGTCTCCAAATTGTCTT
AAACTATTTCCGTAGGTTCAGCCGTTCAGGAGTCGAATCAGCCTCTGCCGGCGT
TTTCTTTGCACGTACGACGGACACACATGGGCATACCATATAGCTGGTCCATGA
CATTAGGAGAGAGAACGTACGTGTTGACCTGTAGCTGAGATATAACAAGGTTGA
TTATAATATCACCAAACATGAAATCATCCAAGGATGACCCATAACTATCACTACTA
TAGTACTGCATCTGGTAAAAGAAATTGTATAGACTCTATTTCGAGCACTACCACA
TAACGCCTGCAATGTGACACCCTACCTATTCACTAATGTGCCTCTTCCCACACG
CTTTCCACCCGTACTGCTCACAGCTTTAAGAACCAGAACAAATGAGTAATATTAG
TGTCGGTTCATGGCTAAAACCAGCACTGATGTACATGACCACATATGTCAAATG
CTGCTTCTAGGCATGACCCGCTCTTACTAATACCTACTCATCGCTAGAAGAATTT
TCGGCTGATAAATTTTCAATTTAAGCAAGAGTTATCTGCGTTGGTTCATAACTCA
AACTGATGGCCCCAACCATATTAGTGCAAATTTCACATATGATCATAACCTTTTC
ATATGAAATCGGATCGAGATGAACTTTATATAAACATTGTAGCTGTCGATGATAC
CTACAATTTTATAGTTCACAACCTTTTTATTTCAAGTCATTTAAATGCCCAAATAG
GTGTTTCAAATCTCAGATAGAAATGTTCAAAAGTAAAAAAGGTCCCTATCATAAC
ATAATTGATATGTAAGTGAGTTGGAAAAAGATAAGTACGTGTGAGAGAGATCGG
GGATCAAATTCTGGTGTAATAATGTATGTATTTCAGTCATAAAAATTGGTAGCAG
TAGTTGGGGCTCTGTATATATACCGGTAAGGATGGGATGGTAGTAGAATAATTC
TTTTTTTGTTTTTAGTTTTTTCTGGTCCAAAATTTCAAATTTGGATCCCTTACTTGT
ACCAACTAATATTAATGAGTGTTGAGGGTAGTAGAGGTGCAACTTTACCATAATC
CCTCTGTTTCAGGTTATAAGACGTTTTGACTTTAAATTTGACCAAGTTTATGCGCA
AATATAGTAATATTTATAATACTATATTAGTTTCATTAAATAAATAATTGAATATATT
TTCATAATAAATTTGTGTTGAGTTCAAAATATTATTAATTTTTTCTACAAACTTGGT
CAAACTTGAAGCAGTTTGACTTTGACCAAAGTCAAAACGTCTTATAACTTGAAAC
GGATGGATTACTTTTTTTGTGGGGACAAGTTTACAATGTTTAATAAAGCACAATC
CATCTTAATGTTTTCAAGCTGAATATTGTAAAATTCATGGATAAACCAGCTTCTAA
ATGTTTAACCGGGAAAATGTCGAACGACAAATTAATATTTTTAAGTGATGGGGAG
TATTAATTAAGGAGTGACAACTCAACTTTCAATATCGTACTAAACTGTGGGATTTA
TTTTCTAAAATTTTATACCCTGCCAATTCACGTGTTGTAGATCTTTTTTTTTCACTA
ACCGACACCAGGTATATCAATTTTATTGAATATAGCAGCAAAAAGAATGTGTTGT
ACTTGTAAACAAAAAGCAAACTGTACATAAAAAAAAATGCACTCCTATATAATTAA
GCTCATAAAGATGCTTTGCTTCGTGAGGGCCCAAGTTTTGATGACCTTTTGCTTG
ATCTCGAAATTAAAATTTAAGTACTGTTAAGGGAGTTCACACCACCATCAATTTTC
AGCCTGAAGAAACAGTTAAACAACGACCCCGATGACCAGTCTACTGCTCTCCAC
ATACTAGCTGCATTATTGATCACAAAACAAAACAAAACGAAATAAAAATCAGCAG
CGAGAGTGTGCAGAGAGAGACAAAGGTGATCTGGCGTGGATATCTCCCCATCC
ATCCTCACCCGCGCTGCCCATCACTCGCCGCCGCATACTCCATCATGTGGAGA
GAGGAAGACGAGGACCACAGCCAGAGCCCGGGTCGAGATGCCACCACGGCCA
CAACCCACGAGCCCGGCGCGACACCACCGCGCGCGCGTGAGCCAGCCACAAA
CGCCCGCGGATAGGCGCGCGCACGCCGGCCAATCCTACCACATCCCCGGCCT
CCGCGGCTCGCGAGCGCCGCTGCCATCCGATCCGCTGAGTTTTGGCTATTTAT
ACGTACCGCGGGAGCCTGTGTGCAGAGCAGTGCATCTCAAGAAGTACTCGAGC
AAAGAAGGAGAGAGCTTGGTGAGCTGCAGCCATGGTAGATCTGAGGGTAAATT
TCTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTG
AGCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAA
ATATTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGATG
ATGATGATAGTTACAGAACCGACGAACTAGT SEQ ID NO: 40 Horeum GS1 coding
sequence GCGCAGGCGGTTGTGCAGGCGATGCAGTGCCAGGTGGGGGTGAGGGGCAGG
ACGGCCGTCCCGGCGAGGCAGCCCGCGGGCAGGGTGTGGGGCGTCAGGAGG
GCCGCCCGCGCCACCTCCGGGTTCAAGGTGCTGGCGCTCGGCCCGGAGACCA
CCGGGGTCATCCAGAGGATGCAGCAGCTGCTCGACATGGACACCACGCCCTTC
ACCGACAAGATCATCGCCGAGTACATCTGGGTTGGAGGATCTGGAATTGACCTC
AGAAGCAAATCAAGGACGATTTCGAAGCCAGTGGAGGACCCGTCAGAGCTGCC
GAAATGGAACTACGACGGATCGAGCACGGGGCAGGCTCCTGGGGAAGACAGT
GAAGTCATCCTATACCCACAGGCCATATTCAAGGACCCATTCCGAGGAGGCAAC
AACATACTGGTTATCTGTGACACCTACACACCACAGGGGGAACCCATCCCTACT
AACAAACGCCACATGGCTGCACAAATCTTCAGTGACCCCAAGGTCACTTCACAA
GTGCCATGGTTCGGAATCGAACAGGAGTACACTCTGATGCAGAGGGATGTGAA
CTGGCCTCTTGGCTGGCCTGTTGGAGGGTACCCTGGCCCCCAGGGTCCATACT
ACTGCGCCGTAGGATCAGACAAGTCATTTGGCCGTGACATATCAGATGCTCACT
ACAAGGCGTGCCTTTACGCTGGAATTGAAATCAGTGGAACAAACGGGGAGGTC
ATGCCTGGTCAGTGGGAGTACCAGGTTGGACCCAGCGTTGGTATTGATGCAGG
AGACCACATATGGGCTTCCAGATACATTCTCGAGAGAATCACGGAGCAAGCTGG
TGTGGTGCTCACCCTTGACCCAAAACCAATCCAGGGTGACTGGAACGGAGCTG
GCTGCCACACAAACTACAGCACATTGAGCATGCGCGAGGATGGAGGTTTCGAC
GTGATCAAGAAGGCAATCCTGAACCTTTCACTTCGCCATGACTTGCACATAGCC
GCATATGGTGAAGGAAACGAGCGGAGGTTGACAGGGCTACACGAGACAGCTAG
CATATCAGACTTCTCATGGGGTGTGGCGAACCGTGGCTGCTCTATTCGTGTGGG
GCGAGACACCGAGGCGAAGGGCAAAGGATACCTGGAGGACCGTCGCCCGGCC
TCCAACATGGACCCGTACACCGTGACGGCGCTGCTGGCCGAGACCACGATCCT
GTGGGAGCCGACCCTCGAGGCGGAGGCCCTCGCTGCCAAGAAGCTGGCGCTG AAGGTATGA SEQ
ID NO: 41 Horeum GS1 amino acid sequence
AQAVVQAMQCQVGVRGRTAVPARQPAGRVWGVRRAARATSGFKVLALGPETTGV
IQRMQQLLDMDTTPFTDKIIAEYIWVGGSGIDLRSKSRTISKPVEDPSELPKWNYDG
SSTGQAPGEDSEVILYPQAIFKDPFRGGNNILVICDTYTPQGEPIPTNKRHMAAQIFS
DPKVTSQVPWFGIEQEYTLMQRDVNWPLGWPVGGYPGPQGPYYCAVGSDKSFG
RDISDAHYKACLYAGIEISGTNGEVMPGQWEYQVGPSVGIDAGDHIWASRYILERIT
EQAGVVLTLDPKPIQGDWNGAGCHTNYSTLSMREDGGFDVIKKAILNLSLRHDLHIA
AYGEGNERRLTGLHETASISDFSWGVANRGCSIRVGRDTEAKGKGYLEDRRPASN
MDPYTVTALLAETTILWEPTLEAEALAAKKLALKV SEQ ID NO: 42: Expression
cassette combining SEQ ID NO: 39 (5') and SEQ ID NO: 40 (3'),
encoding the Rice rubisco promoter, catI intron and part of Gus
plus protein, and hordeum GS1. Features shown as in SEQ ID NO: 39.
Hordeum GS1 coding sequence begins after SpeI cloning site (double
underline). CTGCAGCAAAGAAACGTTATTAGTTGGTGCTTTTGGTGGTAGGAATGTAGTTTTC
TGACAAAGTCAATTACTGAATATAAAAAAAATCTGCACAGCTCTGCGTCAACAGT
TGTCCAAGGGATGCCTCAAAAATCTGTGCAGATTATCAGTCGTCACGCAGAAGC
AGAACATCATGGTGTGCTAGGTCAGCTTCTTGCATTGGGCCATGAATCCGGTTG
GTTGTTAATCTCTCCTCTCTTATTCTCTTATATTAAGATGCATAACTCTTTTATGTA
GTCTAAAAAAAAATCCAGTGGATCGGATAGTAGTACGTCATGGTGCCATTAGGT
ACCGTTGAACCTAACAGATATTTATGCATGTGTATATATATAGCTATATAGACAAA
ATTGATGCCGATTATAGACCCAAAAGCAATAGGTATATATAATATAATACAGACC
ACACCACCAAACTAAGAATCGATCAAATAGACAAGGCATGTCTCCAAATTGTCTT
AAACTATTTCCGTAGGTTCAGCCGTTCAGGAGTCGAATCAGCCTCTGCCGGCGT
TTTCTTTGCACGTACGACGGACACACATGGGCATACCATATAGCTGGTCCATGA
CATTAGGAGAGAGAACGTACGTGTTGACCTGTAGCTGAGATATAACAAGGTTGA
TTATAATATCACCAAACATGAAATCATCCAAGGATGACCCATAACTATCACTACTA
TAGTACTGCATCTGGTAAAAGAAATTGTATAGACTCTATTTCGAGCACTACCACA
TAACGCCTGCAATGTGACACCCTACCTATTCACTAATGTGCCTCTTCCCACACG
CTTTCCACCCGTACTGCTCACAGCTTTAAGAACCAGAACAAATGAGTAATATTAG
TGTCGGTTCATGGCTAAAACCAGCACTGATGTACATGACCACATATGTCAAATG
CTGCTTCTAGGCATGACCCGCTCTTACTAATACCTACTCATCGCTAGAAGAATTT
TCGGCTGATAAATTTTCAATTTAAGCAAGAGTTATCTGCGTTGGTTCATAACTCA
AACTGATGGCCCCAACCATATTAGTGCAAATTTCACATATGATCATAACCTTTTC
ATATGAAATCGGATCGAGATGAACTTTATATAAACATTGTAGCTGTCGATGATAC
CTACAATTTTATAGTTCACAACCTTTTTATTTCAAGTCATTTAAATGCCCAAATAG
GTGTTTCAAATCTCAGATAGAAATGTTCAAAAGTAAAAAAGGTCCCTATCATAAC
ATAATTGATATGTAAGTGAGTTGGAAAAAGATAAGTACGTGTGAGAGAGATCGG
GGATCAAATTCTGGTGTAATAATGTATGTATTTCAGTCATAAAAATTGGTAGCAG
TAGTTGGGGCTCTGTATATATACCGGTAAGGATGGGATGGTAGTAGAATAATTC
TTTTTTTGTTTTTAGTTTTTTCTGGTCCAAAATTTCAAATTTGGATCCCTTACTTGT
ACCAACTAATATTAATGAGTGTTGAGGGTAGTAGAGGTGCAACTTTACCATAATC
CCTCTGTTTCAGGTTATAAGACGTTTTGACTTTAAATTTGACCAAGTTTATGCGCA
AATATAGTAATATTTATAATACTATATTAGTTTCATTAAATAAATAATTGAATATATT
TTCATAATAAATTTGTGTTGAGTTCAAAATATTATTAATTTTTTCTACAAACTTGGT
CAAACTTGAAGCAGTTTGACTTTGACCAAAGTCAAAACGTCTTATAACTTGAAAC
GGATGGATTACTTTTTTTGTGGGGACAAGTTTACAATGTTTAATAAAGCACAATC
CATCTTAATGTTTTCAAGCTGAATATTGTAAAATTCATGGATAAACCAGCTTCTAA
ATGTTTAACCGGGAAAATGTCGAACGACAAATTAATATTTTTAAAGTGATGGGGAG
TATTAATTAAGGAGTGACAACTCAACTTTCAATATCGTACTAAACTGTGGGATTTA
TTTTCTAAAATTTTATACCCTGCCAATTCACGTGTTGTAGATCTTTTTTTTTCACTA
ACCGACACCAGGTATATCAATTTTATTGAATATAGCAGCAAAAAGAATGTGTTGT
ACTTGTAAACAAAAAGCAAACTGTACATAAAAAAAAATGCACTCCTATATAATTAA
GCTCATAAAGATGCTTTGCTTCGTGAGGGCCCAAGTTTTGATGACCTTTTGCTTG
ATCTCGAAATTAAAATTTAAGTACTGTTAAGGGAGTTCACACCACCATCAATTTTC
AGCCTGAAGAAACAGTTAAACAACGACCCCGATGACCAGTCTACTGCTCTCCAC
ATACTAGCTGCATTATTGATCACAAAACAAAACAAAACGAAATAAAAATCAGCAG
CGAGAGTGTGCAGAGAGAGACAAAGGTGATCTGGCGTGGATATCTCCCCATCC
ATCCTCACCCGCGCTGCCCATCACTCGCCGCCGCATACTCCATCATGTGGAGA
GAGGAAGACGAGGACCACAGCCAGAGCCCGGGTCGAGATGCCACCACGGCCA
CAACCCACGAGCCCGGCGCGACACCACCGCGCGCGCGTGAGCCAGCCACAAA
CGCCCGCGGATAGGCGCGCGCACGCCGGCCAATCCTACCACATCCCCGGCCT
CCGCGGCTCGCGAGCGCCGCTGCCATCCGATCCGCTGAGTTTTGGCTATTTAT
ACGTACCGCGGGAGCCTGTGTGCAGAGCAGTGCATCTCAAGAAGTACTCGAGC
AAAGAAGGAGAGAGCTTGGTGAGCTGCAGCCATGGTAGATCTGAGGGTAAATTT
CTAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTTTTTTGA
GCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGTTATGGTGTAAATA
TTACATAGCTTTAACTGATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGA
TGATAGTTACAGAACCGACGAACTAGTGCGCAGGCGGTTGTGCAGGCGATGCA
GTGCCAGGTGGGGGTGAGGGGCAGGACGGCCGTCCCGGCGAGGCAGCCCGC
GGGCAGGGTGTGGGGCGTCAGGAGGGCCGCCCGCGCCACCTCCGGGTTCAA
GGTGCTGGCGCTCGGCCCGGAGACCACCGGGGTCATCCAGAGGATGCAGCAG
CTGCTCGACATGGACACCACGCCCTTCACCGACAAGATCATCGCCGAGTACATC
TGGGTTGGAGGATCTGGAATTGACCTCAGAAGCAAATCAAGGACGATTTCGAAG
CCAGTGGAGGACCCGTCAGAGCTGCCGAAATGGAACTACGACGGATCGAGCAC
GGGGCAGGCTCCTGGGGAAGACAGTGAAGTCATCCTATACCCACAGGCCATAT
TCAAGGACCCATTCCGAGGAGGCAACAACATACTGGTTATCTGTGACACCTACA
CACCACAGGGGGAACCCATCCCTACTAACAAACGCCACATGGCTGCACAAATCT
TCAGTGACCCCAAGGTCACTTCACAAGTGCCATGGTTCGGAATCGAACAGGAGT
ACACTCTGATGCAGAGGGATGTGAACTGGCCTCTTGGCTGGCCTGTTGGAGGG
TACCCTGGCCCCCAGGGTCCATACTACTGCGCCGTAGGATCAGACAAGTCATTT
GGCCGTGACATATCAGATGCTCACTACAAGGCGTGCCTTTACGCTGGAATTGAA
ATCAGTGGAACAAACGGGGAGGTCATGCCTGGTCAGTGGGAGTACCAGGTTGG
ACCCAGCGTTGGTATTGATGCAGGAGACCACATATGGGCTTCCAGATACATTCT
CGAGAGAATCACGGAGCAAGCTGGTGTGGTGCTCACCCTTGACCCAAAACCAA
TCCAGGGTGACTGGAACGGAGCTGGCTGCCACACAAACTACAGCACATTGAGC
ATGCGCGAGGATGGAGGTTTCGACGTGATCAAGAAGGCAATCCTGAACCTTTCA
CTTCGCCATGACTTGCACATAGCCGCATATGGTGAAGGAAACGAGCGGAGGTT
GACAGGGCTACACGAGACAGCTAGCATATCAGACTTCTCATGGGGTGTGGCGA
ACCGTGGCTGCTCTATTCGTGTGGGGCGAGACACCGAGGCGAAGGGCAAAGG
ATACCTGGAGGACCGTCGCCCGGCCTCCAACATGGACCCGTACACCGTGACGG
CGCTGCTGGCCGAGACCACGATCCTGTGGGAGCCGACCCTCGAGGCGGAGGC
CCTCGCTGCCAAGAAGCTGGCGCTGAAGGTATGA SEQ ID NO: 43 Amino acid
sequence of translation product of SEQ ID NO: 42. Amino-terminal
bold residues from Gusplus and SpeI cloning site (intron removed)
MVDLRNRRTSAQAVVQAMQCQVGVRGRTAVPARQPAGRVWGVRRAARATSGFK
VLALGPETTGVIQRMQQLLDMDTTPFTDKIIAEYIWVGGSGIDLRSKSRTISKPVEDP
SELPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRGGNNILVICDTYTPQGEPIPT
NKRHMAAQIFSDPKVTSQVPWFGIEQEYTLMQRDVNWPLGWPVGGYPGPQGPYY
CAVGSDKSFGRDISDAHYKACLYAGIEISGTNGEVMPGQWEYQVGPSVGIDAGDHI
WASRYILERITEQAGVVLTLDPKPIQGDWNGAGCHTNYSTLSMREDGGFDVIKKAIL
NLSLRHDLHIAAYGEGNERRLTGLHETASISDFSWGVANRGCSIRVGRDTEAKGKG
YLEDRRPASNMDPYTVTALLAETTILWEPTLEAEALAAKKLALKV SEQ ID NO: 44 Maize
ubil promoter: 5'UTR intron shown in italics, TATA box at -30 is
underlined, 5' and 3' PstI cloning sites in bold
CTGCAGTGCAGCGTGACCCGGTCGTGCCCCTCTCTAGAGATAATGAGCATTGC
ATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTG
CAGTTTATCTATCTTTATACATATATTTAAACTTTACTCTACGAATAATATAATCTA
TAGTACTACAATAATATCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGACAT
GGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTA
GTGTGCATGTGTTCTCCTTTTTTTTTGCAAATAGCTTCACCTATATAATACTTCAT
CCATTTTATTAGTACATCCATTTAGGGTTTAGGGTTAATGGTTTTTATAGACTAAT
TTTTTTAGTACATCTATTTTATTCTATTTTAGCCTCTAAATTAAGAAAACTAAAACT
CTATTTTAGTTTTTTTATTTAATAATTTAGATATAAAATAGAATAAAATAAAGTGAC
TAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTT
GTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACA
CCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGAAGCAGACGGCACG
GCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGG
ACTTGCTCCGCTGTCGGCATCCAGAAATTGCGTGGCGGAGCGGCAGACGTGAG
CCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACGGCAGCTACGGGGG
ATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAATAAATAGA
CACCCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACA
CACAACCAGATCTCCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACG
CCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTTCTCTAGATCGGCGTTCCGGT
CCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGT
TTGTGTTAGATCCGTGCTGCTAGCGTTCGTACACGGATGCGACCTGTACGTCAG
ACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGGAATCCTGGGATGG
CTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTGTTTCGTTGCAT
AGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTC
GGGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGG
GCGGTCGTTCTAGATCGGAGTAGAATTCTGTTTCAAACTACCTGGTGGATTTATT
AATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATG
ATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGTTTTACTGA
TGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGG
GCGGTCGTTCATTCGTTCTAGATCGGAGTAGAATACTGTTTCAAACTACCTGGT
GTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACGAGT
TTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTTTT
ACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTG
AGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATATA
CTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCA
TACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTG
GTGTTACTTCTGCAG SEQ ID NO: 45 Hordeum GPT DNA coding sequence,
including targeting sequence coding domain
ATGGCATCCGCCCCCGCCTCCGCCTCCGCGGCCCTCTCCACCGCCGCCCCCG
CCGACAACGGGGCCGCCAAGCCCACGGAGCAGCGGCCGGTACAGGTGGCTAA
GCGATTGGAGAAGTTCAAAACAACAATTTTCACACAGATGAGCATGCTCGCAGT
GAAGCATGGAGCAATAAACCTTGGACAGGGGTTTCCCAATTTTGATGGCCCTGA
CTTTGTCAAAGATGCTGCTATTGAGGCTATCAAAGCTGGAAAGAATCAGTATGCA
AGAGGATATGGTGTGCCTGAATTGAACTCAGCTGTTGCTGAGAGATTTCTCAAG
GACAGTGGATTGCACATCGATCCTGATAAGGAAGTTACTGTTACATCTGGGTGC
ACAGAAGCAATAGCTGCAACGATATTGGGTCTGATCAACCCTGGGGATGAAGTC
ATACTGTTTGCTCCATTCTATGATTCTTATGAGGCTACACTGTCCATGGCTGGTG
CGAATGTCAAAGCCATTACACTCCGCCCTCCGGACTTTGCAGTCCCTCTTGAAG
AGCTAAAGGCTGCAGTCTCGAAGAATACCAGAGCAATAATGATTAATACACCTC
ACAACCCTACCGGGAAAATGTTCACAAGGGAGGAACTTGAGTTCATTGCTGATC
TCTGCAAGGAAAATGACGTGTTGCTCTTTGCCGATGAGGTCTACGACAAGCTGG
CGTTTGAGGCGGATCACATATCAATGGCTTCTATTCCTGGCATGTATGAGAGGA
CCGTCACTATGAACTCCCTGGGGAAGACGTTCTCCTTGACCGGATGGAAGATC
GGCTGGGCGATAGCACCACCGCACCTGACATGGGGCGTAAGGCAGGCACACT
CCTTCCTCACATTCGCCACCTCCACGCCGATGCAATCAGCAGCGGCGGCGGCC
CTGAGAGCACCGGACAGCTACTTTGAGGAGCTGAAGAGGGACTACGGCGCAAA
GAAAGCGCTGCTGGTGGACGGGCTCAAGGCGGCGGGCTTCATCGTCTACCCTT
CGAGCGGAACCTACTTCATCATGGTCGACCACACCCCGTTCGGGTTCGACAAC
GACGTCGAGTTCTGCGAGTACTTGATCCGCGAGGTCGGCGTCGTGGCCATCCC
GCCAAGCGTGTTCTACCTGAACCCGGAGGACGGGAAGAACCTGGTGAGGTTCA
CCTTCTGCAAGGACGACGACACGCTAAGGGCGGCGGTGGACAGGATGAAGGC
CAAGCTCAGGAAGAAATGA SEQ ID NO: 46: Hordeum GPT amino acid sequence,
including putative targeting sequence (in italics).
MASAPASASAALSTAAPADNGAAKPTEQRPVQVAKRLEKFKTTIFTQMSMLAVKHG
AINLGQGFPNFDGPDFVKDAAIEAIKAGKNQYARGYGVPELNSAVAERFLKDSGLHI
DPDKEVTVTSGCTEAIAATILGLINPGDEVILFAPFYDSYEATLSMAGANVKAITLRPP
DFAVPLEELKAAVSKNTRAIMINTPHNPTGKMFTREELEFIADLCKENDVLLFADEVY
DKLAFEADHISMASIPGMYERTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAH
SFLTFATSTPMQSAAAAALRAPDSYFEELKRDYGAKKALLVDGLKAAGFIVYPSSGT
YFIMVDHTPFGFDNDVEFCEYLIREVGVVAIPPSVFYLNPEDGKNLVRFTFCKDDDT
LRAAVDRMKAKLRKK SEQ ID NO: 47 Tomato rubisco small subunit (rbcS3C)
promoter + Arabidopsis GS1 DNA coding sequence; NcoI/AflIII splice
site shown in bold, ATG start of GS1 underlined.
GTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGTAATTTTACTTTGTT
GTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTTTTAATACTAATCTGCTTCTG
AAACTTGTAATGTATGTATATCAGTTTCTTATAATTTATCCAAGTAATATCTTCCAT
TCTCTATGCAATTGCCTGCATAAGCTCGACAAAAGAGTACATCAACCCCTCCTCC
TCTGGACTACTCTAGCTAAACTTGAATTTCCCCTTAAGATTATGAAATTGATATAT
CCTTAACAAACGACTCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTCTTTTGG
GTATATATAGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTGAAATG
GATACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAGGAGTA
ACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCTGTCTTAGACA
GATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTTCGCACTGAGGGTAT
ACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAAAGTTCCCATCGAGCTCTA
AAATCACATCTTTGGAATGAAATTCAATTAGAGATAAGTTGCTTCATAGCATAGG
TAAAATGGAAGATGTGAAGTAACCTGCAATAATCAGTGAAATGACATTAATACAC
TAAATACTTCATATGTAATTATCCTTTCCAGGTTAACAATACTCTATAAAGTAAGA
ATTATCAGAAATGGGCTCATCAAACTTTTGTACTATGTATTTCATATAAGGAAGTA
TAACTATACATAAGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAGAGAC
TGTTTTCGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAATTAT
CTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGTTTGTACC
ATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCATAAACAGTTTTCCC
CAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTAACAGTTCAAATATATGGC
GCAGAAATTACTCTATGCCCAAAACCAAACGAGAAAGAAACAAAATACAGGGGT
TGCAGACTTTATTTTCGTGTTAGGGTGTGTTTTTTCATGTAATTAATCAAAAAATA
TTATGACAAAAACATTTATACATATTTTTACTCAACACTCTGGGTATCAGGGTGG
GTTGTGTTCGACAATCAATATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTAAT
ATTTTCAGTTATACCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTCGT
CAGAAAGAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGAAA
AAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGAGTGAG
GGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCACAAAATCCAAT
GGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGTCCGTTAGATAGGA
AGCCTTATCACTATATATACAAGGCGTCCTAATAACCTCTTAGTAACCAATTATTT
CAGCACCATGTCTCTGCTCTCAGATCTCGTTAACCTCAACCTCACCGATGCCAC
CGGGAAAATCATCGCCGAATACATATGGATCGGTGGATCTGGAATGGATATCAG
AAGCAAAGCCAGGACACTACCAGGACCAGTGACTGATCCATCAAAGCTTCCCAA
GTGGAACTACGACGGATCCAGCACCGGTCAGGCTGCTGGAGAAGACAGTGAAG
TCATTCTATACCCTCAGGCAATATTCAAGGATCCCTTCAGGAAAGGCAACAACAT
CCTGGTGATGTGTGATGCTTACACACCAGCTGGTGATCCTATTCCAACCAACAA
GAGGCACAACGCTGCTAAGATCTTCAGCCACCCCGACGTTGCCAAGGAGGAGC
CTTGGTATGGGATTGAGCAAGAATACACTTTGATGCAAAAGGATGTGAACTGGC
CAATTGGTTGGCCTGTTGGTGGCTACCCTGGCCCTCAGGGACCTTACTACTGTG
GTGTGGGAGCTGACAAAGCCATTGGTCGTGACATTGTGGATGCTCACTACAAG
GCCTGTCTTTACGCCGGTATTGGTATTTCTGGTATCAATGGAGAAGTCATGCCA
GGCCAGTGGGAGTTCCAAGTCGGCCCTGTTGAGGGTATTAGTTCTGGTGATCA
AGTCTGGGTTGCTCGATACCTTCTCGAGAGGATCACTGAGATCTCTGGTGTAAT
TGTCAGCTTCGACCCGAAACCAGTCCCGGGTGACTGGAATGGAGCTGGAGCTC
ACTGCAACTACAGCACTAAGACAATGAGAAACGATGGAGGATTAGAAGTGATCA
AGAAAGCGATAGGGAAGCTTCAGCTGAAACACAAAGAACACATTGCTGCTTACG
GTGAAGGAAACGAGCGTCGTCTCACTGGAAAGCACGAAACCGCAGACATCAAC
ACATTCTCTTGGGGAGTCGCGAACCGTGGAGCGTCAGTGAGAGTGGGACGTGA
CACAGAGAAGGAAGGTAAAGGGTACTTCGAAGACAGAAGGCCAGCTTCTAACAT
GGATCCTTACGTTGTCACCTCCATGATCGCTGAGACGACCATACTCGGTTGA SEQ ID NO: 48:
Putative Clementine orange GPT coding sequence Derived from
BioChain (Hayward, CA orange cDNA library, cat# C1634340; Derived
from clementine PCR primers:
5'-ggccacatgtccgttgctaagtgcttggagaagttta-3' (AflIII oligo) [SEQ ID
NO: ] 5'-cgggcacgtgtcattttctcctcagcttctccttcatcct-3' (PmlI oligo)
[SEQ ID NO: ] ATG start site in bold, AflIII oligo binding site
(start of putative mature coding sequence) is underlined;
terminator sequence italicized.
ATGCTTAAGCCGTCCGCCTTCGGGTCTTCTTTTTCTTCCTCAGCTCTGCTTTCGT
TTTCGAAGCATTTGCATACAATAAGCATTACTGATTCTGTCAACACCAGAAGAAG
AGGAATCAGTACCGCTTGCCCTAGGTACCCTTCTCTCATGGCGAGCTTGTCCAC
CGTTTCCACCAATCAAAGCGACACCATCCAGAAGACCAATCTTCAGCCTCAACA
GGTTGCTAAGTGCTTGGAGAAGTTTAAAACTACAATCTTTACACAAATGAGTATG
CTTGCCATCAAACATGGAGCTATAAATCTTGGTCAAGGCTTTCCCAACTTTGATG
GCCCAGATTTTGTTAAAGATGCAGCGATTCAAGCCATAAGGGATGGGAAGAATC
AATATGCTCGTGGACATGGGGTTCCAGAGTTCAACTCTGCCATTGCTTCCCGGT
TTAAGAAAGATTCTGGGCTCGAGGTTGACCCTGAAAAGGAAGTTACTGTTACCT
CTGGGTGCACCGAAGCCATTGCTGCAACCATCTTAGGTTTGATTAATCCTGGAG
ATGAGGTGATCCTTTTTGCACCTTTCTATGATTCCTATGAAGCTACTCTCTCCAT
GGCTGGTGCTAAAATTAAATGCATCACATTGCGCCCTCCAGAATTTGCCATCCC
CATTGAAGAGCTCAAGTCTACAATCTCAAAAAATACTCGTGCAATTCTTATGAAC
ACTCCACATAACCCCACTGGAAAGATGTTCACTAGGGAGGAACTTAATGTTATTG
CATCTCTTTGCATTGAGAATGATGTGTTGGTTTTTAGTGATGAGGTCTATGATAA
GTTGGCTTTTGAAATGGATCACATTTCCATAGCCTCTCTTCCTGGAATGTATGAG
CGTACTGTAACCATGAATTCCTTAGGGAAGACATTCTCTTTAACAGGGTGGAAG
ATCGGGTGGGCAATAGCTCCACCGCACCTTACATGGGGGGTGCGGCAGGCAC
ACTCTTTTCTCACGTTTGCCACATCCACTCCAATGCAGTGGGCAGCTACAGCAG
CCCTTAGAGCTCCGGAGACGTACTATGAGGAGCTAAAGAGAGATTACTCGGCAA
AGAAGGCAATTTTGGTGGAGGGATTGAATGCTGTTGGTTTCAAGGTATTCCCAT
CTAGTGGGACATACTTTGTGGTTGTAGATCACACCCCATTTGGGCACGAAACTG
ATATTGCATTTTGTGAATATCTGATCAAGGAAGTTGGGGTTGTGGCAATTCCGAC
CAGCGTATTTTACTTGAATCCAGAGGATGGAAAGAATTTGGTGAGATTTACCTTC
TGCAAAGATGAAGGAACTTTGAGGTCTGCAGTTGACAGGATGAAGGAGAAGCT GAGGAGAAAATGA
SEQ ID NO: 49: Putative Clementine orange GPT amino acid sequence;
putative mature protein sequence begins at VAK shown in bold
underline.
MLKPSAFGSSFSSSALLSFSKHLHTISITDSVNTRRRGISTACPRYPSLMASLSTVST
NQSDTIQKTNLQPQQVAKCLEKFKTTIFTQMSMLAIKHGAINLGQGFPNFDGPDFVK
DAAIQAIRDGKNQYARGHGVPEFNSAIASRFKKDSGLEVDPEKEVTVTSGCTEAIAA
TILGLINPGDEVILFAPFYDSYEATLSMAGAKIKCITLRPPEFAIPIEELKSTISKNTRAIL
MNTPHNPTGKMFTREELNVIASLCIENDVLVFSDEVYDKLAFEMDHISIASLPGMYE
RTVTMNSLGKTFSLTGWKIGWAIAPPHLTWGVRQAHSFLTFATSTPMQWAATAALR
APETYYEELKRDYSAKKAILVEGLNAVGFKVFPSSGTYFVVVDHTPFGHETDIAFCE
YLIKEVGVVAIPTSVFYLNPEDGKNLVRFTFCKDEGTLRSAVDRMKEKLRRK
Sequence CWU 1
1
5111323DNAAradopsis thaliana 1atgtacctgg acataaatgg tgtgatgatc
aaacagttta gcttcaaagc ctctcttctc 60ccattctctt ctaatttccg acaaagctcc
gccaaaatcc atcgtcctat cggagccacc 120atgaccacag tttcgactca
gaacgagtct actcaaaaac ccgtccaggt ggcgaagaga 180ttagagaagt
tcaagactac tattttcact caaatgagca tattggcagt taaacatgga
240gcgatcaatt taggccaagg ctttcccaat ttcgacggtc ctgattttgt
taaagaagct 300gcgatccaag ctattaaaga tggtaaaaac cagtatgctc
gtggatacgg cattcctcag 360ctcaactctg ctatagctgc gcggtttcgt
gaagatacgg gtcttgttgt tgatcctgag 420aaagaagtta ctgttacatc
tggttgcaca gaagccatag ctgcagctat gttgggttta 480ataaaccctg
gtgatgaagt cattctcttt gcaccgtttt atgattccta tgaagcaaca
540ctctctatgg ctggtgctaa agtaaaagga atcactttac gtccaccgga
cttctccatc 600cctttggaag agcttaaagc tgcggtaact aacaagactc
gagccatcct tatgaacact 660ccgcacaacc cgaccgggaa gatgttcact
agggaggagc ttgaaaccat tgcatctctc 720tgcattgaaa acgatgtgct
tgtgttctcg gatgaagtat acgataagct tgcgtttgaa 780atggatcaca
tttctatagc ttctcttccc ggtatgtatg aaagaactgt gaccatgaat
840tccctgggaa agactttctc tttaaccgga tggaagatcg gctgggcgat
tgcgccgcct 900catctgactt ggggagttcg acaagcacac tcttacctca
cattcgccac atcaacacca 960gcacaatggg cagccgttgc agctctcaag
gcaccagagt cttacttcaa agagctgaaa 1020agagattaca atgtgaaaaa
ggagactctg gttaagggtt tgaaggaagt cggatttaca 1080gtgttcccat
cgagcgggac ttactttgtg gttgctgatc acactccatt tggaatggag
1140aacgatgttg ctttctgtga gtatcttatt gaagaagttg gggtcgttgc
gatcccaacg 1200agcgtctttt atctgaatcc agaagaaggg aagaatttgg
ttaggtttgc gttctgtaaa 1260gacgaagaga cgttgcgtgg tgcaattgag
aggatgaagc agaagcttaa gagaaaagtc 1320tga 13232440PRTAradopsis
thaliana 2Met Tyr Leu Asp Ile Asn Gly Val Met Ile Lys Gln Phe Ser
Phe Lys1 5 10 15Ala Ser Leu Leu Pro Phe Ser Ser Asn Phe Arg Gln Ser
Ser Ala Lys 20 25 30Ile His Arg Pro Ile Gly Ala Thr Met Thr Thr Val
Ser Thr Gln Asn 35 40 45Glu Ser Thr Gln Lys Pro Val Gln Val Ala Lys
Arg Leu Glu Lys Phe 50 55 60Lys Thr Thr Ile Phe Thr Gln Met Ser Ile
Leu Ala Val Lys His Gly65 70 75 80Ala Ile Asn Leu Gly Gln Gly Phe
Pro Asn Phe Asp Gly Pro Asp Phe 85 90 95Val Lys Glu Ala Ala Ile Gln
Ala Ile Lys Asp Gly Lys Asn Gln Tyr 100 105 110Ala Arg Gly Tyr Gly
Ile Pro Gln Leu Asn Ser Ala Ile Ala Ala Arg 115 120 125Phe Arg Glu
Asp Thr Gly Leu Val Val Asp Pro Glu Lys Glu Val Thr 130 135 140Val
Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Ala Met Leu Gly Leu145 150
155 160Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala Pro Phe Tyr Asp
Ser 165 170 175Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Lys Val Lys
Gly Ile Thr 180 185 190Leu Arg Pro Pro Asp Phe Ser Ile Pro Leu Glu
Glu Leu Lys Ala Ala 195 200 205Val Thr Asn Lys Thr Arg Ala Ile Leu
Met Asn Thr Pro His Asn Pro 210 215 220Thr Gly Lys Met Phe Thr Arg
Glu Glu Leu Glu Thr Ile Ala Ser Leu225 230 235 240Cys Ile Glu Asn
Asp Val Leu Val Phe Ser Asp Glu Val Tyr Asp Lys 245 250 255Leu Ala
Phe Glu Met Asp His Ile Ser Ile Ala Ser Leu Pro Gly Met 260 265
270Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe Ser Leu
275 280 285Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu
Thr Trp 290 295 300Gly Val Arg Gln Ala His Ser Tyr Leu Thr Phe Ala
Thr Ser Thr Pro305 310 315 320Ala Gln Trp Ala Ala Val Ala Ala Leu
Lys Ala Pro Glu Ser Tyr Phe 325 330 335Lys Glu Leu Lys Arg Asp Tyr
Asn Val Lys Lys Glu Thr Leu Val Lys 340 345 350Gly Leu Lys Glu Val
Gly Phe Thr Val Phe Pro Ser Ser Gly Thr Tyr 355 360 365Phe Val Val
Ala Asp His Thr Pro Phe Gly Met Glu Asn Asp Val Ala 370 375 380Phe
Cys Glu Tyr Leu Ile Glu Glu Val Gly Val Val Ala Ile Pro Thr385 390
395 400Ser Val Phe Tyr Leu Asn Pro Glu Glu Gly Lys Asn Leu Val Arg
Phe 405 410 415Ala Phe Cys Lys Asp Glu Glu Thr Leu Arg Gly Ala Ile
Glu Arg Met 420 425 430Lys Gln Lys Leu Lys Arg Lys Val 435
44031374DNAMedicago sativa 3atttccgttt tcgttttcat ttgattcatt
gaatcaaatc gaatcgaatc tttaggattc 60aatacagatt ccttagattt tactaagttt
gaaaccaaaa ccaaaacatg tctctccttt 120cagatcttat caaccttgac
ctctccgaaa ccaccgagaa aatcatcgcc gaatacatat 180ggattggtgg
atctggtttg gacttgagga gcaaagcaag gactctacca ggaccagtta
240ctgacccttc acagcttccc aagtggaact atgatggttc cagcacaggt
caagctcctg 300gagaagatag tgaagttatt atctacccac aagccatttt
caaggaccca tttagaaggg 360gtaacaatat cttggttatg tgtgatgcat
acactccagc tggagagccc attcccacca 420acaagagaca tgcagctgcc
aagattttca gccatcctga tgttgttgct gaagtaccat 480ggtatggtat
tgagcaagaa tacaccttgt tgcagaaaga catcaattgg cctcttggtt
540ggccagttgg tggttttcct ggacctcagg gaccatacta ttgtggagct
ggtgctgaca 600aggcatttgg ccgtgacatt gttgactcac attacaaagc
ctgtctttat gccggcatca 660acatcagtgg aatcaatggt gaagtgatgc
ctggtcaatg ggaattccaa gttggtccct 720cagttggtat ctctgctggt
gatgagatat gggttgctcg ttacattttg gagaggatca 780ctgaggttgc
tggtgtggtg ctttcctttg acccaaaacc aattaagggt gattggaatg
840gtgctggtgc tcacacaaat tacagcacca agtctatgag agaagatggt
ggctatgaag 900tcatcttgaa agcaattgag aagcttggga agaagcacaa
ggagcacatt gctgcttatg 960gagaaggcaa cgagcgtaga ttgacagggc
gacatgagac agctgacatt aacaccttct 1020tatggggtgt tgcaaaccgt
ggtgcgtcga ttagagttgg aagggacaca gagaaagcag 1080ggaaaggtta
tttcgaggat aggaggccat catctaacat ggatccatat gttgttactt
1140ccatgattgc agacaccacc attctctgga aaccataagc caccacacac
acatgcattg 1200aagtatttga aagtcattgt tgattccgca ttagaatttg
gtcattgttt tttctaggat 1260ttggatttgt gttattgtta tggttcacac
tttgtttgtt tgaatttgag gccttgttat 1320aggtttcata tttctttctc
ttgttctaag taaatgtcag aataataatg taat 13744356PRTMedicago sativa
4Met Ser Leu Leu Ser Asp Leu Ile Asn Leu Asp Leu Ser Glu Thr Thr1 5
10 15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Leu
Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Thr Asp
Pro Ser 35 40 45Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly
Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Ile Tyr Pro Gln Ala
Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu Val
Met Cys Asp Ala Tyr Thr 85 90 95Pro Ala Gly Glu Pro Ile Pro Thr Asn
Lys Arg His Ala Ala Ala Lys 100 105 110Ile Phe Ser His Pro Asp Val
Val Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr
Leu Leu Gln Lys Asp Ile Asn Trp Pro Leu Gly 130 135 140Trp Pro Val
Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155
160Ala Gly Ala Asp Lys Ala Phe Gly Arg Asp Ile Val Asp Ser His Tyr
165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn
Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro
Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Ile Trp Val Ala Arg
Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Val Ala Gly Val Val Leu
Ser Phe Asp Pro Lys Pro Ile Lys225 230 235 240Gly Asp Trp Asn Gly
Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Glu
Asp Gly Gly Tyr Glu Val Ile Leu Lys Ala Ile Glu Lys 260 265 270Leu
Gly Lys Lys His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280
285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300Leu Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val Gly
Arg Asp305 310 315 320Thr Glu Lys Ala Gly Lys Gly Tyr Phe Glu Asp
Arg Arg Pro Ser Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Ser
Met Ile Ala Asp Thr Thr Ile 340 345 350Leu Trp Lys Pro
35551419DNAMedicago sativa 5atcgatgaat tcgagctcgg tacccatttc
cgttttcgtt ttcatttgat tcattgaatc 60aaatcgaatc gaatctttag gattcaatac
agattcctta gattttacta agtttgaaac 120caaaaccaaa acatgtctct
cctttcagat cttatcaacc ttgacctctc cgaaaccacc 180gagaaaatca
tcgccgaata catatggatt ggtggatctg gtttggactt gaggagcaaa
240gcaaggactc taccaggacc agttactgac ccttcacagc ttcccaagtg
gaactatgat 300ggttccagca caggtcaagc tcctggagaa gatagtgaag
ttattatcta cccacaagcc 360attttcaagg acccatttag aaggggtaac
aatatcttgg ttatgtgtga tgcatacact 420ccagctggag agcccattcc
caccaacaag agacatgcag ctgccaagat tttcagccat 480cctgatgttg
ttgctgaagt accatggtat ggtattgagc aagaatacac cttgttgcag
540aaagacatca attggcctct tggttggcca gttggtggtt ttcctggacc
tcagggacca 600tactattgtg gagctggtgc tgacaaggca tttggccgtg
acattgttga ctcacattac 660aaagcctgtc tttatgccgg catcaacatc
agtggaatca atggtgaagt gatgcctggt 720caatgggaat tccaagttgg
tccctcagtt ggtatctctg ctggtgatga gatatgggtt 780gctcgttaca
ttttggagag gatcactgag gttgctggtg tggtgctttc ctttgaccca
840aaaccaatta agggtgattg gaatggtgct ggtgctcaca caaattacag
caccaagtct 900atgagagaag atggtggcta tgaagtcatc ttgaaagcaa
ttgagaagct tgggaagaag 960cacaaggagc acattgctgc ttatggagaa
ggcaacgagc gtagattgac agggcgacat 1020gagacagctg acattaacac
cttcttatgg ggtgttgcaa accgtggtgc gtcgattaga 1080gttggaaggg
acacagagaa agcagggaaa ggttatttcg aggataggag gccatcatct
1140aacatggatc catatgttgt tacttccatg attgcagaca ccaccattct
ctggaaacca 1200taagccacca cacacacatg cattgaagta tttgaaagtc
attgttgatt ccgcattaga 1260atttggtcat tgttttttct aggatttgga
tttgtgttat tgttatggtt cacactttgt 1320ttgtttgaat ttgaggcctt
gttataggtt tcatatttct ttctcttgtt ctaagtaaat 1380gtcagaataa
taatgtaatg gggatcctct agagtcgag 141961302DNAArtificial
SequenceSynthetic plasmid vector sequence 6aaaaaagaaa aaaaaaacat
atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta aaattcagtg
gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc
ctgtaagatg aggtttgcta actctttttg tccgttagat aggaagcctt
180atcactatat atacaaggcg tcctaataac ctcttagtaa ccaattattt
cagcaccatg 240tctctgctct cagatctcgt taacctcaac ctcaccgatg
ccaccgggaa aatcatcgcc 300gaatacatat ggatcggtgg atctggaatg
gatatcagaa gcaaagccag gacactacca 360ggaccagtga ctgatccatc
aaagcttccc aagtggaact acgacggatc cagcaccggt 420caggctgctg
gagaagacag tgaagtcatt ctataccctc aggcaatatt caaggatccc
480ttcaggaaag gcaacaacat cctggtgatg tgtgatgctt acacaccagc
tggtgatcct 540attccaacca acaagaggca caacgctgct aagatcttca
gccaccccga cgttgccaag 600gaggagcctt ggtatgggat tgagcaagaa
tacactttga tgcaaaagga tgtgaactgg 660ccaattggtt ggcctgttgg
tggctaccct ggccctcagg gaccttacta ctgtggtgtg 720ggagctgaca
aagccattgg tcgtgacatt gtggatgctc actacaaggc ctgtctttac
780gccggtattg gtatttctgg tatcaatgga gaagtcatgc caggccagtg
ggagttccaa 840gtcggccctg ttgagggtat tagttctggt gatcaagtct
gggttgctcg ataccttctc 900gagaggatca ctgagatctc tggtgtaatt
gtcagcttcg acccgaaacc agtcccgggt 960gactggaatg gagctggagc
tcactgcaac tacagcacta agacaatgag aaacgatgga 1020ggattagaag
tgatcaagaa agcgataggg aagcttcagc tgaaacacaa agaacacatt
1080gctgcttacg gtgaaggaaa cgagcgtcgt ctcactggaa agcacgaaac
cgcagacatc 1140aacacattct cttggggagt cgcgaaccgt ggagcgtcag
tgagagtggg acgtgacaca 1200gagaaggaag gtaaagggta cttcgaagac
agaaggccag cttctaacat ggatccttac 1260gttgtcacct ccatgatcgc
tgagacgacc atactcggtt ga 13027364PRTArabidopsis
thalianaSITE(1)..(9)amino terminal 9 amino acids vector encoded
sequence 7Met Val Asp Leu Arg Asn Arg Arg Thr Ser Met Ser Leu Leu
Ser Asp1 5 10 15Leu Val Asn Leu Asn Leu Thr Asp Ala Thr Gly Lys Ile
Ile Ala Glu 20 25 30Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp Ile Arg
Ser Lys Ala Arg 35 40 45Thr Leu Pro Gly Pro Val Thr Asp Pro Ser Lys
Leu Pro Lys Trp Asn 50 55 60Tyr Asp Gly Ser Ser Thr Gly Gln Ala Ala
Gly Glu Asp Ser Glu Val65 70 75 80Ile Leu Tyr Pro Gln Ala Ile Phe
Lys Asp Pro Phe Arg Lys Gly Asn 85 90 95Asn Ile Leu Val Met Cys Asp
Ala Tyr Thr Pro Ala Gly Asp Pro Ile 100 105 110Pro Thr Asn Lys Arg
His Asn Ala Ala Lys Ile Phe Ser His Pro Asp 115 120 125Val Ala Lys
Glu Glu Pro Trp Tyr Gly Ile Glu Gln Glu Tyr Thr Leu 130 135 140Met
Gln Lys Asp Val Asn Trp Pro Ile Gly Trp Pro Val Gly Gly Tyr145 150
155 160Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly Val Gly Ala Asp Lys
Ala 165 170 175Ile Gly Arg Asp Ile Val Asp Ala His Tyr Lys Ala Cys
Leu Tyr Ala 180 185 190Gly Ile Gly Ile Ser Gly Ile Asn Gly Glu Val
Met Pro Gly Gln Trp 195 200 205Glu Phe Gln Val Gly Pro Val Glu Gly
Ile Ser Ser Gly Asp Gln Val 210 215 220Trp Val Ala Arg Tyr Leu Leu
Glu Arg Ile Thr Glu Ile Ser Gly Val225 230 235 240Ile Val Ser Phe
Asp Pro Lys Pro Val Pro Gly Asp Trp Asn Gly Ala 245 250 255Gly Ala
His Cys Asn Tyr Ser Thr Lys Thr Met Arg Asn Asp Gly Gly 260 265
270Leu Glu Val Ile Lys Lys Ala Ile Gly Lys Leu Gln Leu Lys His Lys
275 280 285Glu His Ile Ala Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu
Thr Gly 290 295 300Lys His Glu Thr Ala Asp Ile Asn Thr Phe Ser Trp
Gly Val Ala Asn305 310 315 320Arg Gly Ala Ser Val Arg Val Gly Arg
Asp Thr Glu Lys Glu Gly Lys 325 330 335Gly Tyr Phe Glu Asp Arg Arg
Pro Ala Ser Asn Met Asp Pro Tyr Val 340 345 350Val Thr Ser Met Ile
Ala Glu Thr Thr Ile Leu Gly 355 36081817DNAArtificial
SequenceSynthetic plasmid vector sequence including vitis vinifera
GPT coding sequence 8aaaaaagaaa aaaaaaacat atcttgtttg tcagtatggg
aagtttgaga taaggacgag 60tgaggggtta aaattcagtg gccattgatt ttgtaatgcc
aagaaccaca aaatccaatg 120gttaccattc ctgtaagatg aggtttgcta
actctttttg tccgttagat aggaagcctt 180atcactatat atacaaggcg
tcctaataac ctcttagtaa ccaattattt cagcaccatg 240gtagatctga
gggtaaattt ctagtttttc tccttcattt tcttggttag gacccttttc
300tctttttatt tttttgagct ttgatctttc tttaaactga tctatttttt
aattgattgg 360ttatggtgta aatattacat agctttaact gataatctga
ttactttatt tcgtgtgtct 420atgatgatga tgatagttac agaaccgacg
aactagtatg cagctctctc aatgtacctg 480gacattccca gagttgctta
aaagaccagc ctttttaagg aggagtattg atagtatttc 540gagtagaagt
aggtccagct ccaagtatcc atctttcatg gcgtccgcat caacggtctc
600cgctccaaat acggaggctg agcagaccca taacccccct caacctctac
aggttgcaaa 660gcgcttggag aaattcaaaa caacaatctt tactcaaatg
agcatgcttg ccatcaaaca 720tggagcaata aaccttggcc aagggtttcc
caactttgat ggtcctgagt ttgtcaaaga 780agcagcaatt caagccatta
aggatgggaa aaaccaatat gctcgtggat atggagttcc 840tgatctcaac
tctgctgttg ctgatagatt caagaaggat acaggactcg tggtggaccc
900cgagaaggaa gttactgtta cttctggatg tacagaagca attgctgcta
ctatgctagg 960cttgataaat cctggtgatg aggtgatcct ctttgctcca
ttttatgatt cctatgaagc 1020cactctatcc atggctggtg cccaaataaa
atccatcact ttacgtcctc cggattttgc 1080tgtgcccatg gatgagctca
agtctgcaat ctcaaagaat acccgtgcaa tccttataaa 1140cactccccat
aaccccacag gaaagatgtt cacaagggag gaactgaatg tgattgcatc
1200cctctgcatt gagaatgatg tgttggtgtt tactgatgaa gtttacgaca
agttggcttt 1260cgaaatggat cacatttcca tggcttctct tcctgggatg
tacgagagga ccgtgactat 1320gaattcctta gggaaaactt tctccctgac
tggatggaag attggttgga cagtagctcc 1380cccacacctg acatggggag
tgaggcaagc ccactcattc ctcacgtttg ctacctgcac 1440cccaatgcaa
tgggcagctg caacagccct ccgggcccca gactcttact atgaagagct
1500aaagagagat tacagtgcaa agaaggcaat cctggtggag ggattgaagg
ctgtcggttt 1560cagggtatac ccatcaagtg ggacctattt tgtggtggtg
gatcacaccc catttgggtt 1620gaaagacgat attgcgtttt gtgagtatct
gatcaaggaa gttggggtgg tagcaattcc 1680gacaagcgtt ttctacttac
acccagaaga tggaaagaac cttgtgaggt ttaccttctg 1740taaagacgag
ggaactctga gagctgcagt tgaaaggatg aaggagaaac tgaagcctaa
1800acaatagggg cacgtga 18179459PRTVitis vinifera 9Met Val Asp Leu
Arg Asn Arg Arg Thr Ser Met Gln Leu Ser Gln Cys1 5 10 15Thr Trp Thr
Phe Pro Glu Leu Leu Lys Arg Pro Ala Phe Leu Arg Arg 20 25 30Ser Ile
Asp Ser Ile Ser Ser Arg Ser Arg Ser Ser Ser Lys Tyr Pro 35 40
45Ser Phe Met Ala Ser Ala Ser Thr Val Ser Ala Pro Asn Thr Glu Ala
50 55 60Glu Gln Thr His Asn Pro Pro Gln Pro Leu Gln Val Ala Lys Arg
Leu65 70 75 80Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Met
Leu Ala Ile 85 90 95Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro
Asn Phe Asp Gly 100 105 110Pro Glu Phe Val Lys Glu Ala Ala Ile Gln
Ala Ile Lys Asp Gly Lys 115 120 125Asn Gln Tyr Ala Arg Gly Tyr Gly
Val Pro Asp Leu Asn Ser Ala Val 130 135 140Ala Asp Arg Phe Lys Lys
Asp Thr Gly Leu Val Val Asp Pro Glu Lys145 150 155 160Glu Val Thr
Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Thr Met 165 170 175Leu
Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala Pro Phe 180 185
190Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala Gln Ile Lys
195 200 205Ser Ile Thr Leu Arg Pro Pro Asp Phe Ala Val Pro Met Asp
Glu Leu 210 215 220Lys Ser Ala Ile Ser Lys Asn Thr Arg Ala Ile Leu
Ile Asn Thr Pro225 230 235 240His Asn Pro Thr Gly Lys Met Phe Thr
Arg Glu Glu Leu Asn Val Ile 245 250 255Ala Ser Leu Cys Ile Glu Asn
Asp Val Leu Val Phe Thr Asp Glu Val 260 265 270Tyr Asp Lys Leu Ala
Phe Glu Met Asp His Ile Ser Met Ala Ser Leu 275 280 285Pro Gly Met
Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly Lys Thr 290 295 300Phe
Ser Leu Thr Gly Trp Lys Ile Gly Trp Thr Val Ala Pro Pro His305 310
315 320Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe Leu Thr Phe Ala
Thr 325 330 335Cys Thr Pro Met Gln Trp Ala Ala Ala Thr Ala Leu Arg
Ala Pro Asp 340 345 350Ser Tyr Tyr Glu Glu Leu Lys Arg Asp Tyr Ser
Ala Lys Lys Ala Ile 355 360 365Leu Val Glu Gly Leu Lys Ala Val Gly
Phe Arg Val Tyr Pro Ser Ser 370 375 380Gly Thr Tyr Phe Val Val Val
Asp His Thr Pro Phe Gly Leu Lys Asp385 390 395 400Asp Ile Ala Phe
Cys Glu Tyr Leu Ile Lys Glu Val Gly Val Val Ala 405 410 415Ile Pro
Thr Ser Val Phe Tyr Leu His Pro Glu Asp Gly Lys Asn Leu 420 425
430Val Arg Phe Thr Phe Cys Lys Asp Glu Gly Thr Leu Arg Ala Ala Val
435 440 445Glu Arg Met Lys Glu Lys Leu Lys Pro Lys Gln 450
455101446DNAArtificial SequenceSynthetic DNA encoding Oryza sativa
GPT protein, codons optimized for expression in E. coli
10atgtggatga acctggcagg ctttctggca accccggcaa ccgcaaccgc aacccgtcat
60gaaatgccgc tgaacccgag cagcagcgcg agctttctgc tgagcagcct gcgtcgtagc
120ctggtggcga gcctgcgtaa agcgagcccg gcagcagcag cagcactgag
cccgatggca 180agcgcaagca ccgtggcagc agaaaacggt gcagcaaaag
cagcagcaga aaaacagcag 240cagcagccgg tgcaggtggc gaaacgtctg
gaaaaattta aaaccaccat ttttacccag 300atgagcatgc tggcgattaa
acatggcgcg attaacctgg gccagggctt tccgaacttt 360gatggcccgg
attttgtgaa agaagcggcg attcaggcga ttaacgcggg caaaaaccag
420tatgcgcgtg gctatggcgt gccggaactg aacagcgcga ttgcggaacg
ttttctgaaa 480gatagcggcc tgcaggtgga tccggaaaaa gaagtgaccg
tgaccagcgg ctgcaccgaa 540gcgattgcgg cgaccattct gggcctgatt
aacccgggcg atgaagtgat tctgtttgcg 600ccgttttatg atagctatga
agcgaccctg agcatggcgg gcgcgaacgt gaaagcgatt 660accctgcgtc
cgccggattt tagcgtgccg ctggaagaac tgaaagcggc cgtgagcaaa
720aacacccgtg cgattatgat taacaccccg cataacccga ccggcaaaat
gtttacccgt 780gaagaactgg aatttattgc gaccctgtgc aaagaaaacg
atgtgctgct gtttgcggat 840gaagtgtatg ataaactggc gtttgaagcg
gatcatatta gcatggcgag cattccgggc 900atgtatgaac gtaccgtgac
catgaacagc ctgggcaaaa cctttagcct gaccggctgg 960aaaattggct
gggcgattgc gccgccgcat ctgacctggg gcgtgcgtca ggcacatagc
1020tttctgacct ttgcaacctg caccccgatg caggcagccg ccgcagcagc
actgcgtgca 1080ccggatagct attatgaaga actgcgtcgt gattatggcg
cgaaaaaagc gctgctggtg 1140aacggcctga aagatgcggg ctttattgtg
tatccgagca gcggcaccta ttttgtgatg 1200gtggatcata ccccgtttgg
ctttgataac gatattgaat tttgcgaata tctgattcgt 1260gaagtgggcg
tggtggcgat tccgccgagc gtgttttatc tgaacccgga agatggcaaa
1320aacctggtgc gttttacctt ttgcaaagat gatgaaaccc tgcgtgcggc
ggtggaacgt 1380atgaaaacca aactgcgtaa aaaaaagctt gcggccgcac
tcgagcacca ccaccaccac 1440cactga 144611481PRTArtificial
SequenceOryza sativa GPT protein sequence with amino- and
carboxyl-terminal vector sequences 11Met Trp Met Asn Leu Ala Gly
Phe Leu Ala Thr Pro Ala Thr Ala Thr1 5 10 15Ala Thr Arg His Glu Met
Pro Leu Asn Pro Ser Ser Ser Ala Ser Phe 20 25 30Leu Leu Ser Ser Leu
Arg Arg Ser Leu Val Ala Ser Leu Arg Lys Ala 35 40 45Ser Pro Ala Ala
Ala Ala Ala Leu Ser Pro Met Ala Ser Ala Ser Thr 50 55 60Val Ala Ala
Glu Asn Gly Ala Ala Lys Ala Ala Ala Glu Lys Gln Gln65 70 75 80Gln
Gln Pro Val Gln Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr 85 90
95Ile Phe Thr Gln Met Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn
100 105 110Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro Asp Phe Val
Lys Glu 115 120 125Ala Ala Ile Gln Ala Ile Asn Ala Gly Lys Asn Gln
Tyr Ala Arg Gly 130 135 140Tyr Gly Val Pro Glu Leu Asn Ser Ala Ile
Ala Glu Arg Phe Leu Lys145 150 155 160Asp Ser Gly Leu Gln Val Asp
Pro Glu Lys Glu Val Thr Val Thr Ser 165 170 175Gly Cys Thr Glu Ala
Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro 180 185 190Gly Asp Glu
Val Ile Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala 195 200 205Thr
Leu Ser Met Ala Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro 210 215
220Pro Asp Phe Ser Val Pro Leu Glu Glu Leu Lys Ala Ala Val Ser
Lys225 230 235 240Asn Thr Arg Ala Ile Met Ile Asn Thr Pro His Asn
Pro Thr Gly Lys 245 250 255Met Phe Thr Arg Glu Glu Leu Glu Phe Ile
Ala Thr Leu Cys Lys Glu 260 265 270Asn Asp Val Leu Leu Phe Ala Asp
Glu Val Tyr Asp Lys Leu Ala Phe 275 280 285Glu Ala Asp His Ile Ser
Met Ala Ser Ile Pro Gly Met Tyr Glu Arg 290 295 300Thr Val Thr Met
Asn Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp305 310 315 320Lys
Ile Gly Trp Ala Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg 325 330
335Gln Ala His Ser Phe Leu Thr Phe Ala Thr Cys Thr Pro Met Gln Ala
340 345 350Ala Ala Ala Ala Ala Leu Arg Ala Pro Asp Ser Tyr Tyr Glu
Glu Leu 355 360 365Arg Arg Asp Tyr Gly Ala Lys Lys Ala Leu Leu Val
Asn Gly Leu Lys 370 375 380Asp Ala Gly Phe Ile Val Tyr Pro Ser Ser
Gly Thr Tyr Phe Val Met385 390 395 400Val Asp His Thr Pro Phe Gly
Phe Asp Asn Asp Ile Glu Phe Cys Glu 405 410 415Tyr Leu Ile Arg Glu
Val Gly Val Val Ala Ile Pro Pro Ser Val Phe 420 425 430Tyr Leu Asn
Pro Glu Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys 435 440 445Lys
Asp Asp Glu Thr Leu Arg Ala Ala Val Glu Arg Met Lys Thr Lys 450 455
460Leu Arg Lys Lys Lys Leu Ala Ala Ala Leu Glu His His His His
His465 470 475 480His121251DNAArtificial SequenceSynthetic DNA
encoding Glycine max GPT protein, codons optimized for expression
in E. coli 12atgcatcatc accatcacca tggtaagcct atccctaacc ctctcctcgg
tctcgattct 60acggaaaacc tgtattttca gggaattgat cccttcaccg cgaaacgtct
ggaaaaattt 120cagaccacca tttttaccca gatgagcctg ctggcgatta
aacatggcgc gattaacctg 180ggccagggct ttccgaactt tgatggcccg
gaatttgtga aagaagcggc gattcaggcg 240attcgtgatg gcaaaaacca
gtatgcgcgt ggctatggcg tgccggatct gaacattgcg 300attgcggaac
gttttaaaaa agataccggc ctggtggtgg atccggaaaa agaaattacc
360gtgaccagcg gctgcaccga agcgattgcg gcgaccatga ttggcctgat
taacccgggc 420gatgaagtga ttatgtttgc gccgttttat gatagctatg
aagcgaccct gagcatggcg 480ggcgcgaaag tgaaaggcat taccctgcgt
ccgccggatt ttgcggtgcc gctggaagaa 540ctgaaaagca ccattagcaa
aaacacccgt gcgattctga ttaacacccc gcataacccg 600accggcaaaa
tgtttacccg tgaagaactg aactgcattg cgagcctgtg cattgaaaac
660gatgtgctgg tgtttaccga tgaagtgtat gataaactgg cgtttgatat
ggaacatatt 720agcatggcga gcctgccggg catgtttgaa cgtaccgtga
ccctgaacag cctgggcaaa 780acctttagcc tgaccggctg gaaaattggc
tgggcgattg cgccgccgca tctgagctgg 840ggcgtgcgtc aggcgcatgc
gtttctgacc tttgcaaccg cacatccgtt tcagtgcgca 900gcagcagcag
cactgcgtgc accggatagc tattatgtgg aactgaaacg tgattatatg
960gcgaaacgtg cgattctgat tgaaggcctg aaagcggtgg gctttaaagt
gtttccgagc 1020agcggcacct attttgtggt ggtggatcat accccgtttg
gcctggaaaa cgatgtggcg 1080ttttgcgaat atctggtgaa agaagtgggc
gtggtggcga ttccgaccag cgtgttttat 1140ctgaacccgg aagaaggcaa
aaacctggtg cgttttacct tttgcaaaga tgaagaaacc 1200attcgtagcg
cggtggaacg tatgaaagcg aaactgcgta aagtcgacta a
125113416PRTArtificial SequenceGlycine max GPT amino acid sequence
and amino-terminal vector sequence 13Met His His His His His His
Gly Lys Pro Ile Pro Asn Pro Leu Leu1 5 10 15Gly Leu Asp Ser Thr Glu
Asn Leu Tyr Phe Gln Gly Ile Asp Pro Phe 20 25 30Thr Ala Lys Arg Leu
Glu Lys Phe Gln Thr Thr Ile Phe Thr Gln Met 35 40 45Ser Leu Leu Ala
Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 50 55 60Pro Asn Phe
Asp Gly Pro Glu Phe Val Lys Glu Ala Ala Ile Gln Ala65 70 75 80Ile
Arg Asp Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Asp 85 90
95Leu Asn Ile Ala Ile Ala Glu Arg Phe Lys Lys Asp Thr Gly Leu Val
100 105 110Val Asp Pro Glu Lys Glu Ile Thr Val Thr Ser Gly Cys Thr
Glu Ala 115 120 125Ile Ala Ala Thr Met Ile Gly Leu Ile Asn Pro Gly
Asp Glu Val Ile 130 135 140Met Phe Ala Pro Phe Tyr Asp Ser Tyr Glu
Ala Thr Leu Ser Met Ala145 150 155 160Gly Ala Lys Val Lys Gly Ile
Thr Leu Arg Pro Pro Asp Phe Ala Val 165 170 175Pro Leu Glu Glu Leu
Lys Ser Thr Ile Ser Lys Asn Thr Arg Ala Ile 180 185 190Leu Ile Asn
Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu 195 200 205Glu
Leu Asn Cys Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val 210 215
220Phe Thr Asp Glu Val Tyr Asp Lys Leu Ala Phe Asp Met Glu His
Ile225 230 235 240Ser Met Ala Ser Leu Pro Gly Met Phe Glu Arg Thr
Val Thr Leu Asn 245 250 255Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly
Trp Lys Ile Gly Trp Ala 260 265 270Ile Ala Pro Pro His Leu Ser Trp
Gly Val Arg Gln Ala His Ala Phe 275 280 285Leu Thr Phe Ala Thr Ala
His Pro Phe Gln Cys Ala Ala Ala Ala Ala 290 295 300Leu Arg Ala Pro
Asp Ser Tyr Tyr Val Glu Leu Lys Arg Asp Tyr Met305 310 315 320Ala
Lys Arg Ala Ile Leu Ile Glu Gly Leu Lys Ala Val Gly Phe Lys 325 330
335Val Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp His Thr Pro
340 345 350Phe Gly Leu Glu Asn Asp Val Ala Phe Cys Glu Tyr Leu Val
Lys Glu 355 360 365Val Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr
Leu Asn Pro Glu 370 375 380Glu Gly Lys Asn Leu Val Arg Phe Thr Phe
Cys Lys Asp Glu Glu Thr385 390 395 400Ile Arg Ser Ala Val Glu Arg
Met Lys Ala Lys Leu Arg Lys Val Asp 405 410 415141278DNAHordeum
vulgare 14atggtagatc tgaggaaccg acgaactagt atggcatccg cccccgcctc
cgcctccgcg 60gccctctcca ccgccgcccc cgccgacaac ggggccgcca agcccacgga
gcagcggccg 120gtacaggtgg ctaagcgatt ggagaagttc aaaacaacaa
ttttcacaca gatgagcatg 180ctcgcagtga agcatggagc aataaacctt
ggacaggggt ttcccaattt tgatggccct 240gactttgtca aagatgctgc
tattgaggct atcaaagctg gaaagaatca gtatgcaaga 300ggatatggtg
tgcctgaatt gaactcagct gttgctgaga gatttctcaa ggacagtgga
360ttgcacatcg atcctgataa ggaagttact gttacatctg ggtgcacaga
agcaatagct 420gcaacgatat tgggtctgat caaccctggg gatgaagtca
tactgtttgc tccattctat 480gattcttatg aggctacact gtccatggct
ggtgcgaatg tcaaagccat tacactccgc 540cctccggact ttgcagtccc
tcttgaagag ctaaaggctg cagtctcgaa gaataccaga 600gcaataatga
ttaatacacc tcacaaccct accgggaaaa tgttcacaag ggaggaactt
660gagttcattg ctgatctctg caaggaaaat gacgtgttgc tctttgccga
tgaggtctac 720gacaagctgg cgtttgaggc ggatcacata tcaatggctt
ctattcctgg catgtatgag 780aggaccgtca ctatgaactc cctggggaag
acgttctcct tgaccggatg gaagatcggc 840tgggcgatag caccaccgca
cctgacatgg ggcgtaaggc aggcacactc cttcctcaca 900ttcgccacct
ccacgccgat gcaatcagca gcggcggcgg ccctgagagc accggacagc
960tactttgagg agctgaagag ggactacggc gcaaagaaag cgctgctggt
ggacgggctc 1020aaggcggcgg gcttcatcgt ctacccttcg agcggaacct
acttcatcat ggtcgaccac 1080accccgttcg ggttcgacaa cgacgtcgag
ttctgcgagt acttgatccg cgaggtcggc 1140gtcgtggcca tcccgccaag
cgtgttctac ctgaacccgg aggacgggaa gaacctggtg 1200aggttcacct
tctgcaagga cgacgacacg ctaagggcgg cggtggacag gatgaaggcc
1260aagctcagga agaaatga 127815425PRTHordeum vulgare 15Met Val Asp
Leu Arg Asn Arg Arg Thr Ser Met Ala Ser Ala Pro Ala1 5 10 15Ser Ala
Ser Ala Ala Leu Ser Thr Ala Ala Pro Ala Asp Asn Gly Ala 20 25 30Ala
Lys Pro Thr Glu Gln Arg Pro Val Gln Val Ala Lys Arg Leu Glu 35 40
45Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Met Leu Ala Val Lys
50 55 60His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly
Pro65 70 75 80Asp Phe Val Lys Asp Ala Ala Ile Glu Ala Ile Lys Ala
Gly Lys Asn 85 90 95Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu Leu Asn
Ser Ala Val Ala 100 105 110Glu Arg Phe Leu Lys Asp Ser Gly Leu His
Ile Asp Pro Asp Lys Glu 115 120 125Val Thr Val Thr Ser Gly Cys Thr
Glu Ala Ile Ala Ala Thr Ile Leu 130 135 140Gly Leu Ile Asn Pro Gly
Asp Glu Val Ile Leu Phe Ala Pro Phe Tyr145 150 155 160Asp Ser Tyr
Glu Ala Thr Leu Ser Met Ala Gly Ala Asn Val Lys Ala 165 170 175Ile
Thr Leu Arg Pro Pro Asp Phe Ala Val Pro Leu Glu Glu Leu Lys 180 185
190Ala Ala Val Ser Lys Asn Thr Arg Ala Ile Met Ile Asn Thr Pro His
195 200 205Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Glu Phe
Ile Ala 210 215 220Asp Leu Cys Lys Glu Asn Asp Val Leu Leu Phe Ala
Asp Glu Val Tyr225 230 235 240Asp Lys Leu Ala Phe Glu Ala Asp His
Ile Ser Met Ala Ser Ile Pro 245 250 255Gly Met Tyr Glu Arg Thr Val
Thr Met Asn Ser Leu Gly Lys Thr Phe 260 265 270Ser Leu Thr Gly Trp
Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu 275 280 285Thr Trp Gly
Val Arg Gln Ala His Ser Phe Leu Thr Phe Ala Thr Ser 290 295 300Thr
Pro Met Gln Ser Ala Ala Ala Ala Ala Leu Arg Ala Pro Asp Ser305 310
315 320Tyr Phe Glu Glu Leu Lys Arg Asp Tyr Gly Ala Lys Lys Ala Leu
Leu 325 330 335Val Asp Gly Leu Lys Ala Ala Gly Phe Ile Val Tyr Pro
Ser Ser Gly 340 345 350Thr Tyr Phe Ile Met Val Asp His Thr Pro Phe
Gly Phe Asp Asn Asp 355 360 365Val Glu Phe Cys Glu Tyr Leu Ile Arg
Glu Val Gly Val Val Ala Ile 370 375 380Pro Pro Ser Val Phe Tyr Leu
Asn Pro Glu Asp Gly Lys Asn Leu Val385 390 395 400Arg Phe Thr Phe
Cys Lys Asp Asp Asp Thr Leu Arg Ala Ala Val Asp 405 410 415Arg Met
Lys Ala Lys
Leu Arg Lys Lys 420 425161200DNAArtificial SequenceSynthetic DNA
encoding Danio rerio GPT protein, codons optimized for expression
in E. coli, including 5' and 3' vector sequences 16atgtccgtgg
cgaaacgtct ggaaaaattt aaaaccacca tttttaccca gatgagcatg 60ctggcgatta
aacatggcgc gattaacctg ggccagggct ttccgaactt tgatggcccg
120gattttgtga aagaagcggc gattcaggcg attcgtgatg gcaacaacca
gtatgcgcgt 180ggctatggcg tgccggatct gaacattgcg attagcgaac
gttataaaaa agataccggc 240ctggcggtgg atccggaaaa agaaattacc
gtgaccagcg gctgcaccga agcgattgcg 300gcgaccgtgc tgggcctgat
taacccgggc gatgaagtga ttgtgtttgc gccgttttat 360gatagctatg
aagcgaccct gagcatggcg ggcgcgaaag tgaaaggcat taccctgcgt
420ccgccggatt ttgcgctgcc gattgaagaa ctgaaaagca ccattagcaa
aaacacccgt 480gcgattctgc tgaacacccc gcataacccg accggcaaaa
tgtttacccc ggaagaactg 540aacaccattg cgagcctgtg cattgaaaac
gatgtgctgg tgtttagcga tgaagtgtat 600gataaactgg cgtttgatat
ggaacatatt agcattgcga gcctgccggg catgtttgaa 660cgtaccgtga
ccatgaacag cctgggcaaa acctttagcc tgaccggctg gaaaattggc
720tgggcgattg cgccgccgca tctgacctgg ggcgtgcgtc aggcgcatgc
gtttctgacc 780tttgcaacca gcaacccgat gcagtgggca gcagcagtgg
cactgcgtgc accggatagc 840tattataccg aactgaaacg tgattatatg
gcgaaacgta gcattctggt ggaaggcctg 900aaagcggtgg gctttaaagt
gtttccgagc agcggcacct attttgtggt ggtggatcat 960accccgtttg
gccatgaaaa cgatattgcg ttttgcgaat atctggtgaa agaagtgggc
1020gtggtggcga ttccgaccag cgtgttttat ctgaacccgg aagaaggcaa
aaacctggtg 1080cgttttacct tttgcaaaga tgaaggcacc ctgcgtgcgg
cggtggatcg tatgaaagaa 1140aaactgcgta aagtcgacaa gcttgcggcc
gcactcgagc accaccacca ccaccactga 120017399PRTDanio
rerioMISC_FEATURE(1)..(399)Amino- and carboxy-terminal amino acids
shown 17Met Ser Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe
Thr1 5 10 15Gln Met Ser Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu
Gly Gln 20 25 30Gly Phe Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu
Ala Ala Ile 35 40 45Gln Ala Ile Arg Asp Gly Asn Asn Gln Tyr Ala Arg
Gly Tyr Gly Val 50 55 60Pro Asp Leu Asn Ile Ala Ile Ser Glu Arg Tyr
Lys Lys Asp Thr Gly65 70 75 80Leu Ala Val Asp Pro Glu Lys Glu Ile
Thr Val Thr Ser Gly Cys Thr 85 90 95Glu Ala Ile Ala Ala Thr Val Leu
Gly Leu Ile Asn Pro Gly Asp Glu 100 105 110Val Ile Val Phe Ala Pro
Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser 115 120 125Met Ala Gly Ala
Lys Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe 130 135 140Ala Leu
Pro Ile Glu Glu Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg145 150 155
160Ala Ile Leu Leu Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr
165 170 175Pro Glu Glu Leu Asn Thr Ile Ala Ser Leu Cys Ile Glu Asn
Asp Val 180 185 190Leu Val Phe Ser Asp Glu Val Tyr Asp Lys Leu Ala
Phe Asp Met Glu 195 200 205His Ile Ser Ile Ala Ser Leu Pro Gly Met
Phe Glu Arg Thr Val Thr 210 215 220Met Asn Ser Leu Gly Lys Thr Phe
Ser Leu Thr Gly Trp Lys Ile Gly225 230 235 240Trp Ala Ile Ala Pro
Pro His Leu Thr Trp Gly Val Arg Gln Ala His 245 250 255Ala Phe Leu
Thr Phe Ala Thr Ser Asn Pro Met Gln Trp Ala Ala Ala 260 265 270Val
Ala Leu Arg Ala Pro Asp Ser Tyr Tyr Thr Glu Leu Lys Arg Asp 275 280
285Tyr Met Ala Lys Arg Ser Ile Leu Val Glu Gly Leu Lys Ala Val Gly
290 295 300Phe Lys Val Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Val
Asp His305 310 315 320Thr Pro Phe Gly His Glu Asn Asp Ile Ala Phe
Cys Glu Tyr Leu Val 325 330 335Lys Glu Val Gly Val Val Ala Ile Pro
Thr Ser Val Phe Tyr Leu Asn 340 345 350Pro Glu Glu Gly Lys Asn Leu
Val Arg Phe Thr Phe Cys Lys Asp Glu 355 360 365Gly Thr Leu Arg Ala
Ala Val Asp Arg Met Lys Glu Lys Leu Arg Lys 370 375 380Val Asp Lys
Leu Ala Ala Ala Leu Glu His His His His His His385 390
395181236DNAArabidopsis thaliana 18atggccaaaa tccatcgtcc tatcggagcc
accatgacca cagtttcgac tcagaacgag 60tctactcaaa aacccgtcca ggtggcgaag
agattagaga agttcaagac tactattttc 120actcaaatga gcatattggc
agttaaacat ggagcgatca atttaggcca aggctttccc 180aatttcgacg
gtcctgattt tgttaaagaa gctgcgatcc aagctattaa agatggtaaa
240aaccagtatg ctcgtggata cggcattcct cagctcaact ctgctatagc
tgcgcggttt 300cgtgaagata cgggtcttgt tgttgatcct gagaaagaag
ttactgttac atctggttgc 360acagaagcca tagctgcagc tatgttgggt
ttaataaacc ctggtgatga agtcattctc 420tttgcaccgt tttatgattc
ctatgaagca acactctcta tggctggtgc taaagtaaaa 480ggaatcactt
tacgtccacc ggacttctcc atccctttgg aagagcttaa agctgcggta
540actaacaaga ctcgagccat ccttatgaac actccgcaca acccgaccgg
gaagatgttc 600actagggagg agcttgaaac cattgcatct ctctgcattg
aaaacgatgt gcttgtgttc 660tcggatgaag tatacgataa gcttgcgttt
gaaatggatc acatttctat agcttctctt 720cccggtatgt atgaaagaac
tgtgaccatg aattccctgg gaaagacttt ctctttaacc 780ggatggaaga
tcggctgggc gattgcgccg cctcatctga cttggggagt tcgacaagca
840cactcttacc tcacattcgc cacatcaaca ccagcacaat gggcagccgt
tgcagctctc 900aaggcaccag agtcttactt caaagagctg aaaagagatt
acaatgtgaa aaaggagact 960ctggttaagg gtttgaagga agtcggattt
acagtgttcc catcgagcgg gacttacttt 1020gtggttgctg atcacactcc
atttggaatg gagaacgatg ttgctttctg tgagtatctt 1080attgaagaag
ttggggtcgt tgcgatccca acgagcgtct tttatctgaa tccagaagaa
1140gggaagaatt tggttaggtt tgcgttctgt aaagacgaag agacgttgcg
tggtgcaatt 1200gagaggatga agcagaagct taagagaaaa gtctga
123619411PRTArabidopsis thaliana 19Met Ala Lys Ile His Arg Pro Ile
Gly Ala Thr Met Thr Thr Val Ser1 5 10 15Thr Gln Asn Glu Ser Thr Gln
Lys Pro Val Gln Val Ala Lys Arg Leu 20 25 30Glu Lys Phe Lys Thr Thr
Ile Phe Thr Gln Met Ser Ile Leu Ala Val 35 40 45Lys His Gly Ala Ile
Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly 50 55 60Pro Asp Phe Val
Lys Glu Ala Ala Ile Gln Ala Ile Lys Asp Gly Lys65 70 75 80Asn Gln
Tyr Ala Arg Gly Tyr Gly Ile Pro Gln Leu Asn Ser Ala Ile 85 90 95Ala
Ala Arg Phe Arg Glu Asp Thr Gly Leu Val Val Asp Pro Glu Lys 100 105
110Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Ala Met
115 120 125Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile Leu Phe Ala
Pro Phe 130 135 140Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala Gly
Ala Lys Val Lys145 150 155 160Gly Ile Thr Leu Arg Pro Pro Asp Phe
Ser Ile Pro Leu Glu Glu Leu 165 170 175Lys Ala Ala Val Thr Asn Lys
Thr Arg Ala Ile Leu Met Asn Thr Pro 180 185 190His Asn Pro Thr Gly
Lys Met Phe Thr Arg Glu Glu Leu Glu Thr Ile 195 200 205Ala Ser Leu
Cys Ile Glu Asn Asp Val Leu Val Phe Ser Asp Glu Val 210 215 220Tyr
Asp Lys Leu Ala Phe Glu Met Asp His Ile Ser Ile Ala Ser Leu225 230
235 240Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn Ser Leu Gly Lys
Thr 245 250 255Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala Ile Ala
Pro Pro His 260 265 270Leu Thr Trp Gly Val Arg Gln Ala His Ser Tyr
Leu Thr Phe Ala Thr 275 280 285Ser Thr Pro Ala Gln Trp Ala Ala Val
Ala Ala Leu Lys Ala Pro Glu 290 295 300Ser Tyr Phe Lys Glu Leu Lys
Arg Asp Tyr Asn Val Lys Lys Glu Thr305 310 315 320Leu Val Lys Gly
Leu Lys Glu Val Gly Phe Thr Val Phe Pro Ser Ser 325 330 335Gly Thr
Tyr Phe Val Val Ala Asp His Thr Pro Phe Gly Met Glu Asn 340 345
350Asp Val Ala Phe Cys Glu Tyr Leu Ile Glu Glu Val Gly Val Val Ala
355 360 365Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu Glu Gly Lys
Asn Leu 370 375 380Val Arg Phe Ala Phe Cys Lys Asp Glu Glu Thr Leu
Arg Gly Ala Ile385 390 395 400Glu Arg Met Lys Gln Lys Leu Lys Arg
Lys Val 405 410201194DNAArabidopsis thaliana 20atggcgactc
agaacgagtc tactcaaaaa cccgtccagg tggcgaagag attagagaag 60ttcaagacta
ctattttcac tcaaatgagc atattggcag ttaaacatgg agcgatcaat
120ttaggccaag gctttcccaa tttcgacggt cctgattttg ttaaagaagc
tgcgatccaa 180gctattaaag atggtaaaaa ccagtatgct cgtggatacg
gcattcctca gctcaactct 240gctatagctg cgcggtttcg tgaagatacg
ggtcttgttg ttgatcctga gaaagaagtt 300actgttacat ctggttgcac
agaagccata gctgcagcta tgttgggttt aataaaccct 360ggtgatgaag
tcattctctt tgcaccgttt tatgattcct atgaagcaac actctctatg
420gctggtgcta aagtaaaagg aatcacttta cgtccaccgg acttctccat
ccctttggaa 480gagcttaaag ctgcggtaac taacaagact cgagccatcc
ttatgaacac tccgcacaac 540ccgaccggga agatgttcac tagggaggag
cttgaaacca ttgcatctct ctgcattgaa 600aacgatgtgc ttgtgttctc
ggatgaagta tacgataagc ttgcgtttga aatggatcac 660atttctatag
cttctcttcc cggtatgtat gaaagaactg tgaccatgaa ttccctggga
720aagactttct ctttaaccgg atggaagatc ggctgggcga ttgcgccgcc
tcatctgact 780tggggagttc gacaagcaca ctcttacctc acattcgcca
catcaacacc agcacaatgg 840gcagccgttg cagctctcaa ggcaccagag
tcttacttca aagagctgaa aagagattac 900aatgtgaaaa aggagactct
ggttaagggt ttgaaggaag tcggatttac agtgttccca 960tcgagcggga
cttactttgt ggttgctgat cacactccat ttggaatgga gaacgatgtt
1020gctttctgtg agtatcttat tgaagaagtt ggggtcgttg cgatcccaac
gagcgtcttt 1080tatctgaatc cagaagaagg gaagaatttg gttaggtttg
cgttctgtaa agacgaagag 1140acgttgcgtg gtgcaattga gaggatgaag
cagaagctta agagaaaagt ctga 119421397PRTArabidopsis thaliana 21Met
Ala Thr Gln Asn Glu Ser Thr Gln Lys Pro Val Gln Val Ala Lys1 5 10
15Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Ile Leu
20 25 30Ala Val Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn
Phe 35 40 45Asp Gly Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala Ile
Lys Asp 50 55 60Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Ile Pro Gln
Leu Asn Ser65 70 75 80Ala Ile Ala Ala Arg Phe Arg Glu Asp Thr Gly
Leu Val Val Asp Pro 85 90 95Glu Lys Glu Val Thr Val Thr Ser Gly Cys
Thr Glu Ala Ile Ala Ala 100 105 110Ala Met Leu Gly Leu Ile Asn Pro
Gly Asp Glu Val Ile Leu Phe Ala 115 120 125Pro Phe Tyr Asp Ser Tyr
Glu Ala Thr Leu Ser Met Ala Gly Ala Lys 130 135 140Val Lys Gly Ile
Thr Leu Arg Pro Pro Asp Phe Ser Ile Pro Leu Glu145 150 155 160Glu
Leu Lys Ala Ala Val Thr Asn Lys Thr Arg Ala Ile Leu Met Asn 165 170
175Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu Glu
180 185 190Thr Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu Val Phe
Ser Asp 195 200 205Glu Val Tyr Asp Lys Leu Ala Phe Glu Met Asp His
Ile Ser Ile Ala 210 215 220Ser Leu Pro Gly Met Tyr Glu Arg Thr Val
Thr Met Asn Ser Leu Gly225 230 235 240Lys Thr Phe Ser Leu Thr Gly
Trp Lys Ile Gly Trp Ala Ile Ala Pro 245 250 255Pro His Leu Thr Trp
Gly Val Arg Gln Ala His Ser Tyr Leu Thr Phe 260 265 270Ala Thr Ser
Thr Pro Ala Gln Trp Ala Ala Val Ala Ala Leu Lys Ala 275 280 285Pro
Glu Ser Tyr Phe Lys Glu Leu Lys Arg Asp Tyr Asn Val Lys Lys 290 295
300Glu Thr Leu Val Lys Gly Leu Lys Glu Val Gly Phe Thr Val Phe
Pro305 310 315 320Ser Ser Gly Thr Tyr Phe Val Val Ala Asp His Thr
Pro Phe Gly Met 325 330 335Glu Asn Asp Val Ala Phe Cys Glu Tyr Leu
Ile Glu Glu Val Gly Val 340 345 350Val Ala Ile Pro Thr Ser Val Phe
Tyr Leu Asn Pro Glu Glu Gly Lys 355 360 365Asn Leu Val Arg Phe Ala
Phe Cys Lys Asp Glu Glu Thr Leu Arg Gly 370 375 380Ala Ile Glu Arg
Met Lys Gln Lys Leu Lys Arg Lys Val385 390 395221680DNALycopersicon
esculentum 22ggtaccgttt gaatcctcct taaagttttt ctctggagaa actgtagtaa
ttttactttg 60ttgtgttccc ttcatctttt gaattaatgg catttgtttt aatactaatc
tgcttctgaa 120acttgtaatg tatgtatatc agtttcttat aatttatcca
agtaatatct tccattctct 180atgcaattgc ctgcataagc tcgacaaaag
agtacatcaa cccctcctcc tctggactac 240tctagctaaa cttgaatttc
cccttaagat tatgaaattg atatatcctt aacaaacgac 300tccttctgtt
ggaaaatgta gtacttgtct ttcttctttt gggtatatat agtttatata
360caccatacta tgtacaacat ccaagtagag tgaaatggat acatgtacaa
gacttatttg 420attgattgat gacttgagtt gccttaggag taacaaattc
ttaggtcaat aaatcgttga 480tttgaaatta atctctctgt cttagacaga
taggaattat gacttccaat ggtccagaaa 540gcaaagttcg cactgagggt
atacttggaa ttgagacttg cacaggtcca gaaaccaaag 600ttcccatcga
gctctaaaat cacatctttg gaatgaaatt caattagaga taagttgctt
660catagcatag gtaaaatgga agatgtgaag taacctgcaa taatcagtga
aatgacatta 720atacactaaa tacttcatat gtaattatcc tttccaggtt
aacaatactc tataaagtaa 780gaattatcag aaatgggctc atcaaacttt
tgtactatgt atttcatata aggaagtata 840actatacata agtgtataca
caactttatt cctattttgt aaaggtggag agactgtttt 900cgatggatct
aaagcaatat gtctataaaa tgcattgata taataattat ctgagaaaat
960ccagaattgg cgttggatta tttcagccaa atagaagttt gtaccatact
tgttgattcc 1020ttctaagtta aggtgaagta tcattcataa acagttttcc
ccaaagtact actcaccaag 1080tttccctttg tagaattaac agttcaaata
tatggcgcag aaattactct atgcccaaaa 1140ccaaacgaga aagaaacaaa
atacaggggt tgcagacttt attttcgtgt tagggtgtgt 1200tttttcatgt
aattaatcaa aaaatattat gacaaaaaca tttatacata tttttactca
1260acactctggg tatcagggtg ggttgtgttc gacaatcaat atggaaagga
agtattttcc 1320ttattttttt agttaatatt ttcagttata ccaaacatac
cttgtgatat tatttttaaa 1380aatgaaaaac tcgtcagaaa gaaaaagcaa
aagcaacaaa aaaattgcaa gtatttttta 1440aaaaagaaaa aaaaaacata
tcttgtttgt cagtatggga agtttgagat aaggacgagt 1500gaggggttaa
aattcagtgg ccattgattt tgtaatgcca agaaccacaa aatccaatgg
1560ttaccattcc tgtaagatga ggtttgctaa ctctttttgt ccgttagata
ggaagcctta 1620tcactatata tacaaggcgt cctaataacc tcttagtaac
caattatttc agcaccatgg 1680231230DNAPhyllostachys bambusoides
23atggcctccg cggccgtctc caccgtcgcc accgccgccg acggcgtcgc gaagccgacg
60gagaagcagc cggtacaggt cgcaaagcgt ttggaaaagt ttaagacaac aattttcaca
120cagatgagca tgcttgccat caagcatgga gcaataaacc tcggccaggg
ctttccgaat 180tttgatggcc ctgactttgt gaaagaagct gctattcaag
ctatcaatgc tgggaagaat 240cagtatgcaa gaggatatgg tgtgcctgaa
ctgaactcgg ctgttgctga aaggttcctg 300aaggacagtg gcttgcaagt
cgatcccgag aaggaagtta ctgtcacatc tgggtgcacg 360gaagcgatag
ctgcaacgat attgggtctt atcaaccctg gcgatgaagt gatcttgttt
420gctccattct atgattcata cgaggctacg ctgtcgatgg ctggtgccaa
tgtaaaagcc 480attactctcc gtcctccaga ttttgcagtc cctcttgagg
agctaaaggc cacagtctct 540aagaacacca gagcgataat gataaacaca
ccacacaatc ctactgggaa aatgttttct 600agggaagaac ttgaattcat
tgctactctc tgcaagaaaa atgatgtgtt gctttttgct 660gatgaggtct
atgacaagtt ggcatttgag gcagatcata tatcaatggc ttctattcct
720ggcatgtatg agaggactgt gactatgaac tctctgggga agacattctc
tctaacagga 780tggaagatcg gttgggcaat agcaccacca cacctgacat
ggggtgtaag gcaggcacac 840tcattcctca catttgccac ctgcacacca
atgcaatcgg cggcggcggc ggctcttaga 900gcaccagata gctactatgg
ggagctgaag agggattacg gtgcaaagaa agcgatacta 960gtcgacggac
tcaaggctgc aggttttatt gtttaccctt caagtggaac atactttgtc
1020atggtcgatc acaccccgtt tggtttcgac aatgatattg agttctgcga
gtatttgatc 1080cgcgaagtcg gtgttgtcgc cataccacca agcgtatttt
atctcaaccc tgaggatggg 1140aagaacttgg tgaggttcac cttctgcaag
gatgatgata cgctgagagc cgcagttgag 1200aggatgaaga caaagctcag
gaaaaaatga 123024409PRTPhyllostachys bambusoides 24Met Ala Ser Ala
Ala Val Ser Thr Val Ala Thr Ala Ala Asp Gly Val1 5 10 15Ala Lys Pro
Thr Glu Lys Gln Pro Val Gln Val Ala Lys Arg Leu Glu 20 25 30Lys Phe
Lys Thr Thr Ile Phe Thr Gln Met Ser Met Leu Ala Ile Lys 35 40 45His
Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro 50 55
60Asp Phe Val Lys Glu Ala Ala Ile Gln Ala Ile Asn Ala Gly Lys Asn65
70 75 80Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu Leu Asn Ser Ala Val
Ala 85 90 95Glu Arg Phe Leu Lys Asp Ser Gly Leu Gln Val Asp Pro
Glu Lys Glu 100 105 110Val Thr Val Thr Ser Gly Cys Thr Glu Ala Ile
Ala Ala Thr Ile Leu 115 120 125Gly Leu Ile Asn Pro Gly Asp Glu Val
Ile Leu Phe Ala Pro Phe Tyr 130 135 140Asp Ser Tyr Glu Ala Thr Leu
Ser Met Ala Gly Ala Asn Val Lys Ala145 150 155 160Ile Thr Leu Arg
Pro Pro Asp Phe Ala Val Pro Leu Glu Glu Leu Lys 165 170 175Ala Thr
Val Ser Lys Asn Thr Arg Ala Ile Met Ile Asn Thr Pro His 180 185
190Asn Pro Thr Gly Lys Met Phe Ser Arg Glu Glu Leu Glu Phe Ile Ala
195 200 205Thr Leu Cys Lys Lys Asn Asp Val Leu Leu Phe Ala Asp Glu
Val Tyr 210 215 220Asp Lys Leu Ala Phe Glu Ala Asp His Ile Ser Met
Ala Ser Ile Pro225 230 235 240Gly Met Tyr Glu Arg Thr Val Thr Met
Asn Ser Leu Gly Lys Thr Phe 245 250 255Ser Leu Thr Gly Trp Lys Ile
Gly Trp Ala Ile Ala Pro Pro His Leu 260 265 270Thr Trp Gly Val Arg
Gln Ala His Ser Phe Leu Thr Phe Ala Thr Cys 275 280 285Thr Pro Met
Gln Ser Ala Ala Ala Ala Ala Leu Arg Ala Pro Asp Ser 290 295 300Tyr
Tyr Gly Glu Leu Lys Arg Asp Tyr Gly Ala Lys Lys Ala Ile Leu305 310
315 320Val Asp Gly Leu Lys Ala Ala Gly Phe Ile Val Tyr Pro Ser Ser
Gly 325 330 335Thr Tyr Phe Val Met Val Asp His Thr Pro Phe Gly Phe
Asp Asn Asp 340 345 350Ile Glu Phe Cys Glu Tyr Leu Ile Arg Glu Val
Gly Val Val Ala Ile 355 360 365Pro Pro Ser Val Phe Tyr Leu Asn Pro
Glu Asp Gly Lys Asn Leu Val 370 375 380Arg Phe Thr Phe Cys Lys Asp
Asp Asp Thr Leu Arg Ala Ala Val Glu385 390 395 400Arg Met Lys Thr
Lys Leu Arg Lys Lys 405251858DNAOryza sativa 25aaaaaagaaa
aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta
aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg
120gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat
aggaagcctt 180atcactatat atacaaggcg tcctaataac ctcttagtaa
ccaattattt cagcaccatg 240gtagatctga gggtaaattt ctagtttttc
tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct
ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta
aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct
420atgatgatga tgatagttac agaaccgacg aactagtatg aatctggccg
gctttctcgc 480cacgcccgcg accgcgaccg cgacgcggca tgagatgccg
ttaaatccct cctcctccgc 540ctccttcctc ctctcctcgc tccgccgctc
gctcgtcgcg tcgctccgga aggcctcgcc 600ggcggcggcc gcggcgctct
cccccatggc ctccgcgtcc accgtcgccg ccgagaacgg 660cgccgccaag
gcggcggcgg agaagcagca gcagcagcct gtgcaggttg caaagcggtt
720ggaaaagttt aagacgacca ttttcacaca gatgagtatg cttgccatca
agcatggagc 780aataaacctt ggccagggtt ttccgaattt cgatggccct
gactttgtaa aagaggctgc 840tattcaagct atcaatgctg ggaagaatca
gtacgcaaga ggatatggtg tgcctgaact 900gaactcagct attgctgaaa
gattcctgaa ggacagcgga ctgcaagtcg atccggagaa 960ggaagttact
gtcacatctg gatgcacaga agctatagct gcaacaattt taggtctaat
1020taatccaggc gatgaagtga tattgtttgc tccattctat gattcatatg
aggctaccct 1080gtcaatggct ggtgccaacg taaaagccat tactctccgt
cctccagatt tttcagtccc 1140tcttgaagag ctaaaggctg cagtctcgaa
gaacaccaga gctattatga taaacacccc 1200gcacaatcct actgggaaaa
tgtttacaag ggaagaactt gagtttattg ccactctctg 1260caaggaaaat
gatgtgctgc tttttgctga tgaggtctac gacaagttag cttttgaggc
1320agatcatata tcaatggctt ctattcctgg catgtatgag aggaccgtga
ccatgaactc 1380tcttgggaag acattctctc ttacaggatg gaagatcggt
tgggcaatcg caccgccaca 1440cctgacatgg ggtgtaaggc aggcacactc
attcctcacg tttgcgacct gcacaccaat 1500gcaagcagct gcagctgcag
ctctgagagc accagatagc tactatgagg aactgaggag 1560ggattatgga
gctaagaagg cattgctagt caacggactc aaggatgcag gtttcattgt
1620ctatccttca agtggaacat acttcgtcat ggtcgaccac accccatttg
gtttcgacaa 1680tgatattgag ttctgcgagt atttgattcg cgaagtcggt
gttgtcgcca taccacctag 1740tgtattttat ctcaaccctg aggatgggaa
gaacttggtg aggttcacct tttgcaagga 1800tgatgagacg ctgagagccg
cggttgagag gatgaagaca aagctcagga aaaaatga 1858261724DNAArtificial
SequenceSynthetic DNA encoding Hordeum vulgare GPT protein
26aaaaaagaaa aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag
60tgaggggtta aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg
120gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat
aggaagcctt 180atcactatat atacaaggcg tcctaataac ctcttagtaa
ccaattattt cagcaccatg 240gtagatctga gggtaaattt ctagtttttc
tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct
ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta
aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct
420atgatgatga tgatagttac agaaccgacg aactagtatg gcatccgccc
ccgcctccgc 480ctccgcggcc ctctccaccg ccgcccccgc cgacaacggg
gccgccaagc ccacggagca 540gcggccggta caggtggcta agcgattgga
gaagttcaaa acaacaattt tcacacagat 600gagcatgctc gcagtgaagc
atggagcaat aaaccttgga caggggtttc ccaattttga 660tggccctgac
tttgtcaaag atgctgctat tgaggctatc aaagctggaa agaatcagta
720tgcaagagga tatggtgtgc ctgaattgaa ctcagctgtt gctgagagat
ttctcaagga 780cagtggattg cacatcgatc ctgataagga agttactgtt
acatctgggt gcacagaagc 840aatagctgca acgatattgg gtctgatcaa
ccctggggat gaagtcatac tgtttgctcc 900attctatgat tcttatgagg
ctacactgtc catggctggt gcgaatgtca aagccattac 960actccgccct
ccggactttg cagtccctct tgaagagcta aaggctgcag tctcgaagaa
1020taccagagca ataatgatta atacacctca caaccctacc gggaaaatgt
tcacaaggga 1080ggaacttgag ttcattgctg atctctgcaa ggaaaatgac
gtgttgctct ttgccgatga 1140ggtctacgac aagctggcgt ttgaggcgga
tcacatatca atggcttcta ttcctggcat 1200gtatgagagg accgtcacta
tgaactccct ggggaagacg ttctccttga ccggatggaa 1260gatcggctgg
gcgatagcac caccgcacct gacatggggc gtaaggcagg cacactcctt
1320cctcacattc gccacctcca cgccgatgca atcagcagcg gcggcggccc
tgagagcacc 1380ggacagctac tttgaggagc tgaagaggga ctacggcgca
aagaaagcgc tgctggtgga 1440cgggctcaag gcggcgggct tcatcgtcta
cccttcgagc ggaacctact tcatcatggt 1500cgaccacacc ccgttcgggt
tcgacaacga cgtcgagttc tgcgagtact tgatccgcga 1560ggtcggcgtc
gtggccatcc cgccaagcgt gttctacctg aacccggagg acgggaagaa
1620cctggtgagg ttcaccttct gcaaggacga cgacacgcta agggcggcgg
tggacaggat 1680gaaggccaag ctcaggaaga aatgattgag gggcgcacgt gtga
1724271868DNAArtificial SequenceSynthetic DNA encoding Arabidopsis
thaliana GPT protein 27catggagtca aagattcaaa tagaggacct aacagaactc
gccgtaaaga ctggcgaaca 60gttcatacag agtctcttac gactcaatga caagaagaaa
atcttcgtca acatggtgga 120gcacgacaca cttgtctact ccaaaaatat
caaagataca gtctcagaag accaaagggc 180aattgagact tttcaacaaa
gggtaatatc cggaaacctc ctcggattcc attgcccagc 240tatctgtcac
tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca
300ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc
ccaaagatgg 360acccccaccc acgaggagca tcgtggaaaa agaagacgtt
ccaaccacgt cttcaaagca 420agtggattga tgtgatatct ccactgacgt
aagggatgac gcacaatccc actatccttc 480gcaagaccct tcctctatat
aaggaagttc atttcatttg gagagaacac gggggactct 540tgaccatgta
cctggacata aatggtgtga tgatcaaaca gtttagcttc aaagcctctc
600ttctcccatt ctcttctaat ttccgacaaa gctccgccaa aatccatcgt
cctatcggag 660ccaccatgac cacagtttcg actcagaacg agtctactca
aaaacccgtc caggtggcga 720agagattaga gaagttcaag actactattt
tcactcaaat gagcatattg gcagttaaac 780atggagcgat caatttaggc
caaggctttc ccaatttcga cggtcctgat tttgttaaag 840aagctgcgat
ccaagctatt aaagatggta aaaaccagta tgctcgtgga tacggcattc
900ctcagctcaa ctctgctata gctgcgcggt ttcgtgaaga tacgggtctt
gttgttgatc 960ctgagaaaga agttactgtt acatctggtt gcacagaagc
catagctgca gctatgttgg 1020gtttaataaa ccctggtgat gaagtcattc
tctttgcacc gttttatgat tcctatgaag 1080caacactctc tatggctggt
gctaaagtaa aaggaatcac tttacgtcca ccggacttct 1140ccatcccttt
ggaagagctt aaagctgcgg taactaacaa gactcgagcc atccttatga
1200acactccgca caacccgacc gggaagatgt tcactaggga ggagcttgaa
accattgcat 1260ctctctgcat tgaaaacgat gtgcttgtgt tctcggatga
agtatacgat aagcttgcgt 1320ttgaaatgga tcacatttct atagcttctc
ttcccggtat gtatgaaaga actgtgacca 1380tgaattccct gggaaagact
ttctctttaa ccggatggaa gatcggctgg gcgattgcgc 1440cgcctcatct
gacttgggga gttcgacaag cacactctta cctcacattc gccacatcaa
1500caccagcaca atgggcagcc gttgcagctc tcaaggcacc agagtcttac
ttcaaagagc 1560tgaaaagaga ttacaatgtg aaaaaggaga ctctggttaa
gggtttgaag gaagtcggat 1620ttacagtgtt cccatcgagc gggacttact
ttgtggttgc tgatcacact ccatttggaa 1680tggagaacga tgttgctttc
tgtgagtatc ttattgaaga agttggggtc gttgcgatcc 1740caacgagcgt
cttttatctg aatccagaag aagggaagaa tttggttagg tttgcgttct
1800gtaaagacga agagacgttg cgtggtgcaa ttgagaggat gaagcagaag
cttaagagaa 1860aagtctga 1868281780DNAArtificial SequenceSynthetic
DNA encoding Arabidopsis thaliana GPT protein 28aaaaaagaaa
aaaaaaacat atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta
aaattcagtg gccattgatt ttgtaatgcc aagaaccaca aaatccaatg
120gttaccattc ctgtaagatg aggtttgcta actctttttg tccgttagat
aggaagcctt 180atcactatat atacaaggcg tcctaataac ctcttagtaa
ccaattattt cagcaccatg 240gtagatctga gggtaaattt ctagtttttc
tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct
ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta
aatattacat agctttaact gataatctga ttactttatt tcgtgtgtct
420atgatgatga tgatagttac agaaccgacg aactagtatg tacctggaca
taaatggtgt 480gatgatcaaa cagtttagct tcaaagcctc tcttctccca
ttctcttcta atttccgaca 540aagctccgcc aaaatccatc gtcctatcgg
agccaccatg accacagttt cgactcagaa 600cgagtctact caaaaacccg
tccaggtggc gaagagatta gagaagttca agactactat 660tttcactcaa
atgagcatat tggcagttaa acatggagcg atcaatttag gccaaggctt
720tcccaatttc gacggtcctg attttgttaa agaagctgcg atccaagcta
ttaaagatgg 780taaaaaccag tatgctcgtg gatacggcat tcctcagctc
aactctgcta tagctgcgcg 840gtttcgtgaa gatacgggtc ttgttgttga
tcctgagaaa gaagttactg ttacatctgg 900ttgcacagaa gccatagctg
cagctatgtt gggtttaata aaccctggtg atgaagtcat 960tctctttgca
ccgttttatg attcctatga agcaacactc tctatggctg gtgctaaagt
1020aaaaggaatc actttacgtc caccggactt ctccatccct ttggaagagc
ttaaagctgc 1080ggtaactaac aagactcgag ccatccttat gaacactccg
cacaacccga ccgggaagat 1140gttcactagg gaggagcttg aaaccattgc
atctctctgc attgaaaacg atgtgcttgt 1200gttctcggat gaagtatacg
ataagcttgc gtttgaaatg gatcacattt ctatagcttc 1260tcttcccggt
atgtatgaaa gaactgtgac catgaattcc ctgggaaaga ctttctcttt
1320aaccggatgg aagatcggct gggcgattgc gccgcctcat ctgacttggg
gagttcgaca 1380agcacactct tacctcacat tcgccacatc aacaccagca
caatgggcag ccgttgcagc 1440tctcaaggca ccagagtctt acttcaaaga
gctgaaaaga gattacaatg tgaaaaagga 1500gactctggtt aagggtttga
aggaagtcgg atttacagtg ttcccatcga gcgggactta 1560ctttgtggtt
gctgatcaca ctccatttgg aatggagaac gatgttgctt tctgtgagta
1620tcttattgaa gaagttgggg tcgttgcgat cccaacgagc gtcttttatc
tgaatccaga 1680agaagggaag aatttggtta ggtttgcgtt ctgtaaagac
gaagagacgt tgcgtggtgc 1740aattgagagg atgaagcaga agcttaagag
aaaagtctga 1780291155DNAArabidopsis thaliana 29gtggcgaaga
gattagagaa gttcaagact actattttca ctcaaatgag catattggca 60gttaaacatg
gagcgatcaa tttaggccaa ggctttccca atttcgacgg tcctgatttt
120gttaaagaag ctgcgatcca agctattaaa gatggtaaaa accagtatgc
tcgtggatac 180ggcattcctc agctcaactc tgctatagct gcgcggtttc
gtgaagatac gggtcttgtt 240gttgatcctg agaaagaagt tactgttaca
tctggttgca cagaagccat agctgcagct 300atgttgggtt taataaaccc
tggtgatgaa gtcattctct ttgcaccgtt ttatgattcc 360tatgaagcaa
cactctctat ggctggtgct aaagtaaaag gaatcacttt acgtccaccg
420gacttctcca tccctttgga agagcttaaa gctgcggtaa ctaacaagac
tcgagccatc 480cttatgaaca ctccgcacaa cccgaccggg aagatgttca
ctagggagga gcttgaaacc 540attgcatctc tctgcattga aaacgatgtg
cttgtgttct cggatgaagt atacgataag 600cttgcgtttg aaatggatca
catttctata gcttctcttc ccggtatgta tgaaagaact 660gtgaccatga
attccctggg aaagactttc tctttaaccg gatggaagat cggctgggcg
720attgcgccgc ctcatctgac ttggggagtt cgacaagcac actcttacct
cacattcgcc 780acatcaacac cagcacaatg ggcagccgtt gcagctctca
aggcaccaga gtcttacttc 840aaagagctga aaagagatta caatgtgaaa
aaggagactc tggttaaggg tttgaaggaa 900gtcggattta cagtgttccc
atcgagcggg acttactttg tggttgctga tcacactcca 960tttggaatgg
agaacgatgt tgctttctgt gagtatctta ttgaagaagt tggggtcgtt
1020gcgatcccaa cgagcgtctt ttatctgaat ccagaagaag ggaagaattt
ggttaggttt 1080gcgttctgta aagacgaaga gacgttgcgt ggtgcaattg
agaggatgaa gcagaagctt 1140aagagaaaag tctga 115530384PRTArabidopsis
thaliana 30Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr
Gln Met1 5 10 15Ser Ile Leu Ala Val Lys His Gly Ala Ile Asn Leu Gly
Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu Ala
Ala Ile Gln Ala 35 40 45Ile Lys Asp Gly Lys Asn Gln Tyr Ala Arg Gly
Tyr Gly Ile Pro Gln 50 55 60Leu Asn Ser Ala Ile Ala Ala Arg Phe Arg
Glu Asp Thr Gly Leu Val65 70 75 80Val Asp Pro Glu Lys Glu Val Thr
Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala Ala Met Leu Gly
Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Leu Phe Ala Pro Phe
Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120 125Gly Ala Lys
Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe Ser Ile 130 135 140Pro
Leu Glu Glu Leu Lys Ala Ala Val Thr Asn Lys Thr Arg Ala Ile145 150
155 160Leu Met Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg
Glu 165 170 175Glu Leu Glu Thr Ile Ala Ser Leu Cys Ile Glu Asn Asp
Val Leu Val 180 185 190Phe Ser Asp Glu Val Tyr Asp Lys Leu Ala Phe
Glu Met Asp His Ile 195 200 205Ser Ile Ala Ser Leu Pro Gly Met Tyr
Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly Lys Thr Phe Ser
Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230 235 240Ile Ala Pro Pro
His Leu Thr Trp Gly Val Arg Gln Ala His Ser Tyr 245 250 255Leu Thr
Phe Ala Thr Ser Thr Pro Ala Gln Trp Ala Ala Val Ala Ala 260 265
270Leu Lys Ala Pro Glu Ser Tyr Phe Lys Glu Leu Lys Arg Asp Tyr Asn
275 280 285Val Lys Lys Glu Thr Leu Val Lys Gly Leu Lys Glu Val Gly
Phe Thr 290 295 300Val Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Ala
Asp His Thr Pro305 310 315 320Phe Gly Met Glu Asn Asp Val Ala Phe
Cys Glu Tyr Leu Ile Glu Glu 325 330 335Val Gly Val Val Ala Ile Pro
Thr Ser Val Phe Tyr Leu Asn Pro Glu 340 345 350Glu Gly Lys Asn Leu
Val Arg Phe Ala Phe Cys Lys Asp Glu Glu Thr 355 360 365Leu Arg Gly
Ala Ile Glu Arg Met Lys Gln Lys Leu Lys Arg Lys Val 370 375
38031384PRTVitis vinifera 31Val Ala Lys Arg Leu Glu Lys Phe Lys Thr
Thr Ile Phe Thr Gln Met1 5 10 15Ser Met Leu Ala Ile Lys His Gly Ala
Ile Asn Leu Gly Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Glu Phe
Val Lys Glu Ala Ala Ile Gln Ala 35 40 45Ile Lys Asp Gly Lys Asn Gln
Tyr Ala Arg Gly Tyr Gly Val Pro Asp 50 55 60Leu Asn Ser Ala Val Ala
Asp Arg Phe Lys Lys Asp Thr Gly Leu Val65 70 75 80Val Asp Pro Glu
Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala
Thr Met Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Leu
Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120
125Gly Ala Gln Ile Lys Ser Ile Thr Leu Arg Pro Pro Asp Phe Ala Val
130 135 140Pro Met Asp Glu Leu Lys Ser Ala Ile Ser Lys Asn Thr Arg
Ala Ile145 150 155 160Leu Ile Asn Thr Pro His Asn Pro Thr Gly Lys
Met Phe Thr Arg Glu 165 170 175Glu Leu Asn Val Ile Ala Ser Leu Cys
Ile Glu Asn Asp Val Leu Val 180 185 190Phe Thr Asp Glu Val Tyr Asp
Lys Leu Ala Phe Glu Met Asp His Ile 195 200 205Ser Met Ala Ser Leu
Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly
Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Thr225 230 235
240Val Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe
245 250 255Leu Thr Phe Ala Thr Cys Thr Pro Met Gln Trp Ala Ala Ala
Thr Ala 260 265 270Leu Arg Ala Pro Asp Ser Tyr Tyr Glu Glu Leu Lys
Arg Asp Tyr Ser 275 280 285Ala Lys Lys Ala Ile Leu Val Glu Gly Leu
Lys Ala Val Gly Phe Arg 290 295 300Val Tyr Pro Ser Ser Gly Thr Tyr
Phe Val Val Val Asp His Thr Pro305 310 315 320Phe Gly Leu Lys Asp
Asp Ile Ala Phe Cys Glu Tyr Leu Ile Lys Glu
325 330 335Val Gly Val Val Ala Ile Pro Thr Ser Val Phe Tyr Leu His
Pro Glu 340 345 350Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys
Asp Glu Gly Thr 355 360 365Leu Arg Ala Ala Val Glu Arg Met Lys Glu
Lys Leu Lys Pro Lys Gln 370 375 38032383PRTOryza sativa 32Val Ala
Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln Met1 5 10 15Ser
Met Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25
30Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala
35 40 45Ile Asn Ala Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro
Glu 50 55 60Leu Asn Ser Ala Ile Ala Glu Arg Phe Leu Lys Asp Ser Gly
Leu Gln65 70 75 80Val Asp Pro Glu Lys Glu Val Thr Val Thr Ser Gly
Cys Thr Glu Ala 85 90 95Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro
Gly Asp Glu Val Ile 100 105 110Leu Phe Ala Pro Phe Tyr Asp Ser Tyr
Glu Ala Thr Leu Ser Met Ala 115 120 125Gly Ala Asn Val Lys Ala Ile
Thr Leu Arg Pro Pro Asp Phe Ser Val 130 135 140Pro Leu Glu Glu Leu
Lys Ala Ala Val Ser Lys Asn Thr Arg Ala Ile145 150 155 160Met Ile
Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu 165 170
175Glu Leu Glu Phe Ile Ala Thr Leu Cys Lys Glu Asn Asp Val Leu Leu
180 185 190Phe Ala Asp Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp
His Ile 195 200 205Ser Met Ala Ser Ile Pro Gly Met Tyr Glu Arg Thr
Val Thr Met Asn 210 215 220Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly
Trp Lys Ile Gly Trp Ala225 230 235 240Ile Ala Pro Pro His Leu Thr
Trp Gly Val Arg Gln Ala His Ser Phe 245 250 255Leu Thr Phe Ala Thr
Cys Thr Pro Met Gln Ala Ala Ala Ala Ala Ala 260 265 270Leu Arg Ala
Pro Asp Ser Tyr Tyr Glu Glu Leu Arg Arg Asp Tyr Gly 275 280 285Ala
Lys Lys Ala Leu Leu Val Asn Gly Leu Lys Asp Ala Gly Phe Ile 290 295
300Val Tyr Pro Ser Ser Gly Thr Tyr Phe Val Met Val Asp His Thr
Pro305 310 315 320Phe Gly Phe Asp Asn Asp Ile Glu Phe Cys Glu Tyr
Leu Ile Arg Glu 325 330 335Val Gly Val Val Ala Ile Pro Pro Ser Val
Phe Tyr Leu Asn Pro Glu 340 345 350Asp Gly Lys Asn Leu Val Arg Phe
Thr Phe Cys Lys Asp Asp Glu Thr 355 360 365Leu Arg Ala Ala Val Glu
Arg Met Lys Thr Lys Leu Arg Lys Lys 370 375 38033383PRTGlycine max
33Ala Lys Arg Leu Glu Lys Phe Gln Thr Thr Ile Phe Thr Gln Met Ser1
5 10 15Leu Leu Ala Ile Lys His Gly Ala Ile Asn Leu Gly Gln Gly Phe
Pro 20 25 30Asn Phe Asp Gly Pro Glu Phe Val Lys Glu Ala Ala Ile Gln
Ala Ile 35 40 45Arg Asp Gly Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val
Pro Asp Leu 50 55 60Asn Ile Ala Ile Ala Glu Arg Phe Lys Lys Asp Thr
Gly Leu Val Val65 70 75 80Asp Pro Glu Lys Glu Ile Thr Val Thr Ser
Gly Cys Thr Glu Ala Ile 85 90 95Ala Ala Thr Met Ile Gly Leu Ile Asn
Pro Gly Asp Glu Val Ile Met 100 105 110Phe Ala Pro Phe Tyr Asp Ser
Tyr Glu Ala Thr Leu Ser Met Ala Gly 115 120 125Ala Lys Val Lys Gly
Ile Thr Leu Arg Pro Pro Asp Phe Ala Val Pro 130 135 140Leu Glu Glu
Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg Ala Ile Leu145 150 155
160Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met Phe Thr Arg Glu Glu
165 170 175Leu Asn Cys Ile Ala Ser Leu Cys Ile Glu Asn Asp Val Leu
Val Phe 180 185 190Thr Asp Glu Val Tyr Asp Lys Leu Ala Phe Asp Met
Glu His Ile Ser 195 200 205Met Ala Ser Leu Pro Gly Met Phe Glu Arg
Thr Val Thr Leu Asn Ser 210 215 220Leu Gly Lys Thr Phe Ser Leu Thr
Gly Trp Lys Ile Gly Trp Ala Ile225 230 235 240Ala Pro Pro His Leu
Ser Trp Gly Val Arg Gln Ala His Ala Phe Leu 245 250 255Thr Phe Ala
Thr Ala His Pro Phe Gln Cys Ala Ala Ala Ala Ala Leu 260 265 270Arg
Ala Pro Asp Ser Tyr Tyr Val Glu Leu Lys Arg Asp Tyr Met Ala 275 280
285Lys Arg Ala Ile Leu Ile Glu Gly Leu Lys Ala Val Gly Phe Lys Val
290 295 300Phe Pro Ser Ser Gly Thr Tyr Phe Val Val Val Asp His Thr
Pro Phe305 310 315 320Gly Leu Glu Asn Asp Val Ala Phe Cys Glu Tyr
Leu Val Lys Glu Val 325 330 335Gly Val Val Ala Ile Pro Thr Ser Val
Phe Tyr Leu Asn Pro Glu Glu 340 345 350Gly Lys Asn Leu Val Arg Phe
Thr Phe Cys Lys Asp Glu Glu Thr Ile 355 360 365Arg Ser Ala Val Glu
Arg Met Lys Ala Lys Leu Arg Lys Val Asp 370 375 38034383PRTHordeum
vulgare 34Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr
Gln Met1 5 10 15Ser Met Leu Ala Val Lys His Gly Ala Ile Asn Leu Gly
Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Asp Phe Val Lys Asp Ala
Ala Ile Glu Ala 35 40 45Ile Lys Ala Gly Lys Asn Gln Tyr Ala Arg Gly
Tyr Gly Val Pro Glu 50 55 60 Leu Asn Ser Ala Val Ala Glu Arg Phe
Leu Lys Asp Ser Gly Leu His65 70 75 80Ile Asp Pro Asp Lys Glu Val
Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala Thr Ile Leu
Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Leu Phe Ala Pro
Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120 125Gly Ala
Asn Val Lys Ala Ile Thr Leu Arg Pro Pro Asp Phe Ala Val 130 135
140Pro Leu Glu Glu Leu Lys Ala Ala Val Ser Lys Asn Thr Arg Ala
Ile145 150 155 160Met Ile Asn Thr Pro His Asn Pro Thr Gly Lys Met
Phe Thr Arg Glu 165 170 175Glu Leu Glu Phe Ile Ala Asp Leu Cys Lys
Glu Asn Asp Val Leu Leu 180 185 190Phe Ala Asp Glu Val Tyr Asp Lys
Leu Ala Phe Glu Ala Asp His Ile 195 200 205Ser Met Ala Ser Ile Pro
Gly Met Tyr Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly Lys
Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230 235 240Ile
Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ser Phe 245 250
255Leu Thr Phe Ala Thr Ser Thr Pro Met Gln Ser Ala Ala Ala Ala Ala
260 265 270Leu Arg Ala Pro Asp Ser Tyr Phe Glu Glu Leu Lys Arg Asp
Tyr Gly 275 280 285Ala Lys Lys Ala Leu Leu Val Asp Gly Leu Lys Ala
Ala Gly Phe Ile 290 295 300Val Tyr Pro Ser Ser Gly Thr Tyr Phe Ile
Met Val Asp His Thr Pro305 310 315 320Phe Gly Phe Asp Asn Asp Val
Glu Phe Cys Glu Tyr Leu Ile Arg Glu 325 330 335Val Gly Val Val Ala
Ile Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu 340 345 350Asp Gly Lys
Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Asp Asp Thr 355 360 365Leu
Arg Ala Ala Val Asp Arg Met Lys Ala Lys Leu Arg Lys Lys 370 375
38035382PRTDanio rerio 35Val Ala Lys Arg Leu Glu Lys Phe Lys Thr
Thr Ile Phe Thr Gln Met1 5 10 15Ser Met Leu Ala Ile Lys His Gly Ala
Ile Asn Leu Gly Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly Pro Asp Phe
Val Lys Glu Ala Ala Ile Gln Ala 35 40 45Ile Arg Asp Gly Asn Asn Gln
Tyr Ala Arg Gly Tyr Gly Val Pro Asp 50 55 60Leu Asn Ile Ala Ile Ser
Glu Arg Tyr Lys Lys Asp Thr Gly Leu Ala65 70 75 80Val Asp Pro Glu
Lys Glu Ile Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90 95Ile Ala Ala
Thr Val Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile 100 105 110Val
Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser Met Ala 115 120
125Gly Ala Lys Val Lys Gly Ile Thr Leu Arg Pro Pro Asp Phe Ala Leu
130 135 140Pro Ile Glu Glu Leu Lys Ser Thr Ile Ser Lys Asn Thr Arg
Ala Ile145 150 155 160Leu Leu Asn Thr Pro His Asn Pro Thr Gly Lys
Met Phe Thr Pro Glu 165 170 175Glu Leu Asn Thr Ile Ala Ser Leu Cys
Ile Glu Asn Asp Val Leu Val 180 185 190Phe Ser Asp Glu Val Tyr Asp
Lys Leu Ala Phe Asp Met Glu His Ile 195 200 205Ser Ile Ala Ser Leu
Pro Gly Met Phe Glu Arg Thr Val Thr Met Asn 210 215 220Ser Leu Gly
Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp Ala225 230 235
240Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln Ala His Ala Phe
245 250 255Leu Thr Phe Ala Thr Ser Asn Pro Met Gln Trp Ala Ala Ala
Val Ala 260 265 270Leu Arg Ala Pro Asp Ser Tyr Tyr Thr Glu Leu Lys
Arg Asp Tyr Met 275 280 285Ala Lys Arg Ser Ile Leu Val Glu Gly Leu
Lys Ala Val Gly Phe Lys 290 295 300Val Phe Pro Ser Ser Gly Thr Tyr
Phe Val Val Val Asp His Thr Pro305 310 315 320Phe Gly His Glu Asn
Asp Ile Ala Phe Cys Glu Tyr Leu Val Lys Glu 325 330 335Val Gly Val
Val Ala Ile Pro Thr Ser Val Phe Tyr Leu Asn Pro Glu 340 345 350Glu
Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Glu Gly Thr 355 360
365Leu Arg Ala Ala Val Asp Arg Met Lys Glu Lys Leu Arg Lys 370 375
38036383PRTPhyllostachys bambusoides 36Val Ala Lys Arg Leu Glu Lys
Phe Lys Thr Thr Ile Phe Thr Gln Met1 5 10 15Ser Met Leu Ala Ile Lys
His Gly Ala Ile Asn Leu Gly Gln Gly Phe 20 25 30Pro Asn Phe Asp Gly
Pro Asp Phe Val Lys Glu Ala Ala Ile Gln Ala 35 40 45Ile Asn Ala Gly
Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu 50 55 60Leu Asn Ser
Ala Val Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu Gln65 70 75 80Val
Asp Pro Glu Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 85 90
95Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile
100 105 110Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser
Met Ala 115 120 125Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro Pro
Asp Phe Ala Val 130 135 140Pro Leu Glu Glu Leu Lys Ala Thr Val Ser
Lys Asn Thr Arg Ala Ile145 150 155 160Met Ile Asn Thr Pro His Asn
Pro Thr Gly Lys Met Phe Ser Arg Glu 165 170 175Glu Leu Glu Phe Ile
Ala Thr Leu Cys Lys Lys Asn Asp Val Leu Leu 180 185 190Phe Ala Asp
Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His Ile 195 200 205Ser
Met Ala Ser Ile Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn 210 215
220Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly Trp
Ala225 230 235 240Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln
Ala His Ser Phe 245 250 255Leu Thr Phe Ala Thr Cys Thr Pro Met Gln
Ser Ala Ala Ala Ala Ala 260 265 270Leu Arg Ala Pro Asp Ser Tyr Tyr
Gly Glu Leu Lys Arg Asp Tyr Gly 275 280 285Ala Lys Lys Ala Ile Leu
Val Asp Gly Leu Lys Ala Ala Gly Phe Ile 290 295 300Val Tyr Pro Ser
Ser Gly Thr Tyr Phe Val Met Val Asp His Thr Pro305 310 315 320Phe
Gly Phe Asp Asn Asp Ile Glu Phe Cys Glu Tyr Leu Ile Arg Glu 325 330
335Val Gly Val Val Ala Ile Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu
340 345 350Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Asp
Asp Thr 355 360 365Leu Arg Ala Ala Val Glu Arg Met Lys Thr Lys Leu
Arg Lys Lys 370 375 3803734DNAArtificial SequenceSynthetic primer
sequence 37cccatcgatg tacctggaca taaatggtgt gatg
343837DNAArtificial SequenceSynthetic primer sequence 38gatggtacct
cagacttttc tcttaagctt ctgcttc 37392992DNAArtificial
SequenceSynthetic expression cassette 39ctgcagcaaa gaaacgttat
tagttggtgc ttttggtggt aggaatgtag ttttctgaca 60aagtcaatta ctgaatataa
aaaaaatctg cacagctctg cgtcaacagt tgtccaaggg 120atgcctcaaa
aatctgtgca gattatcagt cgtcacgcag aagcagaaca tcatggtgtg
180ctaggtcagc ttcttgcatt gggccatgaa tccggttggt tgttaatctc
tcctctctta 240ttctcttata ttaagatgca taactctttt atgtagtcta
aaaaaaaatc cagtggatcg 300gatagtagta cgtcatggtg ccattaggta
ccgttgaacc taacagatat ttatgcatgt 360gtatatatat agctatatag
acaaaattga tgccgattat agacccaaaa gcaataggta 420tatataatat
aatacagacc acaccaccaa actaagaatc gatcaaatag acaaggcatg
480tctccaaatt gtcttaaact atttccgtag gttcagccgt tcaggagtcg
aatcagcctc 540tgccggcgtt ttctttgcac gtacgacgga cacacatggg
cataccatat agctggtcca 600tgacattagg agagagaacg tacgtgttga
cctgtagctg agatataaca aggttgatta 660taatatcacc aaacatgaaa
tcatccaagg atgacccata actatcacta ctatagtact 720gcatctggta
aaagaaattg tatagactct atttcgagca ctaccacata acgcctgcaa
780tgtgacaccc tacctattca ctaatgtgcc tcttcccaca cgctttccac
ccgtactgct 840cacagcttta agaaccagaa caaatgagta atattagtgt
cggttcatgg ctaaaaccag 900cactgatgta catgaccaca tatgtcaaat
gctgcttcta ggcatgaccc gctcttacta 960atacctactc atcgctagaa
gaattttcgg ctgataaatt ttcaatttaa gcaagagtta 1020tctgcgttgg
ttcataactc aaactgatgg ccccaaccat attagtgcaa atttcacata
1080tgatcataac cttttcatat gaaatcggat cgagatgaac tttatataaa
cattgtagct 1140gtcgatgata cctacaattt tatagttcac aaccttttta
tttcaagtca tttaaatgcc 1200caaataggtg tttcaaatct cagatagaaa
tgttcaaaag taaaaaaggt ccctatcata 1260acataattga tatgtaagtg
agttggaaaa agataagtac gtgtgagaga gatcggggat 1320caaattctgg
tgtaataatg tatgtatttc agtcataaaa attggtagca gtagttgggg
1380ctctgtatat ataccggtaa ggatgggatg gtagtagaat aattcttttt
ttgtttttag 1440ttttttctgg tccaaaattt caaatttgga tcccttactt
gtaccaacta atattaatga 1500gtgttgaggg tagtagaggt gcaactttac
cataatccct ctgtttcagg ttataagacg 1560ttttgacttt aaatttgacc
aagtttatgc gcaaatatag taatatttat aatactatat 1620tagtttcatt
aaataaataa ttgaatatat tttcataata aatttgtgtt gagttcaaaa
1680tattattaat tttttctaca aacttggtca aacttgaagc agtttgactt
tgaccaaagt 1740caaaacgtct tataacttga aacggatgga ttactttttt
tgtggggaca agtttacaat 1800gtttaataaa gcacaatcca tcttaatgtt
ttcaagctga atattgtaaa attcatggat 1860aaaccagctt ctaaatgttt
aaccgggaaa atgtcgaacg acaaattaat atttttaagt 1920gatggggagt
attaattaag gagtgacaac tcaactttca atatcgtact aaactgtggg
1980atttattttc taaaatttta taccctgcca attcacgtgt tgtagatctt
tttttttcac 2040taaccgacac caggtatatc aattttattg aatatagcag
caaaaagaat gtgttgtact 2100tgtaaacaaa aagcaaactg tacataaaaa
aaaatgcact cctatataat taagctcata 2160aagatgcttt gcttcgtgag
ggcccaagtt ttgatgacct tttgcttgat ctcgaaatta 2220aaatttaagt
actgttaagg gagttcacac caccatcaat tttcagcctg aagaaacagt
2280taaacaacga ccccgatgac cagtctactg ctctccacat actagctgca
ttattgatca 2340caaaacaaaa caaaacgaaa taaaaatcag cagcgagagt
gtgcagagag agacaaaggt 2400gatctggcgt ggatatctcc ccatccatcc
tcacccgcgc tgcccatcac tcgccgccgc 2460atactccatc atgtggagag
aggaagacga ggaccacagc cagagcccgg gtcgagatgc 2520caccacggcc
acaacccacg agcccggcgc gacaccaccg cgcgcgcgtg agccagccac
2580aaacgcccgc ggataggcgc gcgcacgccg gccaatccta ccacatcccc
ggcctccgcg 2640gctcgcgagc gccgctgcca tccgatccgc tgagttttgg
ctatttatac gtaccgcggg 2700agcctgtgtg cagagcagtg catctcaaga
agtactcgag caaagaagga gagagcttgg 2760tgagctgcag
ccatggtaga tctgagggta aatttctagt ttttctcctt cattttcttg
2820gttaggaccc ttttctcttt ttattttttt gagctttgat ctttctttaa
actgatctat 2880tttttaattg attggttatg gtgtaaatat tacatagctt
taactgataa tctgattact 2940ttatttcgtg tgtctatgat gatgatgata
gttacagaac cgacgaacta gt 2992401281DNAHordeum vulgare 40gcgcaggcgg
ttgtgcaggc gatgcagtgc caggtggggg tgaggggcag gacggccgtc 60ccggcgaggc
agcccgcggg cagggtgtgg ggcgtcagga gggccgcccg cgccacctcc
120gggttcaagg tgctggcgct cggcccggag accaccgggg tcatccagag
gatgcagcag 180ctgctcgaca tggacaccac gcccttcacc gacaagatca
tcgccgagta catctgggtt 240ggaggatctg gaattgacct cagaagcaaa
tcaaggacga tttcgaagcc agtggaggac 300ccgtcagagc tgccgaaatg
gaactacgac ggatcgagca cggggcaggc tcctggggaa 360gacagtgaag
tcatcctata cccacaggcc atattcaagg acccattccg aggaggcaac
420aacatactgg ttatctgtga cacctacaca ccacaggggg aacccatccc
tactaacaaa 480cgccacatgg ctgcacaaat cttcagtgac cccaaggtca
cttcacaagt gccatggttc 540ggaatcgaac aggagtacac tctgatgcag
agggatgtga actggcctct tggctggcct 600gttggagggt accctggccc
ccagggtcca tactactgcg ccgtaggatc agacaagtca 660tttggccgtg
acatatcaga tgctcactac aaggcgtgcc tttacgctgg aattgaaatc
720agtggaacaa acggggaggt catgcctggt cagtgggagt accaggttgg
acccagcgtt 780ggtattgatg caggagacca catatgggct tccagataca
ttctcgagag aatcacggag 840caagctggtg tggtgctcac ccttgaccca
aaaccaatcc agggtgactg gaacggagct 900ggctgccaca caaactacag
cacattgagc atgcgcgagg atggaggttt cgacgtgatc 960aagaaggcaa
tcctgaacct ttcacttcgc catgacttgc acatagccgc atatggtgaa
1020ggaaacgagc ggaggttgac agggctacac gagacagcta gcatatcaga
cttctcatgg 1080ggtgtggcga accgtggctg ctctattcgt gtggggcgag
acaccgaggc gaagggcaaa 1140ggatacctgg aggaccgtcg cccggcctcc
aacatggacc cgtacaccgt gacggcgctg 1200ctggccgaga ccacgatcct
gtgggagccg accctcgagg cggaggccct cgctgccaag 1260aagctggcgc
tgaaggtatg a 128141426PRTHordeum vulgare 41Ala Gln Ala Val Val Gln
Ala Met Gln Cys Gln Val Gly Val Arg Gly1 5 10 15Arg Thr Ala Val Pro
Ala Arg Gln Pro Ala Gly Arg Val Trp Gly Val 20 25 30Arg Arg Ala Ala
Arg Ala Thr Ser Gly Phe Lys Val Leu Ala Leu Gly 35 40 45Pro Glu Thr
Thr Gly Val Ile Gln Arg Met Gln Gln Leu Leu Asp Met 50 55 60Asp Thr
Thr Pro Phe Thr Asp Lys Ile Ile Ala Glu Tyr Ile Trp Val65 70 75
80Gly Gly Ser Gly Ile Asp Leu Arg Ser Lys Ser Arg Thr Ile Ser Lys
85 90 95Pro Val Glu Asp Pro Ser Glu Leu Pro Lys Trp Asn Tyr Asp Gly
Ser 100 105 110Ser Thr Gly Gln Ala Pro Gly Glu Asp Ser Glu Val Ile
Leu Tyr Pro 115 120 125Gln Ala Ile Phe Lys Asp Pro Phe Arg Gly Gly
Asn Asn Ile Leu Val 130 135 140Ile Cys Asp Thr Tyr Thr Pro Gln Gly
Glu Pro Ile Pro Thr Asn Lys145 150 155 160Arg His Met Ala Ala Gln
Ile Phe Ser Asp Pro Lys Val Thr Ser Gln 165 170 175Val Pro Trp Phe
Gly Ile Glu Gln Glu Tyr Thr Leu Met Gln Arg Asp 180 185 190Val Asn
Trp Pro Leu Gly Trp Pro Val Gly Gly Tyr Pro Gly Pro Gln 195 200
205Gly Pro Tyr Tyr Cys Ala Val Gly Ser Asp Lys Ser Phe Gly Arg Asp
210 215 220Ile Ser Asp Ala His Tyr Lys Ala Cys Leu Tyr Ala Gly Ile
Glu Ile225 230 235 240Ser Gly Thr Asn Gly Glu Val Met Pro Gly Gln
Trp Glu Tyr Gln Val 245 250 255Gly Pro Ser Val Gly Ile Asp Ala Gly
Asp His Ile Trp Ala Ser Arg 260 265 270Tyr Ile Leu Glu Arg Ile Thr
Glu Gln Ala Gly Val Val Leu Thr Leu 275 280 285Asp Pro Lys Pro Ile
Gln Gly Asp Trp Asn Gly Ala Gly Cys His Thr 290 295 300Asn Tyr Ser
Thr Leu Ser Met Arg Glu Asp Gly Gly Phe Asp Val Ile305 310 315
320Lys Lys Ala Ile Leu Asn Leu Ser Leu Arg His Asp Leu His Ile Ala
325 330 335Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu Thr Gly Leu His
Glu Thr 340 345 350Ala Ser Ile Ser Asp Phe Ser Trp Gly Val Ala Asn
Arg Gly Cys Ser 355 360 365Ile Arg Val Gly Arg Asp Thr Glu Ala Lys
Gly Lys Gly Tyr Leu Glu 370 375 380Asp Arg Arg Pro Ala Ser Asn Met
Asp Pro Tyr Thr Val Thr Ala Leu385 390 395 400Leu Ala Glu Thr Thr
Ile Leu Trp Glu Pro Thr Leu Glu Ala Glu Ala 405 410 415Leu Ala Ala
Lys Lys Leu Ala Leu Lys Val 420 425424273DNAArtificial
SequenceSynthetic expression cassette 42ctgcagcaaa gaaacgttat
tagttggtgc ttttggtggt aggaatgtag ttttctgaca 60aagtcaatta ctgaatataa
aaaaaatctg cacagctctg cgtcaacagt tgtccaaggg 120atgcctcaaa
aatctgtgca gattatcagt cgtcacgcag aagcagaaca tcatggtgtg
180ctaggtcagc ttcttgcatt gggccatgaa tccggttggt tgttaatctc
tcctctctta 240ttctcttata ttaagatgca taactctttt atgtagtcta
aaaaaaaatc cagtggatcg 300gatagtagta cgtcatggtg ccattaggta
ccgttgaacc taacagatat ttatgcatgt 360gtatatatat agctatatag
acaaaattga tgccgattat agacccaaaa gcaataggta 420tatataatat
aatacagacc acaccaccaa actaagaatc gatcaaatag acaaggcatg
480tctccaaatt gtcttaaact atttccgtag gttcagccgt tcaggagtcg
aatcagcctc 540tgccggcgtt ttctttgcac gtacgacgga cacacatggg
cataccatat agctggtcca 600tgacattagg agagagaacg tacgtgttga
cctgtagctg agatataaca aggttgatta 660taatatcacc aaacatgaaa
tcatccaagg atgacccata actatcacta ctatagtact 720gcatctggta
aaagaaattg tatagactct atttcgagca ctaccacata acgcctgcaa
780tgtgacaccc tacctattca ctaatgtgcc tcttcccaca cgctttccac
ccgtactgct 840cacagcttta agaaccagaa caaatgagta atattagtgt
cggttcatgg ctaaaaccag 900cactgatgta catgaccaca tatgtcaaat
gctgcttcta ggcatgaccc gctcttacta 960atacctactc atcgctagaa
gaattttcgg ctgataaatt ttcaatttaa gcaagagtta 1020tctgcgttgg
ttcataactc aaactgatgg ccccaaccat attagtgcaa atttcacata
1080tgatcataac cttttcatat gaaatcggat cgagatgaac tttatataaa
cattgtagct 1140gtcgatgata cctacaattt tatagttcac aaccttttta
tttcaagtca tttaaatgcc 1200caaataggtg tttcaaatct cagatagaaa
tgttcaaaag taaaaaaggt ccctatcata 1260acataattga tatgtaagtg
agttggaaaa agataagtac gtgtgagaga gatcggggat 1320caaattctgg
tgtaataatg tatgtatttc agtcataaaa attggtagca gtagttgggg
1380ctctgtatat ataccggtaa ggatgggatg gtagtagaat aattcttttt
ttgtttttag 1440ttttttctgg tccaaaattt caaatttgga tcccttactt
gtaccaacta atattaatga 1500gtgttgaggg tagtagaggt gcaactttac
cataatccct ctgtttcagg ttataagacg 1560ttttgacttt aaatttgacc
aagtttatgc gcaaatatag taatatttat aatactatat 1620tagtttcatt
aaataaataa ttgaatatat tttcataata aatttgtgtt gagttcaaaa
1680tattattaat tttttctaca aacttggtca aacttgaagc agtttgactt
tgaccaaagt 1740caaaacgtct tataacttga aacggatgga ttactttttt
tgtggggaca agtttacaat 1800gtttaataaa gcacaatcca tcttaatgtt
ttcaagctga atattgtaaa attcatggat 1860aaaccagctt ctaaatgttt
aaccgggaaa atgtcgaacg acaaattaat atttttaagt 1920gatggggagt
attaattaag gagtgacaac tcaactttca atatcgtact aaactgtggg
1980atttattttc taaaatttta taccctgcca attcacgtgt tgtagatctt
tttttttcac 2040taaccgacac caggtatatc aattttattg aatatagcag
caaaaagaat gtgttgtact 2100tgtaaacaaa aagcaaactg tacataaaaa
aaaatgcact cctatataat taagctcata 2160aagatgcttt gcttcgtgag
ggcccaagtt ttgatgacct tttgcttgat ctcgaaatta 2220aaatttaagt
actgttaagg gagttcacac caccatcaat tttcagcctg aagaaacagt
2280taaacaacga ccccgatgac cagtctactg ctctccacat actagctgca
ttattgatca 2340caaaacaaaa caaaacgaaa taaaaatcag cagcgagagt
gtgcagagag agacaaaggt 2400gatctggcgt ggatatctcc ccatccatcc
tcacccgcgc tgcccatcac tcgccgccgc 2460atactccatc atgtggagag
aggaagacga ggaccacagc cagagcccgg gtcgagatgc 2520caccacggcc
acaacccacg agcccggcgc gacaccaccg cgcgcgcgtg agccagccac
2580aaacgcccgc ggataggcgc gcgcacgccg gccaatccta ccacatcccc
ggcctccgcg 2640gctcgcgagc gccgctgcca tccgatccgc tgagttttgg
ctatttatac gtaccgcggg 2700agcctgtgtg cagagcagtg catctcaaga
agtactcgag caaagaagga gagagcttgg 2760tgagctgcag ccatggtaga
tctgagggta aatttctagt ttttctcctt cattttcttg 2820gttaggaccc
ttttctcttt ttattttttt gagctttgat ctttctttaa actgatctat
2880tttttaattg attggttatg gtgtaaatat tacatagctt taactgataa
tctgattact 2940ttatttcgtg tgtctatgat gatgatgata gttacagaac
cgacgaacta gtgcgcaggc 3000ggttgtgcag gcgatgcagt gccaggtggg
ggtgaggggc aggacggccg tcccggcgag 3060gcagcccgcg ggcagggtgt
ggggcgtcag gagggccgcc cgcgccacct ccgggttcaa 3120ggtgctggcg
ctcggcccgg agaccaccgg ggtcatccag aggatgcagc agctgctcga
3180catggacacc acgcccttca ccgacaagat catcgccgag tacatctggg
ttggaggatc 3240tggaattgac ctcagaagca aatcaaggac gatttcgaag
ccagtggagg acccgtcaga 3300gctgccgaaa tggaactacg acggatcgag
cacggggcag gctcctgggg aagacagtga 3360agtcatccta tacccacagg
ccatattcaa ggacccattc cgaggaggca acaacatact 3420ggttatctgt
gacacctaca caccacaggg ggaacccatc cctactaaca aacgccacat
3480ggctgcacaa atcttcagtg accccaaggt cacttcacaa gtgccatggt
tcggaatcga 3540acaggagtac actctgatgc agagggatgt gaactggcct
cttggctggc ctgttggagg 3600gtaccctggc ccccagggtc catactactg
cgccgtagga tcagacaagt catttggccg 3660tgacatatca gatgctcact
acaaggcgtg cctttacgct ggaattgaaa tcagtggaac 3720aaacggggag
gtcatgcctg gtcagtggga gtaccaggtt ggacccagcg ttggtattga
3780tgcaggagac cacatatggg cttccagata cattctcgag agaatcacgg
agcaagctgg 3840tgtggtgctc acccttgacc caaaaccaat ccagggtgac
tggaacggag ctggctgcca 3900cacaaactac agcacattga gcatgcgcga
ggatggaggt ttcgacgtga tcaagaaggc 3960aatcctgaac ctttcacttc
gccatgactt gcacatagcc gcatatggtg aaggaaacga 4020gcggaggttg
acagggctac acgagacagc tagcatatca gacttctcat ggggtgtggc
4080gaaccgtggc tgctctattc gtgtggggcg agacaccgag gcgaagggca
aaggatacct 4140ggaggaccgt cgcccggcct ccaacatgga cccgtacacc
gtgacggcgc tgctggccga 4200gaccacgatc ctgtgggagc cgaccctcga
ggcggaggcc ctcgctgcca agaagctggc 4260gctgaaggta tga
427343436PRTArtificial SequenceTranslation product of SEQ ID NO 42
DNA 43Met Val Asp Leu Arg Asn Arg Arg Thr Ser Ala Gln Ala Val Val
Gln1 5 10 15Ala Met Gln Cys Gln Val Gly Val Arg Gly Arg Thr Ala Val
Pro Ala 20 25 30Arg Gln Pro Ala Gly Arg Val Trp Gly Val Arg Arg Ala
Ala Arg Ala 35 40 45Thr Ser Gly Phe Lys Val Leu Ala Leu Gly Pro Glu
Thr Thr Gly Val 50 55 60Ile Gln Arg Met Gln Gln Leu Leu Asp Met Asp
Thr Thr Pro Phe Thr65 70 75 80Asp Lys Ile Ile Ala Glu Tyr Ile Trp
Val Gly Gly Ser Gly Ile Asp 85 90 95Leu Arg Ser Lys Ser Arg Thr Ile
Ser Lys Pro Val Glu Asp Pro Ser 100 105 110Glu Leu Pro Lys Trp Asn
Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro 115 120 125Gly Glu Asp Ser
Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp 130 135 140Pro Phe
Arg Gly Gly Asn Asn Ile Leu Val Ile Cys Asp Thr Tyr Thr145 150 155
160Pro Gln Gly Glu Pro Ile Pro Thr Asn Lys Arg His Met Ala Ala Gln
165 170 175Ile Phe Ser Asp Pro Lys Val Thr Ser Gln Val Pro Trp Phe
Gly Ile 180 185 190Glu Gln Glu Tyr Thr Leu Met Gln Arg Asp Val Asn
Trp Pro Leu Gly 195 200 205Trp Pro Val Gly Gly Tyr Pro Gly Pro Gln
Gly Pro Tyr Tyr Cys Ala 210 215 220Val Gly Ser Asp Lys Ser Phe Gly
Arg Asp Ile Ser Asp Ala His Tyr225 230 235 240Lys Ala Cys Leu Tyr
Ala Gly Ile Glu Ile Ser Gly Thr Asn Gly Glu 245 250 255Val Met Pro
Gly Gln Trp Glu Tyr Gln Val Gly Pro Ser Val Gly Ile 260 265 270Asp
Ala Gly Asp His Ile Trp Ala Ser Arg Tyr Ile Leu Glu Arg Ile 275 280
285Thr Glu Gln Ala Gly Val Val Leu Thr Leu Asp Pro Lys Pro Ile Gln
290 295 300Gly Asp Trp Asn Gly Ala Gly Cys His Thr Asn Tyr Ser Thr
Leu Ser305 310 315 320Met Arg Glu Asp Gly Gly Phe Asp Val Ile Lys
Lys Ala Ile Leu Asn 325 330 335Leu Ser Leu Arg His Asp Leu His Ile
Ala Ala Tyr Gly Glu Gly Asn 340 345 350Glu Arg Arg Leu Thr Gly Leu
His Glu Thr Ala Ser Ile Ser Asp Phe 355 360 365Ser Trp Gly Val Ala
Asn Arg Gly Cys Ser Ile Arg Val Gly Arg Asp 370 375 380Thr Glu Ala
Lys Gly Lys Gly Tyr Leu Glu Asp Arg Arg Pro Ala Ser385 390 395
400Asn Met Asp Pro Tyr Thr Val Thr Ala Leu Leu Ala Glu Thr Thr Ile
405 410 415Leu Trp Glu Pro Thr Leu Glu Ala Glu Ala Leu Ala Ala Lys
Lys Leu 420 425 430Ala Leu Lys Val 435441992DNAZea mays
44ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta
60agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta
120tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt
actacaataa 180tatcagtgtt ttagagaatc atataaatga acagttagac
atggtctaaa ggacaattga 240gtattttgac aacaggactc tacagtttta
tctttttagt gtgcatgtgt tctccttttt 300ttttgcaaat agcttcacct
atataatact tcatccattt tattagtaca tccatttagg 360gtttagggtt
aatggttttt atagactaat ttttttagta catctatttt attctatttt
420agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat
aatttagata 480taaaatagaa taaaataaag tgactaaaaa ttaaacaaat
accctttaag aaattaaaaa 540aactaaggaa acatttttct tgtttcgagt
agataatgcc agcctgttaa acgccgtcga 600cgagtctaac ggacaccaac
cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660cggcacggca
tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg
720acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt
gagccggcac 780ggcaggcggc ctcctcctcc tctcacggca cggcagctac
gggggattcc tttcccaccg 840ctccttcgct ttcccttcct cgcccgccgt
aataaataga caccccctcc acaccctctt 900tccccaacct cgtgttgttc
ggagcgcaca cacacacaac cagatctccc ccaaatccac 960ccgtcggcac
ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct
1020tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc
tgttcatgtt 1080tgtgttagat ccgtgtttgt gttagatccg tgctgctagc
gttcgtacac ggatgcgacc 1140tgtacgtcag acacgttctg attgctaact
tgccagtgtt tctctttggg gaatcctggg 1200atggctctag ccgttccgca
gacgggatcg atttcatgat tttttttgtt tcgttgcata 1260gggtttggtt
tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca
1320tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg
cggtcgttct 1380agatcggagt agaattctgt ttcaaactac ctggtggatt
tattaatttt ggatctgtat 1440gtgtgtgcca tacatattca tagttacgaa
ttgaagatga tggatggaaa tatcgatcta 1500ggataggtat acatgttgat
gcgggtttta ctgatgcata tacagagatg ctttttgttc 1560gcttggttgt
gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag
1620aatactgttt caaactacct ggtgtattta ttaattttgg aactgtatgt
gtgtgtcata 1680catcttcata gttacgagtt taagatggat ggaaatatcg
atctaggata ggtatacatg 1740ttgatgtggg ttttactgat gcatatacat
gatggcatat gcagcatcta ttcatatgct 1800ctaaccttga gtacctatct
attataataa acaagtatgt tttataatta ttttgatctt 1860gatatacttg
gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc
1920atacgctatt tatttgcttg gtactgtttc ttttgtcgat gctcaccctg
ttgtttggtg 1980ttacttctgc ag 1992451248DNAHordeum vulgare
45atggcatccg cccccgcctc cgcctccgcg gccctctcca ccgccgcccc cgccgacaac
60ggggccgcca agcccacgga gcagcggccg gtacaggtgg ctaagcgatt ggagaagttc
120aaaacaacaa ttttcacaca gatgagcatg ctcgcagtga agcatggagc
aataaacctt 180ggacaggggt ttcccaattt tgatggccct gactttgtca
aagatgctgc tattgaggct 240atcaaagctg gaaagaatca gtatgcaaga
ggatatggtg tgcctgaatt gaactcagct 300gttgctgaga gatttctcaa
ggacagtgga ttgcacatcg atcctgataa ggaagttact 360gttacatctg
ggtgcacaga agcaatagct gcaacgatat tgggtctgat caaccctggg
420gatgaagtca tactgtttgc tccattctat gattcttatg aggctacact
gtccatggct 480ggtgcgaatg tcaaagccat tacactccgc cctccggact
ttgcagtccc tcttgaagag 540ctaaaggctg cagtctcgaa gaataccaga
gcaataatga ttaatacacc tcacaaccct 600accgggaaaa tgttcacaag
ggaggaactt gagttcattg ctgatctctg caaggaaaat 660gacgtgttgc
tctttgccga tgaggtctac gacaagctgg cgtttgaggc ggatcacata
720tcaatggctt ctattcctgg catgtatgag aggaccgtca ctatgaactc
cctggggaag 780acgttctcct tgaccggatg gaagatcggc tgggcgatag
caccaccgca cctgacatgg 840ggcgtaaggc aggcacactc cttcctcaca
ttcgccacct ccacgccgat gcaatcagca 900gcggcggcgg ccctgagagc
accggacagc tactttgagg agctgaagag ggactacggc 960gcaaagaaag
cgctgctggt ggacgggctc aaggcggcgg gcttcatcgt ctacccttcg
1020agcggaacct acttcatcat ggtcgaccac accccgttcg ggttcgacaa
cgacgtcgag 1080ttctgcgagt acttgatccg cgaggtcggc gtcgtggcca
tcccgccaag cgtgttctac 1140ctgaacccgg aggacgggaa gaacctggtg
aggttcacct tctgcaagga cgacgacacg 1200ctaagggcgg cggtggacag
gatgaaggcc aagctcagga agaaatga 124846415PRTHordeum vulgare 46Met
Ala Ser Ala Pro Ala Ser Ala Ser Ala Ala Leu Ser Thr Ala Ala1 5 10
15Pro Ala Asp Asn Gly Ala Ala Lys Pro Thr Glu Gln Arg Pro Val Gln
20 25 30Val Ala Lys Arg Leu Glu Lys Phe Lys Thr Thr Ile Phe Thr Gln
Met 35 40 45Ser Met Leu Ala Val Lys His Gly Ala Ile Asn Leu Gly Gln
Gly Phe 50 55 60Pro Asn Phe Asp Gly Pro
Asp Phe Val Lys Asp Ala Ala Ile Glu Ala65 70 75 80Ile Lys Ala Gly
Lys Asn Gln Tyr Ala Arg Gly Tyr Gly Val Pro Glu 85 90 95Leu Asn Ser
Ala Val Ala Glu Arg Phe Leu Lys Asp Ser Gly Leu His 100 105 110Ile
Asp Pro Asp Lys Glu Val Thr Val Thr Ser Gly Cys Thr Glu Ala 115 120
125Ile Ala Ala Thr Ile Leu Gly Leu Ile Asn Pro Gly Asp Glu Val Ile
130 135 140Leu Phe Ala Pro Phe Tyr Asp Ser Tyr Glu Ala Thr Leu Ser
Met Ala145 150 155 160Gly Ala Asn Val Lys Ala Ile Thr Leu Arg Pro
Pro Asp Phe Ala Val 165 170 175Pro Leu Glu Glu Leu Lys Ala Ala Val
Ser Lys Asn Thr Arg Ala Ile 180 185 190Met Ile Asn Thr Pro His Asn
Pro Thr Gly Lys Met Phe Thr Arg Glu 195 200 205Glu Leu Glu Phe Ile
Ala Asp Leu Cys Lys Glu Asn Asp Val Leu Leu 210 215 220Phe Ala Asp
Glu Val Tyr Asp Lys Leu Ala Phe Glu Ala Asp His Ile225 230 235
240Ser Met Ala Ser Ile Pro Gly Met Tyr Glu Arg Thr Val Thr Met Asn
245 250 255Ser Leu Gly Lys Thr Phe Ser Leu Thr Gly Trp Lys Ile Gly
Trp Ala 260 265 270Ile Ala Pro Pro His Leu Thr Trp Gly Val Arg Gln
Ala His Ser Phe 275 280 285Leu Thr Phe Ala Thr Ser Thr Pro Met Gln
Ser Ala Ala Ala Ala Ala 290 295 300Leu Arg Ala Pro Asp Ser Tyr Phe
Glu Glu Leu Lys Arg Asp Tyr Gly305 310 315 320Ala Lys Lys Ala Leu
Leu Val Asp Gly Leu Lys Ala Ala Gly Phe Ile 325 330 335Val Tyr Pro
Ser Ser Gly Thr Tyr Phe Ile Met Val Asp His Thr Pro 340 345 350Phe
Gly Phe Asp Asn Asp Val Glu Phe Cys Glu Tyr Leu Ile Arg Glu 355 360
365Val Gly Val Val Ala Ile Pro Pro Ser Val Phe Tyr Leu Asn Pro Glu
370 375 380Asp Gly Lys Asn Leu Val Arg Phe Thr Phe Cys Lys Asp Asp
Asp Thr385 390 395 400Leu Arg Ala Ala Val Asp Arg Met Lys Ala Lys
Leu Arg Lys Lys 405 410 415472735DNAArtificial SequenceSynthetic
expression cassette 47gtttgaatcc tccttaaagt ttttctctgg agaaactgta
gtaattttac tttgttgtgt 60tcccttcatc ttttgaatta atggcatttg ttttaatact
aatctgcttc tgaaacttgt 120aatgtatgta tatcagtttc ttataattta
tccaagtaat atcttccatt ctctatgcaa 180ttgcctgcat aagctcgaca
aaagagtaca tcaacccctc ctcctctgga ctactctagc 240taaacttgaa
tttcccctta agattatgaa attgatatat ccttaacaaa cgactccttc
300tgttggaaaa tgtagtactt gtctttcttc ttttgggtat atatagttta
tatacaccat 360actatgtaca acatccaagt agagtgaaat ggatacatgt
acaagactta tttgattgat 420tgatgacttg agttgcctta ggagtaacaa
attcttaggt caataaatcg ttgatttgaa 480attaatctct ctgtcttaga
cagataggaa ttatgacttc caatggtcca gaaagcaaag 540ttcgcactga
gggtatactt ggaattgaga cttgcacagg tccagaaacc aaagttccca
600tcgagctcta aaatcacatc tttggaatga aattcaatta gagataagtt
gcttcatagc 660ataggtaaaa tggaagatgt gaagtaacct gcaataatca
gtgaaatgac attaatacac 720taaatacttc atatgtaatt atcctttcca
ggttaacaat actctataaa gtaagaatta 780tcagaaatgg gctcatcaaa
cttttgtact atgtatttca tataaggaag tataactata 840cataagtgta
tacacaactt tattcctatt ttgtaaaggt ggagagactg ttttcgatgg
900atctaaagca atatgtctat aaaatgcatt gatataataa ttatctgaga
aaatccagaa 960ttggcgttgg attatttcag ccaaatagaa gtttgtacca
tacttgttga ttccttctaa 1020gttaaggtga agtatcattc ataaacagtt
ttccccaaag tactactcac caagtttccc 1080tttgtagaat taacagttca
aatatatggc gcagaaatta ctctatgccc aaaaccaaac 1140gagaaagaaa
caaaatacag gggttgcaga ctttattttc gtgttagggt gtgttttttc
1200atgtaattaa tcaaaaaata ttatgacaaa aacatttata catattttta
ctcaacactc 1260tgggtatcag ggtgggttgt gttcgacaat caatatggaa
aggaagtatt ttccttattt 1320ttttagttaa tattttcagt tataccaaac
ataccttgtg atattatttt taaaaatgaa 1380aaactcgtca gaaagaaaaa
gcaaaagcaa caaaaaaatt gcaagtattt tttaaaaaag 1440aaaaaaaaaa
catatcttgt ttgtcagtat gggaagtttg agataaggac gagtgagggg
1500ttaaaattca gtggccattg attttgtaat gccaagaacc acaaaatcca
atggttacca 1560ttcctgtaag atgaggtttg ctaactcttt ttgtccgtta
gataggaagc cttatcacta 1620tatatacaag gcgtcctaat aacctcttag
taaccaatta tttcagcacc atgtctctgc 1680tctcagatct cgttaacctc
aacctcaccg atgccaccgg gaaaatcatc gccgaataca 1740tatggatcgg
tggatctgga atggatatca gaagcaaagc caggacacta ccaggaccag
1800tgactgatcc atcaaagctt cccaagtgga actacgacgg atccagcacc
ggtcaggctg 1860ctggagaaga cagtgaagtc attctatacc ctcaggcaat
attcaaggat cccttcagga 1920aaggcaacaa catcctggtg atgtgtgatg
cttacacacc agctggtgat cctattccaa 1980ccaacaagag gcacaacgct
gctaagatct tcagccaccc cgacgttgcc aaggaggagc 2040cttggtatgg
gattgagcaa gaatacactt tgatgcaaaa ggatgtgaac tggccaattg
2100gttggcctgt tggtggctac cctggccctc agggacctta ctactgtggt
gtgggagctg 2160acaaagccat tggtcgtgac attgtggatg ctcactacaa
ggcctgtctt tacgccggta 2220ttggtatttc tggtatcaat ggagaagtca
tgccaggcca gtgggagttc caagtcggcc 2280ctgttgaggg tattagttct
ggtgatcaag tctgggttgc tcgatacctt ctcgagagga 2340tcactgagat
ctctggtgta attgtcagct tcgacccgaa accagtcccg ggtgactgga
2400atggagctgg agctcactgc aactacagca ctaagacaat gagaaacgat
ggaggattag 2460aagtgatcaa gaaagcgata gggaagcttc agctgaaaca
caaagaacac attgctgctt 2520acggtgaagg aaacgagcgt cgtctcactg
gaaagcacga aaccgcagac atcaacacat 2580tctcttgggg agtcgcgaac
cgtggagcgt cagtgagagt gggacgtgac acagagaagg 2640aaggtaaagg
gtacttcgaa gacagaaggc cagcttctaa catggatcct tacgttgtca
2700cctccatgat cgctgagacg accatactcg gttga 2735481371DNACitrus
reticulata 48atgcttaagc cgtccgcctt cgggtcttct ttttcttcct cagctctgct
ttcgttttcg 60aagcatttgc atacaataag cattactgat tctgtcaaca ccagaagaag
aggaatcagt 120accgcttgcc ctaggtaccc ttctctcatg gcgagcttgt
ccaccgtttc caccaatcaa 180agcgacacca tccagaagac caatcttcag
cctcaacagg ttgctaagtg cttggagaag 240tttaaaacta caatctttac
acaaatgagt atgcttgcca tcaaacatgg agctataaat 300cttggtcaag
gctttcccaa ctttgatggc ccagattttg ttaaagatgc agcgattcaa
360gccataaggg atgggaagaa tcaatatgct cgtggacatg gggttccaga
gttcaactct 420gccattgctt cccggtttaa gaaagattct gggctcgagg
ttgaccctga aaaggaagtt 480actgttacct ctgggtgcac cgaagccatt
gctgcaacca tcttaggttt gattaatcct 540ggagatgagg tgatcctttt
tgcacctttc tatgattcct atgaagctac tctctccatg 600gctggtgcta
aaattaaatg catcacattg cgccctccag aatttgccat ccccattgaa
660gagctcaagt ctacaatctc aaaaaatact cgtgcaattc ttatgaacac
tccacataac 720cccactggaa agatgttcac tagggaggaa cttaatgtta
ttgcatctct ttgcattgag 780aatgatgtgt tggtttttag tgatgaggtc
tatgataagt tggcttttga aatggatcac 840atttccatag cctctcttcc
tggaatgtat gagcgtactg taaccatgaa ttccttaggg 900aagacattct
ctttaacagg gtggaagatc gggtgggcaa tagctccacc gcaccttaca
960tggggggtgc ggcaggcaca ctcttttctc acgtttgcca catccactcc
aatgcagtgg 1020gcagctacag cagcccttag agctccggag acgtactatg
aggagctaaa gagagattac 1080tcggcaaaga aggcaatttt ggtggaggga
ttgaatgctg ttggtttcaa ggtattccca 1140tctagtggga catactttgt
ggttgtagat cacaccccat ttgggcacga aactgatatt 1200gcattttgtg
aatatctgat caaggaagtt ggggttgtgg caattccgac cagcgtattt
1260tacttgaatc cagaggatgg aaagaatttg gtgagattta ccttctgcaa
agatgaagga 1320actttgaggt ctgcagttga caggatgaag gagaagctga
ggagaaaatg a 137149456PRTCitrus reticulata 49Met Leu Lys Pro Ser
Ala Phe Gly Ser Ser Phe Ser Ser Ser Ala Leu1 5 10 15Leu Ser Phe Ser
Lys His Leu His Thr Ile Ser Ile Thr Asp Ser Val 20 25 30Asn Thr Arg
Arg Arg Gly Ile Ser Thr Ala Cys Pro Arg Tyr Pro Ser 35 40 45Leu Met
Ala Ser Leu Ser Thr Val Ser Thr Asn Gln Ser Asp Thr Ile 50 55 60Gln
Lys Thr Asn Leu Gln Pro Gln Gln Val Ala Lys Cys Leu Glu Lys65 70 75
80Phe Lys Thr Thr Ile Phe Thr Gln Met Ser Met Leu Ala Ile Lys His
85 90 95Gly Ala Ile Asn Leu Gly Gln Gly Phe Pro Asn Phe Asp Gly Pro
Asp 100 105 110Phe Val Lys Asp Ala Ala Ile Gln Ala Ile Arg Asp Gly
Lys Asn Gln 115 120 125Tyr Ala Arg Gly His Gly Val Pro Glu Phe Asn
Ser Ala Ile Ala Ser 130 135 140Arg Phe Lys Lys Asp Ser Gly Leu Glu
Val Asp Pro Glu Lys Glu Val145 150 155 160Thr Val Thr Ser Gly Cys
Thr Glu Ala Ile Ala Ala Thr Ile Leu Gly 165 170 175Leu Ile Asn Pro
Gly Asp Glu Val Ile Leu Phe Ala Pro Phe Tyr Asp 180 185 190Ser Tyr
Glu Ala Thr Leu Ser Met Ala Gly Ala Lys Ile Lys Cys Ile 195 200
205Thr Leu Arg Pro Pro Glu Phe Ala Ile Pro Ile Glu Glu Leu Lys Ser
210 215 220Thr Ile Ser Lys Asn Thr Arg Ala Ile Leu Met Asn Thr Pro
His Asn225 230 235 240Pro Thr Gly Lys Met Phe Thr Arg Glu Glu Leu
Asn Val Ile Ala Ser 245 250 255Leu Cys Ile Glu Asn Asp Val Leu Val
Phe Ser Asp Glu Val Tyr Asp 260 265 270Lys Leu Ala Phe Glu Met Asp
His Ile Ser Ile Ala Ser Leu Pro Gly 275 280 285Met Tyr Glu Arg Thr
Val Thr Met Asn Ser Leu Gly Lys Thr Phe Ser 290 295 300Leu Thr Gly
Trp Lys Ile Gly Trp Ala Ile Ala Pro Pro His Leu Thr305 310 315
320Trp Gly Val Arg Gln Ala His Ser Phe Leu Thr Phe Ala Thr Ser Thr
325 330 335Pro Met Gln Trp Ala Ala Thr Ala Ala Leu Arg Ala Pro Glu
Thr Tyr 340 345 350Tyr Glu Glu Leu Lys Arg Asp Tyr Ser Ala Lys Lys
Ala Ile Leu Val 355 360 365Glu Gly Leu Asn Ala Val Gly Phe Lys Val
Phe Pro Ser Ser Gly Thr 370 375 380Tyr Phe Val Val Val Asp His Thr
Pro Phe Gly His Glu Thr Asp Ile385 390 395 400Ala Phe Cys Glu Tyr
Leu Ile Lys Glu Val Gly Val Val Ala Ile Pro 405 410 415Thr Ser Val
Phe Tyr Leu Asn Pro Glu Asp Gly Lys Asn Leu Val Arg 420 425 430Phe
Thr Phe Cys Lys Asp Glu Gly Thr Leu Arg Ser Ala Val Asp Arg 435 440
445Met Lys Glu Lys Leu Arg Arg Lys 450 4555037DNAArtificial
SequenceSynthetic primer sequence 50ggccacatgt ccgttgctaa
gtgcttggag aagttta 375140DNAArtificial SequenceSynthetic primer
sequence 51cgggcacgtg tcattttctc ctcagcttct ccttcatcct 40
* * * * *
References