U.S. patent application number 11/846475 was filed with the patent office on 2008-04-03 for aromatic methyltransferases and uses thereof.
Invention is credited to Alison Van Eenennaam, Kim Lincoln, Susan R. Norris, Joshua C. Stein, Henry E. Valentin.
Application Number | 20080083045 11/846475 |
Document ID | / |
Family ID | 23290312 |
Filed Date | 2008-04-03 |
United States Patent
Application |
20080083045 |
Kind Code |
A1 |
Norris; Susan R. ; et
al. |
April 3, 2008 |
AROMATIC METHYLTRANSFERASES AND USES THEREOF
Abstract
The present invention relates to genes associated with the
tocopherol biosynthesis pathway. More particularly, the present
invention provides and includes nucleic acid molecules, proteins,
and antibodies associated with genes that encode polypeptides that
have methyltransferase activity. The present invention also
provides methods for utilizing such agents, for example in gene
isolation, gene analysis and the production of transgenic plants.
Moreover, the present invention includes transgenic plants modified
to express the aforementioned polypeptides. In addition, the
present invention includes methods for the production of products
from the tocopherol biosynthesis pathway.
Inventors: |
Norris; Susan R.;
(University City, MO) ; Lincoln; Kim; (University
City, MO) ; Stein; Joshua C.; (Acton, MA) ;
Valentin; Henry E.; (Chesterfield, MO) ; Eenennaam;
Alison Van; (Davis, CA) |
Correspondence
Address: |
SONNENSCHEIN NATH & ROSENTHAL LLP
P.O. BOX 061080
SOUTH WACKER DRIVE STATION, SEARS TOWER
CHICAGO
IL
60606
US
|
Family ID: |
23290312 |
Appl. No.: |
11/846475 |
Filed: |
August 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10279029 |
Oct 24, 2002 |
7262339 |
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11846475 |
Aug 28, 2007 |
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60330563 |
Oct 25, 2001 |
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Current U.S.
Class: |
800/305 ;
800/298; 800/306; 800/312; 800/314; 800/315; 800/316; 800/317.1;
800/317.2; 800/317.4; 800/319; 800/320.2; 800/320.3; 800/322 |
Current CPC
Class: |
C12N 15/8243 20130101;
C12N 9/1007 20130101 |
Class at
Publication: |
800/305 ;
800/298; 800/306; 800/312; 800/314; 800/315; 800/316; 800/317.1;
800/317.2; 800/317.4; 800/319; 800/320.2; 800/320.3; 800/322 |
International
Class: |
A01H 5/00 20060101
A01H005/00 |
Claims
1-26. (canceled)
27. A transformed plant comprising an introduced first nucleic acid
molecule that encodes a polypeptide molecule comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 16,
22 through 28, 33 through 38, and an introduced second nucleic acid
molecule that encodes an enzyme selected from the group consisting
of tyrA, slr1736, HPT, tocopherol cyclase, dxs, dxr, GGPPS, GMT,
HPPD, AANT1, sir 1737, IDI, GGH, and complements thereof.
28. The transformed plant of claim 27, wherein said plant is
selected from the group consisting of alfalfa, Arabidopsis
thaliana, barley, Brassica campestris, Brassica napus, oilseed
rape, broccoli, cabbage, citrus, canola, cotton, garlic, oat,
Allium, flax, an ornamental plant, peanut, pepper, potato,
rapeseed, rice, rye, sorghum, strawberry, sugarcane, sugarbeet,
tomato, wheat, poplar, pine, fir, eucalyptus, apple, lettuce,
lentils, grape, banana, tea, turf grasses, sunflower, soybean,
corn, Phaseolus, crambe, mustard, castor bean, sesame, cottonseed,
linseed, safflower, and oil palm.
29. The transformed plant of claim 27, wherein said introduced
second nucleic acid molecule encodes GMT and wherein said
transformed plant comprises tissue with one or both of an increased
.alpha.-tocopherol level and increased .alpha.-tocotrienol level
relative to a plant with a similar genetic background but lacking
said introduced first nucleic acid molecule and said introduced
second nucleic acid molecule.
30. The transformed plant of claim 27, wherein said transformed
plant produces a seed with one or both of an increased
.gamma.-tocopherol level and increased .gamma.-tocotrienol level
relative to a plant with a similar genetic background but lacking
said introduced first nucleic acid molecule and said introduced
second nucleic acid molecule.
31. The transformed plant of claim 27, wherein at least one of said
introduced first nucleic acid molecule and said introduced second
nucleic acid molecule further comprises, in the 5' to 3' direction,
an operably linked heterologous promoter.
32. The transformed plant of claim 31, wherein said promoter is a
seed specific promoter.
33. A transformed plant comprising an introduced first nucleic acid
molecule comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1, 2, 8 through 15, and complements
thereof, and an introduced second nucleic acid molecule comprising
a sequence selected from the group consisting of SEQ ID NOs: 39
through 54, and complements thereof.
34. The transformed plant of claim 33, wherein said plant is
selected from the group consisting of alfalfa, Arabidopsis
thaliana, barley, Brassica campestris, Brassica napus, oilseed
rape, broccoli, cabbage, citrus, canola, cotton, garlic, oat,
Allium, flax, an ornamental plant, peanut, pepper, potato,
rapeseed, rice, rye, sorghum, strawberry, sugarcane, sugarbeet,
tomato, wheat, poplar, pine, fir, eucalyptus, apple, lettuce,
lentils, grape, banana, tea, turf grasses, sunflower, soybean,
chick peas, corn, Phaseolus, crambe, mustard, castor bean, sesame,
cottonseed, linseed, safflower, and oil palm.
35. The transformed plant of claim 33, wherein said transformed
plant produces a seed with increased .alpha.-tocopherol levels
relative to a plant with a similar genetic background but lacking
said introduced first nucleic acid molecule and said introduced
second nucleic acid molecule.
36. The transformed plant of claim 33, wherein at least one of said
introduced first nucleic acid molecule and said introduced second
nucleic acid molecule comprises, in the 5' to 3' direction, an
operably linked heterologous promoter.
37. The transformed plant of claim 36, wherein said promoter is a
seed specific promoter.
38. A transformed plant comprising an introduced first nucleic acid
molecule that encodes a polypeptide molecule comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 16,
22 through 28, 33 through 38, and an introduced second nucleic acid
molecule having a sequence selected from the group consisting of
SEQ ID NOs: 39 through 54, and complements thereof.
39. The transformed plant of claim 38, wherein said plant is
selected from the group consisting of alfalfa, Arabidopsis
thaliana, barley, Brassica campestris, Brassica napus, oilseed
rape, broccoli, cabbage, citrus, canola, cotton, garlic, oat,
Allium, flax, an ornamental plant, peanut, pepper, potato,
rapeseed, rice, rye, sorghum, strawberry, sugarcane, sugarbeet,
tomato, wheat, poplar, pine, fir, eucalyptus, apple, lettuce,
lentils, grape, banana, tea, turf grasses, sunflower, soybean,
corn, Phaseolus, crambe, mustard, castor bean, sesame, cottonseed,
linseed, safflower, and oil palm.
40. The transformed plant of claim 38, wherein said transformed
plant produces a seed with one or both of an increased
.alpha.-tocopherol level and increased .alpha.-tocotrienol level
relative to a plant with a similar genetic background but lacking
said introduced first nucleic acid molecule and said introduced
second nucleic acid molecule.
41. The transformed plant of claim 38, wherein at least one nucleic
acid molecule further comprises, in the 5' to 3' direction, an
operably linked heterologous promoter.
42. The transformed plant of claim 41, wherein said promoter is a
seed specific promoter.
43-61. (canceled)
62. Seed derived from a transformed plant, wherein said transformed
plant comprises an introduced first nucleic acid molecule
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1, 2, 8 through 15, and complements
thereof, and an introduced second nucleic acid molecule comprising
a sequence selected from the group consisting of SEQ ID NOs: 39
through 54, and complements thereof.
63-65. (canceled)
66. A transformed plant comprising an introduced first nucleic acid
molecule encoding a tMT2 enzyme and an introduced second nucleic
acid molecule encoding a GMT enzyme.
67-71. (canceled)
Description
[0001] This application claims the benefit of and priority to U.S.
Provisional Application No. 60/330,563, filed Oct. 25, 2001, which
is herein incorporated by reference in its entirety.
[0002] The present invention is in the field of plant genetics and
biochemistry. More specifically, the invention relates to genes
associated with the tocopherol biosynthesis pathway, namely those
encoding methyltransferase activity, and uses of such genes.
[0003] Tocopherols are an important component of mammalian diets.
Epidemiological evidence indicates that tocopherol supplementation
can result in decreased risk for cardiovascular disease and cancer,
can aid in immune function, and is associated with prevention or
retardation of a number of degenerative disease processes in humans
(Traber and Sies, Annu. Rev. Nutr. 16:321-347 (1996)). Tocopherol
functions, in part, by stabilizing the lipid bilayer of biological
membranes (Skrypin and Kagan, Biochim. Biophys. Acta 815:209
(1995); Kagan, N.Y. Acad. Sci. p 121, (1989); Gomez-Fernandez et
al., Ann. N.Y. Acad. Sci. p 109 (1989)), reducing polyunsaturated
fatty acid (PUFA) free radicals generated by lipid oxidation
(Fukuzawa et al., Lipids 17:511-513 (1982)), and scavenging oxygen
free radicals, lipid peroxy radicals and singlet oxygen species
(Diplock et al. Ann. N Y Acad. Sci. 570:72 (1989); Fryer, Plant
Cell Environ. 15(4):381-392 (1992)).
[0004] The compound .alpha.-tocopherol, which is often referred to
as vitamin E, belongs to a class of lipid-soluble antioxidants that
includes .alpha., .beta., .gamma., and .delta.-tocopherols and
.alpha., .beta., .gamma., and .delta.-tocotrienols. Although
.alpha., .beta., .gamma., and .delta.-tocopherols and .alpha.,
.beta., .gamma., and .delta.-tocotrienols are sometimes referred to
collectively as "vitamin E", vitamin E is more appropriately
defined chemically as .alpha.-tocopherol. Vitamin E, or
.alpha.-tocopherol, is significant for human health, in part
because it is readily absorbed and retained by the body, and
therefore has a higher degree of bioactivity than other tocopherol
species (Traber and Sies, Annu. Rev. Nutr. 16:321-347 (1996)).
However, other tocopherols such as .beta., .gamma., and
.delta.-tocopherols also have significant health and nutritional
benefits.
[0005] Tocopherols are primarily synthesized only by plants and
certain other photosynthetic organisms, including cyanobacteria. As
a result, mammalian dietary tocopherols are obtained almost
exclusively from these sources. Plant tissues vary considerably in
total tocopherol content and tocopherol composition, with
.alpha.-tocopherol the predominant tocopherol species found in
green, photosynthetic plant tissues. Leaf tissue can contain from
10-50 .mu.g of total tocopherols per gram fresh weight, but most of
the world's major staple crops (e.g., rice, corn, wheat, potato)
produce low to extremely low levels of total tocopherols, of which
only a small percentage is .alpha.-tocopherol (Hess, Vitamin E,
.alpha.-tocopherol, Antioxidants in Higher Plants, R. Alscher and
J. Hess, Eds., CRC Press, Boca Raton. pp. 111-134 (1993)). Oil seed
crops generally contain much higher levels of total tocopherols,
but .alpha.-tocopherol is present only as a minor component in most
oilseeds (Taylor and Barnes, Chemy Ind., October:722-726
(1981)).
[0006] The recommended daily dietary intake of 15-30 mg of vitamin
E is quite difficult to achieve from the average American diet. For
example, it would take over 750 grams of spinach leaves, in which
.alpha.-tocopherol comprises 60% of total tocopherols, or 200-400
grams of soybean oil to satisfy this recommended daily vitamin E
intake. While it is possible to augment the diet with supplements,
most of these supplements contain primarily synthetic vitamin E,
having eight stereoisomers, whereas natural vitamin E is
predominantly composed of only a single isomer. Furthermore,
supplements tend to be relatively expensive, and the general
population is disinclined to take vitamin supplements on a regular
basis. Therefore, there is a need in the art for compositions and
methods that either increase the total tocopherol production or
increase the relative percentage of .alpha.-tocopherol produced by
plants.
[0007] In addition to the health benefits of tocopherols, increased
.alpha.-tocopherol levels in crops have been associated with
enhanced stability and extended shelf life of plant products
(Peterson, Cereal-Chem. 72(1):21-24 (1995); Ball, Fat-soluble
vitamin assays in food analysis. A comprehensive review, London,
Elsevier Science Publishers Ltd. (1988)). Further, tocopherol
supplementation of swine, beef, and poultry feeds has been shown to
significantly increase meat quality and extend the shelf life of
post-processed meat products by retarding post-processing lipid
oxidation, which contributes to the undesirable flavor components
(Sante and Lacourt, J. Sci. Food Agric. 65(4):503-507 (1994);
Buckley et al., J. of Animal Science 73:3122-3130 (1995)).
Tocopherol Biosynthesis
[0008] The plastids of higher plants exhibit interconnected
biochemical pathways leading to secondary metabolites including
tocopherols. The tocopherol biosynthetic pathway in higher plants
involves condensation of homogentisic acid and phytylpyrophosphate
to form 2-methylphytylplastoquinol (Fiedler et al., Planta
155:511-515 (1982); Soll et al., Arch. Biochem. Biophys.
204:544-550 (1980); Marshall et al., Phytochem. 24:1705-1711
(1985)). This plant tocopherol pathway can be divided into four
parts: 1) synthesis of homogentisic acid (HGA), which contributes
to the aromatic ring of tocopherol; 2) synthesis of
phytylpyrophosphate, which contributes to the side chain of
tocopherol; 3) joining of HGA and phytylpyrophosphate via a
prenyltransferase followed by a subsequent cyclization; 4) and
S-adenosyl methionine dependent methylation of an aromatic ring,
which affects the relative abundance of each of the tocopherol
species: See FIG. 1.
[0009] Various genes and their encoded proteins that are involved
in tocopherol biosynthesis are listed in the table below.
TABLE-US-00001 Gene ID or Enzyme Abbreviation Enzyme name tyrA
Bifunctional Prephenate dehydrogenase HPT Homogentisate phytyl
transferase DXS 1-Deoxyxylulose-5-phosphate synthase DXR
1-Deoxyxylulose-5-phosphate reductoisomerase GGPPS Geranylgeranyl
pyrophosphate synthase HPPD p-Hydroxyphenylpyruvate dioxygenase
AANT1 Adenylate transporter IDI Isopentenyl diphosphate isomerase
MT1 Methyl transferase 1 tMT2 Tocopherol methyl transferase 2 GGH
Geranylgeranyl diphosphate reductase slr1737 Tocopherol cyclase GMT
Gamma Methyl Transferase
[0010] As used herein, homogentisate phytyl transferase (HPT),
phytylprenyl transferase (PPT), slr1736, and ATPT2, each refer to
proteins or genes encoding proteins that have the same enzymatic
activity.
Synthesis of Homogentisic Acid
[0011] Homogentisic acid is the common precursor to both
tocopherols and plastoquinones. In at least some bacteria the
synthesis of homogentisic acid is reported to occur via the
conversion of chorismate to prephenate and then to
p-hydroxyphenylpyruvate via a bifunctional prephenate
dehydrogenase. Examples of bifunctional bacterial prephenate
dehydrogenase enzymes include the proteins encoded by the tyrA
genes of Erwinia herbicola and Escherichia coli. The tyrA gene
product catalyzes the production of prephenate from chorismate, as
well as the subsequent dehydrogenation of prephenate to form
p-hydroxyphenylpyruvate (p-HPP), the immediate precursor to
homogentisic acid. p-HPP is then converted to homogentisic acid by
hydroxyphenylpyruvate dioxygenase (HPPD). In contrast, plants are
believed to lack prephenate dehydrogenase activity, and it is
generally believed that the synthesis of homogentisic acid from
chorismate occurs via the synthesis and conversion of the
intermediate arogenate. Since pathways involved in homogentisic
acid synthesis are also responsible for tyrosine formation, any
alterations in these pathways can also result in the alteration in
tyrosine synthesis and the synthesis of other aromatic amino
acids.
Synthesis of Phytylpyrophosphate
[0012] Tocopherols are a member of the class of compounds referred
to as the isoprenoids. Other isoprenoids include carotenoids,
gibberellins, terpenes, chlorophyll and abscisic acid. A central
intermediate in the production of isoprenoids is isopentenyl
diphosphate (IPP). Cytoplasmic and plastid-based pathways to
generate IPP have been reported. The cytoplasmic based pathway
involves the enzymes acetoacetyl CoA thiolase, HMGCoA synthase,
HMGCoA reductase, mevalonate kinase, phosphomevalonate kinase, and
mevalonate pyrophosphate decarboxylase.
[0013] Recently, evidence for the existence of an alternative,
plastid based, isoprenoid biosynthetic pathway emerged from studies
in the research groups of Rohmer and Arigoni (Eisenreich et al.,
Chem. Bio., 5:R221-R233 (1998); Rohmer, Prog. Drug. Res.,
50:135-154 (1998); Rohmer, Comprehensive Natural Products
Chemistry, Vol. 2, pp. 45-68, Barton and Nakanishi (eds.), Pergamon
Press, Oxford, England (1999)), who found that the isotope labeling
patterns observed in studies on certain eubacterial and plant
terpenoids could not be explained in terms of the mevalonate
pathway. Arigoni and coworkers subsequently showed that
1-deoxyxylulose, or a derivative thereof, serves as an intermediate
of the novel pathway, now referred to as the MEP pathway (Rohmer et
al., Biochem. J., 295:517-524 (1993); Schwarz, Ph.D. thesis,
Eidgenossiche Technische Hochschule, Zurich, Switzerland (1994)).
Recent studies showed the formation of 1-deoxyxylulose 5-phosphate
(Broers, Ph.D. thesis (Eidgenossiche Technische Hochschule, Zurich,
Switzerland) (1994)) from one molecule each of glyceraldehyde
3-phosphate (Rohmer, Comprehensive Natural Products Chemistry, Vol.
2, pp. 45-68, Barton and Nakanishi, eds., Pergamon Press, Oxford,
England (1999)) and pyruvate (Eisenreich et al., Chem. Biol.,
5:R223-R233 (1998); Schwarz supra; Rohmer et al., J. Am. Chem.
Soc., 118:2564-2566 (1996); and Sprenger et al., Proc. Natl. Acad.
Sci. USA, 94:12857-12862 (1997)) by an enzyme encoded by the dxs
gene (Lois et al., Proc. Natl. Acad. Sci. USA, 95:2105-2110 (1997);
and Lange et al., Proc. Natl. Acad. Sci. USA, 95:2100-2104 (1998)).
1-Deoxyxylulose 5-phosphate can be further converted into
2-C-methylerythritol 4-phosphate (Arigoni et al., Proc. Natl. Acad.
Sci. USA, 94:10600-10605 (1997)) by a reductoisomerase encoded by
the dxr gene (Bouvier et al., Plant Physiol, 117:1421-1431 (1998);
and Rohdich et al., Proc. Natl. Acad. Sci. USA, 96:11758-11763
(1999)).
[0014] Reported genes in the MEP pathway also include ygbP, which
catalyzes the conversion of 2-C-methylerythritol 4-phosphate into
its respective cytidyl pyrophosphate derivative and ygbB, which
catalyzes the conversion of 4-phosphocytidyl-2C-methyl-D-erythritol
into 2C-methyl-D-erythritol, 3,4-cyclophosphate. These genes are
tightly linked on the E. coli genome (Herz et al., Proc. Natl.
Acad. Sci. U.S.A., 97(6):2485-2490 (2000)).
[0015] Once IPP is formed by the MEP pathway, it is converted to
GGDP by GGDP synthase, and then to phytylpyrophosphate, which is
the central constituent of the tocopherol side chain.
Combination and Cyclization
[0016] Homogentisic acid is combined with either
phytyl-pyrophosphate or solanyl-pyrophosphate by phytyl/prenyl
transferase forming 2-methylphytyl plastoquinol or 2-methylsolanyl
plastoquinol, respectively. 2-methylsolanyl plastoquinol is a
precursor to the biosynthesis of plastoquinones, while
2-methylphytyl plastoquinol is ultimately converted to
tocopherol.
Methylation of the Aromatic Ring
[0017] The major structural difference between each of the
tocopherol subtypes is the position of the methyl groups around the
phenyl ring. Both 2-methylphytyl plastoquinol and 2-methylsolanyl
plastoquinol serve as substrates for the plant enzyme
2-methylphytylplatoquinol/2-methylsolanylplastoquinol
methyltransferase (Tocopherol Methyl Transferase 2; Methyl
Transferase 2; MT2; tMT2), which is capable of methylating a
tocopherol precursor. Subsequent methylation at the 5 position of
.gamma.-tocopherol by .gamma.-tocopherol methyl-transferase (GMT)
generates the biologically active .alpha.-tocopherol.
[0018] A possible alternate pathway for the generation of
.alpha.-tocopherol involves the generation of .delta.-tocopherol
via the cyclization of 2-methylphytylplastoquinol by tocopherol
cyclase. .delta.-tocopherol is then converted to .beta.-tocopherol
via the methylation of the 5 position by GMT. .delta.-tocopherol
can be converted to .alpha.-tocopherol via methylation of the 3
position by tMT2, followed by methylation of the 5 position by GMT.
In a possible alternative pathway, .beta.-tocopherol is directly
converted to .alpha.-tocopherol by tMT2 via the methylation of the
3 position (see, for example, Biochemical Society Transactions,
11:504-510 (1983); Introduction to Plant Biochemistry, 2.sup.nd
edition, chapter 11 (1983); Vitamin Hormone, 29:153-200 (1971);
Biochemical Journal, 109:577 (1968); and, Biochemical and
Biophysical Research Communication, 28(3):295 (1967)). Since all
potential mechanisms for the generation of .alpha.-tocopherol
involve catalysis by tMT2, plants that are deficient in this
activity accumulate .delta.-tocopherol and .beta.-tocopherol.
Plants which have increased tMT2 activity tend to accumulate
.gamma.-tocopherol and .alpha.-tocopherol. Since there is no GMT
activity in the seeds of many plants, these plants tend to
accumulate .gamma.-tocopherol.
[0019] There is a need in the art for nucleic acid molecules
encoding enzymes involved in tocopherol biosynthesis, as well as
related enzymes and antibodies for the enhancement or alteration of
tocopherol production in plants. There is a further need for
transgenic organisms expressing those nucleic acid molecules
involved in tocopherol biosynthesis, which are capable of
nutritionally enhancing food and feed sources.
BRIEF SUMMARY OF THE INVENTION
[0020] The present invention includes and provides a substantially
purified nucleic acid molecule encoding a tMT2 enzyme.
[0021] The present invention includes and provides a substantially
purified nucleic acid molecule comprising a nucleotide sequence
selected from the group consisting of SEQ ID NOs: 1 and 2.
[0022] The present invention includes and provides a substantially
purified nucleic acid molecule comprising a nucleotide sequence
selected from the group consisting of SEQ ID NOs: 3 through 7.
[0023] The present invention includes and provides a substantially
purified nucleic acid molecule comprising a nucleotide sequence
selected from the group consisting of SEQ ID NOs: 8 through 14.
[0024] The present invention includes and provides a substantially
purified nucleic acid molecule encoding a plant polypeptide
molecule having 2-Methylphytylplastoquinol methyltransferase
activity.
[0025] The present invention includes and provides a substantially
purified plant polypeptide molecule having
2-Methylphytylplastoquinol methyltransferase activity.
[0026] The present invention includes and provides a substantially
purified mutant polypeptide molecule having an altered
2-Methylphytylplastoquinol methyltransferase activity relative to a
non-mutant polypeptide.
[0027] The present invention includes and provides a substantially
purified polypeptide molecule comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 16 and 28.
[0028] The present invention includes and provides a substantially
purified polypeptide molecule comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 17 through 21 and
29 through 32.
[0029] The present invention includes and provides a substantially
purified polypeptide molecule comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 22 through 27 and
33 through 38.
[0030] The present invention includes and provides an antibody
capable of specifically binding a polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs: 16
through 38.
[0031] The present invention includes and provides a transformed
plant comprising an introduced nucleic acid molecule comprising a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 1, 2, 8 through 15, and complements thereof.
[0032] The present invention includes and provides a transformed
plant comprising an introduced nucleic acid molecule that encodes a
polypeptide molecule comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 16, 22 through 28, and 33
through 38.
[0033] The present invention includes and provides a transformed
plant comprising a nucleic acid molecule that encodes a polypeptide
molecule comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 17 through 21, and 29 through 32.
[0034] The present invention includes and provides a transformed
plant comprising an introduced first nucleic acid molecule
comprising a sequence selected from the group consisting of SEQ ID
NOs: 1, 2, 8 through 15, and complements thereof, and an introduced
second nucleic acid molecule encoding an enzyme selected from the
group consisting of tyrA, slr1736, HPT, GMT, tocopherol cyclase,
dxs, dxr, GGPPS, HPPD, AANT1, slr1737, IDI, GGH, and complements
thereof, a plant ortholog thereof, and an antisense construct for
homogentisic acid dioxygenase.
[0035] The present invention includes and provides a transformed
plant comprising an introduced first nucleic acid molecule that
encodes a polypeptide molecule comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 16, 22 through
28, 33 through 38, and an introduced second nucleic acid molecule
encoding an enzyme selected from the group consisting of tyrA,
slr1736, HPT, GMT, tocopherol cyclase, dxs, dxr, GGPPS, HPPD,
AANT1, slr1737, IDI, GGH, and complements thereof, a plant ortholog
thereof, and an antisense construct for homogentisic acid
dioxygenase.
[0036] The present invention includes and provides a transformed
plant comprising an introduced first nucleic acid molecule
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1, 2, 8 through 15, and complements
thereof and an introduced second nucleic acid molecule comprising a
sequence selected from the group consisting of SEQ ID NOs: 39
through 54, and complements thereof.
[0037] The present invention includes and provides a transformed
plant comprising an introduced first nucleic acid molecule that
encodes a polypeptide molecule comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 16, 22 through
28, 33 through 38, and an introduced second nucleic acid molecule
having a sequence selected from the group consisting of SEQ ID NOs:
39 through 54, and complements thereof.
[0038] The present invention includes and provides a method for
reducing expression of the tMT2 gene in a plant comprising: (A)
transforming a plant with a nucleic acid molecule, said nucleic
acid molecule having an introduced promoter region which functions
in plant cells to cause the production of a mRNA molecule, wherein
said introduced promoter region is linked to a transcribed nucleic
acid molecule having a transcribed strand and a non-transcribed
strand, wherein said transcribed strand is complementary to a
nucleic acid molecule comprising a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1 through 15, and wherein
said transcribed nucleic acid molecule is linked to a 3'
non-translated sequence that functions in the plant cells to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA sequence; and (B) growing
said transformed plant.
[0039] The present invention includes and provides a transformed
plant comprising a nucleic acid molecule comprising an introduced
promoter region which functions in plant cells to cause the
production of an mRNA molecule, wherein said introduced promoter
region is linked to a transcribed nucleic acid molecule having a
transcribed strand and a non-transcribed strand, wherein said
transcribed strand is complementary to a nucleic acid molecule
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1 through 15, and wherein said
transcribed nucleic acid molecule is linked to a 3' non-translated
sequence that functions in the plant cells to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of the mRNA sequence.
[0040] The present invention includes and provides a method of
producing a plant having a seed with an increased
.gamma.-tocopherol level comprising: (A) transforming said plant
with an introduced nucleic acid molecule, wherein said nucleic acid
molecule comprises a sequence encoding a polypeptide molecule
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 16, 22 through 28, and 33 through 38; and
(B) growing said transformed plant.
[0041] The present invention includes and provides a method of
producing a plant having a seed with an increased
.gamma.-tocopherol level comprising: (A) transforming said plant
with an introduced first nucleic acid molecule, wherein said first
nucleic acid molecule comprises a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1, 2, 8 through 15, and an
introduced second nucleic acid molecule encoding an enzyme selected
from the group consisting of tyrA, slr1736, HPT, GMT, tocopherol
cyclase, dxs, dxr, GGPPS, HPPD, AANT1, slr1737, IDI, GGH, and
complements thereof, a plant ortholog thereof, and an antisense
construct for homogentisic acid dioxygenase; and (B) growing said
transformed plant.
[0042] The present invention includes and provides a method of
producing a plant having a seed with an increased
.gamma.-tocopherol level comprising: (A) transforming said plant
with an introduced first nucleic acid molecule, wherein said first
nucleic acid molecule comprises a sequence encoding a polypeptide
molecule comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 16, 22 through 28, 33 through 38, and an
introduced second nucleic acid molecule encoding an enzyme selected
from the group consisting of tyrA, slr1736, HPT, GMT, tocopherol
cyclase, dxs, dxr, GGPPS, HPPD, AANT1, slr1737, IDI, GGH, and
complements thereof, a plant ortholog thereof, and an antisense
construct for homogentisic acid dioxygenase; and (B) growing said
transformed plant.
[0043] The present invention includes and provides a method of
producing a plant having a seed with an increased
.alpha.-tocopherol level comprising: (A) transforming said plant
with an introduced first nucleic acid molecule, wherein said first
nucleic acid molecule comprises a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1, 2, 8 through 15, and an
introduced second nucleic acid molecule comprising a sequence
selected from the group consisting of SEQ ID NOs: 39 through 54,
and complements thereof; and (B) growing said transformed
plant.
[0044] The present invention includes and provides a method of
producing a plant having a seed with an increased
.alpha.-tocopherol level comprising: (A) transforming said plant
with an introduced first nucleic acid molecule, wherein said first
nucleic acid molecule comprises a sequence encoding a polypeptide
molecule comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 16, 22 through 28, 33 through 38, and an
introduced second nucleic acid molecule comprising a sequence
selected from the group consisting of SEQ ID NOs: 39 through 54,
and complements thereof; and (B) growing said transformed
plant.
[0045] The present invention includes and provides a seed derived
from a transformed plant comprising an introduced nucleic acid
molecule comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1, 2, and 8 through 15.
[0046] The present invention includes and provides a seed derived
from a transformed plant comprising an introduced nucleic acid
molecule comprising an introduced first nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 1, 2, 8 through
15, and an introduced second nucleic acid encoding an enzyme
selected from the group consisting of tyrA, slr1736, HPT, GMT,
tocopherol cyclase, dxs, dxr, GGPPS, HPPD, AANT1, slr1737, IDI,
GGH, and complements thereof, a plant ortholog thereof, and an
antisense construct for homogentisic acid dioxygenase.
[0047] The present invention includes and provides a seed derived
from a transformed plant comprising an introduced first nucleic
acid molecule comprising a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 1, 2, 8 through 15, and an
introduced second nucleic acid molecule comprising a sequence
selected from the group consisting of SEQ ID NOs: 39 through
54.
[0048] The present invention includes and provides a transformed
plant comprising an introduced first nucleic acid molecule
comprising a sequence selected from the group consisting of SEQ ID
NOs: 1, 2, 8 through 15, and complements thereof, and an introduced
second nucleic acid molecule comprising a sequence selected from
the group consisting of SEQ ID NOs: 39 through 54, and complements
thereof, and an introduced third nucleic acid molecule encoding an
enzyme selected from the group consisting of tyrA, slr1736, HPT,
GMT, tocopherol cyclase, dxs, dxr, GGPPS, HPPD, AANT1, slr1737,
IDI, GGH, and complements thereof, a plant ortholog thereof, and an
antisense construct for homogentisic acid dioxygenase.
[0049] The present invention includes and provides a transformed
plant comprising an introduced first nucleic acid molecule that
encodes a polypeptide molecule comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 16, 22 through
28, 33 through 38, an introduced second nucleic acid molecule
having a sequence selected from the group consisting of SEQ ID NOs:
39 through 54, and complements thereof, and an introduced third
nucleic acid molecule encoding an enzyme selected from the group
consisting of tyrA, slr1736, HPT, GMT, tocopherol cyclase, dxs,
dxr, GGPPS, HPPD, AANT1, slr1737, IDI, GGH, and complements
thereof.
[0050] The present invention includes and provides a transformed
plant comprising an introduced first nucleic acid molecule encoding
a tMT2 enzyme, and a second nucleic acid molecule encoding a GMT
enzyme.
[0051] The present invention includes and provides a method of
producing a plant having seed with an increased .alpha.-tocopherol
level comprising: (A) transforming said plant with a nucleic acid
molecule encoding a tMT2 enzyme and a nucleic acid molecule
encoding a GMT enzyme; and (B) growing said plant.
BRIEF DESCRIPTION OF THE NUCLEIC AND AMINO ACID SEQUENCES
[0052] SEQ ID NO: 1 sets forth a nucleic acid sequence of a DNA
molecule that encodes a wild type Arabidopsis thaliana, Columbia
ecotype, tMT2 enzyme.
[0053] SEQ ID NO: 2 sets forth a nucleic acid sequence of a DNA
molecule that encodes a wild type Arabidopsis thaliana, Landsberg
ecotype, tMT2 enzyme.
[0054] SEQ ID NO: 3 sets forth a nucleic acid sequence of a DNA
molecule that encodes an hdt2 mutant of the Arabidopsis thaliana,
Landsberg ecotype, tMT2 enzyme.
[0055] SEQ ID NO: 4 sets forth a nucleic acid sequence of a DNA
molecule that encodes an hdt6 mutant of the Arabidopsis thaliana,
Columbia ecotype, tMT2 enzyme.
[0056] SEQ ID NO: 5 sets forth a nucleic acid sequence of a DNA
molecule that encodes an hdt9 mutant of the Arabidopsis thaliana,
Columbia ecotype, tMT2 enzyme.
[0057] SEQ ID NO: 6 sets forth a nucleic acid sequence of a DNA
molecule that encodes an hdt10 mutant of the Arabidopsis thaliana,
Landsberg ecotype, tMT2 enzyme.
[0058] SEQ ID NO: 7 sets forth a nucleic acid sequence of a DNA
molecule that encodes an hdt16 mutant of the Arabidopsis thaliana,
Columbia ecotype, tMT2 enzyme.
[0059] SEQ ID NO: 8 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Zea mays tMT2 enzyme.
[0060] SEQ ID NO: 9 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Gossypium hirsutum tMT2 enzyme.
[0061] SEQ ID NO: 10 sets forth a nucleic acid sequence of a DNA
molecule that encodes an Allium porrum tMT2 enzyme.
[0062] SEQ ID NO: 11 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Glycine max tMT2 enzyme.
[0063] SEQ ID NO: 12 sets forth a nucleic acid sequence of a DNA
molecule that encodes an Oryza sativa tMT2 enzyme.
[0064] SEQ ID NO: 13 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Brassica napus tMT2 enzyme.
[0065] SEQ ID NO: 14 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Brassica napus tMT2 enzyme different in
sequence from SEQ ID NO: 13.
[0066] SEQ ID NO: 15 sets forth a nucleic acid coding sequence of a
wild type Arabidopsis thaliana tMT2 enzyme.
[0067] SEQ ID NO: 16 sets forth an amino acid sequence of a wild
type Arabidopsis thaliana, Columbia and Landsberg ecotype, tMT2
enzyme.
[0068] SEQ ID NO: 17 sets forth an amino acid sequence of an hdt2
mutant of the Arabidopsis thaliana, Landsberg ecotype, tMT2
enzyme.
[0069] SEQ ID NO: 18 sets forth an amino acid sequence of an hdt6
mutant of the Arabidopsis thaliana, Columbia ecotype, tMT2
enzyme.
[0070] SEQ ID NO: 19 sets forth an amino acid sequence of an hdt9
mutant of the Arabidopsis thaliana, Columbia ecotype, tMT2
enzyme.
[0071] SEQ ID NO: 20 sets forth an amino acid sequence of an hdt10
mutant of the Arabidopsis thaliana, Landsberg ecotype, tMT2
enzyme.
[0072] SEQ ID NO: 21 sets forth an amino acid sequence of an hdt16
mutant of the Arabidopsis thaliana, Columbia ecotype, tMT2
enzyme.
[0073] SEQ ID NO: 22 sets forth an amino acid sequence of a Zea
mays tMT2 enzyme.
[0074] SEQ ID NO: 23 sets forth an amino acid sequence of a
Gossypium hirsutum tMT2 enzyme.
[0075] SEQ ID NO: 24 sets forth an amino acid sequence of an Allium
porrum tMT2 enzyme.
[0076] SEQ ID NO: 25 sets forth an amino acid sequence of a Glycine
max tMT2 enzyme.
[0077] SEQ ID NO: 26 sets forth an amino acid sequence of an Oryza
sativa tMT2 enzyme.
[0078] SEQ ID NO: 27 sets forth an amino acid sequence of a
Brassica napus tMT2 enzyme.
[0079] SEQ ID NO: 28 sets forth an amino acid sequence of a mature
wild type Arabidopsis thaliana, Columbia ecotype, tMT2 enzyme.
[0080] SEQ ID NO: 29 sets forth an amino acid sequence of a mature
hdt2 mutant of the Arabidopsis thaliana, Landsberg ecotype, tMT2
enzyme.
[0081] SEQ ID NO: 30 sets forth an amino acid sequence of a mature
hdt6 mutant of the Arabidopsis thaliana, Columbia ecotype, tMT2
enzyme.
[0082] SEQ ID NO: 31 sets forth an amino acid sequence of a mature
hdt10 mutant of the Arabidopsis thaliana, Landsberg ecotype, tMT2
enzyme.
[0083] SEQ ID NO: 32 sets forth an amino acid sequence of a mature
hdt16 mutant of the Arabidopsis thaliana, Columbia ecotype, tMT2
enzyme.
[0084] SEQ ID NO: 33 sets forth an amino acid sequence of a mature
Brassica napus tMT2 enzyme.
[0085] SEQ ID NO: 34 sets forth an amino acid sequence of a mature
Oryza sativa tMT2 enzyme.
[0086] SEQ ID NO: 35 sets forth an amino acid sequence of a mature
Zea mays tMT2 enzyme.
[0087] SEQ ID NO: 36 sets forth an amino acid sequence of a mature
Glycine max tMT2 enzyme.
[0088] SEQ ID NO: 37 sets forth an amino acid sequence of a mature
Allium porrum tMT2 enzyme.
[0089] SEQ ID NO: 38 sets forth an amino acid sequence of a mature
Gossypium hirsutum tMT2 enzyme.
[0090] SEQ ID NO: 39 sets forth a nucleic acid sequence of a DNA
molecule that encodes an Arabidopsis thaliana .gamma.-tocopherol
methyltransferase.
[0091] SEQ ID NO: 40 sets forth a nucleic acid sequence of a DNA
molecule that encodes an Arabidopsis thaliana, Columbia ecotype,
.gamma.-tocopherol methyltransferase.
[0092] SEQ ID NO: 41 sets forth a nucleic acid sequence of a DNA
molecule that encodes an Oryza sativa .gamma.-tocopherol
methyltransferase.
[0093] SEQ ID NO: 42 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Zea mays .gamma.-tocopherol
methyltransferase.
[0094] SEQ ID NO: 43 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Gossypium hirsutum .gamma.-tocopherol
methyltransferase.
[0095] SEQ ID NO: 44 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Cuphea pulcherrima .gamma.-tocopherol
methyltransferase.
[0096] SEQ ID NO: 45 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Brassica napus S8 .gamma.-tocopherol
methyltransferase.
[0097] SEQ ID NO: 46 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Brassica napus P4 .gamma.-tocopherol
methyltransferase.
[0098] SEQ ID NO: 47 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Lycopersicon esculentum .gamma.-tocopherol
methyltransferase.
[0099] SEQ ID NO: 48 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Glycine max .gamma.-tocopherol
methyltransferase 1.
[0100] SEQ ID NO: 49 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Glycine max .gamma.-tocopherol
methyltransferase 2.
[0101] SEQ ID NO: 50 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Glycine max .gamma.-tocopherol
methyltransferase 3.
[0102] SEQ ID NO: 51 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Tagetes erecta .gamma.-tocopherol
methyltransferase.
[0103] SEQ ID NO: 52 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Sorghum bicolor .gamma.-tocopherol
methyltransferase
[0104] SEQ ID NO: 53 sets forth a nucleic acid sequence of a DNA
molecule that encodes a Nostoc punctiforme .gamma.-tocopherol
methyltransferase.
[0105] SEQ ID NO: 54 sets forth a nucleic acid sequence of a DNA
molecule that encodes an Anabaena .gamma.-tocopherol
methyltransferase.
[0106] SEQ ID NOs: 55 and 56 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--1 primer pair.
[0107] SEQ ID NOs: 57 and 58 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--2 primer pair.
[0108] SEQ ID NOs: 59 and 60 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--3 primer pair.
[0109] SEQ ID NOs: 61 and 62 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--4 primer pair.
[0110] SEQ ID NOs: 63 and 64 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--5 primer pair.
[0111] SEQ ID NOs: 65 and 66 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--6 primer pair.
[0112] SEQ ID NOs: 67 and 68 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--7 primer pair.
[0113] SEQ ID NOs: 69 and 70 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--8 primer pair.
[0114] SEQ ID NOs: 71 and 72 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--9 primer pair.
[0115] SEQ ID NOs: 73 and 74 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--10 primer pair.
[0116] SEQ ID NOs: 75 and 76 set forth nucleic acid sequences of
the MAA21.sub.--40.sub.--11 primer pair.
[0117] SEQ ID NOs: 77 and 78 set forth nucleic acid sequences of
primers for use in amplifying a gene encoding a mature Brassica
napus tMT2 enzyme.
[0118] SEQ ID NOs: 79 and 80 set forth nucleic acid sequences of
primers for use in amplifying a gene encoding a mature Oryza sativa
tMT2 enzyme.
[0119] SEQ ID NOs: 81 and 82 set forth nucleic acid sequences of
primers for use in amplifying a gene encoding a mature Zea mays
tMT2 enzyme.
[0120] SEQ ID NOs: 83 and 84 set forth nucleic acid sequences of
primers for use in amplifying a gene encoding a mature Glycine max
tMT2 enzyme.
[0121] SEQ ID NOs: 85 and 86 set forth nucleic acid sequences of
primers for use in amplifying a gene encoding a mature Allium
porrum tMT2 enzyme.
[0122] SEQ ID NOs: 87 and 88 set forth nucleic acid sequences of
primers for use in amplifying a gene encoding a mature Gossypium
hirsutum tMT2 enzyme.
[0123] SEQ ID NOs: 89 and 90 set forth nucleic acid sequences of
primers #17286 and #17181 for use in amplifying a gene encoding a
full length Arabidopsis thaliana tMT2 enzyme.
[0124] SEQ ID NO: 91 sets forth an amino acid sequence of an
Arabidopsis thaliana .gamma.-tocopherol methyltransferase.
[0125] SEQ ID NO: 92 sets forth an amino acid sequence of an
Arabidopsis thaliana, Columbia ecotype, .gamma.-tocopherol
methyltransferase.
[0126] SEQ ID NO: 93 sets forth an amino acid sequence of an Oryza
sativa .gamma.-tocopherol methyltransferase.
[0127] SEQ ID NO: 94 sets forth an amino acid sequence of a Zea
mays .gamma.-tocopherol methyltransferase.
[0128] SEQ ID NO: 95 sets forth an amino acid sequence of a
Gossypium hirsutum .gamma.-tocopherol methyltransferase.
[0129] SEQ ID NO: 96 sets forth an amino acid sequence of a Cuphea
pulcherrima .gamma.-tocopherol methyltransferase.
[0130] SEQ ID NO: 97 sets forth an amino acid sequence of a
Brassica napus S8 .gamma.-tocopherol methyltransferase.
[0131] SEQ ID NO: 98 sets forth an amino acid sequence of a
Brassica napus P4 .gamma.-tocopherol methyltransferase.
[0132] SEQ ID NO: 99 sets forth an amino acid sequence of a
Lycopersicon esculentum .gamma.-tocopherol methyltransferase.
[0133] SEQ ID NO: 100 sets forth an amino acid sequence of a
Glycine max .gamma.-tocopherol methyltransferase 1.
[0134] SEQ ID NO: 101 sets forth an amino acid sequence of a
Glycine max .gamma.-tocopherol methyltransferase 2.
[0135] SEQ ID NO: 102 sets forth an amino acid sequence of a
Glycine max .gamma.-tocopherol methyltransferase 3.
[0136] SEQ ID NO: 103 sets forth an amino acid sequence of a
Tagetes erecta .gamma.-tocopherol methyltransferase.
[0137] SEQ ID NO: 104 sets forth an amino acid sequence of a
Sorghum bicolor .gamma.tocopherol methyltransferase.
[0138] SEQ ID NO: 105 sets forth an amino acid sequence of a Lilium
asiaticum .gamma.-tocopherol methyltransferase.
[0139] SEQ ID NO: 106 sets forth an amino acid sequence of a Nostoc
punctiforme .gamma.-tocopherol methyltransferase.
[0140] SEQ ID NO: 107 sets forth an amino acid sequence of an
Anabaena .gamma.-tocopherol methyltransferase.
[0141] tocopherol methyltransferase.
[0142] SEQ ID NO: 108 sets forth an amino acid consensus sequence
for the aligned polypeptides shown in FIGS. 3a and 3b.
BRIEF DESCRIPTION OF THE FIGURES
[0143] FIG. 1 is a schematic diagram of the tocopherol biosynthetic
pathway.
[0144] FIG. 2 represents the results of a TBLASTN homology
comparison of the nucleotide sequences of several crop tMT2 genes
to the amino acid sequence of a tMT2 gene from Arabidopsis thaliana
(NCBI General Identifier Number gi7573324).
[0145] FIGS. 3a and 3b represent the Pretty Alignment (Genetics
Computer Group, Madison Wis.) of tMT2 protein sequences from
different plant species.
[0146] FIG. 4 represents a graph depicting the methyltransferase
activity of recombinantly expressed Anabaena MT1 (positive
control). Enzyme activity is monitored on crude cell extracts from
E. coli harboring pMON67174.
[0147] FIG. 5 represents a graph depicting the methyltransferase
activity of recombinantly expressed mature Arabidopsis tMT2. Enzyme
activity is monitored on crude cell extracts from E. coli harboring
pMON67191.
[0148] FIG. 6 represents a graph depicting the methyltransferase
activity of recombinantly expressed mature Arabidopsis tMT2 hdt2
mutant. Enzyme activity is monitored on crude cell extracts from E.
coli harboring pMON67207.
[0149] FIG. 7 represents a graph depicting the methyltransferase
activity of recombinantly expressed Anabaena MT1 without
2-methylphytylplastoquinol substrate (negative control). Enzyme
activity is monitored on crude cell extracts from E. coli harboring
pMON67174.
[0150] FIG. 8 represents a graph depicting the methyltransferase I
activity in isolated pea chloroplasts (positive control).
[0151] FIG. 9 is a plasmid map of pMON67205.
[0152] FIG. 10 is a plasmid map of pMON67220.
[0153] FIG. 11 is a plasmid map of pMON67226.
[0154] FIG. 12 is a plasmid map of pMON67225.
[0155] FIG. 13 is a plasmid map of pMON67227.
[0156] FIG. 14 is a plasmid map of pMON67224.
[0157] FIG. 15 is a plasmid map of pMON67223.
[0158] FIGS. 16a and 16b depict the levels of expression of
.delta.-tocopherol in various types of Arabidopsis.
[0159] FIG. 17 depicts T3 seed .delta.-tocopherol (%) from two
lines expressing tMT2 under the control of the napin promoter
(pMON67205) in the hdt2 mutant line.
[0160] FIGS. 18a-d depict the levels of .alpha., .beta., .gamma.,
and .delta.-tocopherol in tMT2 pools of 10 seeds.
[0161] FIGS. 19a-d depict the levels of .alpha., .beta., .gamma.,
and .delta.-tocopherol in tMT2/GMT pools of 10 seeds.
[0162] FIG. 20 depicts the tocopherol composition of single seeds
from one line of soybean (28072) transformed with pMON67226.
[0163] FIGS. 21a-d depict the levels of .alpha., .beta., .gamma.,
and .delta.-tocopherol in R1 Soy Single Seed from pMON67226.
[0164] FIG. 22 depicts the tocopherol composition of single seeds
from one line of soybean (28906) transformed with pMON67227.
[0165] FIGS. 23a-d depict the levels of .alpha., .beta., .gamma.,
and .delta.-tocopherol in R1 Soy Single Seed from pMON67227.
[0166] FIG. 24 depicts the results of various
2-methylphytylplastoquinol methyltransferase assays.
DETAILED DESCRIPTION OF THE INVENTION
[0167] The present invention provides a number of agents, for
example, nucleic acid molecules and polypeptides associated with
the synthesis of tocopherol, and provides uses of such agents.
Agents
[0168] The agents of the invention will preferably be "biologically
active" with respect to either a structural attribute, such as the
capacity of a nucleic acid to hybridize to another nucleic acid
molecule, or the ability of a protein to be bound by an antibody
(or to compete with another molecule for such binding).
Alternatively, such an attribute may be catalytic and thus involve
the capacity of the agent to mediate a chemical reaction or
response. The agents will preferably be "substantially purified."
The term "substantially purified," as used herein, refers to a
molecule separated from substantially all other molecules normally
associated with it in its native environmental conditions. More
preferably a substantially purified molecule is the predominant
species present in a preparation. A substantially purified molecule
may be greater than 60% free, preferably 75% free, more preferably
90% free, and most preferably 95% free from the other molecules
(exclusive of solvent) present in the natural mixture. The term
"substantially purified" is not intended to encompass molecules
present in their native environmental conditions.
[0169] The agents of the invention may also be recombinant. As used
herein, the term recombinant means any agent (e.g., DNA, peptide
etc.), that is, or results, however indirectly, from human
manipulation of a nucleic acid molecule.
[0170] It is understood that the agents of the invention may be
labeled with reagents that facilitate detection of the agent (e.g.,
fluorescent labels, Prober et al., Science 238:336-340 (1987);
Albarella et al., EP 144914; chemical labels, Sheldon et al., U.S.
Pat. No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417;
modified bases, Miyoshi et al., EP 119448).
Nucleic Acid Molecules
[0171] Agents of the invention include nucleic acid molecules. In a
preferred aspect of the present invention the nucleic acid molecule
comprises a nucleic acid sequence, which encodes a tocopherol
methyltransferase. As used herein, a tocopherol methyltransferase
(tMT2) is any plant protein that is capable of specifically
catalyzing the methylation of the 3 position of the phenyl ring of
2-methylphytylplastoquinol, 2-methyl-5-phytylplastoquinol,
2-methyl-3-phytylplastoquinol, .delta.-tocopherol, or
.beta.-tocopherol (see, Photosyn. Research, 31:99-111 (1992) and
Phytochemistry 19:215-218 (1980)). A preferred tMT2 is found in an
organism selected from the group consisting of Arabidopsis, maize,
cotton, leek, soybean, rice, and oilseed rape. An example of a more
preferred tMT2 is a polypeptide with the amino acid sequence
selected from the group consisting of SEQ ID NOs: 16 through 38. In
a more preferred embodiment, the tMT2 is encoded by any of SEQ ID
NOs: 1 through 15.
[0172] In another preferred aspect of the present invention a
nucleic acid molecule of the present invention comprises a nucleic
acid sequence selected from the group consisting of SEQ ID NOs: 1
through 15, and complements thereof and fragments of either. In
another preferred aspect of the present invention, a nucleic acid
molecule of the present invention comprises a nucleic acid sequence
selected from the group consisting of SEQ ID NOs: 1 and 2, and
complements thereof. In another preferred aspect of the present
invention the nucleic acid molecule of the invention comprises a
nucleic acid sequence selected from the group consisting of SEQ ID
NOs: 3 through 7, and complements thereof. In another preferred
aspect of the present invention the nucleic acid molecule of the
invention comprises a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 8 through 14, and complements thereof. In
another preferred aspect of the present invention the nucleic acid
molecule of the invention comprises the nucleic acid sequence of
SEQ ID NO: 15 and its complement. In a further aspect of the
present invention the nucleic acid molecule comprises a nucleic
acid sequence encoding an amino acid sequence selected from the
group consisting of SEQ ID NOs: 16 through 38, and fragments
thereof. In a further aspect of the present invention the nucleic
acid molecule comprises a nucleic acid sequence encoding amino acid
sequence SEQ ID NO: 16 and fragments thereof.
[0173] In another embodiment, the present invention provides
nucleic acid molecules comprising a sequence encoding SEQ ID NO:
108, and complements thereof. In another aspect, the present
invention provides nucleic acid molecules comprising a sequence
encoding residues 83 through 356 of SEQ ID NO: 108, and its
complement. In another aspect, the present invention provides
nucleic acid molecules comprising a sequence encoding a fragment of
residues 83 through 356 of SEQ ID NO: 108, wherein the fragment has
a length of at least about 25, 50, 75, 100, 150, 200, or 250
residues, and complements thereof. In yet another aspect, the
present invention provides nucleic acid molecules encoding one or
more of the following fragments of SEQ ID NO: 108, and complements
thereof: 82 through 123, 132 through 146, and 269 through 295.
[0174] The present invention includes the use of the
above-described sequences and fragments thereof in transgenic
plants, other organisms, and for other uses as described below.
[0175] In a further aspect of the present invention the nucleic
acid molecule comprises a nucleic acid sequence encoding an amino
acid sequence selected from the group consisting of SEQ ID NOs: 17
through 21, and fragments thereof. In a further aspect of the
present invention the nucleic acid molecule comprises a nucleic
acid sequence encoding an amino acid sequence selected from the
group consisting of SEQ ID NOs: 22 through 27, and fragments
thereof. In a further aspect of the present invention the nucleic
acid molecule comprises a nucleic acid sequence encoding an amino
acid sequence selected from the group consisting of SEQ ID NOs: 28
through 38, and fragments thereof. In a further aspect of the
present invention the nucleic acid molecule comprises a nucleic
acid sequence encoding an amino acid of SEQ ID NO: 28 and fragments
thereof. In a further aspect of the present invention the nucleic
acid molecule comprises a nucleic acid sequence encoding an amino
acid sequence selected from the group consisting of SEQ ID NOs: 29
through 32, and fragments thereof. In a further aspect of the
present invention the nucleic acid molecule comprises a nucleic
acid sequence encoding an amino acid sequence selected from the
group consisting of SEQ ID NOs: 33 through 38, and fragments
thereof.
[0176] In another preferred aspect of the present invention a
nucleic acid molecule comprises nucleotide sequences encoding a
plastid transit peptide operably fused to a nucleic acid molecule
that encodes a protein or fragment of the present invention.
[0177] In another preferred embodiment of the present invention,
the nucleic acid molecules of the invention encode mutant tMT2
enzymes. As used herein, a "mutant" enzyme or polypeptide is any
enzyme or polypeptide that contains an amino acid that is different
from the amino acid in the same position of a wild type enzyme of
the same type. Examples of suitable mutants of the invention
include, but are not limited to, those found in Example 1 of this
application.
[0178] It is understood that in a further aspect of nucleic acid
sequences of the present invention, the nucleic acids can encode a
protein that differs from any of the proteins in that one or more
amino acids have been deleted, substituted or added without
altering the function. For example, it is understood that codons
capable of coding for such conservative amino acid substitutions
are known in the art.
[0179] In one aspect of the present invention the nucleic acids of
the present invention are said to be introduced nucleic acid
molecules. A nucleic acid molecule is said to be "introduced" if it
is inserted into a cell or organism as a result of human
manipulation, no matter how indirect. Examples of introduced
nucleic acid molecules include, without limitation, nucleic acids
that have been introduced into cells via transformation,
transfection, injection, and projection, and those that have been
introduced into an organism via conjugation, endocytosis,
phagocytosis, etc.
[0180] One subset of the nucleic acid molecules of the invention is
fragment nucleic acids molecules. Fragment nucleic acid molecules
may consist of significant portion(s) of, or indeed most of, the
nucleic acid molecules of the invention, such as those specifically
disclosed. Alternatively, the fragments may comprise smaller
oligonucleotides (having from about 15 to about 400 nucleotide
residues and more preferably, about 15 to about 30 nucleotide
residues, or about 50 to about 100 nucleotide residues, or about
100 to about 200 nucleotide residues, or about 200 to about 400
nucleotide residues, or about 275 to about 350 nucleotide
residues).
[0181] A fragment of one or more of the nucleic acid molecules of
the invention may be a probe and specifically a PCR probe. A PCR
probe is a nucleic acid molecule capable of initiating a polymerase
activity while in a double-stranded structure with another nucleic
acid. Various methods for determining the structure of PCR probes
and PCR techniques exist in the art. Computer generated searches
using programs such as Primer3
(www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi), STSPipeline
(www-genome.wi.mit.edu/cgi-bin/www-STS.sub.--Pipeline), or GeneUp
(Pesole et al., BioTechniques 25:112-123 (1998)), for example, can
be used to identify potential PCR primers.
[0182] Nucleic acid molecules or fragments thereof of the present
invention are capable of specifically hybridizing to other nucleic
acid molecules under certain circumstances. Nucleic acid molecules
of the present invention include those that specifically hybridize
to nucleic acid molecules having a nucleic acid sequence selected
from the group consisting of SEQ ID NOs: 1 through 15, and
complements thereof. Nucleic acid molecules of the present
invention also include those that specifically hybridize to nucleic
acid molecules encoding an amino acid sequence selected from SEQ ID
NOs: 16 through 38, and fragments thereof.
[0183] As used herein, two nucleic acid molecules are said to be
capable of specifically hybridizing to one another if the two
molecules are capable of forming an anti-parallel, double-stranded
nucleic acid structure.
[0184] A nucleic acid molecule is said to be the "complement" of
another nucleic acid molecule if they exhibit complete
complementarity. As used herein, molecules are said to exhibit
"complete complementarity" when every nucleotide of one of the
molecules is complementary to a nucleotide of the other. Two
molecules are said to be "minimally complementary" if they can
hybridize to one another with sufficient stability to permit them
to remain annealed to one another under at least conventional
"low-stringency" conditions. Similarly, the molecules are said to
be "complementary" if they can hybridize to one another with
sufficient stability to permit them to remain annealed to one
another under conventional "high-stringency" conditions.
Conventional stringency conditions are described by Sambrook et
al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989), and by Haymes et
al., Nucleic Acid Hybridization, A Practical Approach, IRL Press,
Washington, D.C. (1985). Departures from complete complementarity
are therefore permissible, as long as such departures do not
completely preclude the capacity of the molecules to form a
double-stranded structure. Thus, in order for a nucleic acid
molecule to serve as a primer or probe it need only be sufficiently
complementary in sequence to be able to form a stable
double-stranded structure under the particular solvent and salt
concentrations employed.
[0185] Appropriate stringency conditions which promote DNA
hybridization are, for example, 6.0.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by a wash of
2.0.times.SSC at 20-25.degree. C., are known to those skilled in
the art or can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the
salt concentration in the wash step can be selected from a low
stringency of about 2.0.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 65.degree. C. In addition, the
temperature in the wash step can be increased from low stringency
conditions at room temperature, about 22.degree. C., to high
stringency conditions at about 65.degree. C. Both temperature and
salt may be varied, or either the temperature or the salt
concentration may be held constant while the other variable is
changed.
[0186] In a preferred embodiment, a nucleic acid of the present
invention will specifically hybridize to one or more of the nucleic
acid molecules set forth in SEQ ID NOs: 1 through 15, and
complements thereof under moderately stringent conditions, for
example at about 2.0.times.SSC and about 65.degree. C.
[0187] In a particularly preferred embodiment, a nucleic acid of
the present invention will include those nucleic acid molecules
that specifically hybridize to one or more of the nucleic acid
molecules set forth in SEQ ID NOs: 1 through 15, and complements
thereof under high stringency conditions such as 0.2.times.SSC and
about 65.degree. C.
[0188] In one aspect of the present invention, the nucleic acid
molecules of the present invention have one or more of the nucleic
acid sequences set forth in SEQ ID NOs: 1 through 15, and
complements thereof. In another aspect of the present invention,
one or more of the nucleic acid molecules of the present invention
share between 100% and 90% sequence identity with one or more of
the nucleic acid sequences set forth in SEQ ID NOs: 1 through 15,
and complements thereof and fragments of either. In a further
aspect of the present invention, one or more of the nucleic acid
molecules of the present invention share between 100% and 95%
sequence identity with one or more of the nucleic acid sequences
set forth in SEQ ID NOs: 1 through 15, complements thereof, and
fragments of either. In a more preferred aspect of the present
invention, one or more of the nucleic acid molecules of the present
invention share between 100% and 98% sequence identity with one or
more of the nucleic acid sequences set forth in SEQ ID NOs: 1
through 15, complements thereof and fragments of either. In an even
more preferred aspect of the present invention, one or more of the
nucleic acid molecules of the present invention share between 100%
and 99% sequence identity with one or more of the sequences set
forth in SEQ ID NOs: 1 through 15, complements thereof, and
fragments of either.
[0189] In a preferred embodiment the percent identity calculations
are performed using BLASTN or BLASTP (default, parameters, version
2.0.8, Altschul et al., Nucleic Acids Res. 25:3389-3402
(1997)).
[0190] A nucleic acid molecule of the invention can also encode a
homolog polypeptide. As used herein, a homolog polypeptide molecule
or fragment thereof is a counterpart protein molecule or fragment
thereof in a second species (e.g., corn rubisco small subunit is a
homolog of Arabidopsis rubisco small subunit). A homolog can also
be generated by molecular evolution or DNA shuffling techniques, so
that the molecule retains at least one functional or structure
characteristic of the original polypeptide (see, for example, U.S.
Pat. No. 5,811,238).
[0191] In another embodiment, the homolog is selected from the
group consisting of alfalfa, Arabidopsis, barley, Brassica
campestris, Brassica napus, oilseed rape, broccoli, cabbage,
canola, citrus, cotton, garlic, oat, Allium, flax, an ornamental
plant, peanut, pepper, potato, rapeseed, rice, rye, sorghum,
strawberry, sugarcane, sugarbeet, tomato, wheat, poplar, pine, fir,
eucalyptus, apple, lettuce, lentils, grape, banana, tea, turf
grasses, sunflower, soybean, corn, Phaseolus, crambe, mustard,
castor bean, sesame, cottonseed, linseed, safflower, and oil palm.
More particularly, preferred homologs are selected from canola,
corn, Brassica campestris, Brassica napus, oilseed rape, soybean,
crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed,
rapeseed, safflower, oil palm, flax, and sunflower. In an even more
preferred embodiment, the homolog is selected from the group
consisting of canola, rapeseed, corn, Brassica campestris, Brassica
napus, oilseed rape, soybean, sunflower, safflower, oil palms, and
peanut. In a particularly preferred embodiment, the homolog is
soybean. In a particularly preferred embodiment, the homolog is
canola. In a particularly preferred embodiment, the homolog is
oilseed rape.
[0192] In a preferred embodiment, nucleic acid molecules having SEQ
ID NOs: 1 through 15, complements thereof, and fragments of either;
or more preferably SEQ ID NOs: 1 through 15, and complements
thereof, can be utilized to obtain such homologs.
[0193] In another further aspect of the present invention, nucleic
acid molecules of the present invention can comprise sequences that
differ from those encoding a polypeptide or fragment thereof in SEQ
ID NOs: 1 through 15 due to the fact that a polypeptide can have
one or more conservative amino acid changes, and nucleic acid
sequences coding for the polypeptide can therefore have sequence
differences. It is understood that codons capable of coding for
such conservative amino acid substitutions are known in the
art.
[0194] It is well known in the art that one or more amino acids in
a native sequence can be substituted with other amino acid(s), the
charge and polarity of which are similar to that of the native
amino acid, i.e., a conservative amino acid substitution.
Conservative substitutes for an amino acid within the native
polypeptide sequence can be selected from other members of the
class to which the amino acid belongs. Amino acids can be divided
into the following four groups: (1) acidic amino acids, (2) basic
amino acids, (3) neutral polar amino acids, and (4) neutral,
nonpolar amino acids. Representative amino acids within these
various groups include, but are not limited to, (1) acidic
(negatively charged) amino acids such as aspartic acid and glutamic
acid; (2) basic (positively charged) amino acids such as arginine,
histidine, and lysine; (3) neutral polar amino acids such as
glycine, serine, threonine, cysteine, cystine, tyrosine,
asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic)
amino acids such as alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine.
[0195] Conservative amino acid substitution within the native
polypeptide sequence can be made by replacing one amino acid from
within one of these groups with another amino acid from within the
same group. In a preferred aspect, biologically functional
equivalents of the proteins or fragments thereof of the present
invention can have ten or fewer conservative amino acid changes,
more preferably seven or fewer conservative amino acid changes, and
most preferably five or fewer conservative amino acid changes. The
encoding nucleotide sequence will thus have corresponding base
substitutions, permitting it to encode biologically functional
equivalent forms of the polypeptides of the present invention.
[0196] It is understood that certain amino acids may be substituted
for other amino acids in a protein structure without appreciable
loss of interactive binding capacity with structures such as, for
example, antigen-binding regions of antibodies or binding sites on
substrate molecules. Because it is the interactive capacity and
nature of a protein that defines that protein's biological
functional activity, certain amino acid sequence substitutions can
be made in a protein sequence and, of course, its underlying DNA
coding sequence and, nevertheless, a protein with like properties
can still be obtained. It is thus contemplated by the inventors
that various changes may be made in the peptide sequences of the
proteins or fragments of the present invention, or corresponding
DNA sequences that encode said peptides, without appreciable loss
of their biological utility or activity. It is understood that
codons capable of coding for such amino acid changes are known in
the art.
[0197] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring interactive biological function on a protein is
generally understood in the art (Kyte and Doolittle, J. Mol. Biol.
157, 105-132 (1982)). It is accepted that the relative hydropathic
character of the amino acid contributes to the secondary structure
of the resultant polypeptide, which in turn defines the interaction
of the protein with other molecules, for example, enzymes,
substrates, receptors, DNA, antibodies, antigens, and the like.
[0198] Each amino acid has been assigned a hydropathic index on the
basis of its hydrophobicity and charge characteristics (Kyte and
Doolittle, J. Mol. Biol. 157:105-132 (1982)); these are isoleucine
(+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8),
cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine
(-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9),
tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate
(-3.5), glutamine (-3.5), aspartate (-3.5), asparagine (-3.5),
lysine (-3.9), and arginine (-4.5).
[0199] In making such changes, the substitution of amino acids
whose hydropathic indices are within .+-.2 is preferred, those that
are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0200] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101 states that the greatest
local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with a
biological property of the protein.
[0201] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0), lysine (+3.0), aspartate (+3.0.+-.1), glutamate
(+3.0.+-.1), serine (+0.3), asparagine (+0.2), glutamine (+0.2),
glycine (0), threonine (-0.4), proline (-0.5.+-.1), alanine (-0.5),
histidine (-0.5), cysteine (-1.0), methionine (-1.3), valine
(-1.5), leucine (-1.8), isoleucine (-1.8), tyrosine (-2.3),
phenylalanine (-2.5), and tryptophan (-3.4).
[0202] In making such changes, the substitution of amino acids
whose hydrophilicity values are within .+-.2 is preferred, those
that are within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0203] In a further aspect of the present invention, one or more of
the nucleic acid molecules of the present invention differ in
nucleic acid sequence from those for which a specific sequence is
provided herein because one or more codons has been replaced with a
codon that encodes a conservative substitution of the amino acid
originally encoded.
[0204] Agents of the invention include nucleic acid molecules that
encode at least about a contiguous 10 amino acid region of a
polypeptide of the present invention, more preferably at least
about a contiguous 25, 40, 50, 100, or 125 amino acid region of a
polypeptide of the present invention.
[0205] In a preferred embodiment, any of the nucleic acid molecules
of the present invention can be operably linked to a promoter
region that functions in a plant cell to cause the production of an
mRNA molecule, where the nucleic acid molecule that is linked to
the promoter is heterologous with respect to that promoter. As used
herein, "heterologous" means not naturally occurring together.
Protein and Peptide Molecules
[0206] A class of agents includes one or more of the polypeptide
molecules encoded by a nucleic acid agent of the invention. A
particular preferred class of proteins is that having an amino acid
sequence selected from the group consisting of SEQ ID NOs: 16
through 38, and fragments thereof. In a further aspect of the
present invention the polypeptide molecule comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 17
through 21, and fragments thereof. In a further aspect of the
present invention the polypeptide molecule comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 22
through 27, and fragments thereof. In a further aspect of the
present invention the polypeptide molecule comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 28
through 38, and fragments thereof. In a further aspect of the
present invention the polypeptide molecule comprises an amino acid
sequence encoding an amino acid of SEQ ID NO: 28 and fragments
thereof. In a further aspect of the present invention the
polypeptide molecule comprises an amino acid sequence selected from
the group consisting of SEQ ID NOs: 29 through 32, and fragments
thereof. In a further aspect of the present invention the
polypeptide molecule comprises an amino acid sequence selected from
the group consisting of SEQ ID NOs: 33 through 38, and fragments
thereof.
[0207] In another embodiment, the present invention provides a
polypeptide comprising the amino acid sequence of SEQ ID NO: 108.
In another aspect, the present invention provides a polypeptide
comprising the amino acid sequence of residues 83 through 356 of
SEQ ID NO: 108. In another aspect, the present invention provides a
polypeptide fragment comprising the amino acid sequence of residues
83 through 356 of SEQ ID NO: 108, wherein the fragment has a length
of at least about 25, 50, 75, 100, 150, 200, or 250 residues. In
yet another aspect, the present invention provides a polypeptide
comprising the amino acid sequence of one or more of the following
fragments of SEQ ID NO: 108: 82 through 123, 132 through 146, and
269 through 295.
[0208] Polypeptide agents may have C-terminal or N-terminal amino
acid sequence extensions. One class of N-terminal extensions
employed in a preferred embodiment are plastid transit peptides.
When employed, plastid transit peptides can be operatively linked
to the N-terminal sequence, thereby permitting the localization of
the agent polypeptides to plastids. In an embodiment of the present
invention, any suitable plastid targeting sequence can be used.
Where suitable, a plastid targeting sequence can be substituted for
a native plastid targeting sequence, for example, for the CTP
occurring natively in the tMT2 protein. In a further embodiment, a
plastid targeting sequence that is heterologous to any tMT2 protein
or fragment described herein can be used. In a further embodiment,
any suitable, modified plastid targeting sequence can be used. In
another embodiment, the plastid targeting sequence is a CTP1
sequence (see WO 00/61771).
[0209] In a preferred aspect a protein of the present invention is
targeted to a plastid using either a native transit peptide
sequence or a heterologous transit peptide sequence. In the case of
nucleic acid sequences corresponding to nucleic acid sequences of
non-higher plant organisms such as cynobacteria, such nucleic acid
sequences can be modified to attach the coding sequence of the
protein to a nucleic acid sequence of a plastid targeting
peptide.
[0210] As used herein, the term "protein," "peptide molecule," or
"polypeptide" includes any molecule that comprises five or more
amino acids. It is well known in the art that protein, peptide or
polypeptide molecules may undergo modification, including
post-translational modifications, such as, but not limited to,
disulfide bond formation, glycosylation, phosphorylation, or
oligomerization. Thus, as used herein, the term "protein," "peptide
molecule," or "polypeptide" includes any protein that is modified
by any biological or non-biological process. The terms "amino acid"
and "amino acids" refer to all naturally occurring L-amino acids.
This definition is meant to include norleucine, norvaline,
ornithine, homocysteine, and homoserine.
[0211] One or more of the protein or fragments thereof, peptide
molecules, or polypeptide molecules may be produced via chemical
synthesis, or more preferably, by expression in a suitable
bacterial or eukaryotic host. Suitable methods for expression are
described by Sambrook et al., In: Molecular Cloning, A Laboratory
Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. (1989) or similar texts.
[0212] A "protein fragment" is a peptide or polypeptide molecule
whose amino acid sequence comprises a subset of the amino acid
sequence of that protein. A protein or fragment thereof that
comprises one or more additional peptide regions not derived from
that protein is a "fusion" protein. Such molecules may be
derivatized to contain carbohydrate or other moieties (such as
keyhole limpet hemocyanin). Fusion protein or peptide molecules of
the invention are preferably produced via recombinant means.
[0213] Another class of agents comprise protein, peptide molecules,
or polypeptide molecules or fragments or fusions thereof comprising
SEQ ID NOs: 16 through 38, and fragments thereof in which
conservative, non-essential or non-relevant amino acid residues
have been added, replaced or deleted. Computerized means for
designing modifications in protein structure are known in the art
(Dahiyat and Mayo, Science 278:82-87 (1997)).
[0214] A protein, peptide or polypeptide of the invention can also
be a homolog protein, peptide or polypeptide. As used herein, a
homolog protein, peptide or polypeptide or fragment thereof is a
counterpart protein, peptide or polypeptide or fragment thereof in
a second species. A homolog can also be generated by molecular
evolution or DNA shuffling techniques, so that the molecule retains
at least one functional or structure characteristic of the original
(see, for example, U.S. Pat. No. 5,811,238).
[0215] In another embodiment, the homolog is selected from the
group consisting of alfalfa, Arabidopsis, barley, broccoli,
cabbage, canola, citrus, cotton, garlic, oat, Allium, flax, an
ornamental plant, peanut, pepper, potato, rapeseed, rice, rye,
sorghum, strawberry, sugarcane, sugarbeet, tomato, wheat, poplar,
pine, fir, eucalyptus, apple, lettuce, lentils, grape, banana, tea,
turf grasses, sunflower, soybean, corn, and Phaseolus. More
particularly, preferred homologs are selected from canola,
rapeseed, corn, Brassica campestris, Brassica napus, oilseed rape,
soybean, crambe, mustard, castor bean, peanut, sesame, cottonseed,
linseed, safflower, oil palm, flax, and sunflower. In an even more
preferred embodiment, the homolog is selected from the group
consisting of canola, rapeseed, corn, Brassica campestris, Brassica
napus, oilseed rape, soybean, sunflower, safflower, oil palms, and
peanut. In a preferred embodiment, the homolog is soybean. In a
preferred embodiment, the homolog is canola. In a preferred
embodiment, the homolog is oilseed rape.
[0216] In a preferred embodiment, the nucleic acid molecules of the
present invention or complements and fragments of either can be
utilized to obtain such homologs.
[0217] Agents of the invention include proteins and fragments
thereof comprising at least about a contiguous 10 amino acid region
preferably comprising at least about a contiguous 20 amino acid
region, even more preferably comprising at least about a contiguous
25, 35, 50, 75 or 100 amino acid region of a protein of the present
invention. In another preferred embodiment, the proteins of the
present invention include between about 10 and about 25 contiguous
amino acid region, more preferably between about 20 and about 50
contiguous amino acid region, and even more preferably between
about 40 and about 80 contiguous amino acid region.
Plant Constructs and Plant Transformants
[0218] One or more of the nucleic acid molecules of the invention
may be used in plant transformation or transfection. Exogenous
genetic material may be transferred into a plant cell and the plant
cell regenerated into a whole, fertile or sterile plant. Exogenous
genetic material is any genetic material, whether naturally
occurring or otherwise, from any source that is capable of being
inserted into any organism.
[0219] In a preferred aspect of the present invention the exogenous
genetic material comprises a nucleic acid sequence that encodes
tocopherol methyltransferase. In another preferred aspect of the
present invention the exogenous genetic material of the invention
comprises a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1 through 15, and complements thereof and
fragments of either. In a further aspect of the present invention
the exogenous genetic material comprises a nucleic acid sequence
encoding an amino acid sequence selected from the group consisting
of SEQ ID NOs: 16 through 38, and fragments thereof. In a further
aspect of the present invention the nucleic acid molecule comprises
a nucleic acid sequence encoding an amino acid sequence selected
from the group consisting of SEQ ID NOs: 17 through 21, and
fragments thereof. In a further aspect of the present invention the
nucleic acid molecule comprises a nucleic acid sequence encoding an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 22 through 27, and fragments thereof. In a further aspect of
the present invention the nucleic acid molecule comprises a nucleic
acid sequence encoding an amino acid sequence selected from the
group consisting of SEQ ID NOs: 28 through 38, and fragments
thereof. In a further aspect of the present invention the nucleic
acid molecule comprises a nucleic acid sequence encoding an amino
acid of SEQ ID NO: 28, and fragments thereof. In a further aspect
of the present invention the nucleic acid molecule comprises a
nucleic acid sequence encoding an amino acid sequence selected from
the group consisting of SEQ ID NOs: 29 through 32, and fragments
thereof. In a further aspect of the present invention the nucleic
acid molecule comprises a nucleic acid sequence encoding an amino
acid sequence selected from the group consisting of SEQ ID NOs: 33
through 38, and fragments thereof. In a further aspect of the
present invention, the nucleic acid sequences of the invention also
encode peptides involved in intracellular localization, export, or
post-translational modification.
[0220] In an embodiment of the present invention, exogenous genetic
material comprising a tMT2 enzyme or fragment thereof is introduced
into a plant with one or more additional genes. In one embodiment,
preferred combinations of genes include one or more of the
following genes: tyrA, slr1736, HPT, GMT, tocopherol cyclase, dxs,
dxr, GGPPS, HPPD, GMT, tMT2, AANT1, slr1737, IDI, GGH, or a plant
ortholog thereof, and an antisense construct for homogentisic acid
dioxygenase (Kridl et al., Seed Sci. Res. 1:209:219 (1991);
Keegstra, Cell 56(2):247-53 (1989); Nawrath, et al., Proc. Natl.
Acad. Sci. U.S.A. 91:12760-12764 (1994); Xia et al., J. Gen.
Microbiol. 138:1309-1316 (1992); Cyanobase,
www.kazusa.or.jp/cyanobase; Lois et al., Proc. Natl. Acad. Sci.
U.S.A. 95 (5):2105-2110 (1998); Takahashi et al. Proc. Natl. Acad.
Sci. U.S.A. 95 (17), 9879-9884 (1998); Norris et al., Plant
Physiol. 117:1317-1323 (1998); Bartley and Scolnik, Plant Physiol.
104:1469-1470 (1994), Smith et al., Plant J. 11:83-92 (1997); WO
00/32757; WO 00/10380; Saint Guily, et al., Plant Physiol.,
100(2):1069-1071 (1992); Sato et al., J. DNA Res. 7 (1):31-63
(2000)).
[0221] In another preferred embodiment, tMT2 is combined with GMT.
In any of the embodiments disclosed herein in which a nucleic acid
molecule encoding a GMT is used, the nucleic acid molecule is
preferably selected from the group consisting of nucleic acid
molecules comprising a nucleic acid sequence selected from the
group SEQ ID NOs: 39 and 54, and nucleic acids molecules encoding
GMTs having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 39-54. In another preferred embodiment,
tMT2 is combined with GMT and one or more of the genes listed
above. In such combinations, one or more of the gene products can
be directed to the plastid by the use of a plastid targeting
sequence. Alternatively, one or more of the gene products can be
localized in the cytoplasm. In a preferred aspect the gene products
of tyrA and HPPD are targeted to the cytoplasm. Such genes can be
introduced, for example, with the tMT2 or GMT or both, or fragment
of either or both on a single construct, introduced on different
constructs but the same transformation event, or introduced into
separate plants followed by one or more crosses to generate the
desired combination of genes. In such combinations, a preferred
promoter is a napin promoter and a preferred plastid targeting
sequence is a CTP1 sequence. It is preferred that gene products are
targeted to the plastid.
[0222] In a preferred combination a nucleic acid molecule encoding
a tMT2 polypeptide and a nucleic acid molecule encoding any of the
following enzymes: tyrA, slr1736, HPT, GMT, tocopherol cyclase,
dxs, dxr, GGPPS, HPPD, tMT2, AANT1, slr1737, IDI, GGH or a plant
ortholog thereof, and an antisense construct for homogentisic acid
dioxygenase are introduced into a plant. A particularly preferred
combination that can be introduced is a nucleic acid molecule
encoding a tMT2 polypeptide and a nucleic acid molecule encoding a
GMT polypeptide, where both polypeptides are targeted to the
plastid and where one of such polypeptides is present and the other
is introduced. Both nucleic acid sequences encoding such
polypeptides can be introduced using a single gene construct, or
each polypeptide can be introduced on separate constructs. In a
further embodiment, tMT2 is combined with GMT and one or more of
tyrA, slr1736, HPT tocopherol cyclase, dxs, dxr, GGPPS, HPPD,
AANT1, slr1737, IDI, and GGH.
[0223] In a particularly preferred combination, a nucleic acid
molecule encoding a tMT2 protein and a nucleic acid molecule
encoding a GMT enzyme are introduced into a plant along with a
nucleic acid molecule that encodes one or more of tyrA, slr1736,
HPT tocopherol cyclase, dxs, dxr, GGPPS, HPPD, AANT1, slr1737, IDI,
and GGH.
[0224] Another particularly preferred combination that can be
introduced is a nucleic acid molecule encoding a tMT2 protein and a
nucleic acid molecule that results in the down regulation of a GMT
protein. In such an aspect, it is preferred that the plant
accumulates either .gamma.-tocopherol or .gamma.-tocotrienol or
both.
[0225] Such genetic material may be transferred into either
monocotyledons or dicotyledons including, but not limited to
canola, corn, soybean, Arabidopsis phaseolus, peanut, alfalfa,
wheat, rice, oat, sorghum, rapeseed, rye, tritordeum, millet,
fescue, perennial ryegrass, sugarcane, cranberry, papaya, banana,
safflower, oil palms, flax, muskmelon, apple, cucumber, dendrobium,
gladiolus, chrysanthemum, liliacea, cotton, eucalyptus, sunflower,
Brassica campestris, oilseed rape, turfgrass, sugarbeet, coffee and
dioscorea (Christou, In: Particle Bombardment for Genetic
Engineering of Plants, Biotechnology Intelligence Unit. Academic
Press, San Diego, Calif. (1996)), with canola, corn, Brassica
campestris, Brassica napus, oilseed rape, rapeseed, soybean,
crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed,
safflower, oil palm, flax, and sunflower preferred, and canola,
rapeseed, corn, Brassica campestris, Brassica napus, oilseed rape,
soybean, sunflower, safflower, oil palms, and peanut preferred. In
a more preferred embodiment, the genetic material is transferred
into canola. In another more preferred embodiment, the genetic
material is transferred into oilseed rape. In another particularly
preferred embodiment, the genetic material is transferred into
soybean.
[0226] Transfer of a nucleic acid molecule that encodes a protein
can result in expression or overexpression of that polypeptide in a
transformed cell or transgenic plant. One or more of the proteins
or fragments thereof encoded by nucleic acid molecules of the
invention may be overexpressed in a transformed cell or transformed
plant. Such expression or overexpression may be the result of
transient or stable transfer of the exogenous genetic material.
[0227] In a preferred embodiment, expression or overexpression of a
polypeptide of the present invention in a plant provides in that
plant, relative to an untransformed plant with a similar genetic
background, an increased level of tocopherols.
[0228] In a preferred embodiment, expression or overexpression of a
polypeptide of the present invention in a plant provides in that
plant, relative to an untransformed plant with a similar genetic
background, an increased level of .alpha.-tocopherols.
[0229] In a preferred embodiment, expression or overexpression of a
polypeptide of the present invention in a plant provides in that
plant, relative to an untransformed plant with a similar genetic
background, an increased level of .gamma.-tocopherols.
[0230] In a preferred embodiment, reduction of the expression,
expression or overexpression of a polypeptide of the present
invention in a plant provides in that plant, relative to an
untransformed plant with a similar genetic background, an increased
level of .delta.-tocopherols.
[0231] In a preferred embodiment, reduction of the expression,
expression or overexpression of a polypeptide of the present
invention in a plant provides in that plant, relative to an
untransformed plant with a similar genetic background, an increased
level of .beta.-tocopherols.
[0232] In a preferred embodiment, expression or overexpression of a
polypeptide of the present invention in a plant provides in that
plant, relative to an untransformed plant with a similar genetic
background, an increased level of tocotrienols.
[0233] In a preferred embodiment, expression or overexpression of a
polypeptide of the present invention in a plant provides in that
plant, relative to an untransformed plant with a similar genetic
background, an increased level of .alpha.-tocotrienols.
[0234] In a preferred embodiment, expression or overexpression of a
polypeptide of the present invention in a plant provides in that
plant, relative to an untransformed plant with a similar genetic
background, an increased level of .gamma.-tocotrienols.
[0235] In a preferred embodiment, reduction of the expression,
expression or overexpression of a polypeptide of the present
invention in a plant provides in that plant, relative to an
untransformed plant with a similar genetic background, an increased
level of .delta.-tocotrienols.
[0236] In a preferred embodiment, reduction of the expression,
expression or overexpression of a polypeptide of the present
invention in a plant provides in that plant, relative to an
untransformed plant with a similar genetic background, an increased
level of .beta.-tocotrienols.
[0237] In a preferred embodiment, expression or overexpression of a
polypeptide of the present invention in combination with a nucleic
acid molecule encoding any of the following enzymes: tyra, slr1736,
HPT, GMT, tocopherol cyclase, dxs, dxr, GGPPS, HPPD, tMT2, AANT1,
slr1737, IDI, GGH or a plant ortholog thereof, and an antisense
construct for homogentisic acid dioxygenase in a plant, provides in
that plant, relative to an untransformed plant with a similar
genetic background, an increased level of total tocopherols.
[0238] In a preferred embodiment, expression or overexpression of a
polypeptide of the present invention in a plant provides in that
plant, relative to an untransformed plant with a similar genetic
background, an increased level of plastoquinols.
[0239] In a preferred embodiment, expression or overexpression of a
polypeptide of the present invention in a plant provides in that
plant, relative to an untransformed plant with a similar genetic
background, an increased level of total tocopherols.
[0240] In any of the embodiments described herein, an increase in
.gamma.-tocopherol, .alpha.-tocopherol, or both can lead to a
decrease in the relative proportion of .beta.-tocopherol,
.delta.-tocopherol, or both. Similarly, an increase in
.gamma.-tocotienol, .alpha.-tocotrienol, or both can lead to a
decrease in the relative proportion of .beta.-tocotrienol,
.delta.-tocotrienol, or both.
[0241] In another embodiment, expression, overexpression of a
polypeptide of the present invention in a plant provides in that
plant, or a tissue of that plant, relative to an untransformed
plant or plant tissue, with a similar genetic background, an
increased level of a tMT2 protein or fragment thereof.
[0242] In some embodiments, the levels of one or more products of
the tocopherol biosynthesis pathway, including any one or more of
tocopherols, .alpha.-tocopherols, .gamma.-tocopherols,
.delta.-tocopherols, .beta.-tocopherols, tocotrienols,
.alpha.-tocotrienols, .gamma.-tocotrienols, .delta.-tocotrienols,
.beta.-tocotrienols are increased by greater than about 10%, or
more preferably greater than about 25%, 35%, 50%, 75%, 80%, 90%,
100%, 150%, 200%, 1,000%, 2,000%, or 2,500%. The levels of products
may be increased throughout an organism such as a plant or
localized in one or more specific organs or tissues of the
organism. For example the levels of products may be increased in
one or more of the tissues and organs of a plant including without
limitation: roots, tubers, stems, leaves, stalks, fruit, berries,
nuts, bark, pods, seeds and flowers. A preferred organ is a
seed.
[0243] In some embodiments, the levels of one or more products of
the tocopherol biosynthesis pathway, including any one or more of
tocopherols, .alpha.-tocopherols, .gamma.-tocopherols,
.delta.-tocopherols, .beta.-tocopherols, tocotrienols,
.alpha.-tocotrienols, .gamma.-tocotrienols, .delta.-tocotrienols,
.beta.-tocotrienols are increased so that they constitute greater
than about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the total
tocopherol content of the organism or tissue. The levels of
products may be increased throughout an organism such as a plant or
localized in one or more specific organs or tissues of the
organism. For example the levels of products may be increased in
one or more of the tissues and organs of a plant including without
limitation: roots, tubers, stems, leaves, stalks, fruit, berries,
nuts, bark, pods, seeds and flowers. A preferred organ is a
seed.
[0244] In a preferred embodiment, expression of enzymes involved in
tocopherol, tocotrienol or plastoquinol synthesis in the seed will
result in an increase in .gamma.-tocopherol levels due to the
absence of significant levels of GMT activity in those tissues. In
another preferred embodiment, expression of enzymes involved in
tocopherol, tocotrienol or plastoquinol synthesis in photosynthetic
tissues will result in an increase in .alpha.-tocopherol due to the
higher levels of GMT activity in those tissues relative to the same
activity in seed tissue.
[0245] In another preferred embodiment, the expression of enzymes
involved in tocopherol, tocotrienol or plastoquinol synthesis in
the seed will result in an increase in the total tocopherol,
tocotrienol or plastoquinol level in the plant.
[0246] In some embodiments, the levels of tocopherols or a species
such as .alpha.-tocopherol may be altered. In some embodiments, the
levels of tocotrienols may be altered. Such alteration can be
compared to a plant with a similar background.
[0247] In another embodiment, either the .alpha.-tocopherol level,
.alpha.-tocotrienol level, or both of plants that natively produce
high levels of either .alpha.-tocopherol, .alpha.-tocotrienol or
both (e.g., sunflowers), can be increased by the introduction of a
gene coding for a tMT2 enzyme.
[0248] In a preferred aspect, a similar genetic background is a
background where the organisms being compared share about 50% or
greater of their nuclear genetic material. In a more preferred
aspect a similar genetic background is a background where the
organisms being compared share about 75% or greater, even more
preferably about 90% or greater of their nuclear genetic material.
In another even more preferable aspect, a similar genetic
background is a background where the organisms being compared are
plants, and the plants are isogenic except for any genetic material
originally introduced using plant transformation techniques.
[0249] In another preferred embodiment, reduction of the
expression, expression, overexpression of a polypeptide of the
present invention in a transformed plant may provide tolerance to a
variety of stress, e.g. oxidative stress tolerance such as to
oxygen or ozone, UV tolerance, cold tolerance, or fungal/microbial
pathogen tolerance.
[0250] As used herein in a preferred aspect, a tolerance or
resistance to stress is determined by the ability of a plant, when
challenged by a stress such as cold to produce a plant having a
higher yield than one without such tolerance or resistance to
stress. In a particularly preferred aspect of the present
invention, the tolerance or resistance to stress is measured
relative to a plant with a similar genetic background to the
tolerant or resistance plant except that the plant reduces the
expression, expresses or over expresses a protein or fragment
thereof of the present invention.
[0251] Exogenous genetic material may be transferred into a host
cell by the use of a DNA vector or construct designed for such a
purpose. Design of such a vector is generally within the skill of
the art (See, Plant Molecular Biology: A Laboratory Manual, Clark
(ed.), Springer, N.Y. (1997)).
[0252] A construct or vector may include a plant promoter to
express the polypeptide of choice. In a preferred embodiment, any
nucleic acid molecules described herein can be operably linked to a
promoter region which functions in a plant cell to cause the
production of an mRNA molecule. For example, any promoter that
functions in a plant cell to cause the production of an mRNA
molecule, such as those promoters described herein, without
limitation, can be used. In a preferred embodiment, the promoter is
a plant promoter.
[0253] A number of promoters that are active in plant cells have
been described in the literature. These include the nopaline
synthase (NOS) promoter (Ebert et al., Proc. Natl. Acad. Sci.
(U.S.A.) 84:5745-5749 (1987)), the octopine synthase (OCS) promoter
(which is carried on tumor-inducing plasmids of Agrobacterium
tumefaciens), the caulimovirus promoters such as the cauliflower
mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol. Biol.
9:315-324 (1987)) and the CaMV 35S promoter (Odell et al., Nature
313:810-812 (1985)), the figwort mosaic virus 35S-promoter, the
light-inducible promoter from the small subunit of
ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the Adh
promoter (Walker et al., Proc. Natl. Acad. Sci. (U.S.A.)
84:6624-6628 (1987)), the sucrose synthase promoter (Yang et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 87:4144-4148 (1990)), the R gene
complex promoter (Chandler et al., The Plant Cell 1:1175-1183
(1989)) and the chlorophyll a/b binding protein gene promoter, etc.
These promoters have been used to create DNA constructs that have
been expressed in plants; see, e.g., PCT publication WO 84/02913.
The CaMV 35S promoters are preferred for use in plants. Promoters
known or found to cause transcription of DNA in plant cells can be
used in the invention.
[0254] For the purpose of expression in source tissues of the
plant, such as the leaf, seed, root or stem, it is preferred that
the promoters utilized have relatively high expression in these
specific tissues. Tissue-specific expression of a protein of the
present invention is a particularly preferred embodiment. For this
purpose, one may choose from a number of promoters for genes with
tissue- or cell-specific or enhanced expression. Examples of such
promoters reported in the literature include the chloroplast
glutamine synthetase GS2 promoter from pea (Edwards et al., Proc.
Natl. Acad. Sci. (U.S.A.) 87:3459-3463 (1990)), the chloroplast
fructose-1,6-biphosphatase (FBPase) promoter from wheat (Lloyd et
al., Mol. Gen. Genet. 225:209-216 (1991)), the nuclear
photosynthetic ST-LS1 promoter from potato (Stockhaus et al., EMBO
J. 8:2445-2451 (1989)), the serine/threonine kinase (PAL) promoter
and the glucoamylase (CHS) promoter from Arabidopsis thaliana. Also
reported to be active in photosynthetically active tissues are the
ribulose-1,5-bisphosphate carboxylase (RbcS) promoter from eastern
larch (Larix laricina), the promoter for the cab gene, cab6, from
pine (Yamamoto et al., Plant Cell Physiol. 35:773-778 (1994)), the
promoter for the Cab-1 gene from wheat (Fejes et al., Plant Mol.
Biol. 15:921-932 (1990)), the promoter for the CAB-1 gene from
spinach (Lubberstedt et al., Plant Physiol. 104:997-1006 (1994)),
the promoter for the cab1R gene from rice (Luan et al., Plant Cell.
4:971-981 (1992)), the pyruvate, orthophosphate dikinase (PPDK)
promoter from corn (Matsuoka et al., Proc. Natl. Acad. Sci.
(U.S.A.) 90:9586-9590 (1993)), the promoter for the tobacco Lhcb1*2
gene (Cerdan et al., Plant Mol. Biol. 33:245-255 (1997)), the
Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit
et al., Planta. 196:564-570 (1995)) and the promoter for the
thylakoid membrane proteins from spinach (psaD, psaF, psaE, PC,
FNR, atpC, atpD, cab, rbcS). Other promoters for the chlorophyll
a/b-binding proteins may also be utilized in the invention, such as
the promoters for LhcB gene and PsbP gene from white mustard
(Sinapis alba; Kretsch et al., Plant Mol. Biol. 28:219-229
(1995)).
[0255] For the purpose of expression in sink tissues of the plant,
such as the tuber of the potato plant, the fruit of tomato, or the
seed of corn, wheat, rice and barley, it is preferred that the
promoters utilized in the invention have relatively high expression
in these specific tissues. A number of promoters for genes with
tuber-specific or tuber-enhanced expression are known, including
the class I patatin promoter (Bevan et al., EMBO J. 8:1899-1906
(1986); Jefferson et al., Plant Mol. Biol. 14:995-1006 (1990)), the
promoter for the potato tuber ADPGPP genes, both the large and
small subunits, the sucrose synthase promoter (Salanoubat and
Belliard, Gene 60:47-56 (1987), Salanoubat and Belliard, Gene
84:181-185 (1989)), the promoter for the major tuber proteins
including the 22 kd protein complexes and protease inhibitors
(Hannapel, Plant Physiol. 101:703-704 (1993)), the promoter for the
granule-bound starch synthase gene (GBSS) (Visser et al., Plant
Mol. Biol. 17:691-699 (1991)) and other class I and II patatins
promoters (Koster-Topfer et al., Mol. Gen. Genet. 219:390-396
(1989); Mignery et al., Gene. 62:27-44 (1988)).
[0256] Other promoters can also be used to express a polypeptide in
specific tissues, such as seeds or fruits. Indeed, in a preferred
embodiment, the promoter used is a seed specific promoter. Examples
of such promoters include the 5' regulatory regions from such genes
as napin (Kridl et al., Seed Sci. Res. 1:209:219 (1991)), phaseolin
(Bustos, et al., Plant Cell, 1(9):839-853 (1989)), soybean trypsin
inhibitor (Riggs, et al., Plant Cell 1(6):609-621 (1989)), ACP
(Baerson, et al., Plant Mol. Biol., 22(2):255-267 (1993)),
stearoyl-ACP desaturase (Slocombe, et al., Plant Physiol. 104(4):
167-176 (1994)), soybean a' subunit of b-conglycinin (soy 7s, (Chen
et al., Proc. Natl. Acad. Sci., 83:8560-8564 (1986))), and oleosin
(see, for example, Hong, et al., Plant Mol. Biol., 34(3):549-555
(1997)). Further examples include the promoter for
.beta.-conglycinin (Chen et al., Dev. Genet. 10:112-122 (1989)).
Also included are the zeins, which are a group of storage proteins
found in corn endosperm. Genomic clones for zein genes have been
isolated (Pedersen et al., Cell 29:1015-1026 (1982), and Russell et
al., Transgenic Res. 6(2):157-168) and the promoters from these
clones, including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and genes,
could also be used. Other promoters known to function, for example,
in corn include the promoters for the following genes: waxy,
Brittle, Shrunken 2, Branching enzymes I and II, starch synthases,
debranching enzymes, oleosins, glutelins and sucrose synthases. A
particularly preferred promoter for corn endosperm expression is
the promoter for the glutelin gene from rice, more particularly the
Osgt-1 promoter (Zheng et al., Mol. Cell. Biol. 13:5829-5842
(1993)). Examples of promoters suitable for expression in wheat
include those promoters for the ADPglucose pyrosynthase (ADPGPP)
subunits, the granule bound and other starch synthase, the
branching and debranching enzymes, the embryogenesis-abundant
proteins, the gliadins and the glutenins. Examples of such
promoters in rice include those promoters for the ADPGPP subunits,
the granule bound and other starch synthase, the branching enzymes,
the debranching enzymes, sucrose synthases and the glutelins. A
particularly preferred promoter is the promoter for rice glutelin,
Osgt-1. Examples of such promoters for barley include those for the
ADPGPP subunits, the granule bound and other starch synthase, the
branching enzymes, the debranching enzymes, sucrose synthases, the
hordeins, the embryo globulins and the aleurone specific proteins.
A preferred promoter for expression in the seed is a napin
promoter. Another preferred promoter for expression is an Arcelin5
promoter.
[0257] Root specific promoters may also be used. An example of such
a promoter is the promoter for the acid chitinase gene (Samac et
al., Plant Mol. Biol. 25:587-596 (1994)). Expression in root tissue
could also be accomplished by utilizing the root specific
subdomains of the CaMV35S promoter that have been identified (Lam
et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:7890-7894 (1989)). Other
root cell specific promoters include those reported by Conkling et
al. (Conkling et al., Plant Physiol. 93:1203-1211 (1990)).
[0258] Additional promoters that may be utilized are described, for
example, in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147;
5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435;
and 4,633,436. In addition, a tissue specific enhancer may be used
(Fromm et al., The Plant Cell 1:977-984 (1989)).
[0259] Constructs or vectors may also include, with the coding
region of interest, a nucleic acid sequence that acts, in whole or
in part, to terminate transcription of that region. A number of
such sequences have been isolated, including the Tr7 3' sequence
and the NOS 3' sequence (Ingelbrecht et al., The Plant Cell
1:671-680 (1989); Bevan et al., Nucleic Acids Res. 11:369-385
(1983)). Regulatory transcript termination regions can be provided
in plant expression constructs of this invention as well.
Transcript termination regions can be provided by the DNA sequence
encoding the gene of interest or a convenient transcription
termination region derived from a different gene source, for
example, the transcript termination region that is naturally
associated with the transcript initiation region. The skilled
artisan will recognize that any convenient transcript termination
region that is capable of terminating transcription in a plant cell
can be employed in the constructs of the present invention.
[0260] A vector or construct may also include regulatory elements.
Examples of such include the Adh intron 1 (Callis et al., Genes and
Develop. 1:1183-1200 (1987)), the sucrose synthase intron (Vasil et
al., Plant Physiol. 91:1575-1579 (1989)) and the TMV omega element
(Gallie et al., The Plant Cell 1:301-311 (1989)). These and other
regulatory elements may be included when appropriate.
[0261] A vector or construct may also include a selectable marker.
Selectable markers may also be used to select for plants or plant
cells that contain the exogenous genetic material. Examples of such
include, but are not limited to: a neo gene (Potrykus et al., Mol.
Gen. Genet. 199:183-188 (1985)), which codes for kanamycin
resistance and can be selected for using kanamycin, RptII, G418,
hpt etc.; a bar gene which codes for bialaphos resistance; a mutant
EPSP synthase gene (Hinchee et al., Bio/Technology 6:915-922
(1988); Reynaerts et al., Selectable and Screenable Markers. In
Gelvin and Schilperoort. Plant Molecular Biology Manual, Kluwer,
Dordrecht (1988); Reynaerts et al, Selectable and screenable
markers. In Gelvin and Schilperoort. Plant Molecular Biology
Manual, Kluwer, Dordrecht (1988)), aadA (Jones et al., Mol. Gen.
Genet. (1987)),) which encodes glyphosate resistance; a nitrilase
gene which confers resistance to bromoxynil (Stalker et al., J.
Biol. Chem. 263:6310-6314 (1988)); a mutant acetolactate synthase
gene (ALS) which confers imidazolinone or sulphonylurea resistance
(European Patent Application 154, 204 (Sep. 11, 1985)), ALS
(D'Halluin et al., Bio/Technology 10:309-314 (1992)), and a
methotrexate resistant DHFR gene (Thillet et al., J. Biol. Chem.
263:12500-12508 (1988)).
[0262] A vector or construct may also include a transit peptide.
Incorporation of a suitable chloroplast transit peptide may also be
employed (European Patent Application Publication Number 0218571).
Translational enhancers may also be incorporated as part of the
vector DNA. DNA constructs could contain one or more 5'
non-translated leader sequences, which may serve to enhance
expression of the gene products from the resulting mRNA
transcripts. Such sequences may be derived from the promoter
selected to express the gene or can be specifically modified to
increase translation of the mRNA. Such regions may also be obtained
from viral RNAs, from suitable eukaryotic genes, or from a
synthetic gene sequence. For a review of optimizing expression of
transgenes, see Koziel et al., Plant Mol. Biol. 32:393-405 (1996).
A preferred transit peptide is CTP1.
[0263] A vector or construct may also include a screenable marker.
Screenable markers may be used to monitor expression. Exemplary
screenable markers include: a glucuronidase or uidA gene (GUS)
which encodes an enzyme for which various chromogenic substrates
are known (Jefferson, Plant Mol. Biol., Rep. 5:387-405 (1987);
Jefferson et al., EMBO J. 6:3901-3907 (1987)); an R-locus gene,
which encodes a product that regulates the production of
anthocyanin pigments (red color) in plant tissues (Dellaporta et
al., Stadler Symposium 11:263-282 (1988)); a .beta.-lactamase gene
(Sutcliffe et al., Proc. Natl. Acad. Sci. (U.S.A) 75:3737-3741
(1978)), a gene which encodes an enzyme for which various
chromogenic substrates are known (e.g., PADAC, a chromogenic
cephalosporin); a luciferase gene (Ow et al., Science 234:856-859
(1986)); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci.
(U.S.A.) 80:1101-1105 (1983)) which encodes a catechol dioxygenase
that can convert chromogenic catechols; an .alpha.-amylase gene
(Ikatu et al., Bio/Technol. 8:241-242 (1990)); a tyrosinase gene
(Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983)) which
encodes an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone which in turn condenses to melanin; an
.alpha.-galactosidase, which will turn a chromogenic
.alpha.-galactose substrate.
[0264] Included within the terms "selectable or screenable marker
genes" are also genes that encode a secretable marker whose
secretion can be detected as a means of identifying or selecting
for transformed cells. Examples include markers that encode a
secretable antigen that can be identified by antibody interaction,
or even secretable enzymes that can be detected catalytically.
Secretable proteins fall into a number of classes, including small,
diffusible proteins that are detectable, (e.g., by ELISA), small
active enzymes that are detectable in extracellular solution (e.g.,
.alpha.-amylase, .beta.-lactamase, phosphinothricin transferase),
or proteins that are inserted or trapped in the cell wall (such as
proteins that include a leader sequence such as that found in the
expression unit of extension or tobacco PR-S). Other possible
selectable and/or screenable marker genes will be apparent to those
of skill in the art.
[0265] There are many methods for introducing transforming nucleic
acid molecules into plant cells. Suitable methods are believed to
include virtually any method by which nucleic acid molecules may be
introduced into a cell, such as by Agrobacterium infection or
direct delivery of nucleic acid molecules such as, for example, by
PEG-mediated transformation, by electroporation or by acceleration
of DNA coated particles, and the like. (Potrykus, Ann. Rev. Plant
Physiol. Plant Mol. Biol. 42:205-225 (1991); Vasil, Plant Mol.
Biol. 25:925-937 (1994)). For example, electroporation has been
used to transform corn protoplasts (Fromm et al., Nature
312:791-793 (1986)).
[0266] Other vector systems suitable for introducing transforming
DNA into a host plant cell include but are not limited to binary
artificial chromosome (BIBAC) vectors (Hamilton et al., Gene
200:107-116 (1997)); and transfection with RNA viral vectors
(Della-Cioppa et al., Ann. N.Y. Acad. Sci. (1996), 792 (Engineering
Plants for Commercial Products and Applications), 57-61).
Additional vector systems also include plant selectable YAC vectors
such as those described in Mullen et al., Molecular Breeding
4:449-457 (1988).
[0267] Technology for introduction of DNA into cells is well known
to those of skill in the art. Four general methods for delivering a
gene into cells have been described: (1) chemical methods (Graham
and van der Eb, Virology 54:536-539 (1973)); (2) physical methods
such as microinjection (Capecchi, Cell 22:479-488 (1980)),
electroporation (Wong and Neumann, Biochem. Biophys. Res. Commun.
107:584-587 (1982); Fromm et al., Proc. Natl. Acad. Sci. (U.S.A)
82:5824-5828 (1985); U.S. Pat. No. 5,384,253); the gene gun
(Johnston and Tang, Methods Cell Biol. 43:353-365 (1994)); and
vacuum infiltration (Bechtold et al., C.R. Acad. Sci. Paris, Life
Sci. 316:1194-1199. (1993)); (3) viral vectors (Clapp, Clin.
Perinatol. 20:155-168 (1993); Lu et al., J. Exp. Med. 178:2089-2096
(1993); Eglitis and Anderson, Biotechniques 6:608-614 (1988)); and
(4) receptor-mediated mechanisms (Curiel et al., Hum. Gen. Ther.
3:147-154 (1992), Wagner et al, Proc. Natl. Acad. Sci. (USA)
89:6099-6103 (1992)).
[0268] Acceleration methods that may be used include, for example,
microprojectile bombardment and the like. One example of a method
for delivering transforming nucleic acid molecules into plant cells
is microprojectile bombardment. This method has been reviewed by
Yang and Christou (eds.), Particle Bombardment Technology for Gene
Transfer, Oxford Press, Oxford, England (1994)). Non-biological
particles (microprojectiles) may be coated with nucleic acids and
delivered into cells by a propelling force. Exemplary particles
include those comprised of tungsten, gold, platinum and the
like.
[0269] A particular advantage of microprojectile bombardment, in
addition to it being an effective means of reproducibly
transforming monocots, is that neither the isolation of protoplasts
(Cristou et al., Plant Physiol. 87:671-674 (1988)) nor the
susceptibility to Agrobacterium infection is required. An
illustrative embodiment of a method for delivering DNA into corn
cells by acceleration is a biolistics .alpha.-particle delivery
system, which can be used to propel particles coated with DNA
through a screen, such as a stainless steel or Nytex screen, onto a
filter surface covered with corn cells cultured in suspension.
Gordon-Kamm et al., describes the basic procedure for coating
tungsten particles with DNA (Gordon-Kamm et al., Plant Cell
2:603-618 (1990)). The screen disperses the tungsten nucleic acid
particles so that they are not delivered to the recipient cells in
large aggregates. A particle delivery system suitable for use with
the invention is the helium acceleration PDS-1000/He gun, which is
available from Bio-Rad Laboratories (Bio-Rad, Hercules, Calif.)
(Sanford et al., Technique 3:3-16 (1991)).
[0270] For the bombardment, cells in suspension may be concentrated
on filters. Filters containing the cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the gun and the cells to be bombarded.
[0271] Alternatively, immature embryos or other target cells may be
arranged on solid culture medium. The cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the acceleration device and the cells to be bombarded.
Through the use of techniques set forth herein one may obtain 1000
or more loci of cells transiently expressing a marker gene. The
number of cells in a focus that express the exogenous gene product
48 hours post-bombardment often ranges from one to ten, and average
one to three.
[0272] In bombardment transformation, one may optimize the
pre-bombardment culturing conditions and the bombardment parameters
to yield the maximum numbers of stable transformants. Both the
physical and biological parameters for bombardment are important in
this technology. Physical factors are those that involve
manipulating the DNA/microprojectile precipitate or those that
affect the flight and velocity of either the macro- or
microprojectiles. Biological factors include all steps involved in
manipulation of cells before and immediately after bombardment, the
osmotic adjustment of target cells to help alleviate the trauma
associated with bombardment and also the nature of the transforming
DNA, such as linearized DNA or intact supercoiled plasmids. It is
believed that pre-bombardment manipulations are especially
important for successful transformation of immature embryos.
[0273] In another alternative embodiment, plastids can be stably
transformed. Methods disclosed for plastid transformation in higher
plants include the particle gun delivery of DNA containing a
selectable marker and targeting of the DNA to the plastid genome
through homologous recombination (Svab et al., Proc. Natl. Acad.
Sci. (U.S.A.) 87:8526-8530 (1990); Svab and Maliga, Proc. Natl.
Acad. Sci. (U.S.A.) 90:913-917 (1993); Staub and Maliga, EMBO J.
12:601-606 (1993); U.S. Pat. Nos. 5,451,513 and 5,545,818).
[0274] Accordingly, it is contemplated that one may wish to adjust
various aspects of the bombardment parameters in small scale
studies to fully optimize the conditions. One may particularly wish
to adjust physical parameters such as gap distance, flight
distance, tissue distance and helium pressure. One may also
minimize the trauma reduction factors by modifying conditions that
influence the physiological state of the recipient cells and which
may therefore influence transformation and integration
efficiencies. For example, the osmotic state, tissue hydration and
the subculture stage or cell cycle of the recipient cells may be
adjusted for optimum transformation. The execution of other routine
adjustments will be known to those of skill in the art in light of
the present disclosure.
[0275] Agrobacterium-mediated transfer is a widely applicable
system for introducing genes into plant cells because the DNA can
be introduced into whole plant tissues, thereby bypassing the need
for regeneration of an intact plant from a protoplast. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA
into plant cells is well known in the art. See, for example the
methods described by Fraley et al., Bio/Technology 3:629-635 (1985)
and Rogers et al., Methods Enzymol. 153:253-277 (1987). Further,
the integration of the Ti-DNA is a relatively precise process
resulting in few rearrangements. The region of DNA to be
transferred is defined by the border sequences and intervening DNA
is usually inserted into the plant genome as described (Spielmann
et al., Mol. Gen. Genet. 205:34 (1986)).
[0276] Modern Agrobacterium transformation vectors are capable of
replication in E. coli as well as Agrobacterium, allowing for
convenient manipulations as described (Klee et al., In: Plant DNA
Infectious Agents, Hohn and Schell (eds.), Springer-Verlag, New
York, pp. 179-203 (1985)). Moreover, technological advances in
vectors for Agrobacterium-mediated gene transfer have improved the
arrangement of genes and restriction sites in the vectors to
facilitate construction of vectors capable of expressing various
polypeptide coding genes. The vectors described have convenient
multi-linker regions flanked by a promoter and a polyadenylation
site for direct expression of inserted polypeptide coding genes and
are suitable for present purposes (Rogers et al., Methods Enzymol.
153:253-277 (1987)). In addition, Agrobacterium containing both
armed and disarmed Ti genes can be used for the transformations. In
those plant strains where Agrobacterium-mediated transformation is
efficient, it is the method of choice because of the facile and
defined nature of the gene transfer.
[0277] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single gene on one chromosome. Such
transgenic plants can be referred to as being heterozygous for the
added gene. More preferred is a transgenic plant that is homozygous
for the added structural gene; i.e., a transgenic plant that
contains two added genes, one gene at the same locus on each
chromosome of a chromosome pair. A homozygous transgenic plant can
be obtained by sexually mating (selfing) an independent segregant,
transgenic plant that contains a single added gene, germinating
some of the seed produced and analyzing the resulting plants
produced for the gene of interest.
[0278] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating, exogenous genes. Selfing of appropriate
progeny can produce plants that are homozygous for both added,
exogenous genes that encode a polypeptide of interest.
Back-crossing to a parental plant and out-crossing with a
non-transgenic plant are also contemplated, as is vegetative
propagation.
[0279] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation and combinations of these
treatments (See, for example, Potrykus et al., Mol. Gen. Genet.
205:193-200 (1986); Lorz et al., Mol. Gen. Genet. 199:178 (1985);
Fromm et al., Nature 319:791 (1986); Uchimiya et al., Mol. Gen.
Genet. 204:204 (1986); Marcotte et al., Nature 335:454-457
(1988)).
[0280] Application of these systems to different plant strains
depends upon the ability to regenerate that particular plant strain
from protoplasts. Illustrative methods for the regeneration of
cereals from protoplasts are described (Fujimura et al., Plant
Tissue Culture Letters 2:74 (1985); Toriyama et al., Theor. Appl.
Genet. 205:34 (1986); Yamada et al., Plant Cell Rep. 4:85 (1986);
Abdullah et al., Biotechnology 4:1087 (1986)).
[0281] To transform plant strains that cannot be successfully
regenerated from protoplasts, other ways to introduce DNA into
intact cells or tissues can be utilized. For example, regeneration
of cereals from immature embryos or explants can be effected as
described (Vasil, Biotechnology 6:397 (1988)). In addition,
"particle gun" or high-velocity microprojectile technology can be
utilized (Vasil et al., Bio/Technology 10:667 (1992)).
[0282] Using the latter technology, DNA is carried through the cell
wall and into the cytoplasm on the surface of small metal particles
as described (Klein et al., Nature 328:70 (1987); Klein et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 85:8502-8505 (1988); McCabe et al.,
Bio/Technology 6:923 (1988)). The metal particles penetrate through
several layers of cells and thus allow the transformation of cells
within tissue explants.
[0283] Other methods of cell transformation can also be used and
include but are not limited to introduction of DNA into plants by
direct DNA transfer into pollen Hess et al., Intern Rev. Cytol.
107:367 (1987); Luo et al., Plant Mol. Biol. Reporter 6:165
(1988)), by direct injection of DNA into reproductive organs of a
plant (Pena et al., Nature 325:274 (1987)), or by direct injection
of DNA into the cells of immature embryos followed by the
rehydration of desiccated embryos (Neuhaus et al., Theor. Appl.
Genet. 75:30 (1987)).
[0284] The regeneration, development and cultivation of plants from
single plant protoplast transformants or from various transformed
explants is well known in the art (Weissbach and Weissbach, In:
Methods for Plant Molecular Biology, Academic Press, San Diego,
Calif., (1988)). This regeneration and growth process typically
includes the steps of selection of transformed cells, culturing
those individualized cells through the usual stages of embryonic
development through the rooted plantlet stage. Transgenic embryos
and seeds are similarly regenerated. The resulting transgenic
rooted shoots are thereafter planted in an appropriate plant growth
medium such as soil.
[0285] The development or regeneration of plants containing the
foreign, exogenous gene that encodes a protein of interest is well
known in the art. Preferably, the regenerated plants are
self-pollinated to provide homozygous transgenic plants. Otherwise,
pollen obtained from the regenerated plants is crossed to
seed-grown plants of agronomically important lines. Conversely,
pollen from plants of these important lines is used to pollinate
regenerated plants. A transgenic plant of the invention containing
a desired polypeptide is cultivated using methods well known to one
skilled in the art.
[0286] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated.
[0287] Methods for transforming dicots, primarily by use of
Agrobacterium tumefaciens and obtaining transgenic plants have been
published for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No.
5,159,135; U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No.
5,569,834; U.S. Pat. No. 5,416,011; McCabe et al., Biotechnology
6:923 (1988); Christou et al., Plant Physiol. 87:671-674 (1988));
Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant
Cell Rep. 15:653-657 (1996), McKently et al., Plant Cell Rep.
14:699-703 (1995)); papaya; pea (Grant et al., Plant Cell Rep.
15:254-258 (1995)); and Arabidopsis thaliana (Bechtold et al., C.R.
Acad. Sci. Paris, Life Sci. 316:1194-1199 (1993)). The latter
method for transforming Arabidopsis thaliana is commonly called
"dipping" or vacuum infiltration or germplasm transformation.
[0288] Transformation of monocotyledons using electroporation,
particle bombardment and Agrobacterium have also been reported.
Transformation and plant regeneration have been achieved in
asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA) 84:5354
(1987)); barley (Wan and Lemaux, Plant Physiol 104:37 (1994)); corn
(Rhodes et al., Science 240:204 (1988); Gordon-Kamm et al., Plant
Cell 2:603-618 (1990); Fromm et al., Bio/Technology 8:833 (1990);
Koziel et al., Bio/Technology 11:194 (1993); Armstrong et al., Crop
Science 35:550-557 (1995)); oat (Somers et al., Bio/Technology
10:1589 (1992)); orchard grass (Horn et al., Plant Cell Rep. 7:469
(1988)); rice (Toriyama et al., Theor Appl. Genet. 205:34 (1986);
Part et al., Plant Mol. Biol. 32:1135-1148 (1996); Abedinia et al.,
Aust. J. Plant Physiol. 24:133-141 (1997); Zhang and Wu, Theor.
Appl. Genet. 76:835 (1988); Zhang et al., Plant Cell Rep. 7:379
(1988); Battraw and Hall, Plant Sci. 86:191-202 (1992); Christou et
al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature
325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409
(1992)); tall fescue (Wang et al., Bio/Technology 10:691 (1992))
and wheat (Vasil et al., Bio/Technology 10:667 (1992); U.S. Pat.
No. 5,631,152).
[0289] Assays for gene expression based on the transient expression
of cloned nucleic acid constructs have been developed by
introducing the nucleic acid molecules into plant cells by
polyethylene glycol treatment, electroporation, or particle
bombardment (Marcotte et al., Nature 335:454-457 (1988); Marcotte
et al., Plant Cell 1:523-532 (1989); McCarty et al., Cell
66:895-905 (1991); Hattori et al., Genes Dev. 6:609-618 (1992);
Goff et al., EMBO J. 9:2517-2522 (1990)). Transient expression
systems may be used to functionally dissect gene constructs (see
generally, Mailga et al., Methods in Plant Molecular Biology, Cold
Spring Harbor Press (1995)).
[0290] Any of the nucleic acid molecules of the invention may be
introduced into a plant cell in a permanent or transient manner in
combination with other genetic elements such as vectors, promoters,
enhancers, etc. Further, any of the nucleic acid molecules of the
invention may be introduced into a plant cell in a manner that
allows for expression or overexpression of the protein or fragment
thereof encoded by the nucleic acid molecule.
[0291] Cosuppression is the reduction in expression levels, usually
at the level of RNA, of a particular endogenous gene or gene family
by the expression of a homologous sense construct that is capable
of transcribing mRNA of the same strandedness as the transcript of
the endogenous gene (Napoli et al., Plant Cell 2:279-289 (1990);
van der Krol et al., Plant Cell 2:291-299 (1990)). Cosuppression
may result from stable transformation with a single copy nucleic
acid molecule that is homologous to a nucleic acid sequence found
with the cell (Prolls and Meyer, Plant J. 2:465-475 (1992)) or with
multiple copies of a nucleic acid molecule that is homologous to a
nucleic acid sequence found with the cell (Mittlesten et al., Mol.
Gen. Genet. 244:325-330 (1994)). Genes, even though different,
linked to homologous promoters may result in the cosuppression of
the linked genes (Vaucheret, C.R. Acad. Sci. III 316:1471-1483
(1993); Flavell, Proc. Natl. Acad. Sci. (U.S.A.) 91:3490-3496
(1994)); van Blokland et al., Plant J. 6:861-877 (1994); Jorgensen,
Trends Biotechnol. 8:340-344 (1990); Meins and Kunz, In: Gene
Inactivation and Homologous Recombination in Plants, Paszkowski
(ed.), pp. 335-348, Kluwer Academic, Netherlands (1994)).
[0292] It is understood that one or more of the nucleic acids of
the invention may be introduced into a plant cell and transcribed
using an appropriate promoter with such transcription resulting in
the cosuppression of an endogenous protein.
[0293] Antisense approaches are a way of preventing or reducing
gene function by targeting the genetic material (Mol et al., FEBS
Lett. 268:427-430 (1990)). The objective of the antisense approach
is to use a sequence complementary to the target gene to block its
expression and create a mutant cell line or organism in which the
level of a single chosen protein is selectively reduced or
abolished. Antisense techniques have several advantages over other
`reverse genetic` approaches. The site of inactivation and its
developmental effect can be manipulated by the choice of promoter
for antisense genes or by the timing of external application or
microinjection. Antisense can manipulate its specificity by
selecting either unique regions of the target gene or regions where
it shares homology to other related genes (Hiatt et al., In:
Genetic Engineering, Setlow (ed.), Vol. 11, New York: Plenum 49-63
(1989)).
[0294] Antisense RNA techniques involve introduction of RNA that is
complementary to the target mRNA into cells, which results in
specific RNA:RNA duplexes being formed by base pairing between the
antisense substrate and the target mRNA (Green et al., Annu. Rev.
Biochem. 55:569-597 (1986)). Under one embodiment, the process
involves the introduction and expression of an antisense gene
sequence. Such a sequence is one in which part or all of the normal
gene sequences are placed under a promoter in inverted orientation
so that the `wrong` or complementary strand is transcribed into a
noncoding antisense RNA that hybridizes with the target mRNA and
interferes with its expression (Takayama and Inouye, Crit. Rev.
Biochem. Mol. Biol. 25:155-184 (1990)). An antisense vector is
constructed by standard procedures and introduced into cells by
transformation, transfection, electroporation, microinjection,
infection, etc. The type of transformation and choice of vector
will determine whether expression is transient or stable. The
promoter used for the antisense gene may influence the level,
timing, tissue, specificity, or inducibility of the antisense
inhibition.
[0295] It is understood that the activity of a protein in a plant
cell may be reduced or depressed by growing a transformed plant
cell containing a nucleic acid molecule whose non-transcribed
strand encodes a protein or fragment thereof. A preferred protein
whose activity can be reduced or depressed, by any method, is tMT2.
In such an embodiment of the invention, it is preferred that the
concentration of .delta.-tocopherol or .delta.-tocotrienol is
increased. Another preferred protein whose activity can be reduced
or depressed, by any method, is homogentisic acid dioxygenase.
[0296] Posttranscriptional gene silencing (PTGS) can result in
virus immunity or gene silencing in plants. PTGS is induced by
dsRNA and is mediated by an RNA-dependent RNA polymerase, present
in the cytoplasm, which requires a dsRNA template. The dsRNA is
formed by hybridization of complementary transgene mRNAs or
complementary regions of the same transcript. Duplex formation can
be accomplished by using transcripts from one sense gene and one
antisense gene collocated in the plant genome, a single transcript
that has self-complementarity, or sense and antisense transcripts
from genes brought together by crossing. The dsRNA-dependent RNA
polymerase makes a complementary strand from the transgene mRNA and
RNAse molecules attach to this complementary strand (cRNA). These
cRNA-RNase molecules hybridize to the endogene mRNA and cleave the
single-stranded RNA adjacent to the hybrid. The cleaved
single-stranded RNAs are further degraded by other host RNases
because one will lack a capped 5' end and the other will lack a
poly(A) tail (Waterhouse et al., PNAS 95:13959-13964 (1998)).
[0297] It is understood that one or more of the nucleic acids of
the invention may be introduced into a plant cell and transcribed
using an appropriate promoter with such transcription resulting in
the postranscriptional gene silencing of an endogenous
transcript.
[0298] Antibodies have been expressed in plants (Hiatt et al.,
Nature 342:76-78 (1989); Conrad and Fielder, Plant Mol. Biol.
26:1023-1030 (1994)). Cytoplasmic expression of a scFv
(single-chain Fv antibody) has been reported to delay infection by
artichoke mottled crinkle virus. Transgenic plants that express
antibodies directed against endogenous proteins may exhibit a
physiological effect (Philips et al., EMBO J. 16:4489-4496 (1997);
Marion-Poll, Trends in Plant Science 2:447-448 (1997)). For
example, expressed anti-abscisic antibodies have been reported to
result in a general perturbation of seed development (Philips et
al., EMBO J. 16:4489-4496 (1997)).
[0299] Antibodies that are catalytic may also be expressed in
plants (abzymes). The principle behind abzymes is that since
antibodies may be raised against many molecules, this recognition
ability can be directed toward generating antibodies that bind
transition states to force a chemical reaction forward (Persidas,
Nature Biotechnology 15:1313-1315 (1997); Baca et al., Ann. Rev.
Biophys. Biomol. Struct. 26:461-493 (1997)). The catalytic
abilities of abzymes may be enhanced by site directed mutagenesis.
Examples of abzymes are, for example, set forth in U.S. Pat. No.
5,658,753; U.S. Pat. No. 5,632,990; U.S. Pat. No. 5,631,137; U.S.
Pat. No. 5,602,015; U.S. Pat. No. 5,559,538; U.S. Pat. No.
5,576,174; U.S. Pat. No. 5,500,358; U.S. Pat. No. 5,318,897; U.S.
Pat. No. 5,298,409; U.S. Pat. No. 5,258,289 and U.S. Pat. No.
5,194,585.
[0300] It is understood that any of the antibodies of the invention
may be expressed in plants and that such expression can result in a
physiological effect. It is also understood that any of the
expressed antibodies may be catalytic.
[0301] The present invention also provides for parts of the plants,
particularly reproductive or storage parts, of the present
invention. Plant parts, without limitation, include seed,
endosperm, ovule and pollen. In a particularly preferred embodiment
of the present invention, the plant part is a seed. In one
embodiment the seed is a constituent of animal feed.
[0302] In another embodiment, the plant part is a fruit, more
preferably a fruit with enhanced shelf life. In another preferred
embodiment, the fruit has increased levels of a tocopherol. In
another preferred embodiment, the fruit has increased levels of a
tocotrienol.
[0303] The present invention also provides a container of over
about 10,000, more preferably about 20,000, and even more
preferably about 40,000 seeds where over about 10%, more preferably
about 25%, more preferably about 50% and even more preferably about
75% or 90% of the seeds are seeds derived from a plant of the
present invention.
[0304] The present invention also provides a container of over
about 10 kg, more preferably about 25 kg, and even more preferably
about 50 kg seeds where over about 10%, more preferably about 25%,
more preferably about 50% and even more preferably about 75% or 90%
of the seeds are seeds derived from a plant of the present
invention.
[0305] Any of the plants or parts thereof of the present invention
may be processed to produce a feed, meal, protein, or oil
preparation, including oil preparations high in total tocopherol
content and oil preparations high in any one or more of each
tocopherol component listed herein. A particularly preferred plant
part for this purpose is a seed. In a preferred embodiment the
feed, meal, protein or oil preparation is designed for livestock
animals or humans, or both. Methods to produce feed, meal, protein
and oil preparations are known in the art. See, for example, U.S.
Pat. Nos. 4,957,748, 5,100,679, 5,219,596, 5,936,069, 6,005,076,
6,146,669, and 6,156,227. In a preferred embodiment, the protein
preparation is a high protein preparation. Such a high protein
preparation preferably has a protein content of greater than about
5% w/v, more preferably about 10% w/v, and even more preferably
about 15% w/v. In a preferred oil preparation, the oil preparation
is a high oil preparation with an oil content derived from a plant
or part thereof of the present invention of greater than about 5%
w/v, more preferably about 10% w/v, and even more preferably about
15% w/v. In a preferred embodiment the oil preparation is a liquid
and of a volume greater than about 1, 5, 10 or 50 liters. The
present invention provides for oil produced from plants of the
present invention or generated by a method of the present
invention. Such an oil may exhibit enhanced oxidative stability.
Also, such oil may be a minor or major component of any resultant
product. Moreover, such oil may be blended with other oils. In a
preferred embodiment, the oil produced from plants of the present
invention or generated by a method of the present invention
constitutes greater than about 0.5%, 1%, 5%, 10%, 25%, 50%, 75% or
90% by volume or weight of the oil component of any product. In
another embodiment, the oil preparation may be blended and can
constitute greater than about 10%, 25%, 35%, 50% or 75% of the
blend by volume. Oil produced from a plant of the present invention
can be admixed with one or more organic solvents or petroleum
distillates.
[0306] Plants of the present invention can be part of or generated
from a breeding program. The choice of breeding method depends on
the mode of plant reproduction, the heritability of the trait(s)
being improved, and the type of cultivar used commercially (e.g.,
F.sub.1 hybrid cultivar, pureline cultivar, etc). Selected,
non-limiting approaches, for breeding the plants of the present
invention are set forth below. A breeding program can be enhanced
using marker assisted selection of the progeny of any cross. It is
further understood that any commercial and non-commercial cultivars
can be utilized in a breeding program. Factors such as, for
example, emergence vigor, vegetative vigor, stress tolerance,
disease resistance, branching, flowering, seed set, seed size, seed
density, standability, and threshability etc. will generally
dictate the choice.
[0307] For highly heritable traits, a choice of superior individual
plants evaluated at a single location will be effective, whereas
for traits with low heritability, selection should be based on mean
values obtained from replicated evaluations of families of related
plants. Popular selection methods commonly include pedigree
selection, modified pedigree selection, mass selection, and
recurrent selection. In a preferred embodiment a backcross or
recurrent breeding program is undertaken.
[0308] The complexity of inheritance influences choice of the
breeding method. Backcross breeding can be used to transfer one or
a few favorable genes for a highly heritable trait into a desirable
cultivar. This approach has been used extensively for breeding
disease-resistant cultivars. Various recurrent selection techniques
are used to improve quantitatively inherited traits controlled by
numerous genes. The use of recurrent selection in self-pollinating
crops depends on the ease of pollination, the frequency of
successful hybrids from each pollination, and the number of hybrid
offspring from each successful cross.
[0309] Breeding lines can be tested and compared to appropriate
standards in environments representative of the commercial target
area(s) for two or more generations. The best lines are candidates
for new commercial cultivars; those still deficient in traits may
be used as parents to produce new populations for further
selection.
[0310] One method of identifying a superior plant is to observe its
performance relative to other experimental plants and to a widely
grown standard cultivar. If a single observation is inconclusive,
replicated observations can provide a better estimate of its
genetic worth. A breeder can select and cross two or more parental
lines, followed by repeated selfing and selection, producing many
new genetic combinations.
[0311] The development of new cultivars requires the development
and selection of varieties, the crossing of these varieties and the
selection of superior hybrid crosses. The hybrid seed can be
produced by manual crosses between selected male-fertile parents or
by using male sterility systems. Hybrids are selected for certain
single gene traits such as pod color, flower color, seed yield,
pubescence color, or herbicide resistance, which indicate that the
seed is truly a hybrid. Additional data on parental lines, as well
as the phenotype of the hybrid, influence the breeder's decision
whether to continue with the specific hybrid cross.
[0312] Pedigree breeding and recurrent selection breeding methods
can be used to develop cultivars from breeding populations.
Breeding programs combine desirable traits from two or more
cultivars or various broad-based sources into breeding pools from
which cultivars are developed by selfing and selection of desired
phenotypes. New cultivars can be evaluated to determine which have
commercial potential.
[0313] Pedigree breeding is used commonly for the improvement of
self-pollinating crops. Two parents who possess favorable,
complementary traits are crossed to produce an F.sub.1. A F.sub.2
population is produced by selfing one or several F.sub.1's.
Selection of the best individuals from the best families is carried
out. Replicated testing of families can begin in the F.sub.4
generation to improve the effectiveness of selection for traits
with low heritability. At an advanced stage of inbreeding (i.e.,
F.sub.6 and F.sub.7), the best lines or mixtures of phenotypically
similar lines are tested for potential release as new
cultivars.
[0314] Backcross breeding has been used to transfer genes for a
simply inherited, highly heritable trait into a desirable
homozygous cultivar or inbred line, which is the recurrent parent.
The source of the trait to be transferred is called the donor
parent. The resulting plant is expected to have the attributes of
the recurrent parent (e.g., cultivar) and the desirable trait
transferred from the donor parent. After the initial cross,
individuals possessing the phenotype of the donor parent are
selected and repeatedly crossed (backcrossed) to the recurrent
parent. The resulting parent is expected to have the attributes of
the recurrent parent (e.g., cultivar) and the desirable trait
transferred from the donor parent.
[0315] The single-seed descent procedure in the strict sense refers
to planting a segregating population, harvesting a sample of one
seed per plant, and using the one-seed sample to plant the next
generation. When the population has been advanced from the F.sub.2
to the desired level of inbreeding, the plants from which lines are
derived will each trace to different F.sub.2 individuals. The
number of plants in a population declines each generation due to
failure of some seeds to germinate or some plants to produce at
least one seed. As a result, not all of the F.sub.2 plants
originally sampled in the population will be represented by a
progeny when generation advance is completed.
[0316] In a multiple-seed procedure, breeders commonly harvest one
or more pods from each plant in a population and thresh them
together to form a bulk. Part of the bulk is used to plant the next
generation and part is put in reserve. The procedure has been
referred to as modified single-seed descent or the pod-bulk
technique.
[0317] The multiple-seed procedure has been used to save labor at
harvest. It is considerably faster to thresh pods with a machine
than to remove one seed from each by hand for the single-seed
procedure. The multiple-seed procedure also makes it possible to
plant the same number of seed of a population each generation of
inbreeding.
[0318] Descriptions of other breeding methods that are commonly
used for different traits and crops can be found in one of several
reference books (e.g. Fehr, Principles of Cultivar Development Vol.
1, pp. 2-3 (1987))).
[0319] A transgenic plant of the present invention may also be
reproduced using apomixis. Apomixis is a genetically controlled
method of reproduction in plants where the embryo is formed without
union of an egg and a sperm. There are three basic types of
apomictic reproduction: 1) apospory where the embryo develops from
a chromosomally unreduced egg in an embryo sac derived from the
nucleus, 2) diplospory where the embryo develops from an unreduced
egg in an embryo sac derived from the megaspore mother cell, and 3)
adventitious embryo where the embryo develops directly from a
somatic cell. In most forms of apomixis, pseudogamy or
fertilization of the polar nuclei to produce endosperm is necessary
for seed viability. In apospory, a nurse cultivar can be used as a
pollen source for endosperm formation in seeds. The nurse cultivar
does not affect the genetics of the aposporous apomictic cultivar
since the unreduced egg of the cultivar develops
parthenogenetically, but makes possible endosperm production.
Apomixis is economically important, especially in transgenic
plants, because it causes any genotype, no matter how heterozygous,
to breed true. Thus, with apomictic reproduction, heterozygous
transgenic plants can maintain their genetic fidelity throughout
repeated life cycles. Methods for the production of apomictic
plants are known in the art. See, U.S. Pat. No. 5,811,636.
Other Organisms
[0320] A nucleic acid of the present invention may be introduced
into any cell or organism such as a mammalian cell, mammal, fish
cell, fish, bird cell, bird, algae cell, algae, fungal cell, fungi,
or bacterial cell. A protein of the present invention may be
produced in an appropriate cell or organism. Preferred host and
transformants include: fungal cells such as Aspergillus, yeasts,
mammals, particularly bovine and porcine, insects, bacteria, and
algae. Particularly preferred bacteria are Agrobacteruim
tumefaciens and E. coli.
[0321] Methods to transform such cells or organisms are known in
the art (EP 0 238 023; Yelton et al., Proc. Natl. Acad. Sci.
(U.S.A), 81:1470-1474 (1984); Malardier et al., Gene, 78:147-156
(1989); Becker and Guarente, In: Abelson and Simon (eds.), Guide to
Yeast Genetics and Molecular Biology, Method Enzymol., Vol. 194,
pp. 182-187, Academic Press, Inc., New York; Ito et al., J.
Bacteriology, 153:163 (1983) Hinnen et al, Proc. Natl. Acad. Sci.
(U.S.A.), 75:1920 (1978); Bennett and LaSure (eds.), More Gene
Manipulations in fungi, Academic Press, CA (1991)). Methods to
produce proteins of the present invention are also known (Kudla et
al., EMBO, 9:1355-1364 (1990); Jarai and Buxton, Current Genetics,
26:2238-2244 (1994); Verdier, Yeast, 6:271-297 (1990; MacKenzie et
al., Journal of Gen. Microbiol., 139:2295-2307 (1993); Hartl et
al., TIBS, 19:20-25 (1994); Bergenron et al., TIBS, 19:124-128
(1994); Demolder et al., J. Biotechnology, 32:179-189 (1994);
Craig, Science, 260:1902-1903 (1993); Gething and Sambrook, Nature,
355:33-45 (1992); Puig and Gilbert, J. Biol. Chem., 269:7764-7771
(1994); Wang and Tsou, FASEB Journal, 7:1515-1517 (1993); Robinson
et al., Bio/Technology, 1:381-384 (1994); Enderlin and Ogrydziak,
Yeast, 10:67-79 (1994); Fuller et al., Proc. Natl. Acad. Sci.
(U.S.A.), 86:1434-1438 (1989); Julius et al., Cell, 37:1075-1089
(1984); Julius et al., Cell 32:839-852 (1983).
[0322] In a preferred embodiment, overexpression of a protein or
fragment thereof of the present invention in a cell or organism
provides in that cell or organism, relative to an untransformed
cell or organism with a similar genetic background, an increased
level of tocopherols.
[0323] In a preferred embodiment, overexpression of a protein or
fragment thereof of the present invention in a cell or organism
provides in that cell or organism, relative to an untransformed
cell or organism with a similar genetic background, an increased
level of .alpha.-tocopherols.
[0324] In a preferred embodiment, overexpression of a protein or
fragment thereof of the present invention in a cell or organism
provides in that cell or organism, relative to an untransformed
cell or organism with a similar genetic background, an increased
level of .gamma.-tocopherols.
[0325] In another preferred embodiment, overexpression of a protein
or fragment thereof of the present invention in a cell or organism
provides in that cell or organism, relative to an untransformed
cell or organism with a similar genetic background, an increased
level of .alpha.-tocotrienols.
[0326] In another preferred embodiment, overexpression of a protein
or fragment thereof of the present invention in a cell or organism
provides in that cell or organism, relative to an untransformed
cell or organism with a similar genetic background, an increased
level of .gamma.-tocotrienols.
Antibodies
[0327] One aspect of the invention concerns antibodies,
single-chain antigen binding molecules, or other proteins that
specifically bind to one or more of the protein or peptide
molecules of the invention and their homologs, fusions or
fragments. In a particularly preferred embodiment, the antibody
specifically binds to a protein having the amino acid sequence set
forth in SEQ ID NOs: 16 through 38 or fragments thereof. In another
embodiment, the antibody specifically binds to a fusion protein
comprising an amino acid sequence selected from the amino acid
sequence set forth in SEQ ID NOs: 16 through 38 or fragments
thereof. Antibodies of the invention may be used to quantitatively
or qualitatively detect the protein or peptide molecules of the
invention, or to detect post translational modifications of the
proteins. As used herein, an antibody or peptide is said to
"specifically bind" to a protein or peptide molecule of the
invention if such binding is not competitively inhibited by the
presence of non-related molecules.
[0328] Nucleic acid molecules that encode all or part of the
protein of the invention can be expressed, via recombinant means,
to yield protein or peptides that can in turn be used to elicit
antibodies that are capable of binding the expressed protein or
peptide. Such antibodies may be used in immunoassays for that
protein. Such protein-encoding molecules, or their fragments may be
a "fusion" molecule (i.e., a part of a larger nucleic acid
molecule) such that, upon expression, a fusion protein is produced.
It is understood that any of the nucleic acid molecules of the
invention may be expressed, via recombinant means, to yield
proteins or peptides encoded by these nucleic acid molecules.
[0329] The antibodies that specifically bind proteins and protein
fragments of the invention may be polyclonal or monoclonal and may
comprise intact immunoglobulins, or antigen binding portions of
immunoglobulins fragments (such as (F(ab'), F(ab').sub.2), or
single-chain immunoglobulins producible, for example, via
recombinant means. It is understood that practitioners are familiar
with the standard resource materials that describe specific
conditions and procedures for the construction, manipulation and
isolation of antibodies (see, for example, Harlow and Lane, In:
Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. (1988)).
[0330] As discussed below, such antibody molecules or their
fragments may be used for diagnostic purposes. Where the antibodies
are intended for diagnostic purposes, it may be desirable to
derivatize them, for example with a ligand group (such as biotin)
or a detectable marker group (such as a fluorescent group, a
radioisotope or an enzyme).
[0331] The ability to produce antibodies that bind the protein or
peptide molecules of the invention permits the identification of
mimetic compounds derived from those molecules. These mimetic
compounds may contain a fragment of the protein or peptide or
merely a structurally similar region and nonetheless exhibits an
ability to specifically bind to antibodies directed against that
compound.
Exemplary Uses
[0332] Nucleic acid molecules and fragments thereof of the
invention may be employed to obtain other nucleic acid molecules
from the same species (nucleic acid molecules from corn may be
utilized to obtain other nucleic acid molecules from corn). Such
nucleic acid molecules include the nucleic acid molecules that
encode the complete coding sequence of a protein and promoters and
flanking sequences of such molecules. In addition, such nucleic
acid molecules include nucleic acid molecules that encode for other
isozymes or gene family members. Such molecules can be readily
obtained by using the above-described nucleic acid molecules or
fragments thereof to screen cDNA or genomic libraries. Methods for
forming such libraries are well known in the art.
[0333] Nucleic acid molecules and fragments thereof of the
invention may also be employed to obtain nucleic acid homologs.
Such homologs include the nucleic acid molecules of plants and
other organisms, including bacteria and fungi, including the
nucleic acid molecules that encode, in whole or in part, protein
homologues of other plant species or other organisms, sequences of
genetic elements, such as promoters and transcriptional regulatory
elements. Such molecules can be readily obtained by using the
above-described nucleic acid molecules or fragments thereof to
screen cDNA or genomic libraries obtained from such plant species.
Methods for forming such libraries are well known in the art. Such
homolog molecules may differ in their nucleotide sequences from
those found in one or more of SEQ ID NOs: 1 through 15, and
complements thereof because complete complementarity is not needed
for stable hybridization. The nucleic acid molecules of the
invention therefore also include molecules that, although capable
of specifically hybridizing with the nucleic acid molecules may
lack "complete complementarity."
[0334] Any of a variety of methods may be used to obtain one or
more of the above-described nucleic acid molecules (Zamechik et
al., Proc. Natl. Acad. Sci. (U.S.A) 83:4143-4146 (1986); Goodchild
et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:5507-5511 (1988);
Wickstrom et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:1028-1032
(1988); Holt et al., Molec. Cell. Biol. 8:963-973 (1988); Gerwirtz
et al., Science 242:1303-1306 (1988); Anfossi et al., Proc. Natl.
Acad. Sci. (U.S.A) 86:3379-3383 (1989); Becker et al., EMBO J.
8:3685-3691 (1989)). Automated nucleic acid synthesizers may be
employed for this purpose. In lieu of such synthesis, the disclosed
nucleic acid molecules may be used to define a pair of primers that
can be used with the polymerase chain reaction (Mullis et al., Cold
Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich et al.,
European Patent 50,424; European Patent 84,796; European Patent
258,017; European Patent 237,362; Mullis, European Patent 201,184;
Mullis et al., U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No.
4,582,788; and Saiki et al., U.S. Pat. No. 4,683,194) to amplify
and obtain any desired nucleic acid molecule or fragment.
[0335] Promoter sequences and other genetic elements, including but
not limited to transcriptional regulatory flanking sequences,
associated with one or more of the disclosed nucleic acid sequences
can also be obtained using the disclosed nucleic acid sequence
provided herein. In one embodiment, such sequences are obtained by
incubating nucleic acid molecules of the present invention with
members of genomic libraries and recovering clones that hybridize
to such nucleic acid molecules thereof. In a second embodiment,
methods of "chromosome walking," or inverse PCR may be used to
obtain such sequences (Frohman et al., Proc. Natl. Acad. Sci.
(U.S.A.) 85:8998-9002 (1988); Ohara et al., Proc. Natl. Acad. Sci.
(U.S.A.) 86:5673-5677 (1989); Pang et al., Biotechniques
22:1046-1048 (1977); Huang et al., Methods Mol. Biol. 69:89-96
(1997); Huang et al., Method Mol. Biol. 67:287-294 (1997); Benkel
et al., Genet. Anal. 13:123-127 (1996); Hartl et al., Methods Mol.
Biol. 58:293-301 (1996)). The term "chromosome walking" means a
process of extending a genetic map by successive hybridization
steps.
[0336] The nucleic acid molecules of the invention may be used to
isolate promoters of cell enhanced, cell specific, tissue enhanced,
tissue specific, developmentally or environmentally regulated
expression profiles. Isolation and functional analysis of the 5'
flanking promoter sequences of these genes from genomic libraries,
for example, using genomic screening methods and PCR techniques
would result in the isolation of useful promoters and
transcriptional regulatory elements. These methods are known to
those of skill in the art and have been described (See, for
example, Birren et al., Genome Analysis: Analyzing DNA, 1, (1997),
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Promoters obtained utilizing the nucleic acid molecules of the
invention could also be modified to affect their control
characteristics. Examples of such modifications would include but
are not limited to enhancer sequences. Such genetic elements could
be used to enhance gene expression of new and existing traits for
crop improvement.
[0337] Another subset of the nucleic acid molecules of the
invention includes nucleic acid molecules that are markers. The
markers can be used in a number of conventional ways in the field
of molecular genetics. Such markers include nucleic acid molecules
SEQ ID NOs: 1 through 15, complements thereof, and fragments of
either that can act as markers and other nucleic acid molecules of
the present invention that can act as markers.
[0338] Genetic markers of the invention include "dominant" or
"codominant" markers. "Codominant markers" reveal the presence of
two or more alleles (two per diploid individual) at a locus.
"Dominant markers" reveal the presence of only a single allele per
locus. The presence of the dominant marker phenotype (e.g., a band
of DNA) is an indication that one allele is in either the
homozygous or heterozygous condition. The absence of the dominant
marker phenotype (e.g., absence of a DNA band) is merely evidence
that "some other" undefined allele is present. In the case of
populations where individuals are predominantly homozygous and loci
are predominately dimorphic, dominant and codominant markers can be
equally valuable. As populations become more heterozygous and
multi-allelic, codominant markers often become more informative of
the genotype than dominant markers. Marker molecules can be, for
example, capable of detecting polymorphisms such as single
nucleotide polymorphisms (SNPs).
[0339] The genomes of animals and plants naturally undergo
spontaneous mutation in the course of their continuing evolution
(Gusella, Ann. Rev. Biochem. 55:831-854 (1986)). A "polymorphism"
is a variation or difference in the sequence of the gene or its
flanking regions that arises in some of the members of a species.
The variant sequence and the "original" sequence co-exist in the
species' population. In some instances, such co-existence is in
stable or quasi-stable equilibrium.
[0340] A polymorphism is thus said to be "allelic," in that, due to
the existence of the polymorphism, some members of a population may
have the original sequence (i.e., the original "allele") whereas
other members may have the variant sequence (i.e., the variant
"allele"). In the simplest case, only one variant sequence may
exist and the polymorphism is thus said to be di-allelic. In other
cases, the species' population may contain multiple alleles and the
polymorphism is termed tri-allelic, etc. A single gene may have
multiple different unrelated polymorphisms. For example, it may
have a di-allelic polymorphism at one site and a multi-allelic
polymorphism at another site.
[0341] The variation that defines the polymorphism may range from a
single nucleotide variation to the insertion or deletion of
extended regions within a gene. In some cases, the DNA sequence
variations are in regions of the genome that are characterized by
short tandem repeats (STRs) that include tandem di- or
tri-nucleotide repeated motifs of nucleotides. Polymorphisms
characterized by such tandem repeats are referred to as "variable
number tandem repeat" ("VNTR") polymorphisms. VNTRs have been used
in identity analysis (Weber, U.S. Pat. No. 5,075,217; Armour et
al., FEBS Lett. 307:113-115 (1992); Jones et al., Eur. J. Haematol.
39:144-147 (1987); Horn et al., PCT Patent Application WO91/14003;
Jeffreys, European Patent Application 370,719; Jeffreys, U.S. Pat.
No. 5,175,082; Jeffreys et al., Amer. J. Hum. Genet. 39:11-24
(1986); Jeffreys et al., Nature 316:76-79 (1985); Gray et al.,
Proc. R. Acad. Soc. Lond. 243:241-253 (1991); Moore et al.,
Genomics 10:654-660 (1991); Jeffreys et al., Anim. Genet. 18:1-15
(1987); Hillel et al., Anim. Genet. 20:145-155 (1989); Hillel et
al., Genet. 124:783-789 (1990)).
[0342] The detection of polymorphic sites in a sample of DNA may be
facilitated through the use of nucleic acid amplification methods.
Such methods specifically increase the concentration of
polynucleotides that span the polymorphic site, or include that
site and sequences located either distal or proximal to it. Such
amplified molecules can be readily detected by gel electrophoresis
or other means.
[0343] In an alternative embodiment, such polymorphisms can be
detected through the use of a marker nucleic acid molecule that is
physically linked to such polymorphism(s). For this purpose, marker
nucleic acid molecules comprising a nucleotide sequence of a
polynucleotide located within 1 mb of the polymorphism(s) and more
preferably within 100 kb of the polymorphism(s) and most preferably
within 10 kb of the polymorphism(s) can be employed.
[0344] The identification of a polymorphism can be determined in a
variety of ways. By correlating the presence or absence of it in a
plant with the presence or absence of a phenotype, it is possible
to predict the phenotype of that plant. If a polymorphism creates
or destroys a restriction endonuclease cleavage site, or if it
results in the loss or insertion of DNA (e.g., a VNTR
polymorphism), it will alter the size or profile of the DNA
fragments that are generated by digestion with that restriction
endonuclease. As such, organisms that possess a variant sequence
can be distinguished from those having the original sequence by
restriction fragment analysis. Polymorphisms that can be identified
in this manner are termed "restriction fragment length
polymorphisms" ("RFLPs") (Glassberg, UK Patent Application 2135774;
Skolnick et al., Cytogen. Cell Genet. 32:58-67 (1982); Botstein et
al., Ann. J. Hum. Genet. 32:314-331 (1980); Fischer et al., (PCT
Application WO90/13668; Uhlen, PCT Application WO90/11369).
[0345] Polymorphisms can also be identified by Single Strand
Conformation Polymorphism (SSCP) analysis (Elles, Methods in
Molecular Medicine: Molecular Diagnosis of Genetic Diseases, Humana
Press (1996)); Orita et al., Genomics 5:874-879 (1989)). A number
of protocols have been described for SSCP including, but not
limited to, Lee et al., Anal. Biochem. 205:289-293 (1992); Suzuki
et al., Anal. Biochem. 192:82-84 (1991); Lo et al., Nucleic Acids
Research 20:1005-1009 (1992); Sarkar et al., Genomics 13:441-443
(1992). It is understood that one or more of the nucleic acids of
the invention, may be utilized as markers or probes to detect
polymorphisms by SSCP analysis.
[0346] Polymorphisms may also be found using a DNA fingerprinting
technique called amplified fragment length polymorphism (AFLP),
which is based on the selective PCR amplification of restriction
fragments from a total digest of genomic DNA to profile that DNA
(Vos et al., Nucleic Acids Res. 23:4407-4414 (1995)). This method
allows for the specific co-amplification of high numbers of
restriction fragments, which can be visualized by PCR without
knowledge of the nucleic acid sequence. It is understood that one
or more of the nucleic acids of the invention may be utilized as
markers or probes to detect polymorphisms by AFLP analysis or for
fingerprinting RNA.
[0347] Polymorphisms may also be found using random amplified
polymorphic DNA (RAPD) (Williams et al, Nucl. Acids Res.
18:6531-6535 (1990)) and cleaveable amplified polymorphic sequences
(CAPS) (Lyamichev et al., Science 260:778-783 (1993)). It is
understood that one or more of the nucleic acid molecules of the
invention, may be utilized as markers or probes to detect
polymorphisms by RAPD or CAPS analysis.
[0348] Single Nucleotide Polymorphisms (SNPs) generally occur at
greater frequency than other polymorphic markers and are spaced
with a greater uniformity throughout a genome than other reported
forms of polymorphism. The greater frequency and uniformity of SNPs
means that there is greater probability that such a polymorphism
will be found near or in a genetic locus of interest than would be
the case for other polymorphisms. SNPs are located in
protein-coding regions and noncoding regions of a genome. Some of
these SNPs may result in defective or variant protein expression
(e.g., as a result of mutations or defective splicing). Analysis
(genotyping) of characterized SNPs can require only a plus/minus
assay rather than a lengthy measurement, permitting easier
automation.
[0349] SNPs can be characterized using any of a variety of methods.
Such methods include the direct or indirect sequencing of the site,
the use of restriction enzymes (Botstein et al., Am. J. Hum. Genet.
32:314-331 (1980); Konieczny and Ausubel, Plant J. 4:403-410
(1993)), enzymatic and chemical mismatch assays (Myers et al.,
Nature 313:495-498 (1985)), allele-specific PCR (Newton et al.,
Nucl. Acids Res. 17:2503-2516 (1989); Wu et al., Proc. Natl. Acad.
Sci. USA 86:2757-2760 (1989)), ligase chain reaction (Barany, Proc.
Natl. Acad. Sci. USA 88:189-193 (1991)), single-strand conformation
polymorphism analysis (Labrune et al., Am. J. Hum. Genet.
48:1115-1120 (1991)), single base primer extension (Kuppuswamy et
al., Proc. Natl. Acad. Sci. USA 88:1143-1147 (1991)), Goelet U.S.
Pat. No. 6,004,744; Goelet 5,888,819), solid-phase ELISA-based
oligonucleotide ligation assays (Nikiforov et al., Nucl. Acids Res.
22:4167-4175 (1994), dideoxy fingerprinting (Sarkar et al.,
Genomics 13:441-443 (1992)), oligonucleotide fluorescence-quenching
assays (Livak et al., PCR Methods Appl. 4:357-362 (1995a)),
5'-nuclease allele-specific hybridization TaqMan.TM. assay (Livak
et al., Nature Genet. 9:341-342 (1995)), template-directed
dye-terminator incorporation (TDI) assay (Chen and Kwok, Nucl.
Acids Res. 25:347-353 (1997)), allele-specific molecular beacon
assay (Tyagi et al., Nature Biotech. 16:49-53 (1998)), PinPoint
assay (Haff and Smirnov, Genome Res. 7:378-388 (1997)), dCAPS
analysis (Neff et al., Plant J. 14:387-392 (1998)), pyrosequencing
(Ronaghi et al, Analytical Biochemistry 267:65-71 (1999); Ronaghi
et al PCT application WO 98/13523; Nyren et al PCT application WO
98/28440; www.pyrosequencing.com), using mass spectrometry, e.g.
the Masscode.TM. system (Howbert et al PCT application, WO
99/05319; Howbert et al PCT application WO 97/27331;
www.rapigene.com; Becker et al PCT application WO 98/26095; Becker
et al PCT application; WO 98/12355; Becker et al PCT application WO
97/33000; Monforte et al U.S. Pat. No. 5,965,363), invasive
cleavage of oligonucleotide probes (Lyamichev et al Nature
Biotechnology 17:292-296; www.twt.com), and using high density
oligonucleotide arrays (Hacia et al Nature Genetics 22:164-167;
www.affymetrix.com).
[0350] Polymorphisms may also be detected using allele-specific
oligonucleotides (ASO), which, can be for example, used in
combination with hybridization based technology including Southern,
Northern, and dot blot hybridizations, reverse dot blot
hybridizations and hybridizations performed on microarray and
related technology.
[0351] The stringency of hybridization for polymorphism detection
is highly dependent upon a variety of factors, including length of
the allele-specific oligonucleotide, sequence composition, degree
of complementarity (i.e. presence or absence of base mismatches),
concentration of salts and other factors such as formamide, and
temperature. These factors are important both during the
hybridization itself and during subsequent washes performed to
remove target polynucleotide that is not specifically hybridized.
In practice, the conditions of the final, most stringent wash are
most critical. In addition, the amount of target polynucleotide
that is able to hybridize to the allele-specific oligonucleotide is
also governed by such factors as the concentration of both the ASO
and the target polynucleotide, the presence and concentration of
factors that act to "tie up" water molecules, so as to effectively
concentrate the reagents (e.g., PEG, dextran, dextran sulfate,
etc.), whether the nucleic acids are immobilized or in solution,
and the duration of hybridization and washing steps.
[0352] Hybridizations are preferably performed below the melting
temperature (T.sub.m) of the ASO. The closer the hybridization
and/or washing step is to the T.sub.m, the higher the stringency.
T.sub.m for an oligonucleotide may be approximated, for example,
according to the following formula: T.sub.m=81.5+16.6.times.(log
10[Na+])+0.41.times.(% G+C)-675/n; where [Na+] is the molar salt
concentration of Na+ or any other suitable cation and n=number of
bases in the oligonucleotide. Other formulas for approximating
T.sub.m are available and are known to those of ordinary skill in
the art.
[0353] Stringency is preferably adjusted so as to allow a given ASO
to differentially hybridize to a target polynucleotide of the
correct allele and a target polynucleotide of the incorrect allele.
Preferably, there will be at least a two-fold differential between
the signal produced by the ASO hybridizing to a target
polynucleotide of the correct allele and the level of the signal
produced by the ASO cross-hybridizing to a target polynucleotide of
the incorrect allele (e.g., an ASO specific for a mutant allele
cross-hybridizing to a wild-type allele). In more preferred
embodiments of the present invention, there is at least a five-fold
signal differential. In highly preferred embodiments of the present
invention, there is at least an order of magnitude signal
differential between the ASO hybridizing to a target polynucleotide
of the correct allele and the level of the signal produced by the
ASO cross-hybridizing to a target polynucleotide of the incorrect
allele.
[0354] While certain methods for detecting polymorphisms are
described herein, other detection methodologies may be utilized.
For example, additional methodologies are known and set forth, in
Birren et al., Genome Analysis, 4:135-186, A Laboratory Manual.
Mapping Genomes, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1999); Maliga et al., Methods in Plant Molecular
Biology. A Laboratory Course Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1995); Paterson, Biotechnology
Intelligence Unit: Genome Mapping in Plants, R.G. Landes Co.,
Georgetown, Tex., and Academic Press, San Diego, Calif. (1996); The
Corn Handbook, Freeling and Walbot, eds., Springer-Verlag, New
York, N.Y. (1994); Methods in Molecular Medicine: Molecular
Diagnosis of Genetic Diseases, Elles, ed., Humana Press, Totowa,
N.J. (1996); Clark, ed., Plant Molecular Biology: A Laboratory
Manual, Clark, ed., Springer-Verlag, Berlin, Germany (1997).
[0355] Factors for marker-assisted selection in a plant breeding
program are: (1) the marker(s) should co-segregate or be closely
linked with the desired trait; (2) an efficient means of screening
large populations for the molecular marker(s) should be available;
and (3) the screening technique should have high reproducibility
across laboratories and preferably be economical to use and be
user-friendly.
[0356] The genetic linkage of marker molecules can be established
by a gene mapping model such as, without limitation, the flanking
marker model reported by Lander and Botstein, Genetics 121:185-199
(1989) and the interval mapping, based on maximum likelihood
methods described by Lander and Botstein, Genetics 121:185-199
(1989) and implemented in the software package MAPMAKER/QTL
(Lincoln and Lander, Mapping Genes Controlling Quantitative Traits
Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,
Massachusetts, (1990). Additional software includes Qgene, Version
2.23 (1996), Department of Plant Breeding and Biometry, 266 Emerson
Hall, Cornell University, Ithaca, N.Y.). Use of Qgene software is a
particularly preferred approach.
[0357] A maximum likelihood estimate (MLE) for the presence of a
marker is calculated, together with an MLE assuming no QTL effect,
to avoid false positives. A log.sub.10 of an odds ratio (LOD) is
then calculated as: LOD=log.sub.10(MLE for the presence of a
QTL/MLE given no linked QTL).
[0358] The LOD score essentially indicates how much more likely the
data are to have arisen assuming the presence of a QTL than in its
absence. The LOD threshold value for avoiding a false positive with
a given confidence, say 95%, depends on the number of markers and
the length of the genome. Graphs indicating LOD thresholds are set
forth in Lander and Botstein, Genetics 121:185-199 (1989) and
further described by Ar s and Moreno-Gonzalez, Plant Breeding,
Hayward et al., (eds.) Chapman & Hall, London, pp. 314-331
(1993).
[0359] In a preferred embodiment of the present invention the
nucleic acid marker exhibits a LOD score of greater than 2.0, more
preferably 2.5, even more preferably greater than 3.0 or 4.0 with
the trait or phenotype of interest. In a preferred embodiment, the
trait of interest is altered tocopherol levels or compositions or
altered tocotrienol levels or compositions.
[0360] Additional models can be used. Many modifications and
alternative approaches to interval mapping have been reported,
including the use of non-parametric methods (Kruglyak and Lander,
Genetics 139:1421-1428 (1995)). Multiple regression methods or
models can also be used, in which the trait is regressed on a large
number of markers (Jansen, Biometrics in Plant Breeding, van Oijen
and Jansen (eds.), Proceedings of the Ninth Meeting of the Eucarpia
Section Biometrics in Plant Breeding, The Netherlands, pp. 116-124
(1994); Weber and Wricke, Advances in Plant Breeding, Blackwell,
Berlin, 16 (1994)). Procedures combining interval mapping with
regression analysis, whereby the phenotype is regressed onto a
single putative QTL at a given marker interval and at the same time
onto a number of markers that serve as `cofactors,` have been
reported by Jansen and Stam, Genetics 136:1447-1455 (1994), and
Zeng, Genetics 136:1457-1468 (1994). Generally, the use of
cofactors reduces the bias and sampling error of the estimated QTL
positions (Utz and Melchinger, Biometrics in Plant Breeding, van
Oijen and Jansen (eds.) Proceedings of the Ninth Meeting of the
Eucarpia Section Biometrics in Plant Breeding, The Netherlands, pp.
195-204 (1994), thereby improving the precision and efficiency of
QTL mapping (Zeng, Genetics 136:1457-1468 (1994)). These models can
be extended to multi-environment experiments to analyze
genotype-environment interactions (Jansen et al., Theo. Appl.
Genet. 91:33-37 (1995)).
[0361] It is understood that one or more of the nucleic acid
molecules of the invention may be used as molecular markers. It is
also understood that one or more of the protein molecules of the
invention may be used as molecular markers.
[0362] In a preferred embodiment, the polymorphism is present and
screened for in a mapping population, e.g. a collection of plants
capable of being used with markers such as polymorphic markers to
map genetic position of traits. The choice of appropriate mapping
population often depends on the type of marker systems employed
(Tanksley et al., J.P. Gustafson and R. Appels (eds.). Plenum
Press, New York, pp. 157-173 (1988)). Consideration must be given
to the source of parents (adapted vs. exotic) used in the mapping
population. Chromosome pairing and recombination rates can be
severely disturbed (suppressed) in wide crosses
(adapted.times.exotic) and generally yield greatly reduced linkage
distances. Wide crosses will usually provide segregating
populations with a relatively large number of polymorphisms when
compared to progeny in a narrow cross (adapted.times.adapted).
[0363] An F.sub.2 population is the first generation of selfing
(self-pollinating) after the hybrid seed is produced. Usually a
single F.sub.1 plant is selfed to generate a population segregating
for all the genes in Mendelian (1:2:1) pattern. Maximum genetic
information is obtained from a completely classified F.sub.2
population using a codominant marker system (Mather, Measurement of
Linkage in Heredity: Methuen and Co., (1938)). In the case of
dominant markers, progeny tests (e.g., F.sub.3, BCF.sub.2) are
required to identify the heterozygotes, in order to classify the
population. However, this procedure is often prohibitive because of
the cost and time involved in progeny testing. Progeny testing of
F.sub.2 individuals is often used in map construction where
phenotypes do not consistently reflect genotype (e.g. disease
resistance) or where trait expression is controlled by a QTL.
Segregation data from progeny test populations e.g. F.sub.3 or
BCF.sub.2) can be used in map construction. Marker-assisted
selection can then be applied to cross progeny based on
marker-trait map associations (F.sub.2, F.sub.3), where linkage
groups have not been completely disassociated by recombination
events (i.e., maximum disequilibrium).
[0364] Recombinant inbred lines (RIL) (genetically related lines;
usually >F.sub.5, developed from continuously selfing F.sub.2
lines towards homozygosity) can be used as a mapping population.
Information obtained from dominant markers can be maximized by
using RIL because all loci are homozygous or nearly so. Under
conditions of tight linkage (i.e., about <10% recombination),
dominant and co-dominant markers evaluated in RIL populations
provide more information per individual than either marker type in
backcross populations (Reiter. Proc. Natl. Acad. Sci. (U.S.A)
89:1477-1481 (1992)). However, as the distance between markers
becomes larger (i.e., loci become more independent), the
information in RIL populations decreases dramatically when compared
to codominant markers.
[0365] Backcross populations (e.g., generated from a cross between
a successful variety (recurrent parent) and another variety (donor
parent) carrying a trait not present in the former) can be utilized
as a mapping population. A series of backcrosses to the recurrent
parent can be made to recover most of its desirable traits. Thus a
population is created consisting of individuals nearly like the
recurrent parent but each individual carries varying amounts or
mosaic of genomic regions from the donor parent. Backcross
populations can be useful for mapping dominant markers if all loci
in the recurrent parent are homozygous and the donor and recurrent
parent have contrasting polymorphic marker alleles (Reiter et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481 (1992)). Information
obtained from backcross populations using either codominant or
dominant markers is less than that obtained from F.sub.2
populations because one, rather than two, recombinant gamete is
sampled per plant. Backcross populations, however, are more
informative (at low marker saturation) when compared to RILs as the
distance between linked loci increases in RIL populations (i.e.
about 0.15% recombination). Increased recombination can be
beneficial for resolution of tight linkages, but may be undesirable
in the construction of maps with low marker saturation.
[0366] Near-isogenic lines (NIL) (created by many backcrosses to
produce a collection of individuals that is nearly identical in
genetic composition except for the trait or genomic region under
interrogation) can be used as a mapping population. In mapping with
NILs, only a portion of the polymorphic loci is expected to map to
a selected region.
[0367] Bulk segregant analysis (BSA) is a method developed for the
rapid identification of linkage between markers and traits of
interest (Michelmore et al., Proc. Natl. Acad. Sci. U.S.A.
88:9828-9832 (1991)). In BSA, two bulked DNA samples are drawn from
a segregating population originating from a single cross. These
bulks contain individuals that are identical for a particular trait
(resistant or susceptible to particular disease) or genomic region
but arbitrary at unlinked regions (i.e. heterozygous). Regions
unlinked to the target region will not differ between the bulked
samples of many individuals in BSA.
[0368] In an aspect of the present invention, one or more of the
nucleic molecules of the present invention are used to determine
the level (i.e., the concentration of mRNA in a sample, etc.) in a
plant (preferably canola, corn, Brassica campestris, Brassica
napus, oilseed rape, rapeseed, soybean, crambe, mustard, castor
bean, peanut, sesame, cottonseed, linseed, safflower, oil palm,
flax or sunflower) or pattern (i.e., the kinetics of expression,
rate of decomposition, stability profile, etc.) of the expression
of a protein encoded in part or whole by one or more of the nucleic
acid molecule of the present invention (collectively, the
"Expression Response" of a cell or tissue).
[0369] As used herein, the Expression Response manifested by a cell
or tissue is said to be "altered" if it differs from the Expression
Response of cells or tissues of plants not exhibiting the
phenotype. To determine whether a Expression Response is altered,
the Expression Response manifested by the cell or tissue of the
plant exhibiting the phenotype is compared with that of a similar
cell or tissue sample of a plant not exhibiting the phenotype. As
will be appreciated, it is not necessary to re-determine the
Expression Response of the cell or tissue sample of plants not
exhibiting the phenotype each time such a comparison is made;
rather, the Expression Response of a particular plant may be
compared with previously obtained values of normal plants. As used
herein, the phenotype of the organism is any of one or more
characteristics of an organism (e.g. disease resistance, pest
tolerance, environmental tolerance such as tolerance to abiotic
stress, male sterility, quality improvement or yield etc.). A
change in genotype or phenotype may be transient or permanent. Also
as used herein, a tissue sample is any sample that comprises more
than one cell. In a preferred aspect, a tissue sample comprises
cells that share a common characteristic (e.g. Derived from root,
seed, flower, leaf, stem or pollen etc.).
[0370] In one aspect of the present invention, an evaluation can be
conducted to determine whether a particular mRNA molecule is
present. One or more of the nucleic acid molecules of the present
invention are utilized to detect the presence or quantity of the
mRNA species. Such molecules are then incubated with cell or tissue
extracts of a plant under conditions sufficient to permit nucleic
acid hybridization. The detection of double-stranded probe-mRNA
hybrid molecules is indicative of the presence of the mRNA; the
amount of such hybrid formed is proportional to the amount of mRNA.
Thus, such probes may be used to ascertain the level and extent of
the mRNA production in a plant's cells or tissues. Such nucleic
acid hybridization may be conducted under quantitative conditions
(thereby providing a numerical value of the amount of the mRNA
present). Alternatively, the assay may be conducted as a
qualitative assay that indicates either that the mRNA is present,
or that its level exceeds a user set, predefined value.
[0371] A number of methods can be used to compare the expression
response between two or more samples of cells or tissue. These
methods include hybridization assays, such as northerns, RNAse
protection assays, and in situ hybridization. Alternatively, the
methods include PCR-type assays. In a preferred method, the
expression response is compared by hybridizing nucleic acids from
the two or more samples to an array of nucleic acids. The array
contains a plurality of suspected sequences known or suspected of
being present in the cells or tissue of the samples.
[0372] An advantage of in situ hybridization over more conventional
techniques for the detection of nucleic acids is that it allows an
investigator to determine the precise spatial population (Angerer
et al., Dev. Biol. 101:477-484 (1984); Angerer et al., Dev. Biol.
112:157-166 (1985); Dixon et al., EMBO J. 10:1317-1324 (1991)). In
situ hybridization may be used to measure the steady-state level of
RNA accumulation (Hardin et al., J. Mol. Biol. 202:417-431 (1989)).
A number of protocols have been devised for in situ hybridization,
each with tissue preparation, hybridization and washing conditions
(Meyerowitz, Plant Mol. Biol. Rep. 5:242-250 (1987); Cox and
Goldberg, In: Plant Molecular Biology: A Practical Approach, Shaw
(ed.), pp. 1-35, IRL Press, Oxford (1988); Raikhel et al., In situ
RNA hybridization in plant tissues, In: Plant Molecular Biology
Manual, vol. B9:1-32, Kluwer Academic Publisher, Dordrecht, Belgium
(1989)).
[0373] In situ hybridization also allows for the localization of
proteins within a tissue or cell (Wilkinson, In Situ Hybridization,
Oxford University Press, Oxford (1992); Langdale, In Situ
Hybridization In: The Corn Handbook, Freeling and Walbot (eds.),
pp. 165-179, Springer-Verlag, New York (1994)). It is understood
that one or more of the molecules of the invention, preferably one
or more of the nucleic acid molecules or fragments thereof of the
invention or one or more of the antibodies of the invention may be
utilized to detect the level or pattern of a protein or mRNA
thereof by in situ hybridization.
[0374] Fluorescent in situ hybridization allows the localization of
a particular DNA sequence along a chromosome, which is useful,
among other uses, for gene mapping, following chromosomes in hybrid
lines, or detecting chromosomes with translocations, transversions
or deletions. In situ hybridization has been used to identify
chromosomes in several plant species (Griffor et al., Plant Mol.
Biol. 17:101-109 (1991); Gustafson et al., Proc. Natl. Acad. Sci.
(U.S.A.) 87:1899-1902 (1990); Mukai and Gill, Genome 34:448-452
(1991); Schwarzacher and Heslop-Harrison, Genome 34:317-323 (1991);
Wang et al., Jpn. J. Genet. 66:313-316 (1991); Parra and Windle,
Nature Genetics 5:17-21 (1993)). It is understood that the nucleic
acid molecules of the invention may be used as probes or markers to
localize sequences along a chromosome.
[0375] Another method to localize the expression of a molecule is
tissue printing. Tissue printing provides a way to screen, at the
same time on the same membrane many tissue sections from different
plants or different developmental stages (Yomo and Taylor, Planta
112:35-43 (1973); Harris and Chrispeels, Plant Physiol. 56:292-299
(1975); Cassab and Vamer, J. Cell. Biol. 105:2581-2588 (1987);
Spruce et al., Phytochemistry 26:2901-2903 (1987); Barres et al.,
Neuron 5:527-544 (1990); Reid and Pont-Lezica, Tissue Printing:
Tools for the Study of Anatomy, Histochemistry and Gene Expression,
Academic Press, New York, N.Y. (1992); Reid et al., Plant Physiol.
93:160-165 (1990); Ye et al., Plant J. 1:175-183 (1991)).
[0376] One skilled in the art can refer to general reference texts
for detailed descriptions of known techniques discussed herein or
equivalent techniques. These texts include Current Protocols in
Molecular Biology Ausubel, et al., eds., John Wiley & Sons,
N.Y. (1989), and supplements through September (1998), Molecular
Cloning, A Laboratory Manual, Sambrook et al, 2.sup.nd Ed., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), Genome
Analysis: A Laboratory Manual 1: Analyzing DNA, Birren et al., Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1997); Genome
Analysis: A Laboratory Manual 2: Detecting Genes, Birren et al.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1998); Genome
Analysis: A Laboratory Manual 3: Cloning Systems, Birren et al.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1999); Genome
Analysis: A Laboratory Manual 4: Mapping Genomes, Birren et al.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1999); Plant
Molecular Biology: A Laboratory Manual, Clark, Springer-Verlag,
Berlin, (1997), Methods in Plant Molecular Biology, Maliga et al.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1995). These
texts can, of course, also be referred to in making or using an
aspect of the invention. It is understood that any of the agents of
the invention can be substantially purified and/or be biologically
active and/or recombinant.
[0377] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples that are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE 1
Identification and Characterization of Mutant hdt2 Arabidopsis
thaliana, Ecotype Landsberg Plants
[0378] Mutagenized (M.sub.2) seeds of Arabidopsis thaliana, ecotype
Landsberg are obtained both by purchase from Lehle Seeds (Round
Rock, Tex., U.S.A.) and by standard EMS mutagenesis methodology.
The M.sub.2 plants are grown from the M.sub.2 seeds in greenhouse
conditions with one plant per 2.5 inch pot. The resulting M.sub.3
seeds are collected from individual M.sub.2 plants and analyzed for
tocopherol levels.
[0379] Seeds from approximately 10,000 M.sub.3 lines of Arabidopsis
thaliana, ecotype Landsberg or Col-O are analyzed for individual
tocopherol levels using the following procedure. Five milligrams of
seeds from individual plants are ground to a fine powder using a
1/8'' steel ball bearing and vigorous shaking. 200 Microliters of
99.5% ethanol/0.5% pyrogallol is added, mixed for 30 seconds and
allowed to incubate at 4.degree. C. for 1 h. 50 Microgram/ml of
tocol (Matreya, Inc., Pleasant Gap, Pa.) is added to each sample as
an injection standard. To remove debris following centrifugation,
the supernatant is filtered (PVDF 0.45 .mu.m, Whatman). The
filtrate is then analyzed for tocopherol content using high
performance liquid chromatography (HPLC) using an isocratic
gradient of 90% hexane/10% methyl-t-butyl ether with a Zorbax
silica column (4.6.times.250 mm, Agilent Technologies, Atlanta,
Ga.) and fluorescence detection (model 2790 HPLC with model 474
detector; Waters Corporation, Bedford, Mass.) (excitation at 290
nm, emit at 336 nm, 30 nm bandpass and slits). Levels of .alpha.,
.beta., .gamma., and .delta.-tocopherol are measured in addition to
tocol, the injection standard. Individual plant lines that have
.delta.-tocopherol levels higher than wild type are reanalyzed in
the next generation (M4), to confirm their inheritability. Five
Arabidopsis high .delta.-tocopherol (hdt) mutants possessing
increased levels of .delta.-tocopherols, as compared to wild type,
are isolated.
[0380] Table 1 below shows the percentage, on a dry weight basis,
of .delta.-tocopherol levels and the relative increases over the
appropriate wild type parental ecotype for each of the six mutants.
The results show that the six mutants have significant increases in
.delta.-tocopherol levels when compared to the corresponding wild
type control. The magnitude of the increases ranged from 2-25 fold.
TABLE-US-00002 TABLE 1 Mutant WT ecotype Delta Composition Increase
over WT hdt2 Ler 48% 25 fold hdt6 Col-0 45% 20 fold hdt9 Col-0 6% 2
fold hdt10 Ler 25% 7 fold hdt16 Col-0 50% 17 fold
EXAMPLE 2
Identification and Sequencing of the Mutant hdt2 Gene in the
Arabidopsis thaliana, Landsberg Erecta (Ler) High
.delta.-Tocopherol Mutants
[0381] Using map-based cloning techniques (see, for example, U.S.
Ser. No. 09/803,736, Plant Polymorphic Markers and Uses Thereof,
filed Mar. 12, 2001) the mutant hdt2 gene is mapped to chromosome 3
telomeric marker T12C14.sub.--1563 at 85 cM. This region contains
approximately 60 predicted genes. Our analysis of the genes in this
region revealed that one of the genes, MAA21.sub.--40, possesses
homology to known ubiquinone methyltransferases. Based on this
homology and the prediction that MAA21.sub.--40 is targeted to the
chloroplast, this gene is determined to be likely to contain the
mutation responsible for the high .delta.-tocopherol phenotype in
hdt2 mutants. The sequences of the MAA21.sub.--40 gene locus in the
wild types and hdt2 mutants are PCR amplified, and determined by
standard sequencing methodology. The gene locus, in each case, is
amplified using the sequencing primers as described below:
TABLE-US-00003 Primer Pair Name MAA21_40_1 Forward Primer (SEQ ID
NO: 55) TGTAAAACGACGGCCAGTTGCTGAAAGTTGAAAAGAGCAA Reverse Primer
(SEQ ID NO: 56) CAGGAAACAGCTATGACCCAATTTGATCAATGTTCCACGA Primer
Pair Name MAA21_40_2 Forward Primer (SEQ ID NO: 57)
TGTAAAACGACGGCCAGTAGCTATGCGGATTGATGGTC Reverse Primer (SEQ ID NO:
58) CAGGAAACAGCTATGACCTCCTCCTGGGAACTCTAGCA Primer Pair Name
MAA21_40_3 Forward Primer (SEQ ID NO: 59)
TGTAAAACGACGGCCAGTTGCTGACTTGCGAGTTTTTC Reverse Primer (SEQ ID NO:
60) CAGGAAACAGCTATGACCCCTGTCAACAACCCCTTCTC Primer Pair Name
MAA21_40_4 Forward Primer (SEQ ID NO: 61)
TGTAAAACGACGGCCAGTCCACAAGAGGGGTTTACAATG Reverse Primer (SEQ ID NO:
62) CAGGAAACAGCTATGACCACCCAACCTTCTGGCTCTCT Primer Pair Name
MAA21_40_5 Forward Primer (SEQ ID NO: 63)
TGTAAAACGACGGCCAGTGGTCTTTGGGAACGATCTGA Reverse Primer (SEQ ID NO:
64) CAGGAAACAGCTATGACCAGGGAAGCGTACAGGGTTCT Primer Pair Name
MAA21_40_6 Forward Primer (SEQ ID NO: 65)
TGTAAAACGACGGCCAGTCCTCTTGAGCTGAACGTCCT Reverse Primer (SEQ ID NO:
66) CAGGAAACAGCTATGACCGGCGGAACTGGTTTCACTAC Primer Pair Name
MAA21_40_7 Forward Primer (SEQ ID NO: 67)
TGTAAAACGACGGCCAGTTGTCAGCATAATCGGTTGGA Reverse Primer (SEQ ID NO:
68) CAGGAAACAGCTATGACCTCCCCAAAGGTTTAGGTTCC Primer Pair Name
MAA21_40_8 Forward Primer (SEQ ID NO: 69)
TGTAAAACGACGGCCAGTAAGCCTCCTTCTTGTGCTGA Reverse Primer (SEQ ID NO:
70) CAGGAAACAGCTATGACCCGACTTTTCCCTTCCATTTG Primer Pair Name
MAA21_40_9 Forward Primer (SEQ ID NO: 71)
TGTAAAACGACGGCCAGTTGGAGGTTCGGGTAACTGAG Reverse Primer (SEQ ID NO:
72) CAGGAAACAGCTATGACCCATCCTCTCGCTAGCAGGTC Primer Pair Name
MAA21_40_10 Forward Primer (SEQ ID NO: 73)
TGTAAAACGACGGCCAGTGGAACCAGGGGAACCTAAAC Reverse Primer (SEQ ID NO:
74) CAGGAAACAGCTATGACCGCCGTGAGAAACAGACTCCT Primer Pair Name
MAA21_40_11 Forward Primer (SEQ ID NO: 75)
TGTAAAACGACGGCCAGTCAAATGGAAGGGAAAAGTCG Reverse Primer (SEQ ID NO:
76) CAGGAAACAGCTATGACCGATCCAAAGAGAACCCAGCA
[0382] The following Polymerase Chain Reaction (PCR) mixture is
prepared for each primer pair:
PCR Mixture:
5 .mu.l 10.times. Taq Buffer
5 .mu.l 25 mM MgCl.sub.2
4 .mu.l 10 mM dNTPs
2 .mu.l Template DNA
0.5 .mu.l Taq Gold
5 .mu.l F/R Sequencing Primers
28.5 .mu.l dH.sub.2O
The PCR amplification is carried out using the following
Thermocycler program:
[0383] 1. 94.degree. C. for 10 minutes
[0384] 2. 94.degree. C. for 15 seconds
[0385] 3. 56.degree. C. for 15 seconds
[0386] 4. 72.degree. C. for 1 minute, 30 seconds
[0387] 5. Repeat Steps 2 through 4 an additional 44 times
[0388] 6. 72.degree. C. for 10 minutes
[0389] 7. Hold at 4.degree. C.
[0390] The resulting PCR products are sequenced using standard
sequencing methodologies.
[0391] The wild type Col-0 genomic sequence for the MAA21.sub.--40
locus is set forth in SEQ ID NO: 1. The wild type Ler genomic
sequence for the MAA21.sub.--40 locus is set forth in SEQ ID NO: 2.
The wild type coding DNA and peptide sequence for Columbia and
Landsberg ecotypes are described in SEQ ID NOs: 15 and 16,
respectively.
[0392] Once the sequences of the MAA21.sub.--40 gene from the hdt2
mutant are determined, they are compared to the sequence of the
wild type gene. The high .delta.-tocopherol mutant identified as
hdt2 is determined to have a MAA21.sub.--40 gene with the nucleic
acid sequence set forth in SEQ ID NO: 3. This sequence has a
glutamate to lysine substitution at amino acid position 292,
relative to the ATG of the Arabidopsis MAA21.sub.--40, as shown in
the amino acid sequence of SEQ ID NO: 17.
[0393] Another high .delta.-tocopherol mutant, identified as hdt6,
is determined to have a MAA21.sub.--40 gene with the nucleic acid
sequence set forth in SEQ ID NO: 4. This sequence has a glutamate
to a lysine substitution at amino acid 72, relative to the wild
type Arabidopsis MAA21.sub.--40, as shown in the amino acid
sequence of SEQ ID NO: 18.
[0394] Another high .delta.-tocopherol mutant, identified as hdt9
is determined to have a MAA21.sub.--40 gene with the nucleic acid
sequence set forth in SEQ ID NO: 5. This sequence has a proline to
a serine substitution at amino acid 13, relative to the Arabidopsis
MAA21.sub.--40, as shown in the amino acid sequence of SEQ ID NO:
19.
[0395] Another high .delta.-tocopherol mutant, identified as hdt10
is determined to have a MAA21.sub.--40 gene with the nucleic acid
sequence set forth in SEQ ID NO: 6 which encodes MAA21.sub.--40
with a aspartate to a asparagine substitution at amino acid 116,
relative to the Arabidopsis MAA21.sub.--40, as shown in the amino
acid sequence of SEQ ID NO: 20.
[0396] Another high .delta.-tocopherol mutant hdt16 is determined
to have a MAA21.sub.--40 gene with the nucleic acid sequence set
forth in SEQ ID NO: 7 which encodes MAA21.sub.--40 with a threonine
to an isoleucine substitution at amino acid 94, relative to the
Arabidopsis MAA21.sub.--40, as shown in the amino acid sequence of
SEQ ID NO: 21.
[0397] Table 2 summarizes the mutations described above.
TABLE-US-00004 TABLE 2 Mutant Nucleotide Mutation Ammo Acid Change
hdt2 G1041A E292K hdt6 G214A E72K hdt9 C37T P13S hdt10 G346A D116N
hdt16 C281T T94I
EXAMPLE 3
Identification of Genes from Various Sources Demonstrating Homology
to the tMT2 Gene from Arabidopsis thaliana
[0398] The protein sequence of tMT2 from Arabidopsis thaliana (NCBI
General Identifier Number gi7573324) is used to search databases
for plant sequences with homology to tMT2 using TBLASTN (Altschul
et al., Nucleic Acids Res. 25:3389-3402 (1997); see also
www.ncbi.nlm.nih.gov/BLAST/). Nucleic acid sequences SEQ ID NO: 8
through 15 are found to have high homology with the Arabidopsis
sequence. TABLE-US-00005 >CPR19219 Brassica napus tMT2 homolog 1
- LIB4153- 013-R1-K1-B7 (SEQ ID NO: 13)
ATGGCTTCTCTCATGCTCAACGGGGCCATCACCTTCCCCAAGGGATTAGG
CTTCCCCGCTTCCAATCTACACGCCAGACCAAGTCCTCCGCTGAGTCTCG
TCTCAAACACAGCCACGCGGAGACTCTCCGTGGCGACAAGATGCAGCAGC
AGCAGCAGCGTGTCGGCGTCAAGGCCATCTGCGCAGCCTAGGTTCATCCA
GCACAAGAAAGAGGCCTACTGGTTCTACAGGTTCCTGTCCATCGTGTACG
ACCACATCATCAATCCCGGCCACTGGACGGAGGATATGAGGGACGACGCT
CTCGAGCCTGCGGATCTGAGCCATCCGGACATGCGAGTTGTCGACGTCGG
AGGCGGAACGGGTTTCACCACGCTGGGAATCGTCAAGACGGTGAAGGCTA
AGAACGTGACGATTCTGGACCAGTCGCCGCATCAGCTGGCAAAGGCGAAG
CAGAAGGAGCCGTTGAAGGAGTGCAAGATCGTTGAAGGAGATGCGGAGGA
TCTCCCTTTTCCTACTGATTATGCTGACAGATACGTCTCTGCTGGAAGCA
TTGAGTACTGGCCCGACCCGCAGAGGGGGATAAGGGAAGCGTACAGAGTT
CTCAAGATCGGTGGGAAAGCATGTCTCATTGGCCCTGTCCACCCGACGTT
TTGGCTTTCTCGTTTCTTTGCAGATGTGTGGATGCTTTTCCCCAAGGAGG
AGGAGTACATTGAGTGGTTCAAGAATGCTGGTTTCAAGGACGTTCAGCTT
AAGAGGATTGGCCCCAAGTGGTACCGTGGTGTTCGCAGGCACGGACTTAT
CATGGGATGCTCTGTTACTGGTGTCAAACCTGCCTCTGGAGACTCTCCTC
TCCAGCTTGGACCAAAGGAAGAGGACGTGGAGAAGCCTGTAAACAATCCT
TTCTCCTTCTTGGGACGCTTCCTCTTGGGAACCTTAGCGGCTGCCTGGTT
TGTGTTAATCCCAATCTACATGTGGATCAAGGATCAGATCGTTCCCAAAG ACCAACCCATCTGA
>Protein sequence Brassica napus tMT2 hornolog 1 -
LIB4153-013-R1-K1-B7 (SEQ ID NO: 27)
MASLMLNGAITFPKGLGFPASNLHARPSPPLSLVSNTATRRLSVATRCSS
SSSVSASRPSAQPRFIQHKKEAYWFYRFLSIVYDHIINPGHWTEDMRDDA
LEPADLSHPDMRVVDVGGGTGFTTLGIVKTVKAKNVTILDQSPHQLAKAK
QKEPLKECKIVEGDAEDLPFPTDYADRYVSAGSIEYWPDPQRGIREAYRV
LKIGGKACLIGPVHPTFWLSRFFADVWMLFPKEEEYIEWFKNAGFKDVQL
KRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKEEDVEKPVNNP
FSFLGRFLLGTLAAAWFVLIPIYMWIKDQIVPKDQPI >CPR19220 Brassica napus
tMT2 homolog 2 - LIB80-011-Q1-E1-E9 (SEQ ID NO: 14)
ATGGCTTCTCTCATGCTCAACGGGGCCATCACCTTCCCCAAGGGATTAGG
CTTCCCCGCTTCCAATCTACACGCCAGACCAAGTCCTCCGCTGAGTCTCG
TCTCAAACACAGCCACGCGGAGACTCTCCGTGGCGACAAGATGCAGCAGC
AGCAGCAGCGTGTCGGCGTCAAGGCCATCTGCGCAGCCTAGGTTCATCCA
GCACAAGAAAGAGGCCTACTGGTTCTACAGGTTCCTGTCCATCGTGTACG
ACCACATCATCAATCCCGGCCACTGGACGGAGGATATGAGGGACGACGCT
CTCGAGCCTGCGGATCTGAGCCATCCGGACATGCGAGTTGTCGACGTCGG
AGGCGGAACGGGTTTCACCACGCTGGGAATCGTCAAGACGGTGAAGGCTA
AGAACGTGACGATTCTGGACCAGTCGCCGCATCAGCTGGCAAAGGCGAAG
CAGAAGGAGCCGTTGAAGGAGTGCAAGATCGTGGAAGGAGATGCGGAGGA
TCTCCCTTTTCCTACTGATTATGCTGACAGATACGTCTCTGCTGGAAGCA
TTGAGTACTGGCCCGACCCGCAGAGGGGTATAAGGGAAGCGTACAGAGTT
CTCAAGATCGGTGGGAAAGCATGTCTCATTGGCCCTGTCCACCCGACGTT
TTGGCTTTCACGCTTCTTTGCAGATGTGTGGATGCTTTTCCCCAAGGAGG
AGGAGTACATTGAGTGGTTCAAGAATGCTGGTTTCAAGGACGTTCAGCTT
AAGAGGATTGGCCCCAAGTGGTACCGTGGTGTTCGCAGGCACGGACTTAT
CATGGGATGCTCTGTTACTGGTGTCAAACCTGCCTCTGGAGACTCTCCTC
TCCAGCTTGGACCAAAGGAAGAGGACGTGGAGAAGCCTGTAAACAATCCT
TTCTCCTTCTTGGGACGCTTCCTCTTGGGTACCCTAGCGGCTGCCTGGTT
TGTGTTAATCCCAATCTACATGTGGATCAAGGATCAGATCGTTCCCAAAG ACCAACCCATCTGA
> CPR 193223 Oryza sativa tMT2- LIB4371-041-R1- K1-F7 (SEQ ID
NO: 12) ATGGCGATGGCCTCCTCCGCCTACGCCCCAGCGGGCGGCGTTGGCACCCA
CTCCGCGCCGGGCAGGATCAGGCCGCCGCGCGGCCTCGGCTTCTCCACCA
CCACCACCAAGTCGAGGCCCCTCGTGCTCACCAGGCGTGGGGGAGGCGGC
GGCAACATCTCCGTGGCTCGGCTGAGGTGCGCGGCGTCGTCGTCGTCGGC
GGCGGCGAGGCCGATGTCGCAGCCGCGGTTCATCCAGCACAAGAAGGAGG
CGTTCTGGTTCTACCGCTTCCTCTCCATCGTCTACGACCACGTCATCAAC
CCGGGCCACTGGACGGAGGACATGCGGGACGACGCCCTCGAGCCCGCCGA
CCTCTACAGCCGCAAGCTCAGGGTCGTCGACGTCGGCGGCGGGACGGGGT
TCACCACGCTCGGGATCGTCAAGCGCGTCGACCCGGAGAACGTCACGCTG
CTCGACCAGTCCCCGCACCAGCTCGAGAAGGCCCGGGAGAAGGAGGCCCT
CAAGGGCGTCACCATCATGGAGGGCGACGCCGAGGACCTCCCCTTCCCCA
CCGACACCTTCGACCGCTACGTCTCCGCCGGCAGCATCGAGTATTGGCCC
GATCCGCAGCGAGGAATCAAGGAAGCTTACAGGGTTTTGAGGCTTGGTGG
AGTGGCTTGCATGATTGGCCCCGTGCACCCAACCTTCTGGCTGTCTCGCT
TTTTCGCTGACATGTGGATGCTCTTCCCGAAGGAAGAGGAGTATATTGAG
TGGTTCAAAAAGGCAGGGTTCAAGGATGTCAAGCTCAAAAGGATTGGACC
AAAATGGTACCGTGGTGTCCGAAGGCATGGCCTGATTATGGGATGCTCTG
TGACGGGCGTCAAAAGAGAACATGGAGACTCCCCTTTGCAGCTTGGTCCA
AAGGTTGAGGATGTCAGCAAACCTGTGAATCCTATCACCTTCCTCTTCCG
CTTCCTCATGGGAACAATATGTGCTGCATACTATGTTCTGGTGCCTATCT
ACATGTGGATAAAGGACCAGATTGTGCCCAAAGGCATGCCGATCTAA > Protein
translation Oryza sativa tMT2 - LIB4371- 041-R1-K1-F7 (SEQ ID NO:
26) MAMASSAYAPAGGVGTHSAPGRIRPPRGLGFSTTTTKSRPLVLTRRGGGG
GNISVARLRCAASSSSAAARPMSQPRFIQHKKEAFWFYRFLSIVYDHVIN
PGHWTEDMRDDALEPADLYSRKLRVVDVGGGTGFTTLGIVKRVDPENVTL
LDQSPHQLEKAREKEALKGVTIMEGDAEDLPFPTDTFDRYVSAGSIEYWP
DPQRGIKEAYRVLRLGGVACMIGPVHPTFWLSRFFADMWMLFPKEEEYIE
WFKKAGFKDVKLKRIGPKWYRGVRRHGLIMGCSVTGVKREHGDSPLQLGP
KVEDVSKPVNPITFLFRFLMGTICAAYYVLVPIYMWIKDQIVPKGMPI > CPR193225 and
193226 Zea mays tMT2- LIB3587-273- Q1-K6-C5/ LIB3600-046-Q1-K6-G1
(SEQ ID NO: 8) ATGGCGATGGCCTCCACCTACGCGCCGGGCGGAGGCGCGCGGGCGCTCGC
GCAGGGTAGATGCAGGGTCCGCGGTCCCGCGGGGCTGGGCTTCCTCGGCC
CCTCCAAGGCCGCCGGCCTCCCCCGCCCCCTCGCCCTCGCCCTCGCCAGG
CGGATGAGCAGCCCCGTCGCGGTGGGCGCCAGGCTGCGATGCGCGGCGTC
GTCGTCCCCCGCGGCGGCGCGGCCCGCCACGGCGCCGCGCTTCATCCAGC
ACAAGAAGGAGGCCTTCTGGTTCTACCGCTTCCTCTCCATCGTGTACGAC
CACGTCATCAATCCGGGCCACTGGACCGAGGACATGCGCGACGACGCGCT
GGAACCTGCCGACCTCTTCAGCCGCCACCTCACGGTCGTCGACGTCGGCG
GCGGCACGGGGTTCACCACGCTCGGCATCGTCAAGCACGTCAACCCGGAG
AACGTCACGCTGCTCGACCAGTCCCCGCACCAGCTCGACAAGGCCCGGCA
GAAGGAGGCCCTCAAGGGGGTCACCATCATGGAGGGCGACGCCGAGGACC
TCCCGTTCCCCACCGACTCCTTCGACCGATACATCTCCGCCGGCAGCATC
GAGTACTGGCCAGACCCACAGCGGGGGATCAAGGAAGCCTACAGGGTCCT
GAGATTTGGTGGGCTAGCTTGTGTGATCGGCCCGGTCTACCCGACCTTCT
GGCTGTCCCGCTTCTTCGCCGACATGTGGATGCTCTTCCCCAAGGAGGAA
GAGTACATCGAGTGGTTCAAGAAGGCTGGGTTTAGGGATGTCAAGCTGAA
GAGGATTGGACCGAAGTGGTACCGCGGTGTCCGAAGGCATGGCCTCATCA
TGGGCTGCTCCGTCACAGGCGTCAAGAGAGAGCGCGGTGACTCTCCCTTG
GAGCTTGGTCCCAAGGCGGAGGATGTCAGCAAGCCAGTGAATCCGATCAC
CTTCCTCTTCCGCTTCCTCGTAGGAACGATATGTGCTGCCTACTATGTTC
TGGTGCCTATTTACATGTGGATAAAGGACCAGATCGTGCCAAAAGGCATG CCAATCTGA >
Protein translation Zea mays tMT2- LIB3587-273-
Q1-K6-C5/LIB3600-046-Q1-K6-G1 (SEQ ID NO: 22)
MAMASTYAPGGGARALAQGRCRVRGPAGLGFLGPSKAAGLPRPLALALAR
RMSSPVAVGARLRCAASSSPAAARPATAPRFIQHKKEAFWFYRFLSIVYD
HVINPGHWTEDMRDDALEPADLFSRHLTVVDVGGGTGFTTLGIVKHVNPE
NVTLLDQSPHQLDKARQKEALKGVTIMEGDAEDLPFPTDSFDRYISAGSI
EYWPDPQRGIKEAYRVLRFGGLACVIGPVYPTFWLSRFFADMWMLFPKEE
EYIEWFKKAGFRDVKLKRIGPKWYRGVRRHGLIMGCSVTGVKRERGDSPL
ELGPKAEDVSKPVNPITFLFRFLVGTICAAYYVLVPIYMWIKDQIVPKGM PI >CPR193234
Glycine max tMT2 - LIB3049-032-Q1-E1-G8
(SEQ ID NO: 11) ATGGGTTCAGTAATGCTCAGTGGAACTGAAAACCTCACTCTCAGAACCCT
AACCGGGAACGGCTTAGGTTTCACTGGTTCGGATTTGCACGGTAAGAACT
TCCCAAGAGTGAGTTTCGCTGCTACCACTAGTGCTAAAGTTCCCAACTTT
AGAAGCATAGTAGTACCCAAGTGTAGTGTCTCGGCTTCCAGGCCAAGCTC
GCAGCCAAGGTTCATTCAGCACAAAAAAGAGGCCTTTTGGTTCTATAGGT
TTCTCTCAATTGTGTATGACCATGTCATTAACCCTGGCCATTGGACCGAG
GACATGAGGGATGATGCCCTTGAACCCGCTGATCTCAATGACAGGAACAT
GATTGTGGTGGATGTTGGTGGCGGCACGGGTTTCACCACTCTTGGTATTG
TCAAGCACGTGGATGCCAAGAATGTCACCATTCTTGACCAGTCACCCCAC
CAGCTCGCCAAGGCCAAGCAGAAGGAGCCACTCAAGGAATGCAAAATAAT
CGAAGGGGATGCCGAGGATCTCCCCTTTCGAACTGATTATGCCGATAGAT
ATGTATCCGCAGGAAGTATTGAGTACTGGCCGGATCCACAGCGTGGCATC
AAGGAGGCATACAGGGTTTTGAAACTTGGAGGCAAAGCGTGTCTAATTGG
TCCGGTCTACCCAACATTTTGGTTGTCACGTTTCTTTGCAGATGTTTGGA
TGCTTTTCCCCAAGGAGGAAGAGTATATTGAGTGGTTTCAGAAGGCAGGG
TTTAAGGACGTCCAACTAAAAAGGATTGGCCCAAAATGGTATCGTGGGGT
TCGCCGTCATGGCTTGATTATGGGTTGTTCAGTGACCGGTGTTAAACCTG
CATCTGGAGATTCTCCTTTGCAGCTTGGTCCAAAGGAAGAAGATGTTGAA
AAGCCCGTTAATCCTTTTGTCTTTGCACTGCGCTTCGTTTTGGGTGCCTT
GGCAGCGACATGGTTTGTGTTGGTTCCTATTTACATGTGGCTGAAAGATC
AAGTTGTTCCCAAAGGTCAGCCAATCTAA >Protein translation Glycine max
tMT2 - LIB3049- 032-Q1-E1-G8 (SEQ ID NO: 25)
MGSVMLSGTEKLTLRTLTGNGLGFTGSDLHGKNFPRVSFAATTSAKVPNF
RSIVVPKCSVSASRPSSQPRFIQHKKEAFWFYRFLSIVYDHVINPGHWTE
DMRDDALEPADLNDRNMIVVDVGGGTGFTTLGIVKHVDAKNVTILDQSPH
QLAKAKQKEPLKECKIIEGDAEDLPFRTDYADRYVSAGSIEYWPDPQRGI
KEAYRVLKLGGKACLIGPVYPTFWLSRFFADVWMLFPKEEEYIEWFQKAG
FKDVQLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKEEDVE
KPVNPFVFALRFVLGALAATWFVLVPIYMWLKDQVVPKGQPI >CPR193236 Allium
Porrum - LIB4521-015-Q1-K1-D6 (SEQ ID NO: 10)
ATGGCTTCCTCCATGCTCAGCGGAGCAGAAAGCCTCTCAATGCTCCGAAT
CCACCACCAACCCAAACTCACCTTCTCGAGCCCATCCCTCCATTCCAAAC
CCACAAACCTCAAAATGGATCTCATCCCTTTCGCCACCAAGCATCAAAAA
ACGAAAAAAGCTTCGATCTTTACATGCAGCGCGTCCTCATCATCCCGACC
TGCTTCTCAGCCGAGGTTCATCCAGCACAAGCAGGAGGCGTTCTGGTTCT
ACAGGTTCCTGTCGATAGTGTACGACCATGTGATAAACCCAGGGCACTGG
ACCGAGGACATGAGAGACGATGCGTTGGAGCCAGCCGAGCTGTACGATTC
CAGGATGAAGGTGGTGGACGTAGGAGGAGGAACTGGGTTCACCACCTTGG
GGATTATAAAGCACATCGACCCTAAAAACGTTACGATTCTGGATCAGTCT
CCGCATCAGCTTGAGAAGGCTAGGCAGAAGGAGGCTTTGAAGGAGTGTAC
TATTGTTGAAGGTGATGCTGAGGATCTCCCTTTTCCTACTGATACTTTCG
ATCGATATGTATCTGCTGGCAGCATAGAATACTGGCCAGACCCACAAAGA
GGGATAAAGGAAGCATACCGGGTTCTAAAACTGGGAGGCGTTGCCTGCTT
GATAGGACCCGTGCACCCTACCTTCTGGCTTTCCAGGTTCTTCGCCGACA
TGTGGATGTTGTTCCCCACCGAAGAAGAATACATAGAGTGGTTTAAAAAG
GCCGGGTTCAAAGATGTGAAGTTGAAGAGGATTGGCCCAAAATGGTACCG
TGGTGTGCGTAGACACGGGCTCATCATGGGCTGTTCCGTCACTGGTGTTA
AACGTCTCTCTGGTGACTCCCCTCTTCAGCTTGGACCGAAGGCGGAGGAT
GTGAAGAAGCCGATCAATCCATTCTCGTTCCTTCTGCGCTTCATTTTGGG
TACGATAGCAGCTACTTACTACGTTTTGGTGCCGATATACATGTGGATAA
AGGATCAGATTGTACCGAAAGGCCAGCCCATATGA >Protein translation Allium
Porrum - LIB4521-015- Q1-K1-D6 (SEQ ID NO: 24)
MASSMLSGAESLSMLRIHHQPKLTFSSPSLHSKPTNLKMDLIPFATKHQK
TKKASIFTCSASSSSRPASQPRFIQHKQEAFWFYRFLSIVYDHVINPGHW
TEDMRDDALEPAELYDSRMKVVDVGGGTGFTTLGIIKHIDPKNVTILDQS
PHQLEKARQKEALKECTIVEGDAEDLPFPTDTFDRYVSAGSIEYWPDPQR
GIKEAYRVLKLGGVACLIGPVHPTFWLSRFFADMWMLFPTEEEYIEWFKK
AGFKDVKLKRIGPKWYRGVRRHGLIMGCSVTGVKRLSGDSPLQLGPKAED
VKKPINPFSFLLRFILGTIAATYYVLVPIYMWIKDQIVPKGQPI >CPR204065
Gossypium hirsutum tMT2 - LIB3272-054- P1-K1-C11 (SEQ ID NO: 9)
ATGGCTTCTTCCATGCTGAATGGAGCTGAAACCTTCACTCTCATCCGAGG
TGTTACCCCAAAAAGTATTGGTTTTTTGGGGTCAGGTTTACATGGGAAAC
AGTTTTCCAGTGCGGGTTTAATCTACAGTCCGAAGATGTCCAGGGTAGGA
ACGACGATAGCCCCGAGGTGCAGCTTATCAGCGTCAAGGCCAGCTTCACA
ACCAAGATTCATACAACACAAAAAAGAGGCCTTTTGGTTCTACAGGTTCC
TCTCAATTGTCTATGACCATGTCATAAACCCAGGTCACTGGACTGAAGAC
ATGAGGGATGATGCACTTGAGCCGGCTGATCTCAATGACAGGGACATGGT
AGTTGTAGATGTTGGTGGTGGAACTGGTTTCACTACTTTGGGTATTGTTC
AGCATGTGGATGCTAAGAATGTTACAATCCTTGACCAATCTCCTCACCAG
CTTGCAAAGGCTAAACAGAAGGAGCCTCTCAAGGAATGCAACATAATTGA
AGGTGATGCAGAAGATCTTCCTTTTCCTACTGATTATGCCGATAGATATG
TGTCTGCTGGAAGCATAGAGTACTGGCCAGACCCACAACGGGGGATCAAG
GAAGCATACAGGGTGTTGAAACAAGGAGGAAAAGCTTGCTTAATTGGTCC
TGTGTACCCTACATTTTGGTTGTCTCGTTTCTTTGCAGACGTTTGGATGC
TTTTCCCTAAGGAGGAAGAATATATAGAGTGGTTTGAAAAGGCTGGATTT
AAGGATGTCCAACTCAAAAGGATTGGCCCTAAATGGTATCGTGGAGTTCG
CCGACATGGTTTGATCATGGGGTGCTCTGTAACCGGTGTTAAACCCGCAT
CTGGGGACTCTCCTTTGCAGCTTGGACCTAAGGCAGAGGATGTATCAAAG
CCGGTAAATCCGTTTGTATTTCTCTTACGCTTCATGTTGGGTGCCACTGC
AGCAGCATATTATGTACTGGTTCCTATCTACATGTGGCTCAAAGATCAAA
TTGTACCAGAGGGTCAACCAATCTAA >Protein translation Gossypium
hirsutum tMT2 - LIB3272-054-P1-K1-C11 (SEQ ID NO: 23)
MASSMLNGAETFTLIRGVTPKSIGFLGSGLHGKQFSSAGLIYSPKMSRVG
TTIAPRCSLSASRPASQPRFIQHKKEAFWFYRFLSIVYDHVINPGHWTED
MRDDALEPADLNDRDMVVVDVGGGTGFTTLGIVQHVDAKNVTILDQSPHQ
LAKAKQKEPLKECNIIEGDAEDLPFPTDYADRYVSAGSIEYWPDPQRGIK
EAYRVLKQGGKACLIGPVYPTFWLSRFFADVWMLFPKEEEYIEWFEKAGF
KDVQLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKAEDVSK
PVNPFVFLLRFMLGATAAAYYVLVPIYMWLKDQIVPEGQPI
[0399] The protein sequence of tMT2 from Arabidopsis thaliana is
compared against the tMT2 plant protein sequences listed above
using BLASTP (Altschul et al., Nucleic Acids Res. 25:3389-3402
(1997); see also www.ncbi.nlm.nih.gov/BLAST/). The calculated
protein identity of each sequence compared to the Arabidopsis
sequence is shown in FIG. 2. Also shown is a protein sequence
alignment using the Pretty alignment program (Genetics Computer
Group, Madison Wis.) (FIG. 3).
EXAMPLE 4
Preparation of Constructs to Direct the Expression of the Wild Type
tMT2 and Mutant tMT2 Gene Sequences of Arabidopsis thaliana and
tMT2 Gene Sequences from Other Crop Plant Species in a Prokaryotic
Expression System
[0400] A computer program is used to predict the chloroplast
targeting peptide cleavage site of the plant tMT2 protein
("ChloroP", Center for Biological Sequence Analysis, Lyngby,
Denmark). The result of the search is as follows: TABLE-US-00006
Name Length Score cTP CS-score cTP-length Arabidopsis 338 0.585 Y
6.467 51
[0401] Based on this information, the tMT2 protein from Arabidopsis
thaliana, ecotype Landsberg is engineered to remove the predicted
chloroplast target peptide to allow for the expression of the
mature protein in E. coli. In order for these proteins to be
expressed in a prokaryotic expression system, an amino terminal
methionine is required. To make the addition of a 5' ATG the tMT2
coding sequence is amplified from cDNA of wild type and the high
.delta.-tocopherol hdt6, and hdt16 mutant lines of Arabidopsis
thaliana, ecotype Columbia, and the high .delta.-tocopherol hdt2
and hdt10 mutant lines of Arabidopsis thaliana, ecotype
Landsberg.
[0402] PolyA.sup.+ RNA is isolated from each source using an
adapted biotin/streptavidin procedure based on the "mRNA Capture
Kit" by Roche Molecular Biochemicals (Indianapolis, Ind.). A young
plantlet, approximately 1 cm tall, with root tissue removed is
homogenized in CTAB buffer (500 mM Tris-HCl pH 9, 0.8M NaCl, 0.5%
CTAB, 10 mM EDTA), extracted with chloroform, and pelleted with
centrifugation. As specified by the manufacturer's instructions,
polyA.sup.+ RNA in the soluble fraction is hybridized to
biotin-labeled oligo-dT, immobilized on streptavidin-coated PCR
tubes and washed. The first strand cDNA is synthesized using the
"1.sup.st strand cDNA synthesis kit for RT-PCR" (Roche Molecular
Biochemicals) in a 50 .mu.l volume according to the manufacturer's
protocol. Following the cDNA synthesis, the soluble contents of the
tube are replaced with equal volume amplification reaction mixture.
The components of the mixture at final concentration consist of:
[0403] 1.times. Buffer 2 (Expand.TM. High Fidelity PCR System,
Roche Molecular Biochemicals) [0404] 200 .mu.M dNTPs
[0405] 300 nM each synthetic oligonucleotide primers;
TABLE-US-00007 #17180 FORWARD-NcoI (SEQ ID NO: 79)
5'GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAGGAGATAGAACCAT
GGCTACTAGATGCAGCAGCAGCAGC 3' and #17181 REVERSE-Sse83871 (SEQ ID
NO: 78) 5'GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGCAGGTCAGATGGGTT
GGTCTTTGGGAACG 3'.
[0406] Each primer contains regions for GATEWAY.TM. cloning (Life
Technologies Division, Invitrogen Corporation) as well as
conventional restriction enzyme sites. [0407] 0.4 .mu.l Expand.TM.
High Fidelity Polymerase (Roche Molecular Biochemicals)
[0408] Constructs are also prepared to direct expression of the
engineered Brassica napus, Oryza sativa, Zea mays, Glycine max,
Allium Porrum, and Gossypium hirsutum tMT2 sequences in a
prokaryotic expression vector. The mature protein coding region of
each tMT2 with the aminoterminal methionine, as described above, is
amplified from plasmid DNA using the following oligonucleotide
primers in the polymerase chain reaction.
[0409] The mature Brassica napus tMT2 coding sequence is amplified
from LIB4153-013-R1-K1-B7 (SEQ ID NO: 13) using the synthetic
oligonucleotide primers: TABLE-US-00008 Brassica forward (17509)
(SEQ ID NO: 77) GGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAGGAGATAGAACCATGGC
GACAAGATGCAGCAGCAGCAGCAG. Brassica reverse (17181) (SEQ ID NO: 78)
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGCAGGTCAGATGGGTTGG
TCTTTGGGAACG.
[0410] The mature Oryza sativa tMT2 coding sequence is amplified
from LIB4371-041-R1-K1-F7 (SEQ ID NO: 12) using the synthetic
oligonucleotide primers: TABLE-US-00009 Rice forward (17512) (SEQ
ID NO: 79) GGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAGGAGATAGAACCATGCG
GCTGAGGTGCGCGGCGTCGTCG Rice reverse (17513) (SEQ ID NO: 80)
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGCAGGTTAGATCGGCATG CCTTTGGGCAC
[0411] The mature Zea mays tMT2 coding sequence is amplified from
LIB3587-273-Q1-K6-C5 (SEQ ID NO: 8) using the synthetic
oligonucleotide primers: TABLE-US-00010 Corn forward (17510) (SEQ
ID NO: 81) GGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAGGAGATAGAACCATGAG
GCTGCCATGCGCGGCGTCGTCG. Corn reverse (17511) (SEQ ID NO: 82)
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGCAGGTCAGATTGGCATG
CCTTTTGGCACC.
[0412] The mature Glycine max tMT2 coding sequence is amplified
from LIB3049-032-Q1-E1-G8 (SEQ ID NO: 11) using the synthetic
oligonucleotide primers: TABLE-US-00011 Soy forward (17516) (SEQ ID
NO: 83) GGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAGGAGATAGAACCATGGT
ACCCAAGTGTAGTGTCTCGGC. Soy reverse (17517) (SEQ ID NO: 84)
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGCAGGTTAGATTGGCTGA CCTTTGGGAAC.
[0413] The mature Allium Porrum tMT2 coding sequence is amplified
from LIB4521-015-Q1-K1-D6 (SEQ ID NO: 10) using the synthetic
oligonucleotide primers: TABLE-US-00012 Leek forward (17518) (SEQ
ID NO: 85) GGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAGGAGATAGAACCATGAT
CTTTACATGCAGCGCGTCCT. Leek reverse (17519) (SEQ ID NO: 86)
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGCAGGTCATATGGGCTGG CCTTTCGGTAC.
[0414] The mature Gossypium hirsutum tMT2 coding sequence is
amplified from LIB3272-054-P1-K1-C11 (SEQ ID NO: 9) using the
synthetic oligonucleotide primers: TABLE-US-00013 Cotton forward
(17514) (SEQ ID NO: 87)
GGGACAAGTTTGTACAAAAAAGCAGGCTTAGAAGGAGATAGAACCATGGC
CCCGAGGTGCAGCTTATCAGCG. Cotton reverse (17515) (SEQ ID NO: 88)
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGCAGGTTAGATTGGTTGA CCCTCTGGTAC.
[0415] The components of each 100 .mu.l PCR reaction at final
concentration consisted of: [0416] 0.5 .mu.l plasmid DNA diluted
1:20 with water [0417] 1.times. Buffer 2 (Expand.TM. High Fidelity
PCR System, Roche Molecular Biochemicals) [0418] 200 .mu.M dNTPs
[0419] 300 nM each, synthetic oligonucleotide primers [0420] 0.8
.mu.l Expand.TM. High Fidelity Polymerase (Roche Molecular
Biochemicals)
[0421] The tMT2 gene from each source is PCR amplified for 30
cycles using the following "touchdown" cycling profile. For each
reaction the reaction mix is pre-incubated for 5 minutes at
95.degree. C., during which the polymerase is spiked in. The
product is then amplified for 15 cycles, each cycle consisting of
denaturation at 94.degree. C. for 30 sec, annealing at 60.degree.
C. for 30 sec, and elongation at 72.degree. C. for 1.5 minutes. The
annealing temperature is decreased by 1.degree. C. per cycle for
each of the previous 15 cycles. An additional 15 cycles follow,
consisting of 94.degree. C. for 30 seconds, 45.degree. C. for 30
seconds, and 72.degree. C. for 1.5 minute, followed by a 7 minute
hold at 72.degree. C. The resulting amplification product is
visualized as a clean band of the appropriate size for each species
on a 0.8% agarose gel.
[0422] The resulting PCR products are subcloned into pDONR.TM.201
(Life Technologies, A Division of Invitrogen Corp., Rockville, Md.)
using the GATEWAY cloning system (Life Technologies, A Division of
Invitrogen Corp., Rockville, Md.).
[0423] To verify that no errors are introduced by the PCR
amplification, the double stranded DNA sequence is obtained using
standard sequencing methodology. The tMT2 sequences are then
recombined behind the T7 promoter in the prokaryotic expression
vector pET-DEST42 (Life Technologies, A Division of Invitrogen
Corp., Rockville, Md.) using the GATEWAY cloning system (Life
Technologies, A Division of Invitrogen Corp., Rockville, Md.).
[0424] The following sequences represent the mature amino acid
sequences of the wild type and mutant genes which may be expressed
in E. coli, following the addition of an amino terminal methionine.
The bolded and italicized amino acid residues represent the
location of the substitution in each of the mutants. TABLE-US-00014
Mature wildtype Arabidopsis tMT2 protein as expressed in E. coli:
(SEQ ID NO: 28) ATRCSSSSVSSSRPSAQPRFIQHKKEAYWFYRFLSIVYDHVINPGHWTED
MRDDALEPADLSHPDMRVVDVGGGTGFTTLGIVKTVKAKNVTILDQSPHQ
LAKAKQKEPLKECKIVEGDAEDLPFPTDYADRYVSAGSIEYWPDPQRGIR
EAYRVLKIGGKACLIGPVYPTFWLSRFFSDVWMLFPKEEEYIEWFKNAGF
KDVQLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKEEDVEK
PVNNPFSFLGRFLLGTLAAAWFVLIPIYMWIKDQIVPKDQPI Mature mutant hdt2
Arabidopsis tmt2 protein as expressed in E. coli (SEQ ID NO: 29)
ATRCSSSSVSSSRPSAQPRFIQHKKEAYWFYRFLSIVYDHVINPGHWTED
MRDDALEPADLSHPDMRVVDVGGGTGFTTLGIVKTVKAKNVTILDQSPHQ
LAKAKQKEPLKECKIVEGDAEDLPFPTDYADRYVSAGSIEYWPDPQRGIR
EAYRVLKIGGKACLIGPVYPTFWLSRFFSDVWMLFPKEEEYIEWFKNAGF
KDVQLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKEKDVEK
PVNNPFSFLGRFLLGTLAAAWFVLIPIYMWIKDQIVPKDQPI Mature mutant hdt6
Arabidopsis tmt2 protein as expressed in E. coli (SEQ ID NO: 30)
ATRCSSSSVSSSRPSAQPRFIQHKKKAYWFYRFLSIVYDHVINPGHWTED
MRDDALEPADLSHPDMRVVDVGGGTGFTTLGIVKTVKAKNVTILDQSPHQ
LAKAKQKEPLKECKIVEGDAEDLPFPTDYADRYVSAGSIEYWPDPQRGIR
EAYRVLKIGGKACLIGPVYPTFWLSRFFSDVWMLFPKEEEYIEWFKNAGF
KDVQLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKEEDVEK
PVNNPFSFLGRFLLGTLAAAWFVLIPIYMWIKDQIVPKDQPI Mature mutant hdt10
Arabidopsis tmt2 protein as expressed in E. coli (SEQ ID NO: 31)
ATRCSSSSVSSSRPSAQPRFIQHKKEAYWFYRFLSIVYDHVINPGHWTED
MRDDALEPADLSHPDMRVVNVGGGTGFTTLGIVKTVKAKNVTILDQSPHQ
LAKAKQKEPLKECKIVEGDAEDLPFPTDYADRYVSAGSIEYWPDPQRGIR
EAYRVLKIGGKACLIGPVYPTFWLSRFFSDVWMLFPKEEEYIEWFKNAGF
KDVQLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKEEDVEK
PVNNPFSFLGRFLLGTLAAAWFVLIPIYMWIKDQIVPKDQPI Mature mutant hdt16
Arabidopsis tmt2 protein as expressed in E. coli (SEQ ID NO: 31)
ATRCSSSSVSSSRPSAQPRFIQHKKEAYWFYRFLSIVYDHVINPGHWIED
MRDDALEPADLSHPDMRVVDVGGGTGFTTLGIVKTVKAKNVTILDQSPHQ
LAKAKQKEPLKECKIVEGDAEDLPFPTDYADRYVSAGSIEYWPDPQRGIR
EAYRVLKIGGKACLIGPVYPTFWLSRFFSDVWMLFPKEEEYIEWFKNAGF
KDVQLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKEEDVEK
PVNNPFSFLGRFLLGTLAAAWFVLIPIYMWIKDQIVPKDQPI Mature Brassica napus
tMT2 as expressed in E. coli (SEQ ID NO: 33)
ATRCSSSSSVSASRPSAQPRFIQHKKEAYWFYRFLSIVYDHIINPGHWTE
DMRDDALEPADLSHPDMRVVDVGGGTGFTTLGIVKTVKAKNVTILDQSPH
QLAKAKQKEPLKECKIVEGDAEDLPFPTDYADRYVSAGSIEYWPDPQRGI
REAYRVLKIGGKACLIGPVHPTFWLSRFFADVWMLFPKEEEYIEWFKNAG
FKDVQLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKEEDVE
KPVNNPFSFLGRFLLGTLAAAWFVLIPIYMWIKDQIVPKDQPI. Mature Oryza sativa
tMT2 as expressed in E. coli (SEQ ID NO: 34)
RLRGAASSSSAAARPMSQPRFIQHKKEAFWFYRFLSIVYDHVINPGHWTE
DMRDDALEPADLYSRKLRVVDVGGGTGFTTLGIVKRVDPENVTLLDQSPH
QLEKAREKEALKGVTIMEGDAEDLPFPTDTFDRYVSAGSIEYWPDPQRGI
KEAYRVLRLGGVACMIGPVHPTFWLSRFFADMWMLFPKEEEYIEWFKKAG
FKDVKLKRIGPKWYRGVRRHGLIMGCSVTGVKREHGDSPLQLGPKVEDVS
KPVNPITFLFRFLMGTICAAYYVLVPIYMWIKDQIVPKGMPI Mature Zea mays tMT2 as
expressed in E. coli (SEQ ID NO: 35)
RLRCAASSSPAAARPATAPRFIQHKKEAFWFYRFLSIVYDHVINPGHWTE
DMRDDALEPADLFSRHLTVVDVGGGTGFTTLGIVKHVNPENVTLLDQSPH
QLDKARQKEALKGVTIMEGDAEDLPFPTDSFDRYISAGSIEYWPDPQRGI
KEAYRVLRFGGLACVIGPVYPTFWLSRFFADMWMLFPKEEEYIEWFKKAG
FRDVKLKRIGPKWYRGVRRHGLIMGCSVTGVKRERGDSPLELGPKAEDVS
KPVNPITFLFRFLVGTICAAYYVLVPIYMWIKDQIVPKGMPI Mature Glycine max tMT2
as expressed in E. coli (SEQ ID NO: 36)
VPKCSVSASRPSSQPRFIQHKKEAFWFYRFLSIVYDHVINPGHWTEDMRD
DALEPADLNDRNMIVVDVGGGTGFTTLGIVKHVDAKNVTILDQSPHQLAK
AKQKEPLKECKIIEGDAEDLPFRTDYADRYVSAGSIEYWPDPQRGIKEAY
RVLKLGGKACLIGPVYPTFWLSRFFADVWMLFPKEEEYIEWFQKAGFKDV
QLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKEEDVEKPVN
PFVFALRFVLGALAATWFVLVPIYMWLKDQVVPKGQPI Mature Allium Porrum as
expressed in E. coli (SEQ ID NO: 37)
IFTCSASSSSRPASQPRFIQHKQEAFWFYRFLSIVYDHVINPGHWTEDMR
DDALEPAELYDSRMKVVDVGGGTGFTTLGIIKHIDPKNVTILDQSPHQLE
KARQKEALKECTIVEGDAEDLPFPTDTFDRYVSAGSIEYWPDPQRGIKEA
YRVLKLGGVACLIGPVHPTFWLSRFFADMWMLFPTEEEYIEWFKKAGFKD
VKLKRIGPKWYRGVRRHGLIMGCSVTGVKRLSGDSPLQLGPKAEDVKKPI
NPFSFLLRFILGTIAATYYVLVPIYMWIKDQIVPKGQPI Mature Gossypium hirsutum
tMT2 as expressed in E. coli (SEQ ID NO: 38)
APRCSLSASRPASQPRFIQHKKEAFWFYRFLSIVYDHVINPGHWTEDMRD
DALEPADLNDRDMVVVDVGGGTGFTTLGIVQHVDAKNVTILDQSPHQLAK
AKQKEPLKECNIIEGDAEDLPFPTDYADRYVSAGSIEYWPDPQRGIKEAY
RVLKQGGKACLIGPVYPTFWLSRFFADVWMLFPKEEEYIEWFEKAGFKDV
QLKRIGPKWYRGVRRHGLIMGCSVTGVKPASGDSPLQLGPKAEDVSKPVN
PFVFLLRFMLGATAAAYYVLVPIYMWLKDQIVPEGQPI
EXAMPLE 5
A 2-Methylphytylplastoquinol Methyltransferase Enzymatic Assay is
Performed on the Mature Cloned Genes Expressed in E. coli to Test
for Functionality of the Encoded Proteins
[0425] A culture is started by inoculating 100 mL of LB media with
appropriate antibiotics with an overnight starter culture of E.
coli BL21 (DE3) cells that is previously transformed with
prokaryotic expression constructs described in Example 4. The
initial inoculation results in an optical density of OD.sub.600=0.1
and the culture is grown at 25.degree. C. to a final density of
OD.sub.600=0.6. An amount corresponding to a final concentration of
0.4 mM IPTG is added to induce protein expression, and the cells
are then incubated at 25.degree. C. for 3 hours until harvest.
[0426] The cells are chilled on ice for 5 minutes and then spun
down at 5000.times.g for 10 minutes. The cell pellet is stored at
-80.degree. C. overnight after thoroughly aspirating off the
supernatant.
[0427] The cell pellet is thawed on ice and resuspended in 4 mL of
extraction buffer XB (10 mM HEPES-KOH pH7.8, 5 mM DTT, 1 mM AEBSF,
0.1 mM aprotinin, 1 mg/ml leupeptin). Cells are disrupted using a
French press by making two passes through the pressure cell at
20,000 psi. Triton X-100 is added to a final concentration of 1%
and the extract is incubated on ice for one hour. The cell
homogenate is then centrifuged at 5000.times.g for 10 minutes at
4.degree. C.
[0428] The enzyme assays are run on the same day that the cells are
extracted. The assays are run in 10 mL polypropylene culture tubes
with a final volume of 1 mL. A reaction mixture consisting of the
following is prepared and brought to a final volume of 950 .mu.L
with distilled water.
Reaction Mixture:
50 mM Tris-HCl pH 8.0
5 mM dithiothreitol (DTT, 100 mM stock solution in water)
100 .mu.M 2-methylphytylplastoquinol (404 g/mol)
0.5% Tween 80 (added directly to phytylplastoquinol after
evaporating off solvent)
1.7 .mu.M .sup.14C-SAM (58 .mu.Ci/.mu.mole)
2-Methyl-phytylplastoquinol and 2-methyl-geranylgeranylplastoquinol
are synthesized as follows:
[0429] Fresh BF.sub.3-etherate (0.3 ml) is added drop by drop to a
solution of 400 mg methylquinol, 1000 mg isophytol in 10 ml dry
dioxane. The mixture is stirred under N.sub.2 in the dark and is
maintained at 50.degree. C. for 2 hours. The reaction mixture is
hydrolyzed with ice, extracted with 3.times.15 ml petroleum
ether/diethyl ether (1:1), the extract is washed several times with
water to remove unused methylquinol, and dried with MgSO.sub.4. The
solvent is evaporated off with a rotavapor to yield an oil like
crude reaction product containing a mixture of methylplastoquinols.
At this stage the reaction mixture is either separated into various
methylphytylplastoquinols by flash chromatography followed by HPLC
purification or alternatively oxidized to yield the more stable
methylplastoquinones. This is achieved by addition of a small
amount of Ag.sub.2O (200 mg) to the reaction product dissolved in
diethyl ether for 1 hour. Removal of the Ag.sub.2O by filtration
provides the methylphytylplastoquinone mixture.
[0430] The synthesis of methylphytylplastoquinol as described above
gives six isomers, namely 2'-cis and 2'-trans isomers of
2-methyl-3-phytylplastoquinol, 2-methyl-5-phytylplastoquinol
2-methyl-6-phytylplastoquinol. Purification of the six isomers is
achieved by an initial separation of the methylphytylplastoquinol
mixture into two bands on TLC (PSC-Fertigplatten Kieselgel 60
F.sub.254+366, Merck, Darmstadt), using solvent system petroleum
ether:diethyl ether (7:3). The final purification of isomers of
methylplastoquinols is achieved by semi-preparative HPLC.
[0431] HPLC is performed on a HP1100 series HPLC system consisting
of HP G1329A Auto Sampler, HP G1311A Quaternary Pump, HP G1315A
Diode Array Detector, HP G1321A Fluorescence Detector. Excitation
is performed at 290 nm, emission is measured at 336 nm. In
parallel, absorption is measured using a diode array detector set
at 210 and 254 nm. The flow rate is kept at 5 mL/min. Plastoquinols
are separated on isocratic HPLC using 90%
Hexane:Methyl-Tertbutyl-Ether (90:10) on an Agilent Zorbax Silica
9.4.times.250 mm column.
[0432] Synthesis of 2-methyl-6-geranylgeranylplastoquinol is
performed as the synthesis of 2-methyl-6-phytylplastoquinol, except
geranyllinalool is used instead of isophytol for synthesis. The
pure product is obtained from flash chromatography followed by
repetitive TLC as described above.
[0433] To perform the methyltransferase assay 50 .mu.L of the cell
extract is added to the assay mixture and mixed well. The reaction
is initiated by adding .sup.14C-SAM (ICN) and incubating for one
hour at 30.degree. C. in the dark. The reactions are then
transferred to 15 mL glass screw cap tubes equipped with Teflon
coated caps. The reaction mixture is extracted with 4 mL 2:1
CHCl.sub.3/MeOH with 1 mg/mL butylated hydroxy toluene (BHT) and
mixed by vortex for 30 seconds. The tubes are centrifuged for 5
minutes to separate layers and the organic phase (bottom) is
transferred to fresh 15 mL glass tube. The CHCl.sub.3 is evaporated
off under a stream of nitrogen gas at 37.degree. C. for about 15
minutes. The residue is dissolved in 200 .mu.L of EtOH containing
1% pyrogallol and then mixed by vortex for 30 seconds. The
resuspension is filtered into a brown LC vial equipped with an
insert and analyzed by HPLC using a normal phase column (Agilent
4.6.times.250 mm Zorbax Sil, Agilent Technologies). The elution
program is an isocratic flow of 10% methyl-tert-butyl-ether (MTBE)
in hexane at 1.5 ml/minute for 12 minutes. Prior to each injection,
a clean up run of 75% MTBE in hexane for 3 minutes is done,
followed by a re-equilibration step of 10% MTBE in hexane for 3
minutes.
[0434] As a positive control, a pea chloroplast concentrate, which
is known to have tMT2 activity, is prepared according to the
procedure described by Arango and Heise, Biochem J. 336:531-533
(1998).
[0435] The results of these enzyme assays are shown in FIGS. 4-8.
The series of HPLC chromatograms demonstrate that the cells
transformed with the MT1 from Anabaena, which is known to have tMT2
activity (FIG. 4) and the tMT2 from Arabidopsis (FIG. 5) accumulate
methylated products comigrating with a
2,3-dimethyl-5-phytylplastoquinone standard. The mutated tMT2 gene
from Arabidopsis (hdt2) accumulated significantly less methylated
products (FIG. 6) than the wildtype tMT2 gene (FIG. 5), showing
that it has a decreased tMT2 activity. By way of comparison, the
negative control where substrate is withheld from the cells
transformed with the MT1 from Anabaena did not show a significant
peak corresponding to the methylated products (FIG. 7).
Furthermore, the positive control of pea chloroplasts showed peaks
corresponding to the methylated products contained in the assays
using E. coli extracts from strains harboring the MT1 and tMT2
expression constructs (FIG. 8).
Expression and Enzyme Assay of Crop tMT2 Orthologs
[0436] tMT2 orthologs from Brassica (pMON67233), corn (pMON67234),
leek (pMON67235), soybean (pMON67245), rice (pMON67232), and cotton
(pMON67244), as well as the wild type Arabidopsis tMT2 (pMON67191),
the hdt2 mutant (pMON67207), and the hdt10 mutant (pMON67243) are
expressed as mature proteins in E. coli (Example 4). An Anabaena
hdt2 otholog is expressed from pMON67190. The Anabaena MT1
(pMON67174) and empty vector (pMON67179) are used as positive and
negative controls, respectively. Cell growth, cell harvest, cell
disruption, and enzyme assay are performed as described in Example
5. HPLC-purified 2-methyl-6-phytylplastoquinol is used as methyl
group acceptor. TABLE-US-00015 TABLE 3
2-Methyl-6-phytylplastoquinol activity of recombinant expressed
tMT2 genes pMON # Gene Enzyme activity [.mu.U/mg protein] 67174
Anabaena MT1 6.5 67179 Plasmid control <1 67190 Anabaena tMT2
ortholog <1 67191 Arabidopsis tMT2 10 67207 Arabidopsis hdt2
mutant 1.1 67232 Rice tMT2 ortholog 4 67233 Brassica tMT2 ortholog
2 67234 Corn tMT2 ortholog <1 67235 Leek tMT2 ortholog <1
67243 Arabidopsis hdt10 mutant <1 67244 Cotton tMT2 ortholog
23.4 67245 Soy tMT2 ortholog 16.8
[0437] E. coli extracts expressing the Anabaena MT1, as well as
mature proteins of the Arabidopsis tMT2, rice tMT2, cotton tMT2,
and the soybean tMT2 are assayed as described in Example 5 using
HPLC-purified 2-methyl-6-phytylplastoquinol,
2-methyl-5-phytylplastoquinol, or 2-methyl-3-phytylplastoquinol as
methyl group acceptor. The assay demonstrates that tMT2 orthologs
have a broader substrate range than the bacterial MT1 (FIG.
24).
[0438] Methyltransferase assays are performed using cell free E.
coli extracts used in the experiments described above, expressing
the Anabaena MT1, as well as the mature Arabidopsis, rice, cotton,
and soybean tMT2s and 2-methyl-6-gernanylplastoquinol,
.delta.-tocopherol, .gamma.-tocopherol, or .beta.-tocopherol as
methyl group accepting substrates. Enzyme activities are below the
limit of detection with all four substrates.
EXAMPLE 6
Transformation and Expression of a Wild Type Arabidopsis tMT2 Gene
in Arabidopsis thialiana
[0439] The coding region of tMT2 is amplified from the EST clone
Lib 3177-021-P1-K1-A3 (SEQ ID NO: 1) using the synthetic
oligonucleotide primers; TABLE-US-00016 #17286 FORWARD (SEQ ID NO:
89) GGGGACAAGTTTGTACAAAAAAGCAGGCTGCGGCCGCTGAACAATGGCCT
CTTTGATGCTCAACG and #17181 REVERSE (SEQ ID NO: 90)
GGGGACCACTTTGTACAAGAAAGCTGGGTCCTGCAGGTCAGATGGGTTGG
TCTTTGGGAACG.
[0440] The amplification reaction consists of 1.0 .mu.l of EST
template, 2.5 .mu.l 20.times. dNTPs, 2.5 .mu.l of each
oligonucleotide primers, 5 .mu.l 10.times.PCR buffer, 35.75 .mu.l
H20 and 0.75 .mu.m Expand High Fidelity DNA Polymerase. PCR
conditions for amplification are as follows: [0441] 1 cycle of
94.degree. for 2 minutes, 10 cycles of 94.degree.-15 seconds;
55.degree.-30 seconds; and 72.degree.-1.5 minutes, [0442] 15 cycles
of 94.degree.-15 seconds; 55.degree.-30 seconds; and 72.degree.-1.5
minutes adding 5 seconds to the 72.degree. extension with each
cycle, [0443] 1 cycle of 72.degree. for 7 minutes.
[0444] After amplification, the samples are purified using a Qiagen
PCR cleanup column (Qiagen Company, Valencia, Calif.), suspended in
30 .mu.l water. The PCR reaction products are separated on an
agarose gel and visualized according to standard methodologies. The
resulting PCR products are subcloned into pDONR.TM.201 (Life
Technologies, A Division of Invitrogen Corp., Rockville, Md.) using
the GATEWAY cloning system (Life Technologies, A Division of
Invitrogen Corp., Rockville, Md.). The resultant intermediate
plasmid is named pMON67204 and the tMT2 sequence is confirmed by
DNA sequencing using standard methodologies.
[0445] The wild type Arabidopsis tMT2 sequence is then cloned from
the pMON67204 donor vector into the pMON67150 destination vector
using the GATEWAY Technology kit (Life Technologies, a Division of
Invitrogen Corporation, Rockville, Md.) according to the
manufacturer's instructions. This destination vector is a GATEWAY
compatible binary vector containing the napin cassette derived from
pCGN3223 (described in U.S. Pat. No. 5,639,790). The resultant
expression vector is named pMON67205 (FIG. 9) and is used to drive
the expression of the tMT2 sequence in seeds.
[0446] The plant binary construct described above is used in
Arabidopsis thaliana plant transformation to direct the expression
of the tMT2 gene in the embryo. The binary vector construct is
transformed into ABI strain Agrobacterium cells by the method of
Holsters et al. Mol. Gen. Genet. 163:181-187 (1978). Transgenic
Arabidopsis thaliana plants are obtained by Agrobacterium-mediated
transformation of Arabidopsis wild type and the high
.delta.-tocopherol mutants hdt2, hdt10, and hdt16 as described by
Valverkens et al., Proc. Nat. Acad. Sci. 85:5536-5540 (1988), Bent
et al., Science 265:1856-1860 (1994), and Bechtold et al., C.R.
Acad. Sci., Life Sciences 316:1194-1199 (1993). Transgenic plants
are selected by sprinkling the transformed T.sub.1 seeds directly
onto soil and then vernalizing them at 4.degree. C. in the absence
of light for 4 days. The seeds are transferred to 21.degree. C., 16
hours light and sprayed with a 1:200 dilution of Finale (AgrEvo
Environmental Health, Montvale, N.J.) at 7 days and 14 days after
seeding. Transformed plants are grown to maturity and the T.sub.2
seed that is produced is analyzed for tocopherol content. The
resulting tocopherol data shown in Tables 4 and 5 confirm a
reduction of .delta.-tocopherol in favor of .gamma. and
.alpha.-tocopherol production in the high .delta.-tocopherol
mutants and in wild type Arabidopsis lines. Tables 4 and 5 contain
the results of HPLC analysis using the methodology (with minor
modifications) described in Savidge et al., Plant Phys. 129:321-332
(2000), Isolation and Characterization of Homogentisate
Phytltransferase Genes from Synechocystis sp PCC 6803 and
Arabidopsis.
[0447] Table 4 below details the results of the T.sub.2 seed
analysis. TABLE-US-00017 TABLE 4 ng ng alpha ng beta gamma ng delta
ng total toco./mg toco./mg toco./mg toco./mg toco./mg Serial %
Average seed seed seed seed seed Number Pedigree Line # Delta %
Delta 5.88 0.00 529.64 18.87 554.39 69000076011 9979-AT00002- 1 3.4
3.2 81: @.0001. 5.45 0.00 525.89 17.44 548.78 69000076009
9979-AT00002- 4 3.2 81: @.0004. 5.74 0.00 511.61 16.32 533.67
69000075994 9979-AT00002- 3 3.1 81: @.0003. 5.04 0.00 507.38 16.10
528.52 69000076023 9979-AT00002- 2 3.0 81: @.0002. 7.74 0.00 466.14
11.53 485.41 69000075463 67205- 10 T2 2.4 1.2 AT00002: 0010. 8.76
0.00 460.36 7.00 476.12 69000075540 67205- 1 T2 1.5 AT00002: 0001.
8.33 0.00 445.02 6.71 460.06 69000075564 67205- 4 T2 1.5 AT00002:
0004. 8.46 0.00 443.94 6.67 459.06 69000075502 67205- 14 T2 1.5
AT00002: 0014. 11.13 0.00 447.27 6.35 464.75 69000075526 67205- 16
T2 1.4 AT00002: 0016. 9.07 0.00 470.64 6.49 486.19 69000075552
67205- 3 T2 1.3 AT00002: 0003. 8.10 0.00 422.89 5.82 436.81
69000075538 67205- 2 T2 1.3 AT00002: 0002. 8.64 0.00 473.01 6.47
488.12 69000075603 67205- 8 T2 1.3 AT00002: 0008. 9.25 0.00 488.63
6.43 504.32 69000075590 67205- 7 T2 1.3 AT00002: 0007. 7.71 0.00
475.80 6.21 489.72 69000075588 67205- 6 T2 1.3 AT00002: 0006. 7.77
0.00 458.67 5.71 472.15 69000075475 67205- 11 T2 1.2 AT00002: 0011.
8.85 0.00 455.97 5.59 470.41 69000075576 67205- 5 T2 1.2 AT00002:
0005. 10.27 0.00 349.67 3.05 362.98 69000075514 67205- 15 T2 0.8
AT00002: 0015. 9.22 0.00 371.75 2.84 383.81 69000075499 67205- 13
T2 0.7 AT00002: 0013. 8.68 0.00 348.97 2.53 360.18 69000075451
67205- 9 T2 0.7 AT00002: 0009. 7.96 0.00 413.19 2.40 423.55
69000075487 67205- 12 T2 0.6 AT00002: 0012. 7.00 0.00 277.36 286.49
570.84 69000077835 hdt2: @.0001. 1 50.2 49.7 6.57 0.00 273.89
278.92 559.38 69000077809 hdt2: @.0004. 4 49.9 6.90 0.00 277.90
279.96 564.77 69000077811 hdt2: @.0003. 3 49.6 6.93 0.00 275.20
273.89 556.01 69000077823 hdt2: @.0002. 2 49.3 8.35 0.00 365.85
143.68 517.88 69000075639 67205- 11 T2 27.7 20.5 hdt2: 0011. 7.75
0.00 384.44 127.60 519.79 69000075689 67205- 16 T2 24.5 hdt2: 0016.
7.05 0.00 358.91 105.17 471.13 69000075627 67205- 10 T2 22.3 hdt2:
0010. 8.33 0.00 342.11 98.01 448.45 69000075665 67205- 14 T2 21.9
hdt2: 0014. 6.73 0.00 410.18 112.97 529.88 69000075716 67205- 6 T2
21.3 hdt2: 0006. 6.89 0.00 357.86 98.47 463.22 69000075704 67205- 7
T2 21.3 hdt2: 0007. 6.85 0.00 352.48 96.71 456.04 69000075691
67205- 8 T2 21.2 hdt2: 0008. 8.06 0.00 356.89 96.10 461.05
69000075754 67205- 2 T2 20.8 hdt2: 0002. 7.60 0.00 311.53 82.55
401.68 69000075677 67205- 15 T2 20.6 hdt2: 0015. 7.81 0.00 344.03
88.44 440.28 69000075615 67205- 9 T2 20.1 hdt2: 0009. 7.50 0.00
368.30 88.66 464.46 69000075641 67205- 12 T2 19.1 hdt2: 0012. 7.13
0.00 336.24 80.34 423.71 69000075728 67205- 5 T2 19.0 hdt2: 0005.
7.78 0.00 345.26 81.26 434.30 69000075766 67205- 1 T2 18.7 hdt2:
0001. 8.82 0.00 340.61 72.71 422.15 69000075730 67205- 4 T2 17.2
hdt2: 0004. 8.11 0.00 418.69 81.01 507.81 69000075742 67205- 3 T2
16.0 hdt2: 0003. 6.08 0.00 365.54 69.78 441.40 69000075653 67205-
13 T2 15.8 hdt2: 0013. 3.36 262.76 180.18 446.30 69000157140 hdt16:
@.0007. Control M5 40.4 38.2 3.36 290.12 177.76 471.24 69000157114
hdt16: @.0003. Control M5 37.7 2.54 305.52 178.20 486.25
69000157099 hdt16: @.0005. Control M5 36.6 4.93 248.24 67.78 320.95
69000156403 AT_G119: @. PMON67205 R2 21.1 16.0 3.55 232.71 62.01
298.26 69000156667 AT_G36: @. PMON67205 R2 20.8 5.55 282.81 64.06
352.42 69000156679 AT_G37: @. PMON67205 R2 18.2 6.79 273.40 55.90
336.09 69000156617 AT_G31: @. PMON67205 R2 16.6 5.65 377.29 52.27
435.22 69000156631 AT_G33: @. PMON67205 R2 12.0 5.82 256.67 20.04
282.53 69000156655 AT_G35: @. PMON67205 R2 7.1 4.32 356.41 71.85
432.59 69000157037 hdt10: @.0001. Control M6 16.6 9.6 5.73 469.11
12.79 487.62 69000157049 hdt10: @.0002. Control M6 2.6 3.39 308.41
27.44 339.24 69000156528 AT_G22: @. PMON67205 R2 8.1 2.9 5.53
350.19 28.83 384.55 69000156592 AT_G29: @. PMON67205 R2 7.5 4.33
329.32 23.29 356.94 69000156489 AT_G18: @. PMON67205 R2 6.5 5.20
344.82 19.81 369.84 69000156566 AT_G26: @. PMON67205 R2 5.4 6.14
348.51 19.38 374.03 69000156453 AT_G15: @. PMON67205 R2 5.2 5.12
394.47 14.59 414.19 69000156578 AT_G27: @. PMON67205 R2 3.5 7.01
473.37 13.03 493.40 69000156530 AT_G23: @. PMON67205 R2 2.6 6.82
355.34 3.94 366.10 69000156580 AT_G28: @. PMON67205 R2 1.1 4.41
395.46 3.82 403.69 69000156477 AT_G17: @. PMON67205 R2 0.9 4.64
383.13 2.46 390.23 69000156542 AT_G24: @. PMON67205 R2 0.6 6.21
319.67 1.91 327.79 69000156465 AT_G16: @. PMON67205 R2 0.6 4.79
291.39 1.59 297.77 69000156441 AT_G14: @. PMON67205 R2 0.5 4.72
393.79 1.89 400.40 69000156491 AT_G19: @. PMON67205 R2 0.5 5.97
378.05 1.59 385.62 69000156516 AT_G21: @. PMON67205 R2 0.4 6.16
358.64 0.00 364.80 69000156554 AT_G25: @. PMON67205 R2 0.0 mp:
indicates "metabolic profiling".
[0448] Table 5 below depicts the results of the analysis of T3 seed
data from pMON67205 in hdt2 mutant lines. TABLE-US-00018 TABLE 5
Crop Biotype Serial Number mp: aT mp: gT mp: dT total toco. % delta
Gen Pedigree Construct AT SEED 69000357524 2 280 190 472 40.3 M7
hdt2: @.0001.0001. AT SEED 69000357512 3 262 208 473 44.0 M7 hdt2:
@.0001.0002. AT SEED 69000357625 4 263 204 471 43.3 M7 hdt2:
@.0001.0003. AT SEED 69000357613 4 271 220 495 44.4 M7 hdt2:
@.0001.0004. AT SEED 69000357803 6 436 26 468 5.6 R3 67205-hdt2:
0003.0001. 67205 AT SEED 69000357790 4 336 149 489 30.5 R3
67205-hdt2: 0003.0002. 67205 AT SEED 69000357788 4 332 112 448 25.0
R3 67205-hdt2: 0003.0003. 67205 AT SEED 69000357776 3 334 140 477
29.4 R3 67205-hdt2: 0003.0004. 67205 AT SEED 69000357764 4 324 128
456 28.1 R3 67205-hdt2: 0003.0005. 67205 AT SEED 69000357598 3 363
97 463 21.0 R3 67205-hdt2: 0003.0006. 67205 AT SEED 69000357586 4
339 145 488 29.7 R3 67205-hdt2: 0003.0007. 67205 AT SEED
69000357574 4 372 99 475 20.8 R3 67205-hdt2: 0003.0008. 67205 AT
SEED 69000357562 5 388 72 465 15.5 R3 67205-hdt2: 0003.0009. 67205
AT SEED 69000357550 4 341 63 408 15.4 R3 67205-hdt2: 0013.0001.
67205 AT SEED 69000357548 3 352 60 415 14.5 R3 67205-hdt2:
0013.0002. 67205 AT SEED 69000357536 4 386 54 444 12.2 R3
67205-hdt2: 0013.0003. 67205 AT SEED 69000358209 4 381 54 439 12.3
R3 67205-hdt2: 0013.0004. 67205 AT SEED 69000358196 6 413 73 492
14.8 R3 67205-hdt2: 0013.0005. 67205 AT SEED 69000358184 3 379 62
444 14.0 R3 67205-hdt2: 0013.0006. 67205 AT SEED 69000358172 5 382
63 450 14.0 R3 67205-hdt2: 0013.0007. 67205 AT SEED 69000358160 5
359 49 413 11.9 R3 67205-hdt2: 0013.0008. 67205 AT SEED 69000357601
4 371 4 379 1.1 R3 67205-hdt2: 0013.0009. 67205
EXAMPLE 7
Method to Prepare Double Gene Constructs for Expression in Soybean
and Arabidopsis
[0449] Constructs are made containing promoters that provide
seed-specific expression of the tMT2 gene alone and in combination
with the GMT gene in soybean. Additionally the tMT2 gene is cloned
behind the napin promoter and cloned into a binary vector with the
HPT gene from Arabidopsis and in another double gene construct with
the prenyltransferase (PT) gene (slr1736) from Synechocystis
(pMON67224 and pMON67223 as shown in FIGS. 14 and 15,
respectively).
Soybean Constructs
[0450] The wild type Arabidopsis tMT2 gene is cloned in between the
7S promoter and the pea SSU Rubisco 3' UTR in the vector pCGN3892
to create pMON67220 (FIG. 10). This clone is then digested with Not
I and the expression cassette is subcloned into the plant binary
expression vector pCGN11121 to create pMON67226 (FIG. 11). This
construct is used to transform soybean. Additionally, the
Arabidopsis GMT between the 7S promoter and the pea SSU Rubisco 3'
UTR is cut out from pMON36503 and then cloned into pMON67220 to
create pMON67225 (FIG. 12). These two genes under the control of 7S
promoters are then cut out of pMON67225 with NotI and cloned into
the Not site of pCGN11121 to create pMON67227 (FIG. 13). This
double gene construct is then used to transform soybean according
to the procedure set forth in WO 00/61771 A3 on pages 99-100.
Transformed plants are grown to maturity and seed that is produced
is analyzed for total tocopherol content and composition.
[0451] The tocopherol data presented in Tables 3 and 5 demonstrate
the reduction of .beta.-tocopherol and more so, .delta.-tocopherol
in favor of .gamma. and .alpha.-tocopherol production in soybean
seeds harboring a tMT2 expression construct. Tables 4 and 6
demonstrate a nearly complete (98% in the R0 generation) conversion
of tocopherols into .alpha.-tocopherol in soybean seed harboring a
double gene expression construct for tMT2 and a
.gamma.-methyltransferase.
[0452] Table 6 below depicts the results of the analysis of various
soybean lines transformed with pMon67226 Soy. Tables 6 and 9
contain the results of HPLC analysis using the methodology (with
minor modifications) described in Savidge et al., Plant Phys.
129:321-332 (2000), Isolation and Characterization of Homogentisate
Phytltransferase Genes from Synechocystis sp PCC 6803 and
Arabidopsis. TABLE-US-00019 TABLE 6 % % % % Pedigree delta gamma
alpha beta* mp: aT mp: bT mp: gT mp: dT total toco. A3244 22.90
63.97 10.44 2.69 31 8 190 68 297 A3244 22.85 64.24 10.26 2.65 31 8
194 69 302 A3244 22.88 64.38 10.46 2.29 32 7 197 70 306 A3244 23.08
64.21 10.37 2.34 31 7 192 69 299 A3244 22.97 64.19 10.47 2.36 31 7
190 68 296 GM_A28213: @. 36.92 51.08 8.31 3.69 27 12 166 120 325
GM_A27926: @. 27.51 62.72 7.46 2.31 29 9 244 107 389 GM_A27928: @.
26.56 62.81 8.13 2.50 26 8 201 85 320 GM_A27993: @. 25.70 62.29
9.50 2.51 34 9 223 92 358 GM_A27628: @. 25.07 61.19 10.75 2.99 36
10 205 84 335 GM_A28069: @. 24.66 58.56 13.01 3.77 38 11 171 72 292
GM_A27927: @. 24.41 63.05 10.17 2.37 30 7 186 72 295 GM_A28930: @.
24.14 63.01 10.03 2.82 32 9 201 77 319 GM_A28597: @. 23.89 61.09
11.60 3.41 34 10 179 70 293 GM_A28077: @. 23.73 65.76 8.47 2.03 25
6 194 70 295 GM_A28410: @. 23.70 66.47 7.80 2.02 27 7 230 82 346
GM_A28212: @. 23.37 63.91 10.06 2.66 34 9 216 79 338 GM_A28079: @.
23.10 62.38 11.22 3.30 34 10 189 70 303 GM_A27992: @. 23.05 52.42
19.70 4.83 53 13 141 62 269 GM_A28074: @. 22.52 61.86 12.61 3.00 42
10 206 75 333 GM_A28931: @. 20.66 63.28 13.44 2.62 41 8 193 63 305
GM_A28767: @. 20.20 65.66 11.78 2.36 35 7 195 60 297 GM_A28598: @.
20.14 61.09 15.02 3.75 44 11 179 59 293 GM_A28214: @. 20.07 61.90
14.29 3.74 42 11 182 59 294 GM_A28062: @. 19.80 64.09 13.09 3.02 39
9 191 59 298 GM_A28505: @. 19.69 66.77 11.69 1.85 38 6 217 64 325
GM_A28067: @. 18.18 62.55 15.64 3.64 43 10 172 50 275 GM_A28503: @.
18.06 65.63 14.24 2.08 41 6 189 52 288 GM_A28408: @. 17.97 64.75
14.58 2.71 43 8 191 53 295 GM_A28061: @. 17.87 62.20 16.15 3.78 47
11 181 52 291 GM_A28504: @. 17.73 62.06 16.67 3.55 47 10 175 50 282
GM_A28409: @. 16.79 63.14 16.42 3.65 45 10 173 46 274 GM_A28060: @.
16.16 68.35 13.80 1.68 41 5 203 48 297 GM_A28076: @. 16.04 60.41
19.11 4.44 56 13 177 47 293 GM_A28066: @. 15.36 59.73 20.48 4.44 60
13 175 45 293 GM_A29037: @. 14.49 71.59 12.22 1.70 43 6 252 51 352
GM_A27855: @. 13.64 74.68 10.39 1.30 32 4 230 42 308 GM_A27856: @.
13.46 72.76 12.18 1.60 38 5 227 42 312 GM_A28081: @. 11.11 76.85
10.80 1.23 35 4 249 36 324 GM_A27627: @. 8.33 75.93 14.20 1.54 46 5
246 27 324 GM_A27932: @. 8.13 81.33 9.94 0.60 33 2 270 27 332
GM_A27857: @. 7.28 78.48 13.29 0.95 42 3 248 23 316 GM_A28073: @.
7.22 67.70 23.37 1.72 68 5 197 21 291 GM_A27708: @. 7.06 75.77
16.26 0.92 53 3 247 23 326 GM_A28059: @. 6.99 77.57 14.71 0.74 40 2
211 19 272 GM_A27925: @. 6.95 76.82 15.23 0.99 46 3 232 21 302
GM_A27859: @. 6.83 77.34 14.39 1.44 40 4 215 19 278 GM_A28065: @.
6.44 73.22 18.64 1.69 55 5 216 19 295 GM_A27931: @. 6.33 78.92
13.86 0.90 46 3 262 21 332 GM_A28246: @. 6.31 72.24 19.87 1.58 63 5
229 20 317 GM_A27994: @. 6.29 79.02 13.99 0.70 40 2 226 18 286
GM_A27995: @. 6.08 78.12 14.89 0.91 49 3 257 20 329 GM_A28075: @.
5.61 73.60 19.14 1.65 58 5 223 17 303 GM_A28070: @. 5.47 79.42
14.47 0.64 45 2 247 17 311 GM_A28068: @. 4.76 75.85 18.71 0.68 55 2
223 14 294 GM_A28078: @. 3.72 81.08 14.53 0.68 43 2 240 11 296
GM_A28080: @. 3.69 73.06 21.77 1.48 59 4 198 10 271 GM_A28071: @.
3.64 75.83 19.87 0.66 60 2 229 11 302 GM_A28058: @. 3.51 82.16
13.74 0.58 47 2 281 12 342 GM_A28064: @. 2.23 85.03 12.74 0.00 40 0
267 7 314 GM_A28599: @. 1.47 82.65 15.88 0.00 54 0 281 5 340
GM_A27929: @. 1.23 83.74 13.80 1.23 45 4 273 4 326 GM_A28063: @.
1.22 74.62 23.55 0.61 77 2 244 4 327 GM_A28072: @. 0.95 76.66 22.08
0.32 70 1 243 3 317 GM_A27930: @. 0.68 79.05 20.27 0.00 60 0 234 2
296
[0453] Table 7 below sets forth the results of the analysis of
various soybean lines transformed with pMON 67227. TABLE-US-00020
TABLE 7 Pedigree % alpha % beta* % gamma % delta mp: aT mp: bT mp:
gT mp: dT total toco. A3244 10.4 2.7 64.0 22.9 31 8 190 68 297
A3244 10.3 2.6 64.2 22.8 31 8 194 69 302 A3244 10.5 2.3 64.4 22.9
32 7 197 70 306 A3244 10.4 2.3 64.2 23.1 31 7 192 69 299 A3244 10.5
2.4 64.2 23.0 31 7 190 68 296 GM_A27999: @. 9.5 2.5 62.9 25.2 31 8
205 82 326 GM_A28091: @. 10.5 3.1 61.9 24.5 31 9 182 72 294
GM_A28090: @. 11.3 2.7 63.0 22.9 33 8 184 67 292 GM_A28933: @. 14.4
2.1 65.8 17.7 48 7 219 59 333 GM_A28601: @. 15.7 3.1 62.4 18.8 45 9
179 54 287 GM_A27712: @. 60.4 2.5 26.9 10.2 171 7 76 29 283
GM_A27936: @. 60.6 20.4 13.8 5.2 163 55 37 14 269 GM_A28093: @.
67.2 3.3 21.2 8.3 203 10 64 25 302 GM_A27934: @. 75.4 3.1 16.5 5.0
196 8 43 13 260 GM_A28096: @. 79.1 3.4 12.5 5.0 253 11 40 16 320
GM_A27935: @. 88.5 2.7 6.9 1.9 231 7 18 5 261 GM_A27998: @. 89.6
2.5 6.0 1.9 285 8 19 6 318 GM_A27711: @. 91.4 3.3 4.3 1.0 276 10 13
3 302
[0454] Table 8 below sets for the results of the analysis of single
seeds of soybean transformed with pMON 67226. TABLE-US-00021 TABLE
8 Pedigree % alpha % beta* % gamma % delta mp: aT mp: bT mp: gT mp:
dT total toco. GM_A27930: @. 12.2 3.4 64.1 20.3 29 8 152 48 237
GM_A27930: @. 21.7 0.0 77.9 0.4 55 0 197 1 253 GM_A27930: @. 15.0
0.0 84.0 1.0 46 0 257 3 306 GM_A27930: @. 22.4 0.0 76.8 0.8 58 0
199 2 259 GM_A27930: @. 13.9 0.0 85.7 0.4 33 0 204 1 238 GM_A27930:
@. 21.7 0.0 77.6 0.7 63 0 229 2 290 GM_A27930: @. 21.7 0.0 77.6 0.8
55 0 197 2 254 GM_A27930: @. 25.7 0.0 74.0 0.4 68 0 196 1 265
GM_A28072: @. 22.4 0.0 76.8 0.8 57 0 195 2 254 GM_A28072: @. 31.3
67.6 1.2 80 0 173 3 256 GM_A28072: @. 22.8 0.0 76.5 0.7 64 0 215 2
281 GM_A28072: @. 17.6 0.0 81.5 1.0 55 0 255 3 313 GM_A28072: @.
20.0 0.0 78.9 1.1 55 0 217 3 275 GM_A28072: @. 35.0 0.0 64.6 0.4 97
0 179 1 277 GM_A28072: @. 31.5 0.0 68.1 0.4 80 0 173 1 254
GM_A28072: @. 16.4 0.0 82.6 1.0 51 0 257 3 311
[0455] Table 9 below sets forth the results of the analysis of
single seeds of soybean transformed with pMON 67227. TABLE-US-00022
TABLE 9 Pedigree % alpha % beta* % gamma % delta mp: aT mp: bT mp:
gT mp: dT total toco. GM_A27711: @. 97.8 2.2 0.0 0.0 263 6 0 0 269
GM_A27711: @. 96.7 3.3 0.0 0.0 320 11 0 0 331 GM_A27711: @. 96.5
3.5 0.0 0.0 301 11 0 0 312 GM_A27711: @. 96.7 3.3 0.0 0.0 295 10 0
0 305 GM_A27711: @. 96.9 3.1 0.0 0.0 308 10 0 0 318 GM_A27711: @.
97.3 2.7 0.0 0.0 287 8 0 0 295 GM_A27711: @. 98.2 1.8 0.0 0.0 272 5
0 0 277 GM_A27711: @. 95.7 4.3 0.0 0.0 287 13 0 0 300 GM_A27935: @.
10.3 2.6 65.4 21.7 28 7 178 59 272 GM_A27935: @. 98.5 1.5 0.0 0.0
261 4 0 0 265 GM_A27935: @. 98.3 1.7 0.0 0.0 230 4 0 0 234
GM_A27935: @. 98.6 1.4 0.0 0.0 272 4 0 0 276 GM_A27935: @. 98.2 1.8
0.0 0.0 267 5 0 0 272 GM_A27935: @. 96.9 3.1 0.0 0.0 277 9 0 0 286
GM_A27935: @. 98.3 1.7 0.0 0.0 337 6 0 0 343 GM_A27935: @. 96.5 3.5
0.0 0.0 276 10 0 0 286 GM_A27998: @. 97.0 3.0 0.0 0.0 318 10 0 0
328 GM_A27998: @. 97.1 2.9 0.0 0.0 300 9 0 0 309 GM_A27998: @. 95.9
4.1 0.0 0.0 324 14 0 0 338 GM_A27998: @. 97.0 3.0 0.0 0.0 292 9 0 0
301 GM_A27998: @. 96.9 3.1 0.0 0.0 314 10 0 0 324 GM_A27998: @.
96.5 3.5 0.0 0.0 359 13 0 0 372 GM_A27998: @. 96.5 3.5 0.0 0.0 335
12 0 0 347 GM_A27998: @. 96.6 3.4 0.0 0.0 310 11 0 0 321 GM_A28096:
@. 11.1 3.7 61.0 24.1 36 12 197 78 323 GM_A28096: @. 9.5 3.3 61.4
25.8 29 10 188 79 306 GM_A28096: @. 96.8 3.2 0.0 0.0 299 10 0 0 309
GM_A28096: @. 96.0 4.0 0.0 0.0 288 12 0 0 300 GM_A28096: @. 95.8
4.2 0.0 0.0 319 14 0 0 333 GM_A28096: @. 95.8 4.2 0.0 0.0 295 13 0
0 308 GM_A28096: @. 97.8 2.2 0.0 0.0 316 7 0 0 323 GM_A28096: @.
95.8 4.2 0.0 0.0 300 13 0 0 313 The * next to % beta in Tables 6
through 9 is a label to indicate that .beta.-tocopherol comigrates
with an unknown compound, making it difficult to quantify.
Arabidopsis Double Constructs
[0456] The tMT2 gene is cut out of the vector pMON67204 using the
restriction enzymes Not I (blunt)/Pst I and then cloned into the
napin shuttle vector pCGN3223 which is digested with Sal
(blunt)/Pst I. This napin cassette containing the tMT2 gene is then
cut out from this vector with Not I and the ends are filled in with
dNTPs using a Klenow procedure. The resulting fragment is inserted
into the vectors pMON16602 (digested with PmeI) and pCGN10822
(digested with SnaBI) to make pMON67224 and pMON67223, respectively
(FIGS. 14 and 15). The vectors pMON16602 and pCGN10822 are
described in PCT application WO 0063391.
[0457] These double constructs express the tMT2 gene and the
prenyltransferase from either Arabidopsis (HPT) or Synechocystis
(slr1736) under the control of the napin seed-specific promoter.
These constructs are used to transform Arabidopsis and transformed
plants are grown to maturity, as detailed in Example 6. The
resulting T.sub.2 seed is analyzed for total tocopherol content and
composition using analytical procedures described in Example 1.
Sequence CWU 1
1
108 1 1184 DNA Arabidopsis thaliana 1 atggcctctt tgatgctcaa
cggggccatt accttcccca aaggtttagg ttcccctggt 60 tccaatttgc
atgccagatc gattcctcgg ccgaccttac tctcagttac ccgaacctcc 120
acacctagac tctcggtggc tactagatgc agcagcagca gcgtgtcgtc ttcccggcca
180 tcggcgcaac ctaggttcat tcagcacaag aaggaggctt actggttcta
caggttctta 240 tccatcgtat acgaccatgt catcaatcct gggcattgga
ccgaggatat gagagacgac 300 gctcttgagc cagcggatct cagccatccg
gacatgcgag tggtcgatgt cggcggcgga 360 actggtttca ctactctggg
catagtcaag acagtgaagg ccaagaatgt gaccattctg 420 gaccagtcgc
cacatcagct ggccaaagca aagcaaaagg agccgttgaa agaatgcaag 480
atcgtcgagg gagatgctga ggatcttcct tttccaaccg attatgctga cagatacgtt
540 tctgctggaa ggtatccttt tcttcttctt cttcttcttc ttcttcttct
tcttataatc 600 gtcttctttc cggtgggttt gattgtgtgt ctcatcatca
cacagcattg agtactggcc 660 ggacccgcag aggggaataa gggaagcgta
cagggttctc aagatcggtg gcaaagcgtg 720 tctcatcggc cctgtctacc
caaccttctg gctctctcgc ttcttttctg atgtctggat 780 gctcttcccc
aaggaggaag agtacattga gtggttcaag aatgccggtt tcaaggacgt 840
tcagctcaag aggattggcc ccaagtggta ccgtggtgtt cgcaggcacg gccttatcat
900 gggatgttct gtcactggtg ttaaacctgc ctccggtgac tctcctctcc
aggtctttta 960 cctcccactt cacctttttt actttcttct ctctttgata
cactaaactt atcactcaaa 1020 tgctgcagct tggtccaaag gaagaggacg
tagagaagcc tgtcaacaac cccttctcct 1080 tcttgggacg cttcctcctg
ggaactctag cagctgcctg gtttgtgtta atccctatct 1140 acatgtggat
caaggatcag atcgttccca aagaccaacc catc 1184 2 1181 DNA Arabidopsis
thaliana 2 atggcctctt tgatgctcaa cggggccatt accttcccca aaggtttagg
ttcccctggt 60 tccaatttgc atgccagatc gattcctcgg ccgaccttac
tctcagttac ccgaacctcc 120 acacctagac tctcggtggc tactagatgc
agcagcagca gcgtgtcgtc ttcccggcca 180 tcggcgcaac ctaggttcat
tcagcacaag aaggaggctt actggttcta caggttctta 240 tccatcgtat
acgaccatgt catcaatcct gggcattgga ccgaggatat gagagacgac 300
gctcttgagc cagcggatct cagccatccg gacatgcgag tggtcgatgt cggcggcgga
360 actggtttca ctactctggg catagtcaag acagtgaagg ccaagaatgt
gaccattctg 420 gaccagtcgc cacatcagct ggccaaagca aagcaaaagg
agccgttgaa agaatgcaag 480 atcgtcgagg gagatgctga ggatcttcct
tttccaaccg attatgctga cagatacgtt 540 tctgctggaa ggtatccttt
tcttcttctt cttcttcttc ttcttcttct tataatcgtc 600 ttctttccgg
tgggtttgat tgtgtgtctc atcatcacac agcattgagt actggccgga 660
cccgcagagg ggaataaggg aagcgtacag ggttctcaag atcggtggca aagcgtgtct
720 catcggccct gtctacccaa ccttctggct ctctcgcttc ttttctgatg
tctggatgct 780 cttccccaag gaggaagagt acattgagtg gttcaagaat
gccggtttca aggacgttca 840 gctcaagagg attggcccca agtggtaccg
tggtgttcgc aggcacggcc ttatcatggg 900 atgttctgtc actggtgtta
aacctgcctc cggtgactct cctctccagg tcttttacct 960 cccacttcac
cttttttact ttcttctctc tttgatacac taaacttatc actcaaatgc 1020
tgcagcttgg tccaaaggaa gaggacgtag agaagcctgt caacaacccc ttctccttct
1080 tgggacgctt cctcctggga actctagcag ctgcctggtt tgtgttaatc
cctatctaca 1140 tgtggatcaa ggatcagatc gttcccaaag accaacccat c 1181
3 1181 DNA Arabidopsis thaliana 3 atggcctctt tgatgctcaa cggggccatt
accttcccca aaggtttagg ttcccctggt 60 tccaatttgc atgccagatc
gattcctcgg ccgaccttac tctcagttac ccgaacctcc 120 acacctagac
tctcggtggc tactagatgc agcagcagca gcgtgtcgtc ttcccggcca 180
tcggcgcaac ctaggttcat tcagcacaag aaggaggctt actggttcta caggttctta
240 tccatcgtat acgaccatgt catcaatcct gggcattgga ccgaggatat
gagagacgac 300 gctcttgagc cagcggatct cagccatccg gacatgcgag
tggtcgatgt cggcggcgga 360 actggtttca ctactctggg catagtcaag
acagtgaagg ccaagaatgt gaccattctg 420 gaccagtcgc cacatcagct
ggccaaagca aagcaaaagg agccgttgaa agaatgcaag 480 atcgtcgagg
gagatgctga ggatcttcct tttccaaccg attatgctga cagatacgtt 540
tctgctggaa ggtatccttt tcttcttctt cttcttcttc ttcttcttct tataatcgtc
600 ttctttccgg tgggtttgat tgtgtgtctc atcatcacac agcattgagt
actggccgga 660 cccgcagagg ggaataaggg aagcgtacag ggttctcaag
atcggtggca aagcgtgtct 720 catcggccct gtctacccaa ccttctggct
ctctcgcttc ttttctgatg tctggatgct 780 cttccccaag gaggaagagt
acattgagtg gttcaagaat gccggtttca aggacgttca 840 gctcaagagg
attggcccca agtggtaccg tggtgttcgc aggcacggcc ttatcatggg 900
atgttctgtc actggtgtta aacctgcctc cggtgactct cctctccagg tcttttacct
960 cccacttcac cttttttact ttcttctctc tttgatacac taaacttatc
actcaaatgc 1020 tgcagcttgg tccaaaggaa aaggacgtag agaagcctgt
caacaacccc ttctccttct 1080 tgggacgctt cctcctggga actctagcag
ctgcctggtt tgtgttaatc cctatctaca 1140 tgtggatcaa ggatcagatc
gttcccaaag accaacccat c 1181 4 1184 DNA Arabidopsis thaliana 4
atggcctctt tgatgctcaa cggggccatt accttcccca aaggtttagg ttcccctggt
60 tccaatttgc atgccagatc gattcctcgg ccgaccttac tctcagttac
ccgaacctcc 120 acacctagac tctcggtggc tactagatgc agcagcagca
gcgtgtcgtc ttcccggcca 180 tcggcgcaac ctaggttcat tcagcacaag
aagaaggctt actggttcta caggttctta 240 tccatcgtat acgaccatgt
catcaatcct gggcattgga ccgaggatat gagagacgac 300 gctcttgagc
cagcggatct cagccatccg gacatgcgag tggtcgatgt cggcggcgga 360
actggtttca ctactctggg catagtcaag acagtgaagg ccaagaatgt gaccattctg
420 gaccagtcgc cacatcagct ggccaaagca aagcaaaagg agccgttgaa
agaatgcaag 480 atcgtcgagg gagatgctga ggatcttcct tttccaaccg
attatgctga cagatacgtt 540 tctgctggaa ggtatccttt tcttcttctt
cttcttcttc ttcttcttct tcttataatc 600 gtcttctttc cggtgggttt
gattgtgtgt ctcatcatca cacagcattg agtactggcc 660 ggacccgcag
aggggaataa gggaagcgta cagggttctc aagatcggtg gcaaagcgtg 720
tctcatcggc cctgtctacc caaccttctg gctctctcgc ttcttttctg atgtctggat
780 gctcttcccc aaggaggaag agtacattga gtggttcaag aatgccggtt
tcaaggacgt 840 tcagctcaag aggattggcc ccaagtggta ccgtggtgtt
cgcaggcacg gccttatcat 900 gggatgttct gtcactggtg ttaaacctgc
ctccggtgac tctcctctcc aggtctttta 960 cctcccactt cacctttttt
actttcttct ctctttgata cactaaactt atcactcaaa 1020 tgctgcagct
tggtccaaag gaagaggacg tagagaagcc tgtcaacaac cccttctcct 1080
tcttgggacg cttcctcctg ggaactctag cagctgcctg gtttgtgtta atccctatct
1140 acatgtggat caaggatcag atcgttccca aagaccaacc catc 1184 5 1184
DNA Arabidopsis thaliana 5 atggcctctt tgatgctcaa cggggccatt
accttctcca aaggtttagg ttcccctggt 60 tccaatttgc atgccagatc
gattcctcgg ccgaccttac tctcagttac ccgaacctcc 120 acacctagac
tctcggtggc tactagatgc agcagcagca gcgtgtcgtc ttcccggcca 180
tcggcgcaac ctaggttcat tcagcacaag aaggaggctt actggttcta caggttctta
240 tccatcgtat acgaccatgt catcaatcct gggcattgga ccgaggatat
gagagacgac 300 gctcttgagc cagcggatct cagccatccg gacatgcgag
tggtcgatgt cggcggcgga 360 actggtttca ctactctggg catagtcaag
acagtgaagg ccaagaatgt gaccattctg 420 gaccagtcgc cacatcagct
ggccaaagca aagcaaaagg agccgttgaa agaatgcaag 480 atcgtcgagg
gagatgctga ggatcttcct tttccaaccg attatgctga cagatacgtt 540
tctgctggaa ggtatccttt tcttcttctt cttcttcttc ttcttcttct tcttataatc
600 gtcttctttc cggtgggttt gattgtgtgt ctcatcatca cacagcattg
agtactggcc 660 ggacccgcag aggggaataa gggaagcgta cagggttctc
aagatcggtg gcaaagcgtg 720 tctcatcggc cctgtctacc caaccttctg
gctctctcgc ttcttttctg atgtctggat 780 gctcttcccc aaggaggaag
agtacattga gtggttcaag aatgccggtt tcaaggacgt 840 tcagctcaag
aggattggcc ccaagtggta ccgtggtgtt cgcaggcacg gccttatcat 900
gggatgttct gtcactggtg ttaaacctgc ctccggtgac tctcctctcc aggtctttta
960 cctcccactt cacctttttt actttcttct ctctttgata cactaaactt
atcactcaaa 1020 tgctgcagct tggtccaaag gaagaggacg tagagaagcc
tgtcaacaac cccttctcct 1080 tcttgggacg cttcctcctg ggaactctag
cagctgcctg gtttgtgtta atccctatct 1140 acatgtggat caaggatcag
atcgttccca aagaccaacc catc 1184 6 1181 DNA Arabidopsis thaliana 6
atggcctctt tgatgctcaa cggggccatt accttcccca aaggtttagg ttcccctggt
60 tccaatttgc atgccagatc gattcctcgg ccgaccttac tctcagttac
ccgaacctcc 120 acacctagac tctcggtggc tactagatgc agcagcagca
gcgtgtcgtc ttcccggcca 180 tcggcgcaac ctaggttcat tcagcacaag
aaggaggctt actggttcta caggttctta 240 tccatcgtat acgaccatgt
catcaatcct gggcattgga ccgaggatat gagagacgac 300 gctcttgagc
cagcggatct cagccatccg gacatgcgag tggtcaatgt cggcggcgga 360
actggtttca ctactctggg catagtcaag acagtgaagg ccaagaatgt gaccattctg
420 gaccagtcgc cacatcagct ggccaaagca aagcaaaagg agccgttgaa
agaatgcaag 480 atcgtcgagg gagatgctga ggatcttcct tttccaaccg
attatgctga cagatacgtt 540 tctgctggaa ggtatccttt tcttcttctt
cttcttcttc ttcttcttct tataatcgtc 600 ttctttccgg tgggtttgat
tgtgtgtctc atcatcacac agcattgagt actggccgga 660 cccgcagagg
ggaataaggg aagcgtacag ggttctcaag atcggtggca aagcgtgtct 720
catcggccct gtctacccaa ccttctggct ctctcgcttc ttttctgatg tctggatgct
780 cttccccaag gaggaagagt acattgagtg gttcaagaat gccggtttca
aggacgttca 840 gctcaagagg attggcccca agtggtaccg tggtgttcgc
aggcacggcc ttatcatggg 900 atgttctgtc actggtgtta aacctgcctc
cggtgactct cctctccagg tcttttacct 960 cccacttcac cttttttact
ttcttctctc tttgatacac taaacttatc actcaaatgc 1020 tgcagcttgg
tccaaaggaa gaggacgtag agaagcctgt caacaacccc ttctccttct 1080
tgggacgctt cctcctggga actctagcag ctgcctggtt tgtgttaatc cctatctaca
1140 tgtggatcaa ggatcagatc gttcccaaag accaacccat c 1181 7 1184 DNA
Arabidopsis thaliana 7 atggcctctt tgatgctcaa cggggccatt accttcccca
aaggtttagg ttcccctggt 60 tccaatttgc atgccagatc gattcctcgg
ccgaccttac tctcagttac ccgaacctcc 120 acacctagac tctcggtggc
tactagatgc agcagcagca gcgtgtcgtc ttcccggcca 180 tcggcgcaac
ctaggttcat tcagcacaag aaggaggctt actggttcta caggttctta 240
tccatcgtat acgaccatgt catcaatcct gggcattgga tcgaggatat gagagacgac
300 gctcttgagc cagcggatct cagccatccg gacatgcgag tggtcgatgt
cggcggcgga 360 actggtttca ctactctggg catagtcaag acagtgaagg
ccaagaatgt gaccattctg 420 gaccagtcgc cacatcagct ggccaaagca
aagcaaaagg agccgttgaa agaatgcaag 480 atcgtcgagg gagatgctga
ggatcttcct tttccaaccg attatgctga cagatacgtt 540 tctgctggaa
ggtatccttt tcttcttctt cttcttcttc ttcttcttct tcttataatc 600
gtcttctttc cggtgggttt gattgtgtgt ctcatcatca cacagcattg agtactggcc
660 ggacccgcag aggggaataa gggaagcgta cagggttctc aagatcggtg
gcaaagcgtg 720 tctcatcggc cctgtctacc caaccttctg gctctctcgc
ttcttttctg atgtctggat 780 gctcttcccc aaggaggaag agtacattga
gtggttcaag aatgccggtt tcaaggacgt 840 tcagctcaag aggattggcc
ccaagtggta ccgtggtgtt cgcaggcacg gccttatcat 900 gggatgttct
gtcactggtg ttaaacctgc ctccggtgac tctcctctcc aggtctttta 960
cctcccactt cacctttttt actttcttct ctctttgata cactaaactt atcactcaaa
1020 tgctgcagct tggtccaaag gaagaggacg tagagaagcc tgtcaacaac
cccttctcct 1080 tcttgggacg cttcctcctg ggaactctag cagctgcctg
gtttgtgtta atccctatct 1140 acatgtggat caaggatcag atcgttccca
aagaccaacc catc 1184 8 1059 DNA Arabidopsis thaliana 8 atggcgatgg
cctccaccta cgcgccgggc ggaggcgcgc gggcgctcgc gcagggtaga 60
tgcagggtcc gcggtcccgc ggggctgggc ttcctcggcc cctccaaggc cgccggcctc
120 ccccgccccc tcgccctcgc cctcgccagg cggatgagca gccccgtcgc
ggtgggcgcc 180 aggctgcgat gcgcggcgtc gtcgtccccc gcggcggcgc
ggcccgccac ggcgccgcgc 240 ttcatccagc acaagaagga ggccttctgg
ttctaccgct tcctctccat cgtgtacgac 300 cacgtcatca atccgggcca
ctggaccgag gacatgcgcg acgacgcgct ggaacctgcc 360 gacctcttca
gccgccacct cacggtcgtc gacgtcggcg gcggcacggg gttcaccacg 420
ctcggcatcg tcaagcacgt caacccggag aacgtcacgc tgctcgacca gtccccgcac
480 cagctcgaca aggcccggca gaaggaggcc ctcaaggggg tcaccatcat
ggagggcgac 540 gccgaggacc tcccgttccc caccgactcc ttcgaccgat
acatctccgc cggcagcatc 600 gagtactggc cagacccaca gcgggggatc
aaggaagcct acagggtcct gagatttggt 660 gggctagctt gtgtgatcgg
cccggtctac ccgaccttct ggctgtcccg cttcttcgcc 720 gacatgtgga
tgctcttccc caaggaggaa gagtacatcg agtggttcaa gaaggctggg 780
tttagggatg tcaagctgaa gaggattgga ccgaagtggt accgcggtgt ccgaaggcat
840 ggcctcatca tgggctgctc cgtcacaggc gtcaagagag agcgcggtga
ctctcccttg 900 gagcttggtc ccaaggcgga ggatgtcagc aagccagtga
atccgatcac cttcctcttc 960 cgcttcctcg taggaacgat atgtgctgcc
tactatgttc tggtgcctat ttacatgtgg 1020 ataaaggacc agatcgtgcc
aaaaggcatg ccaatctga 1059 9 1026 DNA Arabidopsis thaliana 9
atggcttctt ccatgctgaa tggagctgaa accttcactc tcatccgagg tgttacccca
60 aaaagtattg gttttttggg gtcaggttta catgggaaac agttttccag
tgcgggttta 120 atctacagtc cgaagatgtc cagggtagga acgacgatag
ccccgaggtg cagcttatca 180 gcgtcaaggc cagcttcaca accaagattc
atacaacaca aaaaagaggc cttttggttc 240 tacaggttcc tctcaattgt
ctatgaccat gtcataaacc caggtcactg gactgaagac 300 atgagggatg
atgcacttga gccggctgat ctcaatgaca gggacatggt agttgtagat 360
gttggtggtg gaactggttt cactactttg ggtattgttc agcatgtgga tgctaagaat
420 gttacaatcc ttgaccaatc tcctcaccag cttgcaaagg ctaaacagaa
ggagcctctc 480 aaggaatgca acataattga aggtgatgca gaagatcttc
cttttcctac tgattatgcc 540 gatagatatg tgtctgctgg aagcatagag
tactggccag acccacaacg ggggatcaag 600 gaagcataca gggtgttgaa
acaaggagga aaagcttgct taattggtcc tgtgtaccct 660 acattttggt
tgtctcgttt ctttgcagac gtttggatgc ttttccctaa ggaggaagaa 720
tatatagagt ggtttgaaaa ggctggattt aaggatgtcc aactcaaaag gattggccct
780 aaatggtatc gtggagttcg ccgacatggt ttgatcatgg ggtgctctgt
aaccggtgtt 840 aaacccgcat ctggggactc tcctttgcag cttggaccta
aggcagagga tgtatcaaag 900 ccggtaaatc cgtttgtatt tctcttacgc
ttcatgttgg gtgccactgc agcagcatat 960 tatgtactgg ttcctatcta
catgtggctc aaagatcaaa ttgtaccaga gggtcaacca 1020 atctaa 1026 10
1035 DNA Arabidopsis thaliana 10 atggcttcct ccatgctcag cggagcagaa
agcctctcaa tgctccgaat ccaccaccaa 60 cccaaactca ccttctcgag
cccatccctc cattccaaac ccacaaacct caaaatggat 120 ctcatccctt
tcgccaccaa gcatcaaaaa acgaaaaaag cttcgatctt tacatgcagc 180
gcgtcctcat catcccgacc tgcttctcag ccgaggttca tccagcacaa gcaggaggcg
240 ttctggttct acaggttcct gtcgatagtg tacgaccatg tgataaaccc
agggcactgg 300 accgaggaca tgagagacga tgcgttggag ccagccgagc
tgtacgattc caggatgaag 360 gtggtggacg taggaggagg aactgggttc
accaccttgg ggattataaa gcacatcgac 420 cctaaaaacg ttacgattct
ggatcagtct ccgcatcagc ttgagaaggc taggcagaag 480 gaggctttga
aggagtgtac tattgttgaa ggtgatgctg aggatctccc ttttcctact 540
gatactttcg atcgatatgt atctgctggc agcatagaat actggccaga cccacaaaga
600 gggataaagg aagcataccg ggttctaaaa ctgggaggcg ttgcctgctt
gataggaccc 660 gtgcacccta ccttctggct ttccaggttc ttcgccgaca
tgtggatgtt gttccccacc 720 gaagaagaat acatagagtg gtttaaaaag
gccgggttca aagatgtgaa gttgaagagg 780 attggcccaa aatggtaccg
tggtgtgcgt agacacgggc tcatcatggg ctgttccgtc 840 actggtgtta
aacgtctctc tggtgactcc cctcttcagc ttggaccgaa ggcggaggat 900
gtgaagaagc cgatcaatcc attctcgttc cttctgcgct tcattttggg tacgatagca
960 gctacttact acgttttggt gccgatatac atgtggataa aggatcagat
tgtaccgaaa 1020 ggccagccca tatga 1035 11 1029 DNA Arabidopsis
thaliana 11 atgggttcag taatgctcag tggaactgaa aagctcactc tcagaaccct
aaccgggaac 60 ggcttaggtt tcactggttc ggatttgcac ggtaagaact
tcccaagagt gagtttcgct 120 gctaccacta gtgctaaagt tcccaacttt
agaagcatag tagtacccaa gtgtagtgtc 180 tcggcttcca ggccaagctc
gcagccaagg ttcattcagc acaaaaaaga ggccttttgg 240 ttctataggt
ttctctcaat tgtgtatgac catgtcatta accctggcca ttggaccgag 300
gacatgaggg atgatgccct tgaacccgct gatctcaatg acaggaacat gattgtggtg
360 gatgttggtg gcggcacggg tttcaccact cttggtattg tcaagcacgt
ggatgccaag 420 aatgtcacca ttcttgacca gtcaccccac cagctcgcca
aggccaagca gaaggagcca 480 ctcaaggaat gcaaaataat cgaaggggat
gccgaggatc tcccctttcg aactgattat 540 gccgatagat atgtatccgc
aggaagtatt gagtactggc cggatccaca gcgtggcatc 600 aaggaggcat
acagggtttt gaaacttgga ggcaaagcgt gtctaattgg tccggtctac 660
ccaacatttt ggttgtcacg tttctttgca gatgtttgga tgcttttccc caaggaggaa
720 gagtatattg agtggtttca gaaggcaggg tttaaggacg tccaactaaa
aaggattggc 780 ccaaaatggt atcgtggggt tcgccgtcat ggcttgatta
tgggttgttc agtgaccggt 840 gttaaacctg catctggaga ttctcctttg
cagcttggtc caaaggaaga agatgttgaa 900 aagcccgtta atccttttgt
ctttgcactg cgcttcgttt tgggtgcctt ggcagcgaca 960 tggtttgtgt
tggttcctat ttacatgtgg ctgaaagatc aagttgttcc caaaggtcag 1020
ccaatctaa 1029 12 1047 DNA Arabidopsis thaliana 12 atggcgatgg
cctcctccgc ctacgcccca gcgggcggcg ttggcaccca ctccgcgccg 60
ggcaggatca ggccgccgcg cggcctcggc ttctccacca ccaccaccaa gtcgaggccc
120 ctcgtgctca ccaggcgtgg gggaggcggc ggcaacatct ccgtggctcg
gctgaggtgc 180 gcggcgtcgt cgtcgtcggc ggcggcgagg ccgatgtcgc
agccgcggtt catccagcac 240 aagaaggagg cgttctggtt ctaccgcttc
ctctccatcg tctacgacca cgtcatcaac 300 ccgggccact ggacggagga
catgcgggac gacgccctcg agcccgccga cctctacagc 360 cgcaagctca
gggtcgtcga cgtcggcggc gggacggggt tcaccacgct cgggatcgtc 420
aagcgcgtcg acccggagaa cgtcacgctg ctcgaccagt ccccgcacca gctcgagaag
480 gcccgggaga aggaggccct caagggcgtc accatcatgg agggcgacgc
cgaggacctc 540 cccttcccca ccgacacctt cgaccgctac gtctccgccg
gcagcatcga gtattggccc 600 gatccgcagc gaggaatcaa ggaagcttac
agggttttga ggcttggtgg agtggcttgc 660 atgattggcc ccgtgcaccc
aaccttctgg ctgtctcgct ttttcgctga catgtggatg 720 ctcttcccga
aggaagagga gtatattgag tggttcaaaa aggcagggtt caaggatgtc 780
aagctcaaaa ggattggacc aaaatggtac cgtggtgtcc gaaggcatgg cctgattatg
840 ggatgctctg tgacgggcgt caaaagagaa catggagact cccctttgca
gcttggtcca 900 aaggttgagg atgtcagcaa acctgtgaat cctatcacct
tcctcttccg cttcctcatg 960 ggaacaatat gtgctgcata ctatgttctg
gtgcctatct acatgtggat aaaggaccag 1020 attgtgccca aaggcatgcc gatctaa
1047 13 1014 DNA Arabidopsis thaliana 13 atggcttctc tcatgctcaa
cggggccatc accttcccca agggattagg cttccccgct 60 tccaatctac
acgccagacc aagtcctccg ctgagtctcg tctcaaacac agccacgcgg 120
agactctccg tggcgacaag atgcagcagc agcagcagcg tgtcggcgtc aaggccatct
180 gcgcagccta ggttcatcca gcacaagaaa gaggcctact ggttctacag
gttcctgtcc 240 atcgtgtacg accacatcat caatcccggc cactggacgg
aggatatgag ggacgacgct 300 ctcgagcctg cggatctgag ccatccggac
atgcgagttg tcgacgtcgg aggcggaacg 360 ggtttcacca cgctgggaat
cgtcaagacg gtgaaggcta agaacgtgac gattctggac 420 cagtcgccgc
atcagctggc aaaggcgaag cagaaggagc cgttgaagga gtgcaagatc 480
gttgaaggag atgcggagga tctccctttt cctactgatt atgctgacag atacgtctct
540 gctggaagca ttgagtactg gcccgacccg cagaggggga taagggaagc
gtacagagtt 600 ctcaagatcg gtgggaaagc atgtctcatt ggccctgtcc
acccgacgtt ttggctttct 660 cgtttctttg cagatgtgtg gatgcttttc
cccaaggagg aggagtacat tgagtggttc 720 aagaatgctg gtttcaagga
cgttcagctt aagaggattg gccccaagtg gtaccgtggt 780 gttcgcaggc
acggacttat catgggatgc tctgttactg gtgtcaaacc tgcctctgga 840
gactctcctc
tccagcttgg accaaaggaa gaggacgtgg agaagcctgt aaacaatcct 900
ttctccttct tgggacgctt cctcttggga accttagcgg ctgcctggtt tgtgttaatc
960 ccaatctaca tgtggatcaa ggatcagatc gttcccaaag accaacccat ctga
1014 14 1014 DNA Arabidopsis thaliana 14 atggcttctc tcatgctcaa
cggggccatc accttcccca agggattagg cttccccgct 60 tccaatctac
acgccagacc aagtcctccg ctgagtctcg tctcaaacac agccacgcgg 120
agactctccg tggcgacaag atgcagcagc agcagcagcg tgtcggcgtc aaggccatct
180 gcgcagccta ggttcatcca gcacaagaaa gaggcctact ggttctacag
gttcctgtcc 240 atcgtgtacg accacatcat caatcccggc cactggacgg
aggatatgag ggacgacgct 300 ctcgagcctg cggatctgag ccatccggac
atgcgagttg tcgacgtcgg aggcggaacg 360 ggtttcacca cgctgggaat
cgtcaagacg gtgaaggcta agaacgtgac gattctggac 420 cagtcgccgc
atcagctggc aaaggcgaag cagaaggagc cgttgaagga gtgcaagatc 480
gtggaaggag atgcggagga tctccctttt cctactgatt atgctgacag atacgtctct
540 gctggaagca ttgagtactg gcccgacccg cagaggggta taagggaagc
gtacagagtt 600 ctcaagatcg gtgggaaagc atgtctcatt ggccctgtcc
acccgacgtt ttggctttca 660 cgcttctttg cagatgtgtg gatgcttttc
cccaaggagg aggagtacat tgagtggttc 720 aagaatgctg gtttcaagga
cgttcagctt aagaggattg gccccaagtg gtaccgtggt 780 gttcgcaggc
acggacttat catgggatgc tctgttactg gtgtcaaacc tgcctctgga 840
gactctcctc tccagcttgg accaaaggaa gaggacgtgg agaagcctgt aaacaatcct
900 ttctccttct tgggacgctt cctcttgggt accctagcgg ctgcctggtt
tgtgttaatc 960 ccaatctaca tgtggatcaa ggatcagatc gttcccaaag
accaacccat ctga 1014 15 1017 DNA Arabidopsis thaliana 15 atggcctctt
tgatgctcaa cggggccatt accttcccca aaggtttagg ttcccctggt 60
tccaatttgc atgccagatc gattcctcgg ccgaccttac tctcagttac ccgaacctcc
120 acacctagac tctcggtggc tactagatgc agcagcagca gcgtgtcgtc
ttcccggcca 180 tcggcgcaac ctaggttcat tcagcacaag aaggaggctt
actggttcta caggttctta 240 tccatcgtat acgaccatgt catcaatcct
gggcattgga ccgaggatat gagagacgac 300 gctcttgagc cagcggatct
cagccatccg gacatgcgag tggtcgatgt cggcggcgga 360 actggtttca
ctactctggg catagtcaag acagtgaagg ccaagaatgt gaccattctg 420
gaccagtcgc cacatcagct ggccaaagca aagcaaaagg agccgttgaa agaatgcaag
480 atcgtcgagg gagatgctga ggatcttcct tttccaaccg attatgctga
cagatacgtt 540 tctgctggaa gcattgagta ctggccggac ccgcagaggg
gaataaggga agcgtacagg 600 gttctcaaga tcggtggcaa agcgtgtctc
atcggccctg tctacccaac cttctggctc 660 tctcgcttct tttctgatgt
ctggatgctc ttccccaagg aggaagagta cattgagtgg 720 ttcaagaatg
ccggtttcaa ggacgttcag ctcaagagga ttggccccaa gtggtaccgt 780
ggtgttcgca ggcacggcct tatcatggga tgttctgtca ctggtgttaa acctgcctcc
840 ggtgactctc ctctccagct tggtccaaag gaagaggacg tagagaagcc
tgtcaacaac 900 cccttctcct tcttgggacg cttcctcctg ggaactctag
cagctgcctg gtttgtgtta 960 atccctatct acatgtggat caaggatcag
atcgttccca aagaccaacc catctga 1017 16 338 PRT Arabidopsis thaliana
16 Met Ala Ser Leu Met Leu Asn Gly Ala Ile Thr Phe Pro Lys Gly Leu
1 5 10 15 Gly Ser Pro Gly Ser Asn Leu His Ala Arg Ser Ile Pro Arg
Pro Thr 20 25 30 Leu Leu Ser Val Thr Arg Thr Ser Thr Pro Arg Leu
Ser Val Ala Thr 35 40 45 Arg Cys Ser Ser Ser Ser Val Ser Ser Ser
Arg Pro Ser Ala Gln Pro 50 55 60 Arg Phe Ile Gln His Lys Lys Glu
Ala Tyr Trp Phe Tyr Arg Phe Leu 65 70 75 80 Ser Ile Val Tyr Asp His
Val Ile Asn Pro Gly His Trp Thr Glu Asp 85 90 95 Met Arg Asp Asp
Ala Leu Glu Pro Ala Asp Leu Ser His Pro Asp Met 100 105 110 Arg Val
Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu Gly Ile 115 120 125
Val Lys Thr Val Lys Ala Lys Asn Val Thr Ile Leu Asp Gln Ser Pro 130
135 140 His Gln Leu Ala Lys Ala Lys Gln Lys Glu Pro Leu Lys Glu Cys
Lys 145 150 155 160 Ile Val Glu Gly Asp Ala Glu Asp Leu Pro Phe Pro
Thr Asp Tyr Ala 165 170 175 Asp Arg Tyr Val Ser Ala Gly Ser Ile Glu
Tyr Trp Pro Asp Pro Gln 180 185 190 Arg Gly Ile Arg Glu Ala Tyr Arg
Val Leu Lys Ile Gly Gly Lys Ala 195 200 205 Cys Leu Ile Gly Pro Val
Tyr Pro Thr Phe Trp Leu Ser Arg Phe Phe 210 215 220 Ser Asp Val Trp
Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile Glu Trp 225 230 235 240 Phe
Lys Asn Ala Gly Phe Lys Asp Val Gln Leu Lys Arg Ile Gly Pro 245 250
255 Lys Trp Tyr Arg Gly Val Arg Arg His Gly Leu Ile Met Gly Cys Ser
260 265 270 Val Thr Gly Val Lys Pro Ala Ser Gly Asp Ser Pro Leu Gln
Leu Gly 275 280 285 Pro Lys Glu Glu Asp Val Glu Lys Pro Val Asn Asn
Pro Phe Ser Phe 290 295 300 Leu Gly Arg Phe Leu Leu Gly Thr Leu Ala
Ala Ala Trp Phe Val Leu 305 310 315 320 Ile Pro Ile Tyr Met Trp Ile
Lys Asp Gln Ile Val Pro Lys Asp Gln 325 330 335 Pro Ile 17 338 PRT
Arabidopsis thaliana 17 Met Ala Ser Leu Met Leu Asn Gly Ala Ile Thr
Phe Pro Lys Gly Leu 1 5 10 15 Gly Ser Pro Gly Ser Asn Leu His Ala
Arg Ser Ile Pro Arg Pro Thr 20 25 30 Leu Leu Ser Val Thr Arg Thr
Ser Thr Pro Arg Leu Ser Val Ala Thr 35 40 45 Arg Cys Ser Ser Ser
Ser Val Ser Ser Ser Arg Pro Ser Ala Gln Pro 50 55 60 Arg Phe Ile
Gln His Lys Lys Glu Ala Tyr Trp Phe Tyr Arg Phe Leu 65 70 75 80 Ser
Ile Val Tyr Asp His Val Ile Asn Pro Gly His Trp Thr Glu Asp 85 90
95 Met Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu Ser His Pro Asp Met
100 105 110 Arg Val Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu
Gly Ile 115 120 125 Val Lys Thr Val Lys Ala Lys Asn Val Thr Ile Leu
Asp Gln Ser Pro 130 135 140 His Gln Leu Ala Lys Ala Lys Gln Lys Glu
Pro Leu Lys Glu Cys Lys 145 150 155 160 Ile Val Glu Gly Asp Ala Glu
Asp Leu Pro Phe Pro Thr Asp Tyr Ala 165 170 175 Asp Arg Tyr Val Ser
Ala Gly Ser Ile Glu Tyr Trp Pro Asp Pro Gln 180 185 190 Arg Gly Ile
Arg Glu Ala Tyr Arg Val Leu Lys Ile Gly Gly Lys Ala 195 200 205 Cys
Leu Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg Phe Phe 210 215
220 Ser Asp Val Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile Glu Trp
225 230 235 240 Phe Lys Asn Ala Gly Phe Lys Asp Val Gln Leu Lys Arg
Ile Gly Pro 245 250 255 Lys Trp Tyr Arg Gly Val Arg Arg His Gly Leu
Ile Met Gly Cys Ser 260 265 270 Val Thr Gly Val Lys Pro Ala Ser Gly
Asp Ser Pro Leu Gln Leu Gly 275 280 285 Pro Lys Glu Lys Asp Val Glu
Lys Pro Val Asn Asn Pro Phe Ser Phe 290 295 300 Leu Gly Arg Phe Leu
Leu Gly Thr Leu Ala Ala Ala Trp Phe Val Leu 305 310 315 320 Ile Pro
Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys Asp Gln 325 330 335
Pro Ile 18 338 PRT Arabidopsis thaliana 18 Met Ala Ser Leu Met Leu
Asn Gly Ala Ile Thr Phe Pro Lys Gly Leu 1 5 10 15 Gly Ser Pro Gly
Ser Asn Leu His Ala Arg Ser Ile Pro Arg Pro Thr 20 25 30 Leu Leu
Ser Val Thr Arg Thr Ser Thr Pro Arg Leu Ser Val Ala Thr 35 40 45
Arg Cys Ser Ser Ser Ser Val Ser Ser Ser Arg Pro Ser Ala Gln Pro 50
55 60 Arg Phe Ile Gln His Lys Lys Lys Ala Tyr Trp Phe Tyr Arg Phe
Leu 65 70 75 80 Ser Ile Val Tyr Asp His Val Ile Asn Pro Gly His Trp
Thr Glu Asp 85 90 95 Met Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu
Ser His Pro Asp Met 100 105 110 Arg Val Val Asp Val Gly Gly Gly Thr
Gly Phe Thr Thr Leu Gly Ile 115 120 125 Val Lys Thr Val Lys Ala Lys
Asn Val Thr Ile Leu Asp Gln Ser Pro 130 135 140 His Gln Leu Ala Lys
Ala Lys Gln Lys Glu Pro Leu Lys Glu Cys Lys 145 150 155 160 Ile Val
Glu Gly Asp Ala Glu Asp Leu Pro Phe Pro Thr Asp Tyr Ala 165 170 175
Asp Arg Tyr Val Ser Ala Gly Ser Ile Glu Tyr Trp Pro Asp Pro Gln 180
185 190 Arg Gly Ile Arg Glu Ala Tyr Arg Val Leu Lys Ile Gly Gly Lys
Ala 195 200 205 Cys Leu Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser
Arg Phe Phe 210 215 220 Ser Asp Val Trp Met Leu Phe Pro Lys Glu Glu
Glu Tyr Ile Glu Trp 225 230 235 240 Phe Lys Asn Ala Gly Phe Lys Asp
Val Gln Leu Lys Arg Ile Gly Pro 245 250 255 Lys Trp Tyr Arg Gly Val
Arg Arg His Gly Leu Ile Met Gly Cys Ser 260 265 270 Val Thr Gly Val
Lys Pro Ala Ser Gly Asp Ser Pro Leu Gln Leu Gly 275 280 285 Pro Lys
Glu Glu Asp Val Glu Lys Pro Val Asn Asn Pro Phe Ser Phe 290 295 300
Leu Gly Arg Phe Leu Leu Gly Thr Leu Ala Ala Ala Trp Phe Val Leu 305
310 315 320 Ile Pro Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys
Asp Gln 325 330 335 Pro Ile 19 338 PRT Arabidopsis thaliana 19 Met
Ala Ser Leu Met Leu Asn Gly Ala Ile Thr Phe Ser Lys Gly Leu 1 5 10
15 Gly Ser Pro Gly Ser Asn Leu His Ala Arg Ser Ile Pro Arg Pro Thr
20 25 30 Leu Leu Ser Val Thr Arg Thr Ser Thr Pro Arg Leu Ser Val
Ala Thr 35 40 45 Arg Cys Ser Ser Ser Ser Val Ser Ser Ser Arg Pro
Ser Ala Gln Pro 50 55 60 Arg Phe Ile Gln His Lys Lys Glu Ala Tyr
Trp Phe Tyr Arg Phe Leu 65 70 75 80 Ser Ile Val Tyr Asp His Val Ile
Asn Pro Gly His Trp Thr Glu Asp 85 90 95 Met Arg Asp Asp Ala Leu
Glu Pro Ala Asp Leu Ser His Pro Asp Met 100 105 110 Arg Val Val Asp
Val Gly Gly Gly Thr Gly Phe Thr Thr Leu Gly Ile 115 120 125 Val Lys
Thr Val Lys Ala Lys Asn Val Thr Ile Leu Asp Gln Ser Pro 130 135 140
His Gln Leu Ala Lys Ala Lys Gln Lys Glu Pro Leu Lys Glu Cys Lys 145
150 155 160 Ile Val Glu Gly Asp Ala Glu Asp Leu Pro Phe Pro Thr Asp
Tyr Ala 165 170 175 Asp Arg Tyr Val Ser Ala Gly Ser Ile Glu Tyr Trp
Pro Asp Pro Gln 180 185 190 Arg Gly Ile Arg Glu Ala Tyr Arg Val Leu
Lys Ile Gly Gly Lys Ala 195 200 205 Cys Leu Ile Gly Pro Val Tyr Pro
Thr Phe Trp Leu Ser Arg Phe Phe 210 215 220 Ser Asp Val Trp Met Leu
Phe Pro Lys Glu Glu Glu Tyr Ile Glu Trp 225 230 235 240 Phe Lys Asn
Ala Gly Phe Lys Asp Val Gln Leu Lys Arg Ile Gly Pro 245 250 255 Lys
Trp Tyr Arg Gly Val Arg Arg His Gly Leu Ile Met Gly Cys Ser 260 265
270 Val Thr Gly Val Lys Pro Ala Ser Gly Asp Ser Pro Leu Gln Leu Gly
275 280 285 Pro Lys Glu Glu Asp Val Glu Lys Pro Val Asn Asn Pro Phe
Ser Phe 290 295 300 Leu Gly Arg Phe Leu Leu Gly Thr Leu Ala Ala Ala
Trp Phe Val Leu 305 310 315 320 Ile Pro Ile Tyr Met Trp Ile Lys Asp
Gln Ile Val Pro Lys Asp Gln 325 330 335 Pro Ile 20 338 PRT
Arabidopsis thaliana 20 Met Ala Ser Leu Met Leu Asn Gly Ala Ile Thr
Phe Pro Lys Gly Leu 1 5 10 15 Gly Ser Pro Gly Ser Asn Leu His Ala
Arg Ser Ile Pro Arg Pro Thr 20 25 30 Leu Leu Ser Val Thr Arg Thr
Ser Thr Pro Arg Leu Ser Val Ala Thr 35 40 45 Arg Cys Ser Ser Ser
Ser Val Ser Ser Ser Arg Pro Ser Ala Gln Pro 50 55 60 Arg Phe Ile
Gln His Lys Lys Glu Ala Tyr Trp Phe Tyr Arg Phe Leu 65 70 75 80 Ser
Ile Val Tyr Asp His Val Ile Asn Pro Gly His Trp Thr Glu Asp 85 90
95 Met Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu Ser His Pro Asp Met
100 105 110 Arg Val Val Asn Val Gly Gly Gly Thr Gly Phe Thr Thr Leu
Gly Ile 115 120 125 Val Lys Thr Val Lys Ala Lys Asn Val Thr Ile Leu
Asp Gln Ser Pro 130 135 140 His Gln Leu Ala Lys Ala Lys Gln Lys Glu
Pro Leu Lys Glu Cys Lys 145 150 155 160 Ile Val Glu Gly Asp Ala Glu
Asp Leu Pro Phe Pro Thr Asp Tyr Ala 165 170 175 Asp Arg Tyr Val Ser
Ala Gly Ser Ile Glu Tyr Trp Pro Asp Pro Gln 180 185 190 Arg Gly Ile
Arg Glu Ala Tyr Arg Val Leu Lys Ile Gly Gly Lys Ala 195 200 205 Cys
Leu Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg Phe Phe 210 215
220 Ser Asp Val Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile Glu Trp
225 230 235 240 Phe Lys Asn Ala Gly Phe Lys Asp Val Gln Leu Lys Arg
Ile Gly Pro 245 250 255 Lys Trp Tyr Arg Gly Val Arg Arg His Gly Leu
Ile Met Gly Cys Ser 260 265 270 Val Thr Gly Val Lys Pro Ala Ser Gly
Asp Ser Pro Leu Gln Leu Gly 275 280 285 Pro Lys Glu Glu Asp Val Glu
Lys Pro Val Asn Asn Pro Phe Ser Phe 290 295 300 Leu Gly Arg Phe Leu
Leu Gly Thr Leu Ala Ala Ala Trp Phe Val Leu 305 310 315 320 Ile Pro
Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys Asp Gln 325 330 335
Pro Ile 21 338 PRT Arabidopsis thaliana 21 Met Ala Ser Leu Met Leu
Asn Gly Ala Ile Thr Phe Pro Lys Gly Leu 1 5 10 15 Gly Ser Pro Gly
Ser Asn Leu His Ala Arg Ser Ile Pro Arg Pro Thr 20 25 30 Leu Leu
Ser Val Thr Arg Thr Ser Thr Pro Arg Leu Ser Val Ala Thr 35 40 45
Arg Cys Ser Ser Ser Ser Val Ser Ser Ser Arg Pro Ser Ala Gln Pro 50
55 60 Arg Phe Ile Gln His Lys Lys Glu Ala Tyr Trp Phe Tyr Arg Phe
Leu 65 70 75 80 Ser Ile Val Tyr Asp His Val Ile Asn Pro Gly His Trp
Ile Glu Asp 85 90 95 Met Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu
Ser His Pro Asp Met 100 105 110 Arg Val Val Asp Val Gly Gly Gly Thr
Gly Phe Thr Thr Leu Gly Ile 115 120 125 Val Lys Thr Val Lys Ala Lys
Asn Val Thr Ile Leu Asp Gln Ser Pro 130 135 140 His Gln Leu Ala Lys
Ala Lys Gln Lys Glu Pro Leu Lys Glu Cys Lys 145 150 155 160 Ile Val
Glu Gly Asp Ala Glu Asp Leu Pro Phe Pro Thr Asp Tyr Ala 165 170 175
Asp Arg Tyr Val Ser Ala Gly Ser Ile Glu Tyr Trp Pro Asp Pro Gln 180
185 190 Arg Gly Ile Arg Glu Ala Tyr Arg Val Leu Lys Ile Gly Gly Lys
Ala 195 200 205 Cys Leu Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser
Arg Phe Phe 210 215 220 Ser Asp Val Trp Met Leu Phe Pro Lys Glu Glu
Glu Tyr Ile Glu Trp 225 230 235 240 Phe Lys Asn Ala Gly Phe Lys Asp
Val Gln Leu Lys Arg Ile Gly Pro 245 250 255 Lys Trp Tyr Arg Gly Val
Arg Arg His Gly Leu Ile Met Gly Cys Ser 260 265 270 Val Thr Gly Val
Lys Pro Ala Ser Gly Asp Ser Pro Leu Gln Leu Gly 275 280 285 Pro Lys
Glu Glu Asp Val Glu Lys Pro Val Asn Asn Pro Phe Ser Phe 290 295 300
Leu Gly Arg Phe Leu Leu Gly Thr Leu Ala Ala Ala Trp Phe Val Leu 305
310 315 320 Ile Pro Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys
Asp Gln 325 330 335 Pro Ile 22 352 PRT Arabidopsis thaliana 22 Met
Ala Met Ala Ser Thr Tyr Ala Pro Gly Gly Gly Ala Arg Ala Leu 1 5 10
15 Ala Gln Gly Arg Cys Arg Val Arg Gly Pro Ala Gly Leu Gly
Phe Leu 20 25 30 Gly Pro Ser Lys Ala Ala Gly Leu Pro Arg Pro Leu
Ala Leu Ala Leu 35 40 45 Ala Arg Arg Met Ser Ser Pro Val Ala Val
Gly Ala Arg Leu Arg Cys 50 55 60 Ala Ala Ser Ser Ser Pro Ala Ala
Ala Arg Pro Ala Thr Ala Pro Arg 65 70 75 80 Phe Ile Gln His Lys Lys
Glu Ala Phe Trp Phe Tyr Arg Phe Leu Ser 85 90 95 Ile Val Tyr Asp
His Val Ile Asn Pro Gly His Trp Thr Glu Asp Met 100 105 110 Arg Asp
Asp Ala Leu Glu Pro Ala Asp Leu Phe Ser Arg His Leu Thr 115 120 125
Val Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu Gly Ile Val 130
135 140 Lys His Val Asn Pro Glu Asn Val Thr Leu Leu Asp Gln Ser Pro
His 145 150 155 160 Gln Leu Asp Lys Ala Arg Gln Lys Glu Ala Leu Lys
Gly Val Thr Ile 165 170 175 Met Glu Gly Asp Ala Glu Asp Leu Pro Phe
Pro Thr Asp Ser Phe Asp 180 185 190 Arg Tyr Ile Ser Ala Gly Ser Ile
Glu Tyr Trp Pro Asp Pro Gln Arg 195 200 205 Gly Ile Lys Glu Ala Tyr
Arg Val Leu Arg Phe Gly Gly Leu Ala Cys 210 215 220 Val Ile Gly Pro
Val Tyr Pro Thr Phe Trp Leu Ser Arg Phe Phe Ala 225 230 235 240 Asp
Met Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile Glu Trp Phe 245 250
255 Lys Lys Ala Gly Phe Arg Asp Val Lys Leu Lys Arg Ile Gly Pro Lys
260 265 270 Trp Tyr Arg Gly Val Arg Arg His Gly Leu Ile Met Gly Cys
Ser Val 275 280 285 Thr Gly Val Lys Arg Glu Arg Gly Asp Ser Pro Leu
Glu Leu Gly Pro 290 295 300 Lys Ala Glu Asp Val Ser Lys Pro Val Asn
Pro Ile Thr Phe Leu Phe 305 310 315 320 Arg Phe Leu Val Gly Thr Ile
Cys Ala Ala Tyr Tyr Val Leu Val Pro 325 330 335 Ile Tyr Met Trp Ile
Lys Asp Gln Ile Val Pro Lys Gly Met Pro Ile 340 345 350 23 341 PRT
Arabidopsis thaliana 23 Met Ala Ser Ser Met Leu Asn Gly Ala Glu Thr
Phe Thr Leu Ile Arg 1 5 10 15 Gly Val Thr Pro Lys Ser Ile Gly Phe
Leu Gly Ser Gly Leu His Gly 20 25 30 Lys Gln Phe Ser Ser Ala Gly
Leu Ile Tyr Ser Pro Lys Met Ser Arg 35 40 45 Val Gly Thr Thr Ile
Ala Pro Arg Cys Ser Leu Ser Ala Ser Arg Pro 50 55 60 Ala Ser Gln
Pro Arg Phe Ile Gln His Lys Lys Glu Ala Phe Trp Phe 65 70 75 80 Tyr
Arg Phe Leu Ser Ile Val Tyr Asp His Val Ile Asn Pro Gly His 85 90
95 Trp Thr Glu Asp Met Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu Asn
100 105 110 Asp Arg Asp Met Val Val Val Asp Val Gly Gly Gly Thr Gly
Phe Thr 115 120 125 Thr Leu Gly Ile Val Gln His Val Asp Ala Lys Asn
Val Thr Ile Leu 130 135 140 Asp Gln Ser Pro His Gln Leu Ala Lys Ala
Lys Gln Lys Glu Pro Leu 145 150 155 160 Lys Glu Cys Asn Ile Ile Glu
Gly Asp Ala Glu Asp Leu Pro Phe Pro 165 170 175 Thr Asp Tyr Ala Asp
Arg Tyr Val Ser Ala Gly Ser Ile Glu Tyr Trp 180 185 190 Pro Asp Pro
Gln Arg Gly Ile Lys Glu Ala Tyr Arg Val Leu Lys Gln 195 200 205 Gly
Gly Lys Ala Cys Leu Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu 210 215
220 Ser Arg Phe Phe Ala Asp Val Trp Met Leu Phe Pro Lys Glu Glu Glu
225 230 235 240 Tyr Ile Glu Trp Phe Glu Lys Ala Gly Phe Lys Asp Val
Gln Leu Lys 245 250 255 Arg Ile Gly Pro Lys Trp Tyr Arg Gly Val Arg
Arg His Gly Leu Ile 260 265 270 Met Gly Cys Ser Val Thr Gly Val Lys
Pro Ala Ser Gly Asp Ser Pro 275 280 285 Leu Gln Leu Gly Pro Lys Ala
Glu Asp Val Ser Lys Pro Val Asn Pro 290 295 300 Phe Val Phe Leu Leu
Arg Phe Met Leu Gly Ala Thr Ala Ala Ala Tyr 305 310 315 320 Tyr Val
Leu Val Pro Ile Tyr Met Trp Leu Lys Asp Gln Ile Val Pro 325 330 335
Glu Gly Gln Pro Ile 340 24 344 PRT Arabidopsis thaliana 24 Met Ala
Ser Ser Met Leu Ser Gly Ala Glu Ser Leu Ser Met Leu Arg 1 5 10 15
Ile His His Gln Pro Lys Leu Thr Phe Ser Ser Pro Ser Leu His Ser 20
25 30 Lys Pro Thr Asn Leu Lys Met Asp Leu Ile Pro Phe Ala Thr Lys
His 35 40 45 Gln Lys Thr Lys Lys Ala Ser Ile Phe Thr Cys Ser Ala
Ser Ser Ser 50 55 60 Ser Arg Pro Ala Ser Gln Pro Arg Phe Ile Gln
His Lys Gln Glu Ala 65 70 75 80 Phe Trp Phe Tyr Arg Phe Leu Ser Ile
Val Tyr Asp His Val Ile Asn 85 90 95 Pro Gly His Trp Thr Glu Asp
Met Arg Asp Asp Ala Leu Glu Pro Ala 100 105 110 Glu Leu Tyr Asp Ser
Arg Met Lys Val Val Asp Val Gly Gly Gly Thr 115 120 125 Gly Phe Thr
Thr Leu Gly Ile Ile Lys His Ile Asp Pro Lys Asn Val 130 135 140 Thr
Ile Leu Asp Gln Ser Pro His Gln Leu Glu Lys Ala Arg Gln Lys 145 150
155 160 Glu Ala Leu Lys Glu Cys Thr Ile Val Glu Gly Asp Ala Glu Asp
Leu 165 170 175 Pro Phe Pro Thr Asp Thr Phe Asp Arg Tyr Val Ser Ala
Gly Ser Ile 180 185 190 Glu Tyr Trp Pro Asp Pro Gln Arg Gly Ile Lys
Glu Ala Tyr Arg Val 195 200 205 Leu Lys Leu Gly Gly Val Ala Cys Leu
Ile Gly Pro Val His Pro Thr 210 215 220 Phe Trp Leu Ser Arg Phe Phe
Ala Asp Met Trp Met Leu Phe Pro Thr 225 230 235 240 Glu Glu Glu Tyr
Ile Glu Trp Phe Lys Lys Ala Gly Phe Lys Asp Val 245 250 255 Lys Leu
Lys Arg Ile Gly Pro Lys Trp Tyr Arg Gly Val Arg Arg His 260 265 270
Gly Leu Ile Met Gly Cys Ser Val Thr Gly Val Lys Arg Leu Ser Gly 275
280 285 Asp Ser Pro Leu Gln Leu Gly Pro Lys Ala Glu Asp Val Lys Lys
Pro 290 295 300 Ile Asn Pro Phe Ser Phe Leu Leu Arg Phe Ile Leu Gly
Thr Ile Ala 305 310 315 320 Ala Thr Tyr Tyr Val Leu Val Pro Ile Tyr
Met Trp Ile Lys Asp Gln 325 330 335 Ile Val Pro Lys Gly Gln Pro Ile
340 25 342 PRT Arabidopsis thaliana 25 Met Gly Ser Val Met Leu Ser
Gly Thr Glu Lys Leu Thr Leu Arg Thr 1 5 10 15 Leu Thr Gly Asn Gly
Leu Gly Phe Thr Gly Ser Asp Leu His Gly Lys 20 25 30 Asn Phe Pro
Arg Val Ser Phe Ala Ala Thr Thr Ser Ala Lys Val Pro 35 40 45 Asn
Phe Arg Ser Ile Val Val Pro Lys Cys Ser Val Ser Ala Ser Arg 50 55
60 Pro Ser Ser Gln Pro Arg Phe Ile Gln His Lys Lys Glu Ala Phe Trp
65 70 75 80 Phe Tyr Arg Phe Leu Ser Ile Val Tyr Asp His Val Ile Asn
Pro Gly 85 90 95 His Trp Thr Glu Asp Met Arg Asp Asp Ala Leu Glu
Pro Ala Asp Leu 100 105 110 Asn Asp Arg Asn Met Ile Val Val Asp Val
Gly Gly Gly Thr Gly Phe 115 120 125 Thr Thr Leu Gly Ile Val Lys His
Val Asp Ala Lys Asn Val Thr Ile 130 135 140 Leu Asp Gln Ser Pro His
Gln Leu Ala Lys Ala Lys Gln Lys Glu Pro 145 150 155 160 Leu Lys Glu
Cys Lys Ile Ile Glu Gly Asp Ala Glu Asp Leu Pro Phe 165 170 175 Arg
Thr Asp Tyr Ala Asp Arg Tyr Val Ser Ala Gly Ser Ile Glu Tyr 180 185
190 Trp Pro Asp Pro Gln Arg Gly Ile Lys Glu Ala Tyr Arg Val Leu Lys
195 200 205 Leu Gly Gly Lys Ala Cys Leu Ile Gly Pro Val Tyr Pro Thr
Phe Trp 210 215 220 Leu Ser Arg Phe Phe Ala Asp Val Trp Met Leu Phe
Pro Lys Glu Glu 225 230 235 240 Glu Tyr Ile Glu Trp Phe Gln Lys Ala
Gly Phe Lys Asp Val Gln Leu 245 250 255 Lys Arg Ile Gly Pro Lys Trp
Tyr Arg Gly Val Arg Arg His Gly Leu 260 265 270 Ile Met Gly Cys Ser
Val Thr Gly Val Lys Pro Ala Ser Gly Asp Ser 275 280 285 Pro Leu Gln
Leu Gly Pro Lys Glu Glu Asp Val Glu Lys Pro Val Asn 290 295 300 Pro
Phe Val Phe Ala Leu Arg Phe Val Leu Gly Ala Leu Ala Ala Thr 305 310
315 320 Trp Phe Val Leu Val Pro Ile Tyr Met Trp Leu Lys Asp Gln Val
Val 325 330 335 Pro Lys Gly Gln Pro Ile 340 26 348 PRT Arabidopsis
thaliana 26 Met Ala Met Ala Ser Ser Ala Tyr Ala Pro Ala Gly Gly Val
Gly Thr 1 5 10 15 His Ser Ala Pro Gly Arg Ile Arg Pro Pro Arg Gly
Leu Gly Phe Ser 20 25 30 Thr Thr Thr Thr Lys Ser Arg Pro Leu Val
Leu Thr Arg Arg Gly Gly 35 40 45 Gly Gly Gly Asn Ile Ser Val Ala
Arg Leu Arg Cys Ala Ala Ser Ser 50 55 60 Ser Ser Ala Ala Ala Arg
Pro Met Ser Gln Pro Arg Phe Ile Gln His 65 70 75 80 Lys Lys Glu Ala
Phe Trp Phe Tyr Arg Phe Leu Ser Ile Val Tyr Asp 85 90 95 His Val
Ile Asn Pro Gly His Trp Thr Glu Asp Met Arg Asp Asp Ala 100 105 110
Leu Glu Pro Ala Asp Leu Tyr Ser Arg Lys Leu Arg Val Val Asp Val 115
120 125 Gly Gly Gly Thr Gly Phe Thr Thr Leu Gly Ile Val Lys Arg Val
Asp 130 135 140 Pro Glu Asn Val Thr Leu Leu Asp Gln Ser Pro His Gln
Leu Glu Lys 145 150 155 160 Ala Arg Glu Lys Glu Ala Leu Lys Gly Val
Thr Ile Met Glu Gly Asp 165 170 175 Ala Glu Asp Leu Pro Phe Pro Thr
Asp Thr Phe Asp Arg Tyr Val Ser 180 185 190 Ala Gly Ser Ile Glu Tyr
Trp Pro Asp Pro Gln Arg Gly Ile Lys Glu 195 200 205 Ala Tyr Arg Val
Leu Arg Leu Gly Gly Val Ala Cys Met Ile Gly Pro 210 215 220 Val His
Pro Thr Phe Trp Leu Ser Arg Phe Phe Ala Asp Met Trp Met 225 230 235
240 Leu Phe Pro Lys Glu Glu Glu Tyr Ile Glu Trp Phe Lys Lys Ala Gly
245 250 255 Phe Lys Asp Val Lys Leu Lys Arg Ile Gly Pro Lys Trp Tyr
Arg Gly 260 265 270 Val Arg Arg His Gly Leu Ile Met Gly Cys Ser Val
Thr Gly Val Lys 275 280 285 Arg Glu His Gly Asp Ser Pro Leu Gln Leu
Gly Pro Lys Val Glu Asp 290 295 300 Val Ser Lys Pro Val Asn Pro Ile
Thr Phe Leu Phe Arg Phe Leu Met 305 310 315 320 Gly Thr Ile Cys Ala
Ala Tyr Tyr Val Leu Val Pro Ile Tyr Met Trp 325 330 335 Ile Lys Asp
Gln Ile Val Pro Lys Gly Met Pro Ile 340 345 27 337 PRT Arabidopsis
thaliana 27 Met Ala Ser Leu Met Leu Asn Gly Ala Ile Thr Phe Pro Lys
Gly Leu 1 5 10 15 Gly Phe Pro Ala Ser Asn Leu His Ala Arg Pro Ser
Pro Pro Leu Ser 20 25 30 Leu Val Ser Asn Thr Ala Thr Arg Arg Leu
Ser Val Ala Thr Arg Cys 35 40 45 Ser Ser Ser Ser Ser Val Ser Ala
Ser Arg Pro Ser Ala Gln Pro Arg 50 55 60 Phe Ile Gln His Lys Lys
Glu Ala Tyr Trp Phe Tyr Arg Phe Leu Ser 65 70 75 80 Ile Val Tyr Asp
His Ile Ile Asn Pro Gly His Trp Thr Glu Asp Met 85 90 95 Arg Asp
Asp Ala Leu Glu Pro Ala Asp Leu Ser His Pro Asp Met Arg 100 105 110
Val Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu Gly Ile Val 115
120 125 Lys Thr Val Lys Ala Lys Asn Val Thr Ile Leu Asp Gln Ser Pro
His 130 135 140 Gln Leu Ala Lys Ala Lys Gln Lys Glu Pro Leu Lys Glu
Cys Lys Ile 145 150 155 160 Val Glu Gly Asp Ala Glu Asp Leu Pro Phe
Pro Thr Asp Tyr Ala Asp 165 170 175 Arg Tyr Val Ser Ala Gly Ser Ile
Glu Tyr Trp Pro Asp Pro Gln Arg 180 185 190 Gly Ile Arg Glu Ala Tyr
Arg Val Leu Lys Ile Gly Gly Lys Ala Cys 195 200 205 Leu Ile Gly Pro
Val His Pro Thr Phe Trp Leu Ser Arg Phe Phe Ala 210 215 220 Asp Val
Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile Glu Trp Phe 225 230 235
240 Lys Asn Ala Gly Phe Lys Asp Val Gln Leu Lys Arg Ile Gly Pro Lys
245 250 255 Trp Tyr Arg Gly Val Arg Arg His Gly Leu Ile Met Gly Cys
Ser Val 260 265 270 Thr Gly Val Lys Pro Ala Ser Gly Asp Ser Pro Leu
Gln Leu Gly Pro 275 280 285 Lys Glu Glu Asp Val Glu Lys Pro Val Asn
Asn Pro Phe Ser Phe Leu 290 295 300 Gly Arg Phe Leu Leu Gly Thr Leu
Ala Ala Ala Trp Phe Val Leu Ile 305 310 315 320 Pro Ile Tyr Met Trp
Ile Lys Asp Gln Ile Val Pro Lys Asp Gln Pro 325 330 335 Ile 28 292
PRT Arabidopsis thaliana 28 Ala Thr Arg Cys Ser Ser Ser Ser Val Ser
Ser Ser Arg Pro Ser Ala 1 5 10 15 Gln Pro Arg Phe Ile Gln His Lys
Lys Glu Ala Tyr Trp Phe Tyr Arg 20 25 30 Phe Leu Ser Ile Val Tyr
Asp His Val Ile Asn Pro Gly His Trp Thr 35 40 45 Glu Asp Met Arg
Asp Asp Ala Leu Glu Pro Ala Asp Leu Ser His Pro 50 55 60 Asp Met
Arg Val Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu 65 70 75 80
Gly Ile Val Lys Thr Val Lys Ala Lys Asn Val Thr Ile Leu Asp Gln 85
90 95 Ser Pro His Gln Leu Ala Lys Ala Lys Gln Lys Glu Pro Leu Lys
Glu 100 105 110 Cys Lys Ile Val Glu Gly Asp Ala Glu Asp Leu Pro Phe
Pro Thr Asp 115 120 125 Tyr Ala Asp Arg Tyr Val Ser Ala Gly Ser Ile
Glu Tyr Trp Pro Asp 130 135 140 Pro Gln Arg Gly Ile Arg Glu Ala Tyr
Arg Val Leu Lys Ile Gly Gly 145 150 155 160 Lys Ala Cys Leu Ile Gly
Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg 165 170 175 Phe Phe Ser Asp
Val Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile 180 185 190 Glu Trp
Phe Lys Asn Ala Gly Phe Lys Asp Val Gln Leu Lys Arg Ile 195 200 205
Gly Pro Lys Trp Tyr Arg Gly Val Arg Arg His Gly Leu Ile Met Gly 210
215 220 Cys Ser Val Thr Gly Val Lys Pro Ala Ser Gly Asp Ser Pro Leu
Gln 225 230 235 240 Leu Gly Pro Lys Glu Glu Asp Val Glu Lys Pro Val
Asn Asn Pro Phe 245 250 255 Ser Phe Leu Gly Arg Phe Leu Leu Gly Thr
Leu Ala Ala Ala Trp Phe 260 265 270 Val Leu Ile Pro Ile Tyr Met Trp
Ile Lys Asp Gln Ile Val Pro Lys 275 280 285 Asp Gln Pro Ile 290 29
292 PRT Arabidopsis thaliana 29 Ala Thr Arg Cys Ser Ser Ser Ser Val
Ser Ser Ser Arg Pro Ser Ala 1 5 10 15 Gln Pro Arg Phe Ile Gln His
Lys Lys Glu Ala Tyr Trp Phe Tyr Arg 20 25 30 Phe Leu Ser Ile Val
Tyr Asp His Val Ile Asn Pro Gly His Trp Thr 35 40 45 Glu Asp Met
Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu Ser His Pro 50 55 60 Asp
Met Arg Val Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu 65 70
75 80 Gly Ile Val Lys Thr Val Lys Ala Lys Asn Val Thr Ile Leu
Asp
Gln 85 90 95 Ser Pro His Gln Leu Ala Lys Ala Lys Gln Lys Glu Pro
Leu Lys Glu 100 105 110 Cys Lys Ile Val Glu Gly Asp Ala Glu Asp Leu
Pro Phe Pro Thr Asp 115 120 125 Tyr Ala Asp Arg Tyr Val Ser Ala Gly
Ser Ile Glu Tyr Trp Pro Asp 130 135 140 Pro Gln Arg Gly Ile Arg Glu
Ala Tyr Arg Val Leu Lys Ile Gly Gly 145 150 155 160 Lys Ala Cys Leu
Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg 165 170 175 Phe Phe
Ser Asp Val Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile 180 185 190
Glu Trp Phe Lys Asn Ala Gly Phe Lys Asp Val Gln Leu Lys Arg Ile 195
200 205 Gly Pro Lys Trp Tyr Arg Gly Val Arg Arg His Gly Leu Ile Met
Gly 210 215 220 Cys Ser Val Thr Gly Val Lys Pro Ala Ser Gly Asp Ser
Pro Leu Gln 225 230 235 240 Leu Gly Pro Lys Glu Lys Asp Val Glu Lys
Pro Val Asn Asn Pro Phe 245 250 255 Ser Phe Leu Gly Arg Phe Leu Leu
Gly Thr Leu Ala Ala Ala Trp Phe 260 265 270 Val Leu Ile Pro Ile Tyr
Met Trp Ile Lys Asp Gln Ile Val Pro Lys 275 280 285 Asp Gln Pro Ile
290 30 292 PRT Arabidopsis thaliana 30 Ala Thr Arg Cys Ser Ser Ser
Ser Val Ser Ser Ser Arg Pro Ser Ala 1 5 10 15 Gln Pro Arg Phe Ile
Gln His Lys Lys Lys Ala Tyr Trp Phe Tyr Arg 20 25 30 Phe Leu Ser
Ile Val Tyr Asp His Val Ile Asn Pro Gly His Trp Thr 35 40 45 Glu
Asp Met Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu Ser His Pro 50 55
60 Asp Met Arg Val Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu
65 70 75 80 Gly Ile Val Lys Thr Val Lys Ala Lys Asn Val Thr Ile Leu
Asp Gln 85 90 95 Ser Pro His Gln Leu Ala Lys Ala Lys Gln Lys Glu
Pro Leu Lys Glu 100 105 110 Cys Lys Ile Val Glu Gly Asp Ala Glu Asp
Leu Pro Phe Pro Thr Asp 115 120 125 Tyr Ala Asp Arg Tyr Val Ser Ala
Gly Ser Ile Glu Tyr Trp Pro Asp 130 135 140 Pro Gln Arg Gly Ile Arg
Glu Ala Tyr Arg Val Leu Lys Ile Gly Gly 145 150 155 160 Lys Ala Cys
Leu Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg 165 170 175 Phe
Phe Ser Asp Val Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile 180 185
190 Glu Trp Phe Lys Asn Ala Gly Phe Lys Asp Val Gln Leu Lys Arg Ile
195 200 205 Gly Pro Lys Trp Tyr Arg Gly Val Arg Arg His Gly Leu Ile
Met Gly 210 215 220 Cys Ser Val Thr Gly Val Lys Pro Ala Ser Gly Asp
Ser Pro Leu Gln 225 230 235 240 Leu Gly Pro Lys Glu Glu Asp Val Glu
Lys Pro Val Asn Asn Pro Phe 245 250 255 Ser Phe Leu Gly Arg Phe Leu
Leu Gly Thr Leu Ala Ala Ala Trp Phe 260 265 270 Val Leu Ile Pro Ile
Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys 275 280 285 Asp Gln Pro
Ile 290 31 292 PRT Arabidopsis thaliana 31 Ala Thr Arg Cys Ser Ser
Ser Ser Val Ser Ser Ser Arg Pro Ser Ala 1 5 10 15 Gln Pro Arg Phe
Ile Gln His Lys Lys Glu Ala Tyr Trp Phe Tyr Arg 20 25 30 Phe Leu
Ser Ile Val Tyr Asp His Val Ile Asn Pro Gly His Trp Thr 35 40 45
Glu Asp Met Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu Ser His Pro 50
55 60 Asp Met Arg Val Val Asn Val Gly Gly Gly Thr Gly Phe Thr Thr
Leu 65 70 75 80 Gly Ile Val Lys Thr Val Lys Ala Lys Asn Val Thr Ile
Leu Asp Gln 85 90 95 Ser Pro His Gln Leu Ala Lys Ala Lys Gln Lys
Glu Pro Leu Lys Glu 100 105 110 Cys Lys Ile Val Glu Gly Asp Ala Glu
Asp Leu Pro Phe Pro Thr Asp 115 120 125 Tyr Ala Asp Arg Tyr Val Ser
Ala Gly Ser Ile Glu Tyr Trp Pro Asp 130 135 140 Pro Gln Arg Gly Ile
Arg Glu Ala Tyr Arg Val Leu Lys Ile Gly Gly 145 150 155 160 Lys Ala
Cys Leu Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg 165 170 175
Phe Phe Ser Asp Val Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile 180
185 190 Glu Trp Phe Lys Asn Ala Gly Phe Lys Asp Val Gln Leu Lys Arg
Ile 195 200 205 Gly Pro Lys Trp Tyr Arg Gly Val Arg Arg His Gly Leu
Ile Met Gly 210 215 220 Cys Ser Val Thr Gly Val Lys Pro Ala Ser Gly
Asp Ser Pro Leu Gln 225 230 235 240 Leu Gly Pro Lys Glu Glu Asp Val
Glu Lys Pro Val Asn Asn Pro Phe 245 250 255 Ser Phe Leu Gly Arg Phe
Leu Leu Gly Thr Leu Ala Ala Ala Trp Phe 260 265 270 Val Leu Ile Pro
Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys 275 280 285 Asp Gln
Pro Ile 290 32 292 PRT Arabidopsis thaliana 32 Ala Thr Arg Cys Ser
Ser Ser Ser Val Ser Ser Ser Arg Pro Ser Ala 1 5 10 15 Gln Pro Arg
Phe Ile Gln His Lys Lys Glu Ala Tyr Trp Phe Tyr Arg 20 25 30 Phe
Leu Ser Ile Val Tyr Asp His Val Ile Asn Pro Gly His Trp Ile 35 40
45 Glu Asp Met Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu Ser His Pro
50 55 60 Asp Met Arg Val Val Asp Val Gly Gly Gly Thr Gly Phe Thr
Thr Leu 65 70 75 80 Gly Ile Val Lys Thr Val Lys Ala Lys Asn Val Thr
Ile Leu Asp Gln 85 90 95 Ser Pro His Gln Leu Ala Lys Ala Lys Gln
Lys Glu Pro Leu Lys Glu 100 105 110 Cys Lys Ile Val Glu Gly Asp Ala
Glu Asp Leu Pro Phe Pro Thr Asp 115 120 125 Tyr Ala Asp Arg Tyr Val
Ser Ala Gly Ser Ile Glu Tyr Trp Pro Asp 130 135 140 Pro Gln Arg Gly
Ile Arg Glu Ala Tyr Arg Val Leu Lys Ile Gly Gly 145 150 155 160 Lys
Ala Cys Leu Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg 165 170
175 Phe Phe Ser Asp Val Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile
180 185 190 Glu Trp Phe Lys Asn Ala Gly Phe Lys Asp Val Gln Leu Lys
Arg Ile 195 200 205 Gly Pro Lys Trp Tyr Arg Gly Val Arg Arg His Gly
Leu Ile Met Gly 210 215 220 Cys Ser Val Thr Gly Val Lys Pro Ala Ser
Gly Asp Ser Pro Leu Gln 225 230 235 240 Leu Gly Pro Lys Glu Glu Asp
Val Glu Lys Pro Val Asn Asn Pro Phe 245 250 255 Ser Phe Leu Gly Arg
Phe Leu Leu Gly Thr Leu Ala Ala Ala Trp Phe 260 265 270 Val Leu Ile
Pro Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys 275 280 285 Asp
Gln Pro Ile 290 33 293 PRT Arabidopsis thaliana 33 Ala Thr Arg Cys
Ser Ser Ser Ser Ser Val Ser Ala Ser Arg Pro Ser 1 5 10 15 Ala Gln
Pro Arg Phe Ile Gln His Lys Lys Glu Ala Tyr Trp Phe Tyr 20 25 30
Arg Phe Leu Ser Ile Val Tyr Asp His Ile Ile Asn Pro Gly His Trp 35
40 45 Thr Glu Asp Met Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu Ser
His 50 55 60 Pro Asp Met Arg Val Val Asp Val Gly Gly Gly Thr Gly
Phe Thr Thr 65 70 75 80 Leu Gly Ile Val Lys Thr Val Lys Ala Lys Asn
Val Thr Ile Leu Asp 85 90 95 Gln Ser Pro His Gln Leu Ala Lys Ala
Lys Gln Lys Glu Pro Leu Lys 100 105 110 Glu Cys Lys Ile Val Glu Gly
Asp Ala Glu Asp Leu Pro Phe Pro Thr 115 120 125 Asp Tyr Ala Asp Arg
Tyr Val Ser Ala Gly Ser Ile Glu Tyr Trp Pro 130 135 140 Asp Pro Gln
Arg Gly Ile Arg Glu Ala Tyr Arg Val Leu Lys Ile Gly 145 150 155 160
Gly Lys Ala Cys Leu Ile Gly Pro Val His Pro Thr Phe Trp Leu Ser 165
170 175 Arg Phe Phe Ala Asp Val Trp Met Leu Phe Pro Lys Glu Glu Glu
Tyr 180 185 190 Ile Glu Trp Phe Lys Asn Ala Gly Phe Lys Asp Val Gln
Leu Lys Arg 195 200 205 Ile Gly Pro Lys Trp Tyr Arg Gly Val Arg Arg
His Gly Leu Ile Met 210 215 220 Gly Cys Ser Val Thr Gly Val Lys Pro
Ala Ser Gly Asp Ser Pro Leu 225 230 235 240 Gln Leu Gly Pro Lys Glu
Glu Asp Val Glu Lys Pro Val Asn Asn Pro 245 250 255 Phe Ser Phe Leu
Gly Arg Phe Leu Leu Gly Thr Leu Ala Ala Ala Trp 260 265 270 Phe Val
Leu Ile Pro Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro 275 280 285
Lys Asp Gln Pro Ile 290 34 292 PRT Arabidopsis thaliana 34 Arg Leu
Arg Cys Ala Ala Ser Ser Ser Ser Ala Ala Ala Arg Pro Met 1 5 10 15
Ser Gln Pro Arg Phe Ile Gln His Lys Lys Glu Ala Phe Trp Phe Tyr 20
25 30 Arg Phe Leu Ser Ile Val Tyr Asp His Val Ile Asn Pro Gly His
Trp 35 40 45 Thr Glu Asp Met Arg Asp Asp Ala Leu Glu Pro Ala Asp
Leu Tyr Ser 50 55 60 Arg Lys Leu Arg Val Val Asp Val Gly Gly Gly
Thr Gly Phe Thr Thr 65 70 75 80 Leu Gly Ile Val Lys Arg Val Asp Pro
Glu Asn Val Thr Leu Leu Asp 85 90 95 Gln Ser Pro His Gln Leu Glu
Lys Ala Arg Glu Lys Glu Ala Leu Lys 100 105 110 Gly Val Thr Ile Met
Glu Gly Asp Ala Glu Asp Leu Pro Phe Pro Thr 115 120 125 Asp Thr Phe
Asp Arg Tyr Val Ser Ala Gly Ser Ile Glu Tyr Trp Pro 130 135 140 Asp
Pro Gln Arg Gly Ile Lys Glu Ala Tyr Arg Val Leu Arg Leu Gly 145 150
155 160 Gly Val Ala Cys Met Ile Gly Pro Val His Pro Thr Phe Trp Leu
Ser 165 170 175 Arg Phe Phe Ala Asp Met Trp Met Leu Phe Pro Lys Glu
Glu Glu Tyr 180 185 190 Ile Glu Trp Phe Lys Lys Ala Gly Phe Lys Asp
Val Lys Leu Lys Arg 195 200 205 Ile Gly Pro Lys Trp Tyr Arg Gly Val
Arg Arg His Gly Leu Ile Met 210 215 220 Gly Cys Ser Val Thr Gly Val
Lys Arg Glu His Gly Asp Ser Pro Leu 225 230 235 240 Gln Leu Gly Pro
Lys Val Glu Asp Val Ser Lys Pro Val Asn Pro Ile 245 250 255 Thr Phe
Leu Phe Arg Phe Leu Met Gly Thr Ile Cys Ala Ala Tyr Tyr 260 265 270
Val Leu Val Pro Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys 275
280 285 Gly Met Pro Ile 290 35 292 PRT Arabidopsis thaliana 35 Arg
Leu Arg Cys Ala Ala Ser Ser Ser Pro Ala Ala Ala Arg Pro Ala 1 5 10
15 Thr Ala Pro Arg Phe Ile Gln His Lys Lys Glu Ala Phe Trp Phe Tyr
20 25 30 Arg Phe Leu Ser Ile Val Tyr Asp His Val Ile Asn Pro Gly
His Trp 35 40 45 Thr Glu Asp Met Arg Asp Asp Ala Leu Glu Pro Ala
Asp Leu Phe Ser 50 55 60 Arg His Leu Thr Val Val Asp Val Gly Gly
Gly Thr Gly Phe Thr Thr 65 70 75 80 Leu Gly Ile Val Lys His Val Asn
Pro Glu Asn Val Thr Leu Leu Asp 85 90 95 Gln Ser Pro His Gln Leu
Asp Lys Ala Arg Gln Lys Glu Ala Leu Lys 100 105 110 Gly Val Thr Ile
Met Glu Gly Asp Ala Glu Asp Leu Pro Phe Pro Thr 115 120 125 Asp Ser
Phe Asp Arg Tyr Ile Ser Ala Gly Ser Ile Glu Tyr Trp Pro 130 135 140
Asp Pro Gln Arg Gly Ile Lys Glu Ala Tyr Arg Val Leu Arg Phe Gly 145
150 155 160 Gly Leu Ala Cys Val Ile Gly Pro Val Tyr Pro Thr Phe Trp
Leu Ser 165 170 175 Arg Phe Phe Ala Asp Met Trp Met Leu Phe Pro Lys
Glu Glu Glu Tyr 180 185 190 Ile Glu Trp Phe Lys Lys Ala Gly Phe Arg
Asp Val Lys Leu Lys Arg 195 200 205 Ile Gly Pro Lys Trp Tyr Arg Gly
Val Arg Arg His Gly Leu Ile Met 210 215 220 Gly Cys Ser Val Thr Gly
Val Lys Arg Glu Arg Gly Asp Ser Pro Leu 225 230 235 240 Glu Leu Gly
Pro Lys Ala Glu Asp Val Ser Lys Pro Val Asn Pro Ile 245 250 255 Thr
Phe Leu Phe Arg Phe Leu Val Gly Thr Ile Cys Ala Ala Tyr Tyr 260 265
270 Val Leu Val Pro Ile Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys
275 280 285 Gly Met Pro Ile 290 36 288 PRT Arabidopsis thaliana 36
Val Pro Lys Cys Ser Val Ser Ala Ser Arg Pro Ser Ser Gln Pro Arg 1 5
10 15 Phe Ile Gln His Lys Lys Glu Ala Phe Trp Phe Tyr Arg Phe Leu
Ser 20 25 30 Ile Val Tyr Asp His Val Ile Asn Pro Gly His Trp Thr
Glu Asp Met 35 40 45 Arg Asp Asp Ala Leu Glu Pro Ala Asp Leu Asn
Asp Arg Asn Met Ile 50 55 60 Val Val Asp Val Gly Gly Gly Thr Gly
Phe Thr Thr Leu Gly Ile Val 65 70 75 80 Lys His Val Asp Ala Lys Asn
Val Thr Ile Leu Asp Gln Ser Pro His 85 90 95 Gln Leu Ala Lys Ala
Lys Gln Lys Glu Pro Leu Lys Glu Cys Lys Ile 100 105 110 Ile Glu Gly
Asp Ala Glu Asp Leu Pro Phe Arg Thr Asp Tyr Ala Asp 115 120 125 Arg
Tyr Val Ser Ala Gly Ser Ile Glu Tyr Trp Pro Asp Pro Gln Arg 130 135
140 Gly Ile Lys Glu Ala Tyr Arg Val Leu Lys Leu Gly Gly Lys Ala Cys
145 150 155 160 Leu Ile Gly Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg
Phe Phe Ala 165 170 175 Asp Val Trp Met Leu Phe Pro Lys Glu Glu Glu
Tyr Ile Glu Trp Phe 180 185 190 Gln Lys Ala Gly Phe Lys Asp Val Gln
Leu Lys Arg Ile Gly Pro Lys 195 200 205 Trp Tyr Arg Gly Val Arg Arg
His Gly Leu Ile Met Gly Cys Ser Val 210 215 220 Thr Gly Val Lys Pro
Ala Ser Gly Asp Ser Pro Leu Gln Leu Gly Pro 225 230 235 240 Lys Glu
Glu Asp Val Glu Lys Pro Val Asn Pro Phe Val Phe Ala Leu 245 250 255
Arg Phe Val Leu Gly Ala Leu Ala Ala Thr Trp Phe Val Leu Val Pro 260
265 270 Ile Tyr Met Trp Leu Lys Asp Gln Val Val Pro Lys Gly Gln Pro
Ile 275 280 285 37 289 PRT Arabidopsis thaliana 37 Ile Phe Thr Cys
Ser Ala Ser Ser Ser Ser Arg Pro Ala Ser Gln Pro 1 5 10 15 Arg Phe
Ile Gln His Lys Gln Glu Ala Phe Trp Phe Tyr Arg Phe Leu 20 25 30
Ser Ile Val Tyr Asp His Val Ile Asn Pro Gly His Trp Thr Glu Asp 35
40 45 Met Arg Asp Asp Ala Leu Glu Pro Ala Glu Leu Tyr Asp Ser Arg
Met 50 55 60 Lys Val Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr
Leu Gly Ile 65 70 75 80 Ile Lys His Ile Asp Pro Lys Asn Val Thr Ile
Leu Asp Gln Ser Pro 85 90 95 His Gln Leu Glu Lys Ala Arg Gln Lys
Glu Ala Leu Lys Glu Cys Thr 100 105 110 Ile Val Glu Gly Asp Ala Glu
Asp Leu Pro Phe Pro Thr Asp Thr Phe 115 120 125 Asp Arg Tyr Val Ser
Ala Gly Ser Ile Glu Tyr Trp Pro Asp Pro Gln 130 135 140 Arg Gly Ile
Lys Glu Ala Tyr Arg Val Leu Lys Leu Gly Gly Val Ala 145 150 155 160
Cys Leu Ile Gly Pro Val His Pro Thr Phe Trp Leu Ser Arg Phe Phe
165
170 175 Ala Asp Met Trp Met Leu Phe Pro Thr Glu Glu Glu Tyr Ile Glu
Trp 180 185 190 Phe Lys Lys Ala Gly Phe Lys Asp Val Lys Leu Lys Arg
Ile Gly Pro 195 200 205 Lys Trp Tyr Arg Gly Val Arg Arg His Gly Leu
Ile Met Gly Cys Ser 210 215 220 Val Thr Gly Val Lys Arg Leu Ser Gly
Asp Ser Pro Leu Gln Leu Gly 225 230 235 240 Pro Lys Ala Glu Asp Val
Lys Lys Pro Ile Asn Pro Phe Ser Phe Leu 245 250 255 Leu Arg Phe Ile
Leu Gly Thr Ile Ala Ala Thr Tyr Tyr Val Leu Val 260 265 270 Pro Ile
Tyr Met Trp Ile Lys Asp Gln Ile Val Pro Lys Gly Gln Pro 275 280 285
Ile 38 288 PRT Arabidopsis thaliana 38 Ala Pro Arg Cys Ser Leu Ser
Ala Ser Arg Pro Ala Ser Gln Pro Arg 1 5 10 15 Phe Ile Gln His Lys
Lys Glu Ala Phe Trp Phe Tyr Arg Phe Leu Ser 20 25 30 Ile Val Tyr
Asp His Val Ile Asn Pro Gly His Trp Thr Glu Asp Met 35 40 45 Arg
Asp Asp Ala Leu Glu Pro Ala Asp Leu Asn Asp Arg Asp Met Val 50 55
60 Val Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu Gly Ile Val
65 70 75 80 Gln His Val Asp Ala Lys Asn Val Thr Ile Leu Asp Gln Ser
Pro His 85 90 95 Gln Leu Ala Lys Ala Lys Gln Lys Glu Pro Leu Lys
Glu Cys Asn Ile 100 105 110 Ile Glu Gly Asp Ala Glu Asp Leu Pro Phe
Pro Thr Asp Tyr Ala Asp 115 120 125 Arg Tyr Val Ser Ala Gly Ser Ile
Glu Tyr Trp Pro Asp Pro Gln Arg 130 135 140 Gly Ile Lys Glu Ala Tyr
Arg Val Leu Lys Gln Gly Gly Lys Ala Cys 145 150 155 160 Leu Ile Gly
Pro Val Tyr Pro Thr Phe Trp Leu Ser Arg Phe Phe Ala 165 170 175 Asp
Val Trp Met Leu Phe Pro Lys Glu Glu Glu Tyr Ile Glu Trp Phe 180 185
190 Glu Lys Ala Gly Phe Lys Asp Val Gln Leu Lys Arg Ile Gly Pro Lys
195 200 205 Trp Tyr Arg Gly Val Arg Arg His Gly Leu Ile Met Gly Cys
Ser Val 210 215 220 Thr Gly Val Lys Pro Ala Ser Gly Asp Ser Pro Leu
Gln Leu Gly Pro 225 230 235 240 Lys Ala Glu Asp Val Ser Lys Pro Val
Asn Pro Phe Val Phe Leu Leu 245 250 255 Arg Phe Met Leu Gly Ala Thr
Ala Ala Ala Tyr Tyr Val Leu Val Pro 260 265 270 Ile Tyr Met Trp Leu
Lys Asp Gln Ile Val Pro Glu Gly Gln Pro Ile 275 280 285 39 1047 DNA
Arabidopsis thaliana 39 atgaaagcaa ctctagcagc accctcttct ctcacaagcc
tcccttatcg aaccaactct 60 tctttcggct caaagtcatc gcttctcttt
cggtctccat cctcctcctc ctcagtctct 120 atgacgacaa cgcgtggaaa
cgtggctgtg gcggctgctg ctacatccac tgaggcgcta 180 agaaaaggaa
tagcggagtt ctacaatgaa acttcgggtt tgtgggaaga gatttgggga 240
gatcatatgc atcatggctt ttatgaccct gattcttctg ttcaactttc tgattctggt
300 cacaaggaag ctcagatccg tatgattgaa gagtctctcc gtttcgccgg
tgttactgat 360 gaagaggagg agaaaaagat aaagaaagta gtggatgttg
ggtgtgggat tggaggaagc 420 tcaagatatc ttgcctctaa atttggagct
gaatgcattg gcattactct cagccctgtt 480 caggccaaga gagccaatga
tctcgcggct gctcaatcac tctctcataa ggcttccttc 540 caagttgcgg
atgcgttgga tcagccattc gaagatggaa aattcgatct agtgtggtcg 600
atggagagtg gtgagcatat gcctgacaag gccaagtttg taaaagagtt ggtacgtgtg
660 gcggctccag gaggtaggat aataatagtg acatggtgcc atagaaatct
atctgcgggg 720 gaggaagctt tgcagccgtg ggagcaaaac atcttggaca
aaatctgtaa gacgttctat 780 ctcccggctt ggtgctccac cgatgattat
gtcaacttgc ttcaatccca ttctctccag 840 gatattaagt gtgcggattg
gtcagagaac gtagctcctt tctggcctgc ggttatacgg 900 actgcattaa
catggaaggg ccttgtgtct ctgcttcgta gtggtatgaa aagtattaaa 960
ggagcattga caatgccatt gatgattgaa ggttacaaga aaggtgtcat taagtttggt
1020 atcatcactt gccagaagcc actctaa 1047 40 1047 DNA Arabidopsis
thaliana 40 atgaaagcaa ctctagcagc accctcttct ctcacaagcc tcccttatcg
aaccaactct 60 tctttcggct caaagtcatc gcttctcttt cggtctccat
cctcctcctc ctcagtctct 120 atgacgacaa cgcgtggaaa cgtggctgtg
gcggctgctg ctacatccac tgaggcgcta 180 agaaaaggaa tagcggagtt
ctacaatgaa acttcgggtt tgtgggaaga gatttgggga 240 gatcatatgc
atcatggctt ttatgaccct gattcttctg ttcaactttc tgattctggt 300
cacaaggaag ctcagatccg tatgattgaa gagtctctcc gttttgccgg tgttactgat
360 gaagaggagg agaaaaagat aaagaaagta gtggatgttg ggtgtgggat
tggaggaagc 420 tcaagatatc ttgcctctaa atttggagct gaatgcattg
gcattactct cagccctgtt 480 caggccaaga gagccaatga tctcgcggct
gctcaatcac tcgctcataa ggcttccttc 540 caagttgcgg atgcgttgga
tcagccattc gaagatggaa aattcgatct agtgtggtcg 600 atggagagtg
gtgagcatat gcctgacaag gccaagtttg taaaagagtt ggtacgtgtg 660
gcggctccag gaggtaggat aataatagtg acatggtgcc atagaaatct atctgcgggg
720 gaggaagctt tgcagccgtg ggagcaaaac atcttggaca aaatctgtaa
gacgttctat 780 ctcccggctt ggtgctccac cgatgattat gtcaacttgc
ttcaatccca ttctctccag 840 gatattaagt gtgcggattg gtcagagaac
gtagctcctt tctggcctgc ggttatacgg 900 actgcattaa catggaaggg
ccttgtgtct ctgcttcgta gtggtatgaa aagtattaaa 960 ggagcattga
caatgccatt gatgattgaa ggttacaaga aaggtgtcat taagtttggt 1020
atcatcactt gccagaagcc actctaa 1047 41 1095 DNA Arabidopsis thaliana
41 atggcccacg ccgccgcggc cacgggcgca ctggcaccgc tgcatccact
gctccgctgc 60 acgagccgtc atctctgcgc ctcggcttcc cctcgcgccg
gcctctgcct ccaccaccac 120 cgccgccgcc gccgcagcag ccggaggacg
aaactcgccg tgcgcgcgat ggcaccgacg 180 ttgtcctcgt cgtcgacggc
ggcggcagct cccccggggc tgaaggaggg catcgcgggg 240 ctctacgacg
agtcgtccgg cgtgtgggag agcatctggg gcgagcacat gcaccacggc 300
ttctacgacg ccggcgaggc cgcctccatg tccgaccacc gccgcgccca gatccgcatg
360 atcgaggaat ccctcgcctt cgccgccgtc cccggtgcag atgatgcgga
gaagaaaccc 420 aaaagtgtag ttgatgttgg ctgtggcatt ggtggtagct
caagatactt ggcgaacaaa 480 tacggagcgc aatgctacgg catcacgttg
agtccggtgc aggctgaaag aggaaatgcc 540 ctcgcggcag agcaagggtt
atcagacaag gtgcgtattc aagttggtga tgcattggag 600 cagccttttc
ctgatgggca gtttgatctt gtctggtcca tggagagtgg cgagcacatg 660
ccagacaaac ggcagtttgt aagcgagctg gcacgcgtcg cagctcctgg ggcgagaata
720 atcattgtga cctggtgcca taggaacctc gagccatccg aagagtccct
gaaacctgat 780 gagctgaatc tcctgaaaag gatatgcgat gcatattatc
tcccagactg gtgctctcct 840 tctgattatg tcaaaattgc cgagtcactg
tctcttgagg atataaggac agctgattgg 900 tcagagaacg tcgccccatt
ctggcctgcg gttataaaat cagcattgac atggaaaggt 960 ttaacttctc
tgctaagaag tgggtggaag acgataagag gtgcaatggt gatgcctctg 1020
atgatcgaag gatacaagaa agggctcatc aaattcacca tcatcacctg tcgcaagccc
1080 gaaacaacgc agtag 1095 42 1059 DNA Arabidopsis thaliana 42
atggctcacg cggcgctgct ccattgctcc cagtcctcca ggagcctcgc agcctgccgc
60 cgcggcagcc actaccgcgc cccttcgcac gtcccgcgcc actcccgccg
tctccgacgc 120 gccgtcgtca gcctgcgtcc gatggcctcg tcgacggctc
aggcccccgc gacggcgccg 180 ccgggtctga aggagggcat cgcggggctg
tacgacgagt cgtcggggct gtgggagaac 240 atctggggcg accacatgca
ccacggcttc tacgactcga gcgaggccgc ctccatggcc 300 gatcaccgcc
gcgcccagat ccgcatgatc gaggaggcgc tcgccttcgc cggtgtccca 360
gcctcagatg atccagagaa gacaccaaaa acaatagtcg atgtcggatg tggcattggt
420 ggtagctcaa ggtacttggc gaagaaatac ggagcgcagt gcactgggat
cacgttgagc 480 cctgttcaag ccgagagagg aaatgctctc gctgcagcgc
aggggttgtc ggatcaggtt 540 actctgcaag ttgctgatgc tctggagcaa
ccgtttcctg acgggcagtt cgatctggtg 600 tggtccatgg agagtggcga
gcacatgccg gacaagagaa agtttgttag tgagctagca 660 cgcgtggcgg
ctcctggagg gacaataatc atcgtgacat ggtgccatag gaacctggat 720
ccatccgaaa cctcgctaaa gcccgatgaa ctgagcctcc tgaggaggat atgcgacgcg
780 tactacctcc cggactggtg ctcaccttca gactatgtga acattgccaa
gtcactgtct 840 ctcgaggata tcaagacagc tgactggtcg gagaacgtgg
ccccgttttg gcccgccgtg 900 ataaaatcag cgctaacatg gaagggcttc
acctctctgc tgacgaccgg atggaagacg 960 atcagaggcg cgatggtgat
gccgctaatg atccagggct acaagaaggg gctcatcaaa 1020 ttcaccatca
tcacctgtcg caagcctgga gccgcgtag 1059 43 1038 DNA Arabidopsis
thaliana 43 atggctgccg cgttacaatt acaaacacac ccttgcttcc atggcacgtg
ccaactctca 60 cctccgccac gaccttccgt ttccttccct tcttcctccc
gctcgtttcc atctagcaga 120 cgttccctgt ccgcgcatgt gaaggcggcg
gcgtcgtctt tgtccaccac caccttgcag 180 gaagggatag cggagtttta
cgatgagtcg tcggggattt gggaagacat atggggtgac 240 catatgcacc
atggatatta cgagccgggt tccgatattt cgggttcaga tcatcgtgcc 300
gctcagattc gaatggtcga agaatcgctc cgttttgctg gaatatcaga ggacccagca
360 aacaggccca agagaatagt tgatgttggg tgtgggatag gaggcagttc
taggtatcta 420 gcaaggaaat atggggcaaa atgccaaggc attactttga
gccctgttca agctggaaga 480 gccaatgctc ttgctaatgc tcaaggacta
gcagaacagg tttgttttga agttgcagat 540 gccttgaacc aaccattccc
tgatgaccaa tttgatcttg tttggtctat ggaaagcgga 600 gaacacatgc
ctgacaaacc caagtttgtt aaagagctgg tgcgagtggc agctccagga 660
ggcacaataa tagtagtgac atggtgccat agggatcttg gtccatctga agagtctttg
720 cagccatggg agcaaaagct tttaaacaga atatgtgatg cttactattt
accagagtgg 780 tgttctactt ctgattatgt caaattattt cagtccctat
ctctccagga tataaaggca 840 ggagactgga ctgagaatgt agcacccttt
tggccagcag tgatacgttc agcattgaca 900 tggaagggct tcacatcgct
gctacgaagt ggattaaaaa caataaaagg tgcactggtg 960 atgccattga
tgatcgaagg tttccagaaa ggggtgataa agtttgccat cattgcttgc 1020
cggaagccag ctgagtag 1038 44 1131 DNA Arabidopsis thaliana 44
atgccgataa catctatttc cgcaaaccaa aggccattct tcccctcacc ttatagaggc
60 agctccaaga acatggcacc gcccgaactg gctcagtcgc aagtacctat
gggaagtaac 120 aagagcaaca agaaccacgg cttggtcggt tcggtttctg
gttggagaag gatgtttggg 180 acatgggcta ctgccgacaa gactcagagt
accgatacgt ctaatgaagg cgtggttagt 240 tacgatactc aggtcttgca
gaagggtata gcggagttct atgacgagtc gtcgggtata 300 tgggaggata
tatggggaga tcacatgcat catggctact atgatggttc cactcctgtc 360
tccctcccag accatcgctc tgcgcagatc cgaatgattg acgaggctct ccgctttgcc
420 tcggttcctt caggagaaga agatgagtcc aagtctaaga ttccaaagag
gatagtggat 480 gtcgggtgtg ggataggggg aagctccaga tacctggcta
gaaaatatgg cgccgagtgt 540 cggggcatca ctctcagtcc tgtccaggct
gagaggggca attcacttgc acggtctcaa 600 ggtctttctg acaaggtctc
ctttcaagtc gccgatgctt tggcacagcc atttcccgat 660 ggacagtttg
atttggtctg gtccatggag agcggggaac acatgcccga caagagcaag 720
tttgtcaatg agctagtaag agtagcagct ccgggtggca cgataataat tgtcacatgg
780 tgccatagag atctcaggga agacgaagat gcgctgcagc ctcgggagaa
agagatattg 840 gacaagatat gcaacccctt ttatcttccc gcctggtgtt
ctgctgccga ctatgttaag 900 ttgctccagt cacttgatgt cgaggacatt
aaatctgcgg actggactcc atatgttgcc 960 ccattttggc cagctgtgct
gaagtccgct ttcactataa agggcttcgt gtctctattg 1020 aggagcggaa
tgaagaccat aaagggagca tttgcaatgc cgctgatgat cgaaggatac 1080
aagaaaggtg tcatcaagtt ttccatcatc acatgccgta agcccgaata g 1131 45
2045 DNA Arabidopsis thaliana 45 atgaaagcga ctctcgcacc ctcctctctc
ataagcctcc ccaggcacaa agtatcttct 60 ctccgttcac cgtcgcttct
ccttcagtcc caacggccat cctcagcctt aatgacgacg 120 acgacggcat
cacgtggaag cgtggctgtg acggctgctg ctacctcctc cgttgaggcg 180
ctgcgggaag gaatagcgga attctacaac gagacgtcgg gattatggga ggagatttgg
240 ggagatcata tgcatcacgg cttctacgat cctgattcct ctgttcaact
ttcagattcc 300 ggtcaccggg aagctcagat ccggatgatc gaagagtctc
tacgtttcgc cggcgttact 360 ggttcgcttc tcatgctata cagttagagt
ttgattcgtt gtttgttatg aatgataaac 420 ctacacatga acactttcta
gatttattat aaacattctt tttgaactta tattataaac 480 aattcttaca
aacaaaatgc tctttgaact cttaaaaata tataacaatg gtttagtttt 540
gatttgtcgg taagagaaat gagtagggat gtttgaagcc agataaagcc tttcttttat
600 ccctggggag aggcttacag taagccacgt cccatccaga agcagaccca
ttccctaact 660 aggctggatg atgataaata agttcttcct catttcaaga
ttaagaaaac aatctaaact 720 gaaataataa cgcgcagtcg gtgaaaatat
ctttatgctt gggattgttg ttgttattat 780 taatttatat tataaacaca
tgaccttttt aaagaagagg agaaaaagat aaagagagta 840 gtggatgttg
ggtgtgggat cggcggaagc tcaaggtata ttgcctctaa atttggtgcc 900
gaatgcattg gcatcacact cagtcccgtt caagccaaga gagccaatga tctcgccgcc
960 gctcaatcac tctctcataa ggtgtcttct tgtacattcg accatttttt
tctgcggaat 1020 ctgagctaac tgagacgcca ctggaccagg tttccttcca
agttgcagat gcactggagc 1080 aaccatttga agatggtata ttcgatcttg
tgtggtcaat ggaaagcggt gagcatatgc 1140 ctgacaaggc caaggtatac
tacctagctc accataatct ttatactaga tttagtagac 1200 aatatccatc
ttttggatgt caatgatgtc cattaatttt taaataaaca aaataaaaaa 1260
tgagagtaaa attttttttt gtcaaactta tctaataaat attatgtaat aataccacgt
1320 ttttctattt aattatggca tggtttcttt tttttttgtc taaaaaaaat
tgtagtatct 1380 gttagaaaac agaatctaag tatgatattt ttgaaactca
ttcagtcttc gttgtggaag 1440 tatatttacc gtgtgtgcga aatgagtgta
gttcgtgaag gaattggtac gtgtggcggc 1500 tccaggagga aggataataa
tagtgacatg gtgccacaga aatctatctc caggggaaga 1560 ggctttgcag
ccatgggagc agaacctctt ggacagaatc tgcaaaacat tttatctccc 1620
agcctggtgc tccacctcgg attatgtcga tttgcttcag tccctctcgc tccaggttat
1680 tatatttctc acgctccaat tgctaaaatt agtacttgga gctagttaag
tagtgtctca 1740 aatatatgtg tgtttgtagg atattaagtg tgcagattgg
tcagagaacg tagctccttt 1800 ctggccggcg gttatacgaa ccgcattaac
gtggaagggc cttgtgtctc tgcttcgtag 1860 tggtatgttt ccgcaatgtt
gttcacattc atgattttta taagattaga actaaggttg 1920 ttgggtgtcg
gaaacgcaca ggtatgaaga gtataaaagg agcattgaca atgccattga 1980
tgattgaagg gtacaagaaa ggtgtcatta agtttggcat catcacttgc cagaagcctc
2040 tctaa 2045 46 2973 DNA Arabidopsis thaliana 46 atgaaagcga
cactcgcacc accctcctct ctcataagcc tccccaggca caaagtatct 60
tccctccgtt caccgtcgct tctccttcag tcccaacggc gatcctcagc cttaatgacg
120 acgacggcat cacgtggaag cgtggctgtg acggctgctg ctacctcctc
cgctgaggcg 180 ctgcgagaag gaatagcgga attctacaac gagacgtcgg
gattatggga ggagatttgg 240 ggagatcata tgcatcacgg cttctacgat
cccgattcct ctgttcaact ttcagattcc 300 ggtcaccggg aagctcagat
ccggatgatt gaagagtctc tacgtttcgc cggcgttact 360 ggttcgcttc
tcatgctcta cacttgagtt tgatacgttg tttattataa acattttttt 420
gaacttttat tataaacaat tcttacaaac aaattactct ttgaactctt taaaatctat
480 aacaaaggtt tagttttact ttttatttgt tgttggtaac agaaatgagt
agggatgttt 540 gaagtcagat atagcctttc tgtttatccc ttgggaagaa
aggcttacag taagccacgt 600 cccatccaga agcagaccca ttccctaact
aatcattttt atgaacaatt tgtaacacta 660 ttattcctag atattttttt
tttacgttta gttaccctaa ctctttgtat ataagacaag 720 aggtgatttt
tcacattata tatcaaaaca tagacatagt ttttttgaga aaatatatca 780
tacatagttg taacttagaa ttatatattt ttgagaaaaa aactcagtaa taattttctt
840 ataattattc atagttttat atttattaat aataagattt tgtaagctct
ttttgaaact 900 attatggata atgaataagt tccccatttc aagattaaga
aaacaattta aactgaaata 960 ataatgcgca ttcggtgaaa atatctttct
gcttgggatt gttgttgtta atctatatta 1020 ttaaaactga agtacatttt
ggtactgttt ggaaacttag atagtagatt aaatgaaaat 1080 tgtttggaaa
caaggatagc agattaaata tttttttatt tacatattta gtcactgtat 1140
ttctttctca tttacagatt ctgtcgtttg gaaacttgga tagcagatta aatgaaaaat
1200 gtttggaaac acagttaaca tattaaatat ctatttttat ttcatattta
gccattgcat 1260 ttctttctta tttacaaatc tgccacttca cttaaaataa
aaaaattaaa ttaattacaa 1320 tgaattgtta tttctttttg ctgaaaataa
aaacgcaaac tgcaatatat agtatatatt 1380 aatctgctac aatacaattt
tcaagaaaac caaatatcat aaaattaata ataatttata 1440 aaaacctaca
gtaaaaaaat aaatcatttt taaataaata aacaaaaaaa atcaataggt 1500
tgatatatga atattacaat tacatcaaat tgcatcaagt tataaaatta taaatataat
1560 attacgtaca aataaaaatt attatcaaac atctatttta taatataata
tattctactc 1620 taaatatatt tacaaaacat aaaaatataa atggacattt
tataaaatca atggtttata 1680 agtttacatt gaacgcaagt taaattccaa
catccgcgcg gggcgcgggt caagatctag 1740 tattaattta tattataaac
acatgacttt ttttaaagaa gaggagaaaa agataaagag 1800 agtggtggat
gttgggtgtg ggatcggagg aagctcaagg tatattgcct ctaaatttgg 1860
tgccgaatgc attggcatca cactcagtcc cgttcaagcc aagagagcaa atgatctcgc
1920 caccgctcaa tcactctctc ataaggtgtc ttctcgtaca ttcgaccatt
ctttctgcgg 1980 ataatctgat ctaactgaga cgccattgga ccaggtttcc
ttccaagttg cagatgcatt 2040 ggaccaacca tttgaagatg gtatatccga
tcttgtttgg tcaatggaaa gcggtgagca 2100 tatgcctgac aaggccaagg
tatactagct cagcataact tttatactag atttactaga 2160 caatatctat
cttttcatgt caatgatgtc caataatttt aaaataaaca aaagaaggat 2220
gtgagggtaa aattttgtca aatttatata acaacacgtt ttctatttag ttatgtcatg
2280 gtttcttttt gtctaaaaaa ttttaggcag agtttacaaa aagaaaattg
tagtatctgt 2340 tcgaaaacag aatcttagtg tggtatttta gaaactcatt
cagtcttcct tgtggaagca 2400 tatttactgt gtgtgcgaaa tgagtgtagt
tcgtgaagga attggtacgt gtgacggctc 2460 caggaggaag gataataata
gtgacatggt gccacagaaa tctatctcaa ggggaagaat 2520 ctttgcagcc
atgggagcag aacctcttgg acagaatctg caaaacattt tatctcccgg 2580
cctggtgctc caccactgat tatgtcgagt tgcttcaatc cctctcgctc caggttatta
2640 tatttctcac gctccgatgc taaaatcagt aagtattgtc tcaaatatat
gtgtgtttgt 2700 aggatattaa gtatgcagat tggtcagaga acgtagctcc
tttctggccg gcggttatac 2760 gaaccgcatt aacgtggaag ggccttgtgt
ctctgcttcg tagtggtatg tttccgcaat 2820 gttgtttaca ttcatgattc
caaatgttta taagattaga aacatacagg tatgaagagt 2880 ataaaaggag
cattgacaat gccattgatg attgaagggt acaagaaagg tgtcattaag 2940
tttggcatca tcacttgcca gaagcctcta taa 2973 47 933 DNA Arabidopsis
thaliana 47 atggctagtg ttgctgcgat gaatgctgtg tcttcgtcat ctgtagaagt
tggaatacag 60 aatcaacagg agctgaaaaa aggaattgca gatttatatg
atgagtcttc tgggatttgg 120 gaagatattt ggggtgacca tatgcatcat
ggatattatg aacctaaatc ctctgtggaa 180 ctttcagatc atcgtgctgc
tcagatccgt atgattgaac aggctctaag ttttgctgct 240 atttctgaag
atccagcgaa gaaaccaacg tccatagttg atgttggatg tggcatcggt 300
ggcagttcta ggtaccttgc aaagaaatat ggcgctacag ctaaaggtat cactttgagt
360 cctgtacaag cagagagggc tcaagctctt gctgatgctc aaggattagg
tgataaggtt 420 tcatttcaag tagcagacgc cttgaatcag ccttttccag
atgggcaatt cgacttggtt 480 tggtccatgg agagtggaga acacatgccg
aacaaagaaa agtttgttgg cgaattagct 540
cgagtggcag caccaggagg cacaatcatc cttgtcacat ggtgccacag ggacctttcc
600 ccttcggagg aatctctgac tccagaggag aaagagctgt taaataagat
atgcaaagcc 660 ttctatcttc cggcttggtg ttccactgct gattatgtga
agttacttca atccaattct 720 cttcaggata tcaaggcaga agactggtct
gagaatgttg ctccattttg gccagcagtc 780 ataaagtcag cactgacatg
gaagggcttc acatcagtac tacgcagtgg atggaagaca 840 atcaaagctg
cactggcaat gccactgatg attgaaggat acaagaaagg tctcatcaaa 900
tttgccatca tcacatgtcg aaaacctgaa taa 933 48 909 DNA Arabidopsis
thaliana 48 atgtcggtgg agcagaaagc agcagggaag gaggaggagg gaaaactgca
gaagggaatt 60 gcagagttct acgacgagtc gtctggcata tgggagaaca
tttggggcga tcacatgcac 120 cacggctttt atgacccgga ttccaccgtt
tctgtttctg atcatcgcgc tgctcagatc 180 cgaatgatcc aagaatctct
tcgttttgcc tctctgcttt ctgagaaccc ttctaaatgg 240 cccaagagta
tagttgatgt tgggtgtggc atagggggca gctccagata cctggccaag 300
aaatttggag caacgagcgt aggcattact ctgagtcctg ttcaagctca aagagcaaat
360 gctcttgctg ctgctcaagg attggctgat aaggtttcct ttcaggttgc
tgacgctcta 420 cagcaaccat tctctgacgg ccagtttgat ctggtgtggt
ccatggagag tggagagcat 480 atgcctgaca aagctaagtt tgttggagag
ttagctcggg tagcagcacc aggtgccact 540 ataataatag taacatggtg
ccacagggat cttggccctg acgaacaatc cttacatcca 600 tgggagcaag
atctcttaaa gaagatttgc gatgcatatt acctccctgc ctggtgctca 660
acttctgatt atgttaagtt gctccaatcc ctgtcacttc aggacatcaa gtcagaagat
720 tggtctcgct ttgttgctcc attttggcca gcagtgatac gctcagcctt
cacatggaag 780 ggtctaactt cactcttgag cagtggacaa aaaacgataa
aaggagcttt ggctatgcca 840 ttgatgatag agggatacaa gaaagatcta
attaagtttg ccatcattac atgtcgaaaa 900 cctgaataa 909 49 1053 DNA
Arabidopsis thaliana 49 atggccaccg tggtgaggat cccaacaatc tcatgcatcc
acatccacac gttccgttcc 60 caatcccctc gcactttcgc cagaatccgg
gtcggaccca ggtcgtgggc tcctattcgg 120 gcatcggcag cgagctcgga
gagaggggag atagtattgg agcagaagcc gaagaaggag 180 gaggagggga
aactgcagaa gggaatcgca gagttctacg acgagtcgtc tggcttatgg 240
gagaacattt ggggcgacca catgcaccat ggcttttatg acccggattc cactgtttct
300 gtttctgatc atcgcgctgc tcagatccga atgatccaag agtctcttcg
ctttgcctct 360 gtttctgagg agcgtagtaa atggcccaag agtatagttg
atgttgggtg tggcataggt 420 ggcagctcca gatacctggc caagaaattt
ggagcaacca gcgtaggcat tactctgagt 480 cctgttcaag ctcaaagagc
aaatgctctt gctgctgctc aaggattggc tgataaggtt 540 tcctttcagg
ttgctgacgc tctacagcaa ccattctctg acggccagtt tgatctggtg 600
tggtccatgg agagtggaga gcatatgcct gacaaagcta agtttgttgg agagttagct
660 cgggtagcag caccaggtgc cactataata atagtaacat ggtgccacag
ggatcttggc 720 cctgacgaac aatccttaca tccatgggag caagatctct
taaagaagat ttgcgatgca 780 tattaccttc ctgcctggtg ctcaacttct
gattatgtta agttgctcca atccctgtca 840 cttcaggaca tcaagtcaga
agattggtct cgctttgttg ctccattttg gccagcagtg 900 atacgctcag
ccttcacatg gaagggtcta acttcactct tgagcagtgg acttaaaacc 960
ataaaaggag ctttggctat gccattgatg atagagggat acaagaaaga tctaattaag
1020 tttgccatca ttacatgtcg aaaacctgaa taa 1053 50 1053 DNA
Arabidopsis thaliana 50 atggccaccg tggtgaggat cccaacaatc tcatgcatcc
acatccacac gttccgttcc 60 caatcccctc gcactttcgc cagaatccgg
gtcggaccca ggtcgtgggc tcctattcgg 120 gcatcggcag cgagctcgga
gagaggggag atagtattgg agcagaagcc gaagaaggat 180 gacaaggaga
aactgcagaa gggaatcgca gagttttacg acgagtcttc tggcttatgg 240
gagaacattt ggggcgacca catgcaccat ggcttttatg acccggattc cactgtttcg
300 ctttcggatc atcgtgctgc tcagatccga atgatccaag agtctcttcg
ctttgcctct 360 gtttctgagg agcgtagtaa atggcccaag agtatagttg
atgttgggtg tggcataggt 420 ggcagctcca gatacctggc caagaaattt
ggagcaacca gtgtaggcat cactctgagt 480 cctgttcaag ctcaaagagc
aaatgctctt gctgctgctc aaggattggc tgataaggtt 540 tcctttcagg
ttgctgacgc tctacagcaa ccattctctg acggccagtt tgatctggtg 600
tggtccatgg agagtggaga gcatatgcct gacaaagcta agtttgttgg agagttagct
660 cgggtagcag caccaggtgc cactataata atagtaacat ggtgccacag
ggatcttggc 720 cctgacgaac aatccttaca tccatgggag caagatctct
taaagaagat ttgcgatgca 780 tattacctcc ctgcctggtg ctcaacttct
gattatgtta agttgctcca atccctgtca 840 cttcaggaca tcaagtcaga
agattggtct cgctttggtg ctccattttg gccagcagtg 900 atacgctcag
ccttcacatg gaagggtcta acttcactct tgagcagtgg ccaaaaaacg 960
ataaaaggag ctttggctat gccattgatg atagagggat acaagaaaga tctaattaag
1020 tttgccatca ttacatgtcg aaaacctgaa taa 1053 51 933 DNA
Arabidopsis thaliana 51 gcccttagcg tggtcgcggc cgaggtacca gttacggtta
ctccggcgac gacgaaggcg 60 gaggatgtgg agctgaagaa aggaattgca
gagttctacg atgaatcgtc ggagatgtgg 120 gagaatatat ggggagaaca
catgcatcat ggatactata acactaatgc cgttgttgaa 180 ctctccgatc
atcgttctgc tcagatccgt atgattgaac aagccctact tttcgcatct 240
gtttcagatg atccagtaaa gaaacctaga agcatcgttg atgttgggtg tggcataggt
300 ggtagctcaa ggtatctggc aaagaaatac gaagctgaat gccatggaat
cactctcagc 360 cctgtgcaag ctgagagagc tcaagctcta gctgctgctc
aaggattggc cgataaggct 420 tcatttcaag ttgctgatgc tttagaccaa
ccatttcctg atggaaagtt tgatctggtc 480 tggtcaatgg agagtggtga
acacatgcct gacaaactaa agtttgttag tgagttggtt 540 cgggttgctg
ccccaggagc cacgattatc atagttacat ggtgccatag ggatctttct 600
cctggtgaaa agtcccttcg acccgatgaa gaaaaaatct tgaaaaagat ttgttccagc
660 ttttatcttc ctgcttggtg ttcaacatct gattatgtaa aattactaga
gtccctttct 720 cttcaggaca tcaaagctgc agactggtca gcaaacgtgg
ctccattttg gcctgctgta 780 ataaaaacag cattatcttg gaagggcatt
acttcgctac ttcgtagtgg atggaagtca 840 ataagagggg caatggtaat
gccattgatg attgaaggat ttaagaagga tataatcaaa 900 ttctccatca
tcacatgcaa aaagcctgaa taa 933 52 1230 DNA Sorghum bicolor 52
cgaacggcga gcagcaggag ggcgtcgcga acccttgggc ggcggatcgg tacccgtagg
60 cagccactac tactaccgcg ccccttcgca cgtcccgcgc cgctcccgcc
cccgcggacg 120 cggcggcgtc gtcagcctgc gtccgatggc ctcgtcgacg
gcggctcagc cccccgcgcc 180 ggcgcccccg ggcctgaagg agggcatcgc
ggggctgtac gacgagtctt cggggctgtg 240 ggagaacatc tggggcgacc
acatgcacca cggcttctac gactcgggcg aggccgcgtc 300 catggccgac
caccgacgcg cccagatccg catgatcgag gaggcgctcg ccttcgccgc 360
cgtcccatcc ccagatgatc cggagaaggc accaaaaacc atagtagatg ttggatgtgg
420 cattggtggt agctcaaggt acttggctaa gaaatacgga gcacagtgca
aggggatcac 480 attgagccct gttcaagctg aaagaggaaa tgctcttgct
acagcgcagg ggttgtcgga 540 tcaggttact ctgcaagttg ctgatgctct
ggagcaaccg tttcctgatg ggcagtttga 600 tctggtatgg tccatggaga
gtggcgagca catgccggac aagagaaagt ttgttagtga 660 gctggcacgc
gtcgctgctc ctggagggac aataatcatc gtgacatggt gccataggaa 720
cctcgaacca tctgagactt cgctaaaacc cgatgaactg agtctcttga agaggatttg
780 cgatgcgtac tacctcccag actggtgctc accttcagac tatgtgaaca
tcgccaaatc 840 actgtctctg gaggatatca aggcagctga ttggtcagag
aatgtggccc cattttggcc 900 cgctgtgata aaatcagcac taacatggaa
gggcctcacc tctctactga caagcggatg 960 gaagacgatc agaggggcga
tggtgatgcc gctgatgatc caaggttaca agaaggggct 1020 catcaaattc
accatcatca cctgtcgcaa gcctggagca gcgtaggtga ccaaggggca 1080
gaagttactg tcaaagcacc tctgctaagt ccaataatgt agatccatgg ccccatcacc
1140 gtctattgta ctgtactgta ctgtaccaga atgaacagtc tcctgggaca
tgttttccaa 1200 ttgccatgac atgtcaaatg atcttctacc 1230 53 843 DNA
Arabidopsis thaliana 53 atgagtgcaa cactttacca gcaaattcag caattttacg
atgcttcatc tggtctgtgg 60 gaacagatat ggggcgaaca catgcaccac
ggctattacg gcgctgatgg tacccagaaa 120 aaagaccgcc gtcaggctca
aattgattta atcgaagaat tgcttaattg ggcaggggta 180 caagcagcag
aagatatact agatgtgggt tgtggaattg gcggtagttc tttatacctg 240
gcgcaaaagt ttaatgctaa agctacaggg attacattga gtcctgtaca agctgcaaga
300 gcaacagaac gcgcattgga agctaatttg agtctgagaa cacagttcca
agtcgctaat 360 gctcaagcaa tgccctttgc tgacgattct tttgacttgg
tttggtcgct ggaaagtggc 420 gaacacatgc cagataaaac caagtttctt
caggagtgct atcgagtact gaagcctggt 480 ggcaagttaa ttatggtgac
ttggtgtcat cgaccaactg atgaatctcc attaacggca 540 gatgaggaaa
agcacttgca ggatatttat cgggtgtatt gtttgcctta tgtgatttct 600
ttgccagagt atgaagcgat cgcacatcaa ctaccattac ataatatccg cactgctgat
660 tggtcaactg ctgtcgcccc cttttggaat gtggtaattg attctgcatt
cactccccaa 720 gcgctttggg gtttactaaa tgctggttgg actaccattc
aaggggcatt atcactggga 780 ttaatgcgtc gcggttatga acgtgggtta
attcggtttg gcttactgtg cggcaataag 840 tag 843 54 843 DNA Arabidopsis
thaliana 54 atgagtgcaa cactttacca acaaattcag caattttacg atgcttcctc
tgggctgtgg 60 gaagagattt ggggcgaaca tatgcaccac ggctattatg
gtgcagacgg tactgaacaa 120 aaaaaccgcc gtcaggcgca aattgattta
attgaagaat tactcacttg ggcaggagta 180 caaacagcag aaaatatact
agatgtgggt tgtggtattg gtggtagttc tctgtatttg 240 gcaggaaagt
tgaatgctaa agctacagga attaccctga gtccagtgca agccgctaga 300
gccacagaaa gagccaagga agctggttta agtggtagaa gtcagttttt agtggcaaat
360 gcccaagcaa tgccttttga tgataattct tttgacttgg tgtggtcgct
agaaagtggc 420 gaacatatgc cagataaaac caagtttttg caagagtgtt
atcgagtctt gaaaccgggc 480 ggtaagttaa tcatggtgac atggtgtcat
cgtcccactg ataaaacacc actgacggct 540 gatgaaaaaa aacacctaga
agatatttat cgggtgtatt gtttgcctta tgtaatttcg 600 ttgccggagt
atgaagcgat cgcacgtcaa ctaccattaa ataatatccg caccgccgac 660
tggtcgcaat ccgtcgccca attttggaac atagtcatcg attccgcctt taccccccaa
720 gcaatattcg gcttactccg cgcaggttgg actaccatcc aaggagcctt
atcactaggc 780 ttaatgcgtc gcggctatga gcgcgggtta attcggtttg
ggttgctttg tggggataag 840 tga 843 55 40 DNA Arabidopsis thaliana 55
tgtaaaacga cggccagttg ctgaaagttg aaaagagcaa 40 56 40 DNA
Arabidopsis thaliana 56 caggaaacag ctatgaccca atttgatcaa tgttccacga
40 57 38 DNA Arabidopsis thaliana 57 tgtaaaacga cggccagtag
ctatgcggat tgatggtc 38 58 38 DNA Arabidopsis thaliana 58 caggaaacag
ctatgacctc ctcctgggaa ctctagca 38 59 38 DNA Arabidopsis thaliana 59
tgtaaaacga cggccagttg ctgacttgcg agtttttg 38 60 38 DNA Arabidopsis
thaliana 60 caggaaacag ctatgacccc tgtcaacaac cccttctc 38 61 39 DNA
Arabidopsis thaliana 61 tgtaaaacga cggccagtcc acaagagggg tttacaatg
39 62 38 DNA Arabidopsis thaliana 62 caggaaacag ctatgaccac
ccaaccttct ggctctct 38 63 38 DNA Arabidopsis thaliana 63 tgtaaaacga
cggccagtgg tctttgggaa cgatctga 38 64 38 DNA Arabidopsis thaliana 64
caggaaacag ctatgaccag ggaagcgtac agggttct 38 65 38 DNA Arabidopsis
thaliana 65 tgtaaaacga cggccagtcc tcttgagctg aacgtcct 38 66 38 DNA
Arabidopsis thaliana 66 caggaaacag ctatgaccgg cggaactggt ttcactac
38 67 38 DNA Arabidopsis thaliana 67 tgtaaaacga cggccagttg
tcagcataat cggttgga 38 68 38 DNA Arabidopsis thaliana 68 caggaaacag
ctatgacctc cccaaaggtt taggttcc 38 69 38 DNA Arabidopsis thaliana 69
tgtaaaacga cggccagtaa gcctccttct tgtgctga 38 70 38 DNA Arabidopsis
thaliana 70 caggaaacag ctatgacccg acttttccct tccatttg 38 71 38 DNA
Arabidopsis thaliana 71 tgtaaaacga cggccagttg gaggttcggg taactgag
38 72 38 DNA Arabidopsis thaliana 72 caggaaacag ctatgaccca
tcctctcgct agcaggtc 38 73 38 DNA Arabidopsis thaliana 73 tgtaaaacga
cggccagtgg aaccagggga acctaaac 38 74 38 DNA Arabidopsis thaliana 74
caggaaacag ctatgaccgc cgtgagaaac agactcct 38 75 38 DNA Arabidopsis
thaliana 75 tgtaaaacga cggccagtca aatggaaggg aaaagtcg 38 76 38 DNA
Arabidopsis thaliana 76 caggaaacag ctatgaccga tccaaagaga acccagca
38 77 74 DNA Arabidopsis thaliana 77 gggacaagtt tgtacaaaaa
agcaggctta gaaggagata gaaccatggc gacaagatgc 60 agcagcagca gcag 74
78 62 DNA Arabidopsis thaliana 78 ggggaccact ttgtacaaga aagctgggtc
ctgcaggtca gatgggttgg tctttgggaa 60 cg 62 79 72 DNA Arabidopsis
thaliana 79 gggacaagtt tgtacaaaaa agcaggctta gaaggagata gaaccatgcg
gctgaggtgc 60 gcggcgtcgt cg 72 80 61 DNA Arabidopsis thaliana 80
ggggaccact ttgtacaaga aagctgggtc ctgcaggtta gatcggcatg cctttgggca
60 c 61 81 72 DNA Arabidopsis thaliana 81 gggacaagtt tgtacaaaaa
agcaggctta gaaggagata gaaccatgag gctgcgatgc 60 gcggcgtcgt cg 72 82
62 DNA Arabidopsis thaliana 82 ggggaccact ttgtacaaga aagctgggtc
ctgcaggtca gattggcatg ccttttggca 60 cg 62 83 71 DNA Arabidopsis
thaliana 83 gggacaagtt tgtacaaaaa agcaggctta gaaggagata gaaccatggt
acccaagtgt 60 agtgtctcgg c 71 84 61 DNA Arabidopsis thaliana 84
ggggaccact ttgtacaaga aagctgggtc ctgcaggtta gattggctga cctttgggaa
60 c 61 85 70 DNA Arabidopsis thaliana 85 gggacaagtt tgtacaaaaa
agcaggctta gaaggagata gaaccatgat ctttacatgc 60 agcgcgtcct 70 86 61
DNA Arabidopsis thaliana 86 ggggaccact ttgtacaaga aagctgggtc
ctgcaggtca tatgggctgg cctttcggta 60 c 61 87 72 DNA Arabidopsis
thaliana 87 gggacaagtt tgtacaaaaa agcaggctta gaaggagata gaaccatggc
cccgaggtgc 60 agcttatcag cg 72 88 61 DNA Arabidopsis thaliana 88
ggggaccact ttgtacaaga aagctgggtc ctgcaggtta gattggttga ccctctggta
60 c 61 89 65 DNA Arabidopsis thaliana 89 ggggacaagt ttgtacaaaa
aagcaggctg cggccgctga acaatggcct ctttgatgct 60 caacg 65 90 62 DNA
Arabidopsis thaliana 90 ggggaccact ttgtacaaga aagctgggtc ctgcaggtca
gatgggttgg tctttgggaa 60 cg 62 91 348 PRT Arabidopsis thaliana 91
Met Lys Ala Thr Leu Ala Ala Pro Ser Ser Leu Thr Ser Leu Pro Tyr 1 5
10 15 Arg Thr Asn Ser Ser Phe Gly Ser Lys Ser Ser Leu Leu Phe Arg
Ser 20 25 30 Pro Ser Ser Ser Ser Ser Val Ser Met Thr Thr Thr Arg
Gly Asn Val 35 40 45 Ala Val Ala Ala Ala Ala Thr Ser Thr Glu Ala
Leu Arg Lys Gly Ile 50 55 60 Ala Glu Phe Tyr Asn Glu Thr Ser Gly
Leu Trp Glu Glu Ile Trp Gly 65 70 75 80 Asp His Met His His Gly Phe
Tyr Asp Pro Asp Ser Ser Val Gln Leu 85 90 95 Ser Asp Ser Gly His
Lys Glu Ala Gln Ile Arg Met Ile Glu Glu Ser 100 105 110 Leu Arg Phe
Ala Gly Val Thr Asp Glu Glu Glu Glu Lys Lys Ile Lys 115 120 125 Lys
Val Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Tyr Leu 130 135
140 Ala Ser Lys Phe Gly Ala Glu Cys Ile Gly Ile Thr Leu Ser Pro Val
145 150 155 160 Gln Ala Lys Arg Ala Asn Asp Leu Ala Ala Ala Gln Ser
Leu Ser His 165 170 175 Lys Ala Ser Phe Gln Val Ala Asp Ala Leu Asp
Gln Pro Phe Glu Asp 180 185 190 Gly Lys Phe Asp Leu Val Trp Ser Met
Glu Ser Gly Glu His Met Pro 195 200 205 Asp Lys Ala Lys Phe Val Lys
Glu Leu Val Arg Val Ala Ala Pro Gly 210 215 220 Gly Arg Ile Ile Ile
Val Thr Trp Cys His Arg Asn Leu Ser Ala Gly 225 230 235 240 Glu Glu
Ala Leu Gln Pro Trp Glu Gln Asn Ile Leu Asp Lys Ile Cys 245 250 255
Lys Thr Phe Tyr Leu Pro Ala Trp Cys Ser Thr Asp Asp Tyr Val Asn 260
265 270 Leu Leu Gln Ser His Ser Leu Gln Asp Ile Lys Cys Ala Asp Trp
Ser 275 280 285 Glu Asn Val Ala Pro Phe Trp Pro Ala Val Ile Arg Thr
Ala Leu Thr 290 295 300 Trp Lys Gly Leu Val Ser Leu Leu Arg Ser Gly
Met Lys Ser Ile Lys 305 310 315 320 Gly Ala Leu Thr Met Pro Leu Met
Ile Glu Gly Tyr Lys Lys Gly Val 325 330 335 Ile Lys Phe Gly Ile Ile
Thr Cys Gln Lys Pro Leu 340 345 92 348 PRT Arabidopsis thaliana 92
Met Lys Ala Thr Leu Ala Ala Pro Ser Ser Leu Thr Ser Leu Pro Tyr 1 5
10 15 Arg Thr Asn Ser Ser Phe Gly Ser Lys Ser Ser Leu Leu Phe Arg
Ser 20 25 30 Pro Ser Ser Ser Ser Ser Val Ser Met Thr Thr Thr Arg
Gly Asn Val 35 40 45 Ala Val Ala Ala Ala Ala Thr Ser Thr Glu Ala
Leu Arg Lys Gly Ile 50 55 60 Ala Glu Phe Tyr Asn Glu Thr Ser Gly
Leu Trp Glu Glu Ile Trp Gly 65 70 75 80 Asp His Met His His Gly Phe
Tyr Asp Pro Asp Ser Ser Val Gln Leu 85 90 95 Ser Asp Ser Gly His
Lys Glu Ala Gln Ile Arg Met Ile Glu Glu Ser 100 105 110 Leu Arg Phe
Ala Gly Val Thr Asp Glu Glu Glu Glu Lys Lys Ile Lys 115 120 125 Lys
Val Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Tyr Leu 130 135
140 Ala Ser Lys Phe Gly Ala Glu Cys Ile Gly Ile Thr Leu Ser Pro Val
145 150 155 160 Gln Ala
Lys Arg Ala Asn Asp Leu Ala Ala Ala Gln Ser Leu Ala His 165 170 175
Lys Ala Ser Phe Gln Val Ala Asp Ala Leu Asp Gln Pro Phe Glu Asp 180
185 190 Gly Lys Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His Met
Pro 195 200 205 Asp Lys Ala Lys Phe Val Lys Glu Leu Val Arg Val Ala
Ala Pro Gly 210 215 220 Gly Arg Ile Ile Ile Val Thr Trp Cys His Arg
Asn Leu Ser Ala Gly 225 230 235 240 Glu Glu Ala Leu Gln Pro Trp Glu
Gln Asn Ile Leu Asp Lys Ile Cys 245 250 255 Lys Thr Phe Tyr Leu Pro
Ala Trp Cys Ser Thr Asp Asp Tyr Val Asn 260 265 270 Leu Leu Gln Ser
His Ser Leu Gln Asp Ile Lys Cys Ala Asp Trp Ser 275 280 285 Glu Asn
Val Ala Pro Phe Trp Pro Ala Val Ile Arg Thr Ala Leu Thr 290 295 300
Trp Lys Gly Leu Val Ser Leu Leu Arg Ser Gly Met Lys Ser Ile Lys 305
310 315 320 Gly Ala Leu Thr Met Pro Leu Met Ile Glu Gly Tyr Lys Lys
Gly Val 325 330 335 Ile Lys Phe Gly Ile Ile Thr Cys Gln Lys Pro Leu
340 345 93 364 PRT Oryza sativa 93 Met Ala His Ala Ala Ala Ala Thr
Gly Ala Leu Ala Pro Leu His Pro 1 5 10 15 Leu Leu Arg Cys Thr Ser
Arg His Leu Cys Ala Ser Ala Ser Pro Arg 20 25 30 Ala Gly Leu Cys
Leu His His His Arg Arg Arg Arg Arg Ser Ser Arg 35 40 45 Arg Thr
Lys Leu Ala Val Arg Ala Met Ala Pro Thr Leu Ser Ser Ser 50 55 60
Ser Thr Ala Ala Ala Ala Pro Pro Gly Leu Lys Glu Gly Ile Ala Gly 65
70 75 80 Leu Tyr Asp Glu Ser Ser Gly Val Trp Glu Ser Ile Trp Gly
Glu His 85 90 95 Met His His Gly Phe Tyr Asp Ala Gly Glu Ala Ala
Ser Met Ser Asp 100 105 110 His Arg Arg Ala Gln Ile Arg Met Ile Glu
Glu Ser Leu Ala Phe Ala 115 120 125 Ala Val Pro Gly Ala Asp Asp Ala
Glu Lys Lys Pro Lys Ser Val Val 130 135 140 Asp Val Gly Cys Gly Ile
Gly Gly Ser Ser Arg Tyr Leu Ala Asn Lys 145 150 155 160 Tyr Gly Ala
Gln Cys Tyr Gly Ile Thr Leu Ser Pro Val Gln Ala Glu 165 170 175 Arg
Gly Asn Ala Leu Ala Ala Glu Gln Gly Leu Ser Asp Lys Val Arg 180 185
190 Ile Gln Val Gly Asp Ala Leu Glu Gln Pro Phe Pro Asp Gly Gln Phe
195 200 205 Asp Leu Val Trp Ser Met Glu Ser Gly Glu His Met Pro Asp
Lys Arg 210 215 220 Gln Phe Val Ser Glu Leu Ala Arg Val Ala Ala Pro
Gly Ala Arg Ile 225 230 235 240 Ile Ile Val Thr Trp Cys His Arg Asn
Leu Glu Pro Ser Glu Glu Ser 245 250 255 Leu Lys Pro Asp Glu Leu Asn
Leu Leu Lys Arg Ile Cys Asp Ala Tyr 260 265 270 Tyr Leu Pro Asp Trp
Cys Ser Pro Ser Asp Tyr Val Lys Ile Ala Glu 275 280 285 Ser Leu Ser
Leu Glu Asp Ile Arg Thr Ala Asp Trp Ser Glu Asn Val 290 295 300 Ala
Pro Phe Trp Pro Ala Val Ile Lys Ser Ala Leu Thr Trp Lys Gly 305 310
315 320 Leu Thr Ser Leu Leu Arg Ser Gly Trp Lys Thr Ile Arg Gly Ala
Met 325 330 335 Val Met Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly Leu
Ile Lys Phe 340 345 350 Thr Ile Ile Thr Cys Arg Lys Pro Glu Thr Thr
Gln 355 360 94 352 PRT Zea mays 94 Met Ala His Ala Ala Leu Leu His
Cys Ser Gln Ser Ser Arg Ser Leu 1 5 10 15 Ala Ala Cys Arg Arg Gly
Ser His Tyr Arg Ala Pro Ser His Val Pro 20 25 30 Arg His Ser Arg
Arg Leu Arg Arg Ala Val Val Ser Leu Arg Pro Met 35 40 45 Ala Ser
Ser Thr Ala Gln Ala Pro Ala Thr Ala Pro Pro Gly Leu Lys 50 55 60
Glu Gly Ile Ala Gly Leu Tyr Asp Glu Ser Ser Gly Leu Trp Glu Asn 65
70 75 80 Ile Trp Gly Asp His Met His His Gly Phe Tyr Asp Ser Ser
Glu Ala 85 90 95 Ala Ser Met Ala Asp His Arg Arg Ala Gln Ile Arg
Met Ile Glu Glu 100 105 110 Ala Leu Ala Phe Ala Gly Val Pro Ala Ser
Asp Asp Pro Glu Lys Thr 115 120 125 Pro Lys Thr Ile Val Asp Val Gly
Cys Gly Ile Gly Gly Ser Ser Arg 130 135 140 Tyr Leu Ala Lys Lys Tyr
Gly Ala Gln Cys Thr Gly Ile Thr Leu Ser 145 150 155 160 Pro Val Gln
Ala Glu Arg Gly Asn Ala Leu Ala Ala Ala Gln Gly Leu 165 170 175 Ser
Asp Gln Val Thr Leu Gln Val Ala Asp Ala Leu Glu Gln Pro Phe 180 185
190 Pro Asp Gly Gln Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His
195 200 205 Met Pro Asp Lys Arg Lys Phe Val Ser Glu Leu Ala Arg Val
Ala Ala 210 215 220 Pro Gly Gly Thr Ile Ile Ile Val Thr Trp Cys His
Arg Asn Leu Asp 225 230 235 240 Pro Ser Glu Thr Ser Leu Lys Pro Asp
Glu Leu Ser Leu Leu Arg Arg 245 250 255 Ile Cys Asp Ala Tyr Tyr Leu
Pro Asp Trp Cys Ser Pro Ser Asp Tyr 260 265 270 Val Asn Ile Ala Lys
Ser Leu Ser Leu Glu Asp Ile Lys Thr Ala Asp 275 280 285 Trp Ser Glu
Asn Val Ala Pro Phe Trp Pro Ala Val Ile Lys Ser Ala 290 295 300 Leu
Thr Trp Lys Gly Phe Thr Ser Leu Leu Thr Thr Gly Trp Lys Thr 305 310
315 320 Ile Arg Gly Ala Met Val Met Pro Leu Met Ile Gln Gly Tyr Lys
Lys 325 330 335 Gly Leu Ile Lys Phe Thr Ile Ile Thr Cys Arg Lys Pro
Gly Ala Ala 340 345 350 95 345 PRT Gossypium hirsutum 95 Met Ala
Ala Ala Leu Gln Leu Gln Thr His Pro Cys Phe His Gly Thr 1 5 10 15
Cys Gln Leu Ser Pro Pro Pro Arg Pro Ser Val Ser Phe Pro Ser Ser 20
25 30 Ser Arg Ser Phe Pro Ser Ser Arg Arg Ser Leu Ser Ala His Val
Lys 35 40 45 Ala Ala Ala Ser Ser Leu Ser Thr Thr Thr Leu Gln Glu
Gly Ile Ala 50 55 60 Glu Phe Tyr Asp Glu Ser Ser Gly Ile Trp Glu
Asp Ile Trp Gly Asp 65 70 75 80 His Met His His Gly Tyr Tyr Glu Pro
Gly Ser Asp Ile Ser Gly Ser 85 90 95 Asp His Arg Ala Ala Gln Ile
Arg Met Val Glu Glu Ser Leu Arg Phe 100 105 110 Ala Gly Ile Ser Glu
Asp Pro Ala Asn Arg Pro Lys Arg Ile Val Asp 115 120 125 Val Gly Cys
Gly Ile Gly Gly Ser Ser Arg Tyr Leu Ala Arg Lys Tyr 130 135 140 Gly
Ala Lys Cys Gln Gly Ile Thr Leu Ser Pro Val Gln Ala Gly Arg 145 150
155 160 Ala Asn Ala Leu Ala Asn Ala Gln Gly Leu Ala Glu Gln Val Cys
Phe 165 170 175 Glu Val Ala Asp Ala Leu Asn Gln Pro Phe Pro Asp Asp
Gln Phe Asp 180 185 190 Leu Val Trp Ser Met Glu Ser Gly Glu His Met
Pro Asp Lys Pro Lys 195 200 205 Phe Val Lys Glu Leu Val Arg Val Ala
Ala Pro Gly Gly Thr Ile Ile 210 215 220 Val Val Thr Trp Cys His Arg
Asp Leu Gly Pro Ser Glu Glu Ser Leu 225 230 235 240 Gln Pro Trp Glu
Gln Lys Leu Leu Asn Arg Ile Cys Asp Ala Tyr Tyr 245 250 255 Leu Pro
Glu Trp Cys Ser Thr Ser Asp Tyr Val Lys Leu Phe Gln Ser 260 265 270
Leu Ser Leu Gln Asp Ile Lys Ala Gly Asp Trp Thr Glu Asn Val Ala 275
280 285 Pro Phe Trp Pro Ala Val Ile Arg Ser Ala Leu Thr Trp Lys Gly
Phe 290 295 300 Thr Ser Leu Leu Arg Ser Gly Leu Lys Thr Ile Lys Gly
Ala Leu Val 305 310 315 320 Met Pro Leu Met Ile Glu Gly Phe Gln Lys
Gly Val Ile Lys Phe Ala 325 330 335 Ile Ile Ala Cys Arg Lys Pro Ala
Glu 340 345 96 376 PRT cuphea pulcherrima 96 Met Pro Ile Thr Ser
Ile Ser Ala Asn Gln Arg Pro Phe Phe Pro Ser 1 5 10 15 Pro Tyr Arg
Gly Ser Ser Lys Asn Met Ala Pro Pro Glu Leu Ala Gln 20 25 30 Ser
Gln Val Pro Met Gly Ser Asn Lys Ser Asn Lys Asn His Gly Leu 35 40
45 Val Gly Ser Val Ser Gly Trp Arg Arg Met Phe Gly Thr Trp Ala Thr
50 55 60 Ala Asp Lys Thr Gln Ser Thr Asp Thr Ser Asn Glu Gly Val
Val Ser 65 70 75 80 Tyr Asp Thr Gln Val Leu Gln Lys Gly Ile Ala Glu
Phe Tyr Asp Glu 85 90 95 Ser Ser Gly Ile Trp Glu Asp Ile Trp Gly
Asp His Met His His Gly 100 105 110 Tyr Tyr Asp Gly Ser Thr Pro Val
Ser Leu Pro Asp His Arg Ser Ala 115 120 125 Gln Ile Arg Met Ile Asp
Glu Ala Leu Arg Phe Ala Ser Val Pro Ser 130 135 140 Gly Glu Glu Asp
Glu Ser Lys Ser Lys Ile Pro Lys Arg Ile Val Asp 145 150 155 160 Val
Gly Cys Gly Ile Gly Gly Ser Ser Arg Tyr Leu Ala Arg Lys Tyr 165 170
175 Gly Ala Glu Cys Arg Gly Ile Thr Leu Ser Pro Val Gln Ala Glu Arg
180 185 190 Gly Asn Ser Leu Ala Arg Ser Gln Gly Leu Ser Asp Lys Val
Ser Phe 195 200 205 Gln Val Ala Asp Ala Leu Ala Gln Pro Phe Pro Asp
Gly Gln Phe Asp 210 215 220 Leu Val Trp Ser Met Glu Ser Gly Glu His
Met Pro Asp Lys Ser Lys 225 230 235 240 Phe Val Asn Glu Leu Val Arg
Val Ala Ala Pro Gly Gly Thr Ile Ile 245 250 255 Ile Val Thr Trp Cys
His Arg Asp Leu Arg Glu Asp Glu Asp Ala Leu 260 265 270 Gln Pro Arg
Glu Lys Glu Ile Leu Asp Lys Ile Cys Asn Pro Phe Tyr 275 280 285 Leu
Pro Ala Trp Cys Ser Ala Ala Asp Tyr Val Lys Leu Leu Gln Ser 290 295
300 Leu Asp Val Glu Asp Ile Lys Ser Ala Asp Trp Thr Pro Tyr Val Ala
305 310 315 320 Pro Phe Trp Pro Ala Val Leu Lys Ser Ala Phe Thr Ile
Lys Gly Phe 325 330 335 Val Ser Leu Leu Arg Ser Gly Met Lys Thr Ile
Lys Gly Ala Phe Ala 340 345 350 Met Pro Leu Met Ile Glu Gly Tyr Lys
Lys Gly Val Ile Lys Phe Ser 355 360 365 Ile Ile Thr Cys Arg Lys Pro
Glu 370 375 97 347 PRT Brassica napus 97 Met Lys Ala Thr Leu Ala
Pro Ser Ser Leu Ile Ser Leu Pro Arg His 1 5 10 15 Lys Val Ser Ser
Leu Arg Ser Pro Ser Leu Leu Leu Gln Ser Gln Arg 20 25 30 Pro Ser
Ser Ala Leu Met Thr Thr Thr Thr Ala Ser Arg Gly Ser Val 35 40 45
Ala Val Thr Ala Ala Ala Thr Ser Ser Val Glu Ala Leu Arg Glu Gly 50
55 60 Ile Ala Glu Phe Tyr Asn Glu Thr Ser Gly Leu Trp Glu Glu Ile
Trp 65 70 75 80 Gly Asp His Met His His Gly Phe Tyr Asp Pro Asp Ser
Ser Val Gln 85 90 95 Leu Ser Asp Ser Gly His Arg Glu Ala Gln Ile
Arg Met Ile Glu Glu 100 105 110 Ser Leu Arg Phe Ala Gly Val Thr Glu
Glu Glu Lys Lys Ile Lys Arg 115 120 125 Val Val Asp Val Gly Cys Gly
Ile Gly Gly Ser Ser Arg Tyr Ile Ala 130 135 140 Ser Lys Phe Gly Ala
Glu Cys Ile Gly Ile Thr Leu Ser Pro Val Gln 145 150 155 160 Ala Lys
Arg Ala Asn Asp Leu Ala Ala Ala Gln Ser Leu Ser His Lys 165 170 175
Val Ser Phe Gln Val Ala Asp Ala Leu Glu Gln Pro Phe Glu Asp Gly 180
185 190 Ile Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His Met Pro
Asp 195 200 205 Lys Ala Lys Phe Val Lys Glu Leu Val Arg Val Ala Ala
Pro Gly Gly 210 215 220 Arg Ile Ile Ile Val Thr Trp Cys His Arg Asn
Leu Ser Pro Gly Glu 225 230 235 240 Glu Ala Leu Gln Pro Trp Glu Gln
Asn Leu Leu Asp Arg Ile Cys Lys 245 250 255 Thr Phe Tyr Leu Pro Ala
Trp Cys Ser Thr Ser Asp Tyr Val Asp Leu 260 265 270 Leu Gln Ser Leu
Ser Leu Gln Asp Ile Lys Cys Ala Asp Trp Ser Glu 275 280 285 Asn Val
Ala Pro Phe Trp Pro Ala Val Ile Arg Thr Ala Leu Thr Trp 290 295 300
Lys Gly Leu Val Ser Leu Leu Arg Ser Gly Met Lys Ser Ile Lys Gly 305
310 315 320 Ala Leu Thr Met Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly
Val Ile 325 330 335 Lys Phe Gly Ile Ile Thr Cys Gln Lys Pro Leu 340
345 98 347 PRT Brassica napus 98 Met Lys Ala Thr Leu Ala Pro Pro
Ser Ser Leu Ile Ser Leu Pro Arg 1 5 10 15 His Lys Val Ser Ser Leu
Arg Ser Pro Ser Leu Leu Leu Gln Ser Gln 20 25 30 Arg Arg Ser Ser
Ala Leu Met Thr Thr Thr Ala Ser Arg Gly Ser Val 35 40 45 Ala Val
Thr Ala Ala Ala Thr Ser Ser Ala Glu Ala Leu Arg Glu Gly 50 55 60
Ile Ala Glu Phe Tyr Asn Glu Thr Ser Gly Leu Trp Glu Glu Ile Trp 65
70 75 80 Gly Asp His Met His His Gly Phe Tyr Asp Pro Asp Ser Ser
Val Gln 85 90 95 Leu Ser Asp Ser Gly His Arg Glu Ala Gln Ile Arg
Met Ile Glu Glu 100 105 110 Ser Leu Arg Phe Ala Gly Val Thr Glu Glu
Glu Lys Lys Ile Lys Arg 115 120 125 Val Val Asp Val Gly Cys Gly Ile
Gly Gly Ser Ser Arg Tyr Ile Ala 130 135 140 Ser Lys Phe Gly Ala Glu
Cys Ile Gly Ile Thr Leu Ser Pro Val Gln 145 150 155 160 Ala Lys Arg
Ala Asn Asp Leu Ala Thr Ala Gln Ser Leu Ser His Lys 165 170 175 Val
Ser Phe Gln Val Ala Asp Ala Leu Asp Gln Pro Phe Glu Asp Gly 180 185
190 Ile Ser Asp Leu Val Trp Ser Met Glu Ser Gly Glu His Met Pro Asp
195 200 205 Lys Ala Lys Phe Val Lys Glu Leu Val Arg Val Thr Ala Pro
Gly Gly 210 215 220 Arg Ile Ile Ile Val Thr Trp Cys His Arg Asn Leu
Ser Gln Gly Glu 225 230 235 240 Glu Ser Leu Gln Pro Trp Glu Gln Asn
Leu Leu Asp Arg Ile Cys Lys 245 250 255 Thr Phe Tyr Leu Pro Ala Trp
Cys Ser Thr Thr Asp Tyr Val Glu Leu 260 265 270 Leu Gln Ser Leu Ser
Leu Gln Asp Ile Lys Tyr Ala Asp Trp Ser Glu 275 280 285 Asn Val Ala
Pro Phe Trp Pro Ala Val Ile Arg Thr Ala Leu Thr Trp 290 295 300 Lys
Gly Leu Val Ser Leu Leu Arg Ser Gly Met Lys Ser Ile Lys Gly 305 310
315 320 Ala Leu Thr Met Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly Val
Ile 325 330 335 Lys Phe Gly Ile Ile Thr Cys Gln Lys Pro Leu 340 345
99 310 PRT Lycopersicon esculentum 99 Met Ala Ser Val Ala Ala Met
Asn Ala Val Ser Ser Ser Ser Val Glu 1 5 10 15 Val Gly Ile Gln Asn
Gln Gln Glu Leu Lys Lys Gly Ile Ala Asp Leu 20 25 30 Tyr Asp Glu
Ser Ser Gly Ile Trp Glu Asp Ile Trp Gly Asp His Met 35 40 45 His
His Gly Tyr Tyr Glu Pro Lys Ser Ser Val Glu Leu Ser Asp His 50 55
60 Arg Ala Ala Gln Ile Arg Met Ile Glu Gln Ala Leu Ser Phe Ala Ala
65 70 75 80 Ile Ser Glu Asp Pro Ala Lys Lys Pro Thr Ser Ile Val Asp
Val Gly 85 90 95 Cys Gly Ile Gly Gly Ser Ser Arg Tyr Leu Ala
Lys
Lys Tyr Gly Ala 100 105 110 Thr Ala Lys Gly Ile Thr Leu Ser Pro Val
Gln Ala Glu Arg Ala Gln 115 120 125 Ala Leu Ala Asp Ala Gln Gly Leu
Gly Asp Lys Val Ser Phe Gln Val 130 135 140 Ala Asp Ala Leu Asn Gln
Pro Phe Pro Asp Gly Gln Phe Asp Leu Val 145 150 155 160 Trp Ser Met
Glu Ser Gly Glu His Met Pro Asn Lys Glu Lys Phe Val 165 170 175 Gly
Glu Leu Ala Arg Val Ala Ala Pro Gly Gly Thr Ile Ile Leu Val 180 185
190 Thr Trp Cys His Arg Asp Leu Ser Pro Ser Glu Glu Ser Leu Thr Pro
195 200 205 Glu Glu Lys Glu Leu Leu Asn Lys Ile Cys Lys Ala Phe Tyr
Leu Pro 210 215 220 Ala Trp Cys Ser Thr Ala Asp Tyr Val Lys Leu Leu
Gln Ser Asn Ser 225 230 235 240 Leu Gln Asp Ile Lys Ala Glu Asp Trp
Ser Glu Asn Val Ala Pro Phe 245 250 255 Trp Pro Ala Val Ile Lys Ser
Ala Leu Thr Trp Lys Gly Phe Thr Ser 260 265 270 Val Leu Arg Ser Gly
Trp Lys Thr Ile Lys Ala Ala Leu Ala Met Pro 275 280 285 Leu Met Ile
Glu Gly Tyr Lys Lys Gly Leu Ile Lys Phe Ala Ile Ile 290 295 300 Thr
Cys Arg Lys Pro Glu 305 310 100 302 PRT GLYCINE MAX 100 Met Ser Val
Glu Gln Lys Ala Ala Gly Lys Glu Glu Glu Gly Lys Leu 1 5 10 15 Gln
Lys Gly Ile Ala Glu Phe Tyr Asp Glu Ser Ser Gly Ile Trp Glu 20 25
30 Asn Ile Trp Gly Asp His Met His His Gly Phe Tyr Asp Pro Asp Ser
35 40 45 Thr Val Ser Val Ser Asp His Arg Ala Ala Gln Ile Arg Met
Ile Gln 50 55 60 Glu Ser Leu Arg Phe Ala Ser Leu Leu Ser Glu Asn
Pro Ser Lys Trp 65 70 75 80 Pro Lys Ser Ile Val Asp Val Gly Cys Gly
Ile Gly Gly Ser Ser Arg 85 90 95 Tyr Leu Ala Lys Lys Phe Gly Ala
Thr Ser Val Gly Ile Thr Leu Ser 100 105 110 Pro Val Gln Ala Gln Arg
Ala Asn Ala Leu Ala Ala Ala Gln Gly Leu 115 120 125 Ala Asp Lys Val
Ser Phe Gln Val Ala Asp Ala Leu Gln Gln Pro Phe 130 135 140 Ser Asp
Gly Gln Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His 145 150 155
160 Met Pro Asp Lys Ala Lys Phe Val Gly Glu Leu Ala Arg Val Ala Ala
165 170 175 Pro Gly Ala Thr Ile Ile Ile Val Thr Trp Cys His Arg Asp
Leu Gly 180 185 190 Pro Asp Glu Gln Ser Leu His Pro Trp Glu Gln Asp
Leu Leu Lys Lys 195 200 205 Ile Cys Asp Ala Tyr Tyr Leu Pro Ala Trp
Cys Ser Thr Ser Asp Tyr 210 215 220 Val Lys Leu Leu Gln Ser Leu Ser
Leu Gln Asp Ile Lys Ser Glu Asp 225 230 235 240 Trp Ser Arg Phe Val
Ala Pro Phe Trp Pro Ala Val Ile Arg Ser Ala 245 250 255 Phe Thr Trp
Lys Gly Leu Thr Ser Leu Leu Ser Ser Gly Gln Lys Thr 260 265 270 Ile
Lys Gly Ala Leu Ala Met Pro Leu Met Ile Glu Gly Tyr Lys Lys 275 280
285 Asp Leu Ile Lys Phe Ala Ile Ile Thr Cys Arg Lys Pro Glu 290 295
300 101 350 PRT Glycine max 101 Met Ala Thr Val Val Arg Ile Pro Thr
Ile Ser Cys Ile His Ile His 1 5 10 15 Thr Phe Arg Ser Gln Ser Pro
Arg Thr Phe Ala Arg Ile Arg Val Gly 20 25 30 Pro Arg Ser Trp Ala
Pro Ile Arg Ala Ser Ala Ala Ser Ser Glu Arg 35 40 45 Gly Glu Ile
Val Leu Glu Gln Lys Pro Lys Lys Glu Glu Glu Gly Lys 50 55 60 Leu
Gln Lys Gly Ile Ala Glu Phe Tyr Asp Glu Ser Ser Gly Leu Trp 65 70
75 80 Glu Asn Ile Trp Gly Asp His Met His His Gly Phe Tyr Asp Pro
Asp 85 90 95 Ser Thr Val Ser Val Ser Asp His Arg Ala Ala Gln Ile
Arg Met Ile 100 105 110 Gln Glu Ser Leu Arg Phe Ala Ser Val Ser Glu
Glu Arg Ser Lys Trp 115 120 125 Pro Lys Ser Ile Val Asp Val Gly Cys
Gly Ile Gly Gly Ser Ser Arg 130 135 140 Tyr Leu Ala Lys Lys Phe Gly
Ala Thr Ser Val Gly Ile Thr Leu Ser 145 150 155 160 Pro Val Gln Ala
Gln Arg Ala Asn Ala Leu Ala Ala Ala Gln Gly Leu 165 170 175 Ala Asp
Lys Val Ser Phe Gln Val Ala Asp Ala Leu Gln Gln Pro Phe 180 185 190
Ser Asp Gly Gln Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His 195
200 205 Met Pro Asp Lys Ala Lys Phe Val Gly Glu Leu Ala Arg Val Ala
Ala 210 215 220 Pro Gly Ala Thr Ile Ile Ile Val Thr Trp Cys His Arg
Asp Leu Gly 225 230 235 240 Pro Asp Glu Gln Ser Leu His Pro Trp Glu
Gln Asp Leu Leu Lys Lys 245 250 255 Ile Cys Asp Ala Tyr Tyr Leu Pro
Ala Trp Cys Ser Thr Ser Asp Tyr 260 265 270 Val Lys Leu Leu Gln Ser
Leu Ser Leu Gln Asp Ile Lys Ser Glu Asp 275 280 285 Trp Ser Arg Phe
Val Ala Pro Phe Trp Pro Ala Val Ile Arg Ser Ala 290 295 300 Phe Thr
Trp Lys Gly Leu Thr Ser Leu Leu Ser Ser Gly Leu Lys Thr 305 310 315
320 Ile Lys Gly Ala Leu Ala Met Pro Leu Met Ile Glu Gly Tyr Lys Lys
325 330 335 Asp Leu Ile Lys Phe Ala Ile Ile Thr Cys Arg Lys Pro Glu
340 345 350 102 350 PRT Glycine max 102 Met Ala Thr Val Val Arg Ile
Pro Thr Ile Ser Cys Ile His Ile His 1 5 10 15 Thr Phe Arg Ser Gln
Ser Pro Arg Thr Phe Ala Arg Ile Arg Val Gly 20 25 30 Pro Arg Ser
Trp Ala Pro Ile Arg Ala Ser Ala Ala Ser Ser Glu Arg 35 40 45 Gly
Glu Ile Val Leu Glu Gln Lys Pro Lys Lys Asp Asp Lys Glu Lys 50 55
60 Leu Gln Lys Gly Ile Ala Glu Phe Tyr Asp Glu Ser Ser Gly Leu Trp
65 70 75 80 Glu Asn Ile Trp Gly Asp His Met His His Gly Phe Tyr Asp
Pro Asp 85 90 95 Ser Thr Val Ser Leu Ser Asp His Arg Ala Ala Gln
Ile Arg Met Ile 100 105 110 Gln Glu Ser Leu Arg Phe Ala Ser Val Ser
Glu Glu Arg Ser Lys Trp 115 120 125 Pro Lys Ser Ile Val Asp Val Gly
Cys Gly Ile Gly Gly Ser Ser Arg 130 135 140 Tyr Leu Ala Lys Lys Phe
Gly Ala Thr Ser Val Gly Ile Thr Leu Ser 145 150 155 160 Pro Val Gln
Ala Gln Arg Ala Asn Ala Leu Ala Ala Ala Gln Gly Leu 165 170 175 Ala
Asp Lys Val Ser Phe Gln Val Ala Asp Ala Leu Gln Gln Pro Phe 180 185
190 Ser Asp Gly Gln Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His
195 200 205 Met Pro Asp Lys Ala Lys Phe Val Gly Glu Leu Ala Arg Val
Ala Ala 210 215 220 Pro Gly Ala Thr Ile Ile Ile Val Thr Trp Cys His
Arg Asp Leu Gly 225 230 235 240 Pro Asp Glu Gln Ser Leu His Pro Trp
Glu Gln Asp Leu Leu Lys Lys 245 250 255 Ile Cys Asp Ala Tyr Tyr Leu
Pro Ala Trp Cys Ser Thr Ser Asp Tyr 260 265 270 Val Lys Leu Leu Gln
Ser Leu Ser Leu Gln Asp Ile Lys Ser Glu Asp 275 280 285 Trp Ser Arg
Phe Gly Ala Pro Phe Trp Pro Ala Val Ile Arg Ser Ala 290 295 300 Phe
Thr Trp Lys Gly Leu Thr Ser Leu Leu Ser Ser Gly Gln Lys Thr 305 310
315 320 Ile Lys Gly Ala Leu Ala Met Pro Leu Met Ile Glu Gly Tyr Lys
Lys 325 330 335 Asp Leu Ile Lys Phe Ala Ile Ile Thr Cys Arg Lys Pro
Glu 340 345 350 103 310 PRT Tagetes erecta 103 Ala Leu Ser Val Val
Ala Ala Glu Val Pro Val Thr Val Thr Pro Ala 1 5 10 15 Thr Thr Lys
Ala Glu Asp Val Glu Leu Lys Lys Gly Ile Ala Glu Phe 20 25 30 Tyr
Asp Glu Ser Ser Glu Met Trp Glu Asn Ile Trp Gly Glu His Met 35 40
45 His His Gly Tyr Tyr Asn Thr Asn Ala Val Val Glu Leu Ser Asp His
50 55 60 Arg Ser Ala Gln Ile Arg Met Ile Glu Gln Ala Leu Leu Phe
Ala Ser 65 70 75 80 Val Ser Asp Asp Pro Val Lys Lys Pro Arg Ser Ile
Val Asp Val Gly 85 90 95 Cys Gly Ile Gly Gly Ser Ser Arg Tyr Leu
Ala Lys Lys Tyr Glu Ala 100 105 110 Glu Cys His Gly Ile Thr Leu Ser
Pro Val Gln Ala Glu Arg Ala Gln 115 120 125 Ala Leu Ala Ala Ala Gln
Gly Leu Ala Asp Lys Ala Ser Phe Gln Val 130 135 140 Ala Asp Ala Leu
Asp Gln Pro Phe Pro Asp Gly Lys Phe Asp Leu Val 145 150 155 160 Trp
Ser Met Glu Ser Gly Glu His Met Pro Asp Lys Leu Lys Phe Val 165 170
175 Ser Glu Leu Val Arg Val Ala Ala Pro Gly Ala Thr Ile Ile Ile Val
180 185 190 Thr Trp Cys His Arg Asp Leu Ser Pro Gly Glu Lys Ser Leu
Arg Pro 195 200 205 Asp Glu Glu Lys Ile Leu Lys Lys Ile Cys Ser Ser
Phe Tyr Leu Pro 210 215 220 Ala Trp Cys Ser Thr Ser Asp Tyr Val Lys
Leu Leu Glu Ser Leu Ser 225 230 235 240 Leu Gln Asp Ile Lys Ala Ala
Asp Trp Ser Ala Asn Val Ala Pro Phe 245 250 255 Trp Pro Ala Val Ile
Lys Thr Ala Leu Ser Trp Lys Gly Ile Thr Ser 260 265 270 Leu Leu Arg
Ser Gly Trp Lys Ser Ile Arg Gly Ala Met Val Met Pro 275 280 285 Leu
Met Ile Glu Gly Phe Lys Lys Asp Ile Ile Lys Phe Ser Ile Ile 290 295
300 Thr Cys Lys Lys Pro Glu 305 310 104 354 PRT Sorghum bicolor 104
Glu Arg Arg Ala Ala Gly Gly Arg Arg Glu Pro Leu Gly Gly Gly Ser 1 5
10 15 Val Pro Val Gly Ser His Tyr Tyr Tyr Arg Ala Pro Ser His Val
Pro 20 25 30 Arg Arg Ser Arg Pro Arg Gly Arg Gly Gly Val Val Ser
Leu Arg Pro 35 40 45 Met Ala Ser Ser Thr Ala Ala Gln Pro Pro Ala
Pro Ala Pro Pro Gly 50 55 60 Leu Lys Glu Gly Ile Ala Gly Leu Tyr
Asp Glu Ser Ser Gly Leu Trp 65 70 75 80 Glu Asn Ile Trp Gly Asp His
Met His His Gly Phe Tyr Asp Ser Gly 85 90 95 Glu Ala Ala Ser Met
Ala Asp His Arg Arg Ala Gln Ile Arg Met Ile 100 105 110 Glu Glu Ala
Leu Ala Phe Ala Ala Val Pro Ser Pro Asp Asp Pro Glu 115 120 125 Lys
Ala Pro Lys Thr Ile Val Asp Val Gly Cys Gly Ile Gly Gly Ser 130 135
140 Ser Arg Tyr Leu Ala Lys Lys Tyr Gly Ala Gln Cys Lys Gly Ile Thr
145 150 155 160 Leu Ser Pro Val Gln Ala Glu Arg Gly Asn Ala Leu Ala
Thr Ala Gln 165 170 175 Gly Leu Ser Asp Gln Val Thr Leu Gln Val Ala
Asp Ala Leu Glu Gln 180 185 190 Pro Phe Pro Asp Gly Gln Phe Asp Leu
Val Trp Ser Met Glu Ser Gly 195 200 205 Glu His Met Pro Asp Lys Arg
Lys Phe Val Ser Glu Leu Ala Arg Val 210 215 220 Ala Ala Pro Gly Gly
Thr Ile Ile Ile Val Thr Trp Cys His Arg Asn 225 230 235 240 Leu Glu
Pro Ser Glu Thr Ser Leu Lys Pro Asp Glu Leu Ser Leu Leu 245 250 255
Lys Arg Ile Cys Asp Ala Tyr Tyr Leu Pro Asp Trp Cys Ser Pro Ser 260
265 270 Asp Tyr Val Asn Ile Ala Lys Ser Leu Ser Leu Glu Asp Ile Lys
Ala 275 280 285 Ala Asp Trp Ser Glu Asn Val Ala Pro Phe Trp Pro Ala
Val Ile Lys 290 295 300 Ser Ala Leu Thr Trp Lys Gly Leu Thr Ser Leu
Leu Thr Ser Gly Trp 305 310 315 320 Lys Thr Ile Arg Gly Ala Met Val
Met Pro Leu Met Ile Gln Gly Tyr 325 330 335 Lys Lys Gly Leu Ile Lys
Phe Thr Ile Ile Thr Cys Arg Lys Pro Gly 340 345 350 Ala Ala 105 128
PRT Lilium asiaticum 105 Glu Ser Gly Glu His Met Pro Asp Lys Thr
Lys Phe Val Gly Glu Leu 1 5 10 15 Ala Arg Val Ala Ala Pro Gly Ala
Thr Ile Ile Ile Val Thr Trp Cys 20 25 30 His Arg Asp Leu Leu Pro
Ser Glu Asp Ser Leu Arg Pro Asp Glu Ile 35 40 45 Ser Leu Leu Asn
Lys Ile Cys Asp Ala Tyr Tyr Leu Pro Lys Trp Cys 50 55 60 Ser Ala
Val Asp Tyr Val Lys Ile Ala Glu Ser Tyr Ser Leu Glu Lys 65 70 75 80
Ile Arg Thr Ala Asp Trp Ser Glu Asn Val Ala Pro Phe Trp Pro Ala 85
90 95 Val Ile Arg Ser Ala Leu Thr Trp Lys Gly Phe Thr Ser Leu Leu
Arg 100 105 110 Ser Gly Trp Lys Thr Ile Arg Gly Ala Leu Val Met Pro
Leu Met Ile 115 120 125 106 280 PRT Nostoc punctiforme 106 Met Ser
Ala Thr Leu Tyr Gln Gln Ile Gln Gln Phe Tyr Asp Ala Ser 1 5 10 15
Ser Gly Leu Trp Glu Gln Ile Trp Gly Glu His Met His His Gly Tyr 20
25 30 Tyr Gly Ala Asp Gly Thr Gln Lys Lys Asp Arg Arg Gln Ala Gln
Ile 35 40 45 Asp Leu Ile Glu Glu Leu Leu Asn Trp Ala Gly Val Gln
Ala Ala Glu 50 55 60 Asp Ile Leu Asp Val Gly Cys Gly Ile Gly Gly
Ser Ser Leu Tyr Leu 65 70 75 80 Ala Gln Lys Phe Asn Ala Lys Ala Thr
Gly Ile Thr Leu Ser Pro Val 85 90 95 Gln Ala Ala Arg Ala Thr Glu
Arg Ala Leu Glu Ala Asn Leu Ser Leu 100 105 110 Arg Thr Gln Phe Gln
Val Ala Asn Ala Gln Ala Met Pro Phe Ala Asp 115 120 125 Asp Ser Phe
Asp Leu Val Trp Ser Leu Glu Ser Gly Glu His Met Pro 130 135 140 Asp
Lys Thr Lys Phe Leu Gln Glu Cys Tyr Arg Val Leu Lys Pro Gly 145 150
155 160 Gly Lys Leu Ile Met Val Thr Trp Cys His Arg Pro Thr Asp Glu
Ser 165 170 175 Pro Leu Thr Ala Asp Glu Glu Lys His Leu Gln Asp Ile
Tyr Arg Val 180 185 190 Tyr Cys Leu Pro Tyr Val Ile Ser Leu Pro Glu
Tyr Glu Ala Ile Ala 195 200 205 His Gln Leu Pro Leu His Asn Ile Arg
Thr Ala Asp Trp Ser Thr Ala 210 215 220 Val Ala Pro Phe Trp Asn Val
Val Ile Asp Ser Ala Phe Thr Pro Gln 225 230 235 240 Ala Leu Trp Gly
Leu Leu Asn Ala Gly Trp Thr Thr Ile Gln Gly Ala 245 250 255 Leu Ser
Leu Gly Leu Met Arg Arg Gly Tyr Glu Arg Gly Leu Ile Arg 260 265 270
Phe Gly Leu Leu Cys Gly Asn Lys 275 280 107 280 PRT Anabaena sp.
107 Met Ser Ala Thr Leu Tyr Gln Gln Ile Gln Gln Phe Tyr Asp Ala Ser
1 5 10 15 Ser Gly Leu Trp Glu Glu Ile Trp Gly Glu His Met His His
Gly Tyr 20 25 30 Tyr Gly Ala Asp Gly Thr Glu Gln Lys Asn Arg Arg
Gln Ala Gln Ile 35 40 45 Asp Leu Ile Glu Glu Leu Leu Thr Trp Ala
Gly Val Gln Thr Ala Glu 50 55 60 Asn Ile Leu Asp Val Gly Cys Gly
Ile Gly Gly Ser Ser Leu Tyr Leu 65 70 75 80 Ala Gly Lys Leu Asn Ala
Lys Ala Thr Gly Ile Thr Leu Ser Pro Val 85 90 95 Gln Ala Ala Arg
Ala Thr Glu Arg Ala Lys Glu Ala Gly Leu Ser Gly 100 105 110 Arg Ser
Gln Phe Leu Val Ala Asn Ala Gln Ala Met Pro Phe Asp Asp 115 120 125
Asn Ser Phe Asp Leu Val Trp Ser Leu Glu Ser Gly Glu His Met Pro
130
135 140 Asp Lys Thr Lys Phe Leu Gln Glu Cys Tyr Arg Val Leu Lys Pro
Gly 145 150 155 160 Gly Lys Leu Ile Met Val Thr Trp Cys His Arg Pro
Thr Asp Lys Thr 165 170 175 Pro Leu Thr Ala Asp Glu Lys Lys His Leu
Glu Asp Ile Tyr Arg Val 180 185 190 Tyr Cys Leu Pro Tyr Val Ile Ser
Leu Pro Glu Tyr Glu Ala Ile Ala 195 200 205 Arg Gln Leu Pro Leu Asn
Asn Ile Arg Thr Ala Asp Trp Ser Gln Ser 210 215 220 Val Ala Gln Phe
Trp Asn Ile Val Ile Asp Ser Ala Phe Thr Pro Gln 225 230 235 240 Ala
Ile Phe Gly Leu Leu Arg Ala Gly Trp Thr Thr Ile Gln Gly Ala 245 250
255 Leu Ser Leu Gly Leu Met Arg Arg Gly Tyr Glu Arg Gly Leu Ile Arg
260 265 270 Phe Gly Leu Leu Cys Gly Asp Lys 275 280 108 356 PRT
Artificial Sequence Consensus Sequence misc_feature (1)..(2)
Unknown residue. misc_feature (4)..(4) Unknown residue.
misc_feature (6)..(12) Unknown residue. misc_feature (14)..(66)
Unknown residue. misc_feature (68)..(71) Unknown residue.
misc_feature (73)..(76) Unknown residue. misc_feature (79)..(81)
Unknown residue. misc_feature (89)..(89) Unknown residue.
misc_feature (92)..(92) Unknown residue. misc_feature (105)..(105)
Unknown residue. misc_feature (124)..(124) Unknown residue.
misc_feature (126)..(131) Unknown residue. misc_feature
(147)..(153) Unknown residue. misc_feature (157)..(157) Unknown
residue. misc_feature (166)..(166) Unknown residue. misc_feature
(169)..(170) Unknown residue. misc_feature (173)..(173) Unknown
residue. misc_feature (176)..(178) Unknown residue. misc_feature
(180)..(180) Unknown residue. misc_feature (190)..(190) Unknown
residue. misc_feature (193)..(194) Unknown residue. misc_feature
(198)..(198) Unknown residue. misc_feature (214)..(214) Unknown
residue. misc_feature (221)..(222) Unknown residue. misc_feature
(225)..(225) Unknown residue. misc_feature (228)..(228) Unknown
residue. misc_feature (233)..(233) Unknown residue. misc_feature
(243)..(243) Unknown residue. misc_feature (245)..(245) Unknown
residue. misc_feature (251)..(251) Unknown residue. misc_feature
(260)..(261) Unknown residue. misc_feature (265)..(265) Unknown
residue. misc_feature (268)..(268) Unknown residue. misc_feature
(296)..(298) Unknown residue. misc_feature (304)..(304) Unknown
residue. misc_feature (309)..(309) Unknown residue. misc_feature
(313)..(313) Unknown residue. misc_feature (316)..(317) Unknown
residue. misc_feature (320)..(321) Unknown residue. misc_feature
(323)..(324) Unknown residue. misc_feature (327)..(328) Unknown
residue. misc_feature (330)..(332) Unknown residue. misc_feature
(334)..(336) Unknown residue. misc_feature (339)..(339) Unknown
residue. misc_feature (345)..(345) Unknown residue. misc_feature
(349)..(349) Unknown residue. misc_feature (352)..(352) Unknown
residue. misc_feature (353)..(354) Unknown residue. 108 Xaa Xaa Met
Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 50 55 60 Xaa Xaa Cys Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa
Arg Pro Xaa Xaa 65 70 75 80 Xaa Pro Arg Phe Ile Gln His Lys Xaa Glu
Ala Xaa Trp Phe Tyr Arg 85 90 95 Phe Leu Ser Ile Val Tyr Asp His
Xaa Ile Asn Pro Gly His Trp Thr 100 105 110 Glu Asp Met Arg Asp Asp
Ala Leu Glu Pro Ala Xaa Leu Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Val
Val Asp Val Gly Gly Gly Thr Gly Phe Thr Thr Leu 130 135 140 Gly Ile
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Val Thr Xaa Leu Asp Gln 145 150 155
160 Ser Pro His Gln Leu Xaa Lys Ala Xaa Xaa Lys Glu Xaa Leu Lys Xaa
165 170 175 Xaa Xaa Ile Xaa Glu Gly Asp Ala Glu Asp Leu Pro Phe Xaa
Thr Asp 180 185 190 Xaa Xaa Asp Arg Tyr Xaa Ser Ala Gly Ser Ile Glu
Tyr Trp Pro Asp 195 200 205 Pro Gln Arg Gly Ile Xaa Glu Ala Tyr Arg
Val Leu Xaa Xaa Gly Gly 210 215 220 Xaa Ala Cys Xaa Ile Gly Pro Val
Xaa Pro Thr Phe Trp Leu Ser Arg 225 230 235 240 Phe Phe Xaa Asp Xaa
Trp Met Leu Phe Pro Xaa Glu Glu Glu Tyr Ile 245 250 255 Glu Trp Phe
Xaa Xaa Ala Gly Phe Xaa Asp Val Xaa Leu Lys Arg Ile 260 265 270 Gly
Pro Lys Trp Tyr Arg Gly Val Arg Arg His Gly Leu Ile Met Gly 275 280
285 Cys Ser Val Thr Gly Val Lys Xaa Xaa Xaa Gly Asp Ser Pro Leu Xaa
290 295 300 Leu Gly Pro Lys Xaa Glu Asp Val Xaa Lys Pro Xaa Xaa Asn
Pro Xaa 305 310 315 320 Xaa Phe Xaa Xaa Arg Phe Xaa Xaa Gly Xaa Xaa
Xaa Ala Xaa Xaa Xaa 325 330 335 Val Leu Xaa Pro Ile Tyr Met Trp Xaa
Lys Asp Gln Xaa Val Pro Xaa 340 345 350 Xaa Xaa Pro Ile 355
* * * * *
References