U.S. patent application number 11/500769 was filed with the patent office on 2006-12-07 for enzymes involved in triterpene synthesis.
Invention is credited to Gregory T. Bryan, Der-Chyan Hwang, Carl A. Maxwell, Brian McGonigle, Susan M. Potter.
Application Number | 20060277636 11/500769 |
Document ID | / |
Family ID | 29270199 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060277636 |
Kind Code |
A1 |
Bryan; Gregory T. ; et
al. |
December 7, 2006 |
Enzymes involved in triterpene synthesis
Abstract
This invention relates to an isolated nucleic acid fragment
encoding an oxidosqualene cyclase. The invention also relates to
the construction of a chimeric gene encoding all or a portion of
the isolated polynucleotides of the invention, in sense or
antisense orientation, operably linked to a suitable regulatory
sequence.
Inventors: |
Bryan; Gregory T.;
(Manawatu, NZ) ; McGonigle; Brian; (Wilmington,
DE) ; Maxwell; Carl A.; (Elkton, MD) ; Potter;
Susan M.; (Decatur, IL) ; Hwang; Der-Chyan;
(Chesterfield, MO) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
29270199 |
Appl. No.: |
11/500769 |
Filed: |
August 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10221074 |
Sep 9, 2002 |
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PCT/US01/07611 |
Mar 9, 2001 |
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11500769 |
Aug 8, 2006 |
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60188054 |
Mar 9, 2000 |
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Current U.S.
Class: |
800/298 ;
435/193; 435/419; 435/468; 536/23.2 |
Current CPC
Class: |
C12N 15/8209 20130101;
A23J 3/16 20130101; C12N 9/90 20130101; C12N 15/8242 20130101; A23L
33/185 20160801; C12N 15/8243 20130101 |
Class at
Publication: |
800/298 ;
435/468; 435/419; 536/023.2; 435/193 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07H 21/04 20060101 C07H021/04; C12N 9/10 20060101
C12N009/10; C12N 5/04 20060101 C12N005/04; C12N 15/82 20060101
C12N015/82 |
Claims
1. An isolated polynucleotide comprising a nucleotide sequence
selected from the group consisting of: (a) first nucleotide
sequence encoding a polypeptide of at least 100 amino acids having
at least 80% identity based on the Clustal method of alignment when
compared to a polypeptide selected from the group consisting of SEQ
ID NOs: 2, 4, 6, and 8; and (b) a second nucleotide sequence
comprising the complement of the first nucleotide sequence.
2-42. (canceled)
43. A soybean grain produced by a transgenic plant comprising a
polynucleotide selected from the group consisting of: (a) a
nucleotide sequence encoding a polypeptide having oxidosqualene
cyclase activity, wherein the polypeptide has an amino acid
sequence of at least 80% sequence identity based on the Clustal
method of alignment when compared to a polypeptide of SEQ ID NO:2,
4, 6, or 8; wherein the default parameters for pairwise alignment
are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5; and
(b) a full complement of the nucleotide sequence of (a). wherein
said polynucleotide is under the control of a heterologous promoter
and wherein said plant has an altered level of a triterpene.
44. A food product prepared from the soybean grain of claim 43.
45. The food product of claim 44 wherein the food product is
selected from the group consisting of a soy protein product,
soybean meal, soy flour, soy protein concentrate, soy milk, a
dietary supplement, beans, nuts, tofu, natto, miso, and tempeh.
46. A method of making a soy protein product comprising processing
the grain of claim 43, said processing including: (a) cracking said
grain to remove the meats from the hulls; and (b) flaking the meats
obtained in step (a) to obtain a desired flake thickness.
47. A method of making a soy product comprising inactivating an
antinutrient of the grain produced by a transgenic plant comprising
a polynucleotide comprising: (a) a nucleotide sequence encoding a
polypeptide having oxidosqualene cyclase activity, wherein the
polypeptide has an amino acid sequence of at least 80% sequence
identity based on the Clustal method of alignment when compared to
a polypeptide of SEQ ID NO:2, 4, 6, or 8; wherein the default
parameters for pairwise alignment are KTUPLE=1, GAP PENALTY=3,
WINDOW=5 and DIAGONALS SAVED=5; and (b) a full complement of the
nucleotide sequence of (a) wherein said polynucleotide is under the
control of a heterologous promoter and wherein said plant has an
altered level of a triterpene.
48. The method of claim 47 wherein inactivating is toasting.
49. The method of claim 47 wherein the anitnutrient is a trypsin
inhibitor.
50. A method of extracting bioactive compounds from the grain of
claim 43 comprising exposing the grain or portions thereof to a
solvent.
51. The method of claim 50 wherein said bioactive compound is a
saponin.
52. A nutritional supplement produced by the method of claim
50.
53. A pharmaceutical composition produced by the method of claim
50.
Description
FIELD OF TEE INVENTION
[0001] This invention is in the field of plant molecular biology.
More specifically, this invention pertains to nucleic acid
sequences encoding enzymes involved in the cyclization of squalene
epoxide to form the ring structure precursor to triterpenes,
including sterols and saponins, in plants and seeds. This invention
also includes transgenic plants where the expression of the nucleic
acids of the present invention results in altered levels of
triterpenes, including sterols and saponins. Also included in the
invention are protein products and food and dietary supplement
applications.
BACKGROUND OF THE INVENTION
[0002] The terpenoids, also called isoprenoids, constitute the
largest family of natural products with over 22,000 individual
compounds of this class having been described. The triterpenes or
terpenoids (hemiterpenes, monoterpenes, sesquiterpenes, diterpenes,
triterpenes, tetraterpenes, polyprenols, and the like) play diverse
functional roles in plants as hormones, photosynthetic pigments,
electron carriers, mediators of polysaccharide assembly, and
structural components of membranes. The majority of plant
terpenoids are found in resins, latex, waxes, and oils.
[0003] Two molecules of farnesyl pyrophosphate are joined
head-to-head to form squalene, a triterpene, in the first dedicated
step towards sterol biosynthesis. Squalene is then converted to
2,3-oxidosqualene which, in photosynthetic organisms, may be
converted to the 30 carbon, 4 ring structure, cycloartenol or to
the 5 ring homolog, .beta.-amyrin, a sapinogenin precursor. This
conversion step is catalyzed by one of at least two oxidosqualene
cyclases: cycloartenol synthase or .beta.-amyrin synthase.
[0004] Cycloartenol is formed by the enzyme cycloartenol synthase
(EC 5.4.99.8), also called 2,3-epoxysqualene-cycloartenol cyclase.
The basic nucleus of cycloartenol can be further modified by
reactions such as desaturation or demethylation to form the common
sterol backbones such as stigmasterol and sitosterol, which can be
modified further.
[0005] The .beta.-amyrin-cyclization activity is distinct from
cycloartenol synthase (Kushiro, T., et al. (1998) Eur. J. Biochem.
256:238-244). .beta.-amyrin synthase catalyzes the cyclization of
2,3-oxidosqualene to .beta.-amyrin. Yet, the basic .beta.-amyrin
ring structure may be modified in much the same manner as is the
cycloartenol structure to give classes of sapogenins. Saponins are
glycosylated sapogenins and may play a pathogen defense role in
plant tissues.
[0006] Soybean seeds, for example, contain several classes of
saponins, all of which are formed from one sapogenin ring structure
that is modified by hydroxylation and by different carbohydrate
moieties. Total saponin content varies somewhat by soybean cultivar
but is in the range of 0.25% of the seed dry weight (Shiraiwa, M.,
et al. (1991) Agric. Biol. Chem. 55:323-331).
[0007] The name saponin was derived from their strong foaming
power. The physiological function of saponins in soybean seeds is
not clear, but they do contribute to the bitter or astringent
flavor of soybean seeds (Okubo, K., et al. (1992) Biosci.
Biotechnol. Biochem. 56:99-103). Saponins are thought to have
cholesterol-lowering effects and reduction in colon cancer risk.
Besides imparting undesirable flavors to feed and foods, saponins
have been shown to have hemolytic action against red blood cells.
Soybeans are also involved in the reduction of hot flashes in
postmenopausal women, lowering the risk of hormone-related cancer,
slowdown of bone loss in osteoporosis and improvement in vascular
health Saponins are believed to be involved in these beneficial
soybean effects.
[0008] A variety of processed vegetable protein products are
produced from soybean. These range from minimally processed, soy
beans and soy nuts such as toasted soy nuts and defatted items such
as soybean meal, grits, and flours to more highly processed items
such as soy protein concentrates and soy protein isolates. In other
soy protein products, such as fall-fat soy flour, the oil is not
extracted. In addition to these processed products, there are also
a number of specialty products based on traditional Oriental
processes, which utilize the entire bean as the starting material.
Examples include soy milk, soy sauce, tofu, natto, miso, tempeh,
and yuba.
[0009] Examples of use of soy protein products in human foods
include applying soy protein concentrates and soy protein isolates
in nutritional beverage, emulsified meat and whole muscle meat
applications; textured soy protein in meat analogues; soy protein
isolates in infant formula. Facilities and methods to produce
protein concentrates and isolates from soybeans are available
across the world. To the extent that they are retained in these
processed soy fractions and the foods prepared from them, the
saponin content of the starting beans influences the flavor of the
food.
[0010] Sequences of two different .beta.-amyrin synthase isoforms
has been described for Korean ginseng (Panax ginseng, NCBI General
Identifier Nos. 3721856 and 3688600). A soybean EST having NCBI
General Identification No. 5606831 has been identified with and is
"similar to the ginseng sequence," according to the NCBI
entrez.
[0011] Identification of the genes encoding oxidosqualene cyclases
in a variety of crops will allow the manipulation of the same.
Interference with triterpenoid ring synthesis during plant
development may be expected to decrease the total content of
saponins in plant parts resulting in foods with increased
nutritional value, and better flavor.
SUMMARY OF THE INVENTION
[0012] The instant invention relates to isolated nucleic acid
sequences encoding enzymes involved in triterpene synthesis.
Specifically, this invention concerns isolated nucleic acid
sequences encoding oxidosqualene cyclase enzymes.
[0013] The present invention relates to an isolated polynucleotide
comprising a nucleotide sequence selected from the group consisting
of: a first nucleotide sequence encoding a polypeptide of at least
100 amino acids having at least 80% identity based on the Clustal
method of alignment when compared to a polypeptide selected from
the group consisting of SEQ ID NOs: 2, 4, 6, and 8; and a second
nucleotide sequence comprising the complement of the first
nucleotide sequence. The present invention also relates to an
isolated polynucleotide comprising a nucleotide sequence having at
least 30 contiguous nucleotides derived from a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,
and 7 and the complement of such sequences. An isolated polypeptide
of at least 100 amino acids that has at least 80% identity based on
the Clustal method of alignment when compared to a polypeptide
selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8 is
also an embodiment of the subject invention.
[0014] In another embodiment, the instant invention relates to a
chimeric polynucleotide comprising the isolated polynucleotide of
the present invention operably linked to suitable a regulatory
sequence.
[0015] In a further embodiment, the instant invention concerns an
isolated host cell comprising the chimeric polynucleotide of the
present invention. The host cell may be selected from the group
consisting of a yeast cell, a bacterial cell; and a plant cell. The
present invention also relates to a virus comprising a chimeric
gene encoding the polypeptide of the present invention.
[0016] Compositions, including plants and plant parts, comprising
the isolated poypeptide or polynucleotide of the present invention
are also embodied by the present invention. The invention also
includes transformed plants that arise from transformed host cells
of higher plants and seeds or grains derived from such transformed
plants. Such transgenic plant includes those having an altered
level of a triterpenoid, such as .beta.-amyrin.
[0017] The present invention is also directed to an isolated soy
protein and a food product. The food product may be selected from
the group consisting of a soy protein product, soybean meal, soy
flour, soy protein concentrate, soy milk, a dietary supplement,
nuts, tofu, natto, miso, and tempeh.
[0018] The present invention also relates to a method of altering
the level of expression of an oxidosqualene cyclase polypeptide in
a plant cell, which comprises: constructing an isolated
polynucleotide comprising a nucleotide sequence of at least 30
contiguous nucleotides derived from an isolated polynucleotide of
the present invention; introducing the isolated polynucleotide into
a plant cell; measuring the level of oxidosqualene cyclase in the
plant cell containing the polynucleotide; and comparing the level
of oxidosqualene cyclase in the plant cell containing the isolated
polynucleotide with the level of oxidosqualene cyclase in a plant
cell of the same species as the plant cell of step (b) that does
not contain the isolated polynucleotide.
[0019] Another embodiment of the present invention is a method of
producing a plant with altered levels of oxidosqualene cyclase
comprising: transforming a plant cell with the chimeric
polynucleotide of the present invention; growing the transformed
plant cell from step (a) under conditions that promote the
regeneration of a whole plant from the transformed cell, wherein
the plant regenerated from the transformed cell produces an amount
of oxidosqualene cyclase that is greater than the amount of the
oxidosqualene cyclase that is produced in a plant that is
regenerated from a plant cell of the same species as the plant of
step (a) that is not transformed with the chimeric polynucleotide;
and optionally transforming the plant cell of step (a) with a
second chimeric polynucleotide comprising a nucleic acid sequence
encoding a polypeptide that regulates expression of at least one
enzyme of the triterpenoid pathway; and growing the transformed
plant cell from step (c) under conditions that promote the
regeneration of a whole plant from the transformed cell; wherein
the plant regenerated from the transformed cell produces an amount
of oxidosqualene cyclase that is greater than the amount of the
oxidosqualene cyclase that is produced in a plant that is
regenerated from a plant cell of the same species as the plant of
step (c) that is not transformed with the chimeric polynucleotide
of claim 5 and a second chimeric polynucleotide.
[0020] The present invention also relates to: (1) a method of
making a soy protein product comprising processing the grain of the
present invention, said processing including: toasting or cracking
said grain to remove the meats from the hulls; and flaking the
meats obtained in step (a) to obtain a desired flake thickness, and
(2) a method for positive selection of a transformed cell
comprising: transforming a host cell with the chimeric gene of the
present invention; and growing the transformed host cell under
conditions which allow expression of the oxidosqualene cyclase
polynucleotide in an amount sufficient to complement a null mutant
to provide a positive selection means.
[0021] The improved palatability due to the suppression of saponin
expression is also included. Included are also products, with
improved palatability, prepared using soybeans having lower amounts
of saponins due to the presence of a chimeric gene containing the
sequences of the present invention.
BRIEF DESCRIPTION OF THE FIGURE AND SEQUENCE LISTINGS
[0022] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing which form a part of this application.
[0023] The following sequence descriptions and Sequence Listing
attached hereto comply with the rules governing nucleotide and/or
amino acid sequence disclosures in patent applications as set forth
in 37 C.F.R. .sctn. 1.821-1.825.
[0024] FIG. 1 depicts the amino acid sequence alignment between the
oxidosqualene cyclases encoded by soybean clones sdp3c.pk020.o10
(SEQ ID NO:2), src3c.pk024.m11 (SEQ ID NO:4), sah1c.pk002.n23 (SEQ
ID NO:6), wheat clone wdk1c.pk010.o10 (SEQ ED NO:8) and the
.beta.-amyrin synthases from Panax ginseng (NCBI General Identifier
No. 3721856; SEQ ID NO:12, and NCBI General Identifier No. 3688600;
SEQ ID NO:13). The top row indicates with asterisks (*) the amino
acids conserved among all sequences. Dashes are used by the program
to maximize the alignment of the sequences.
[0025] SEQ ID NO:1 is the nucleotide sequence comprising the
soybean cDNA insert in clone sdp3c.pk020.o10 encoding a soybean
oxidosqualene cyclase.
[0026] SEQ ID NO:2 is the deduced amino acid sequence of a soybean
oxidosqualene cyclase derived from the nucleotide sequence of SEQ
ID NO: 1.
[0027] SEQ ID NO:3 is the nucleotide sequence comprising the
soybean cDNA insert in clone src3c.pk024.m11 encoding a soybean
.beta.-amyrin synthase.
[0028] SEQ ID NO:4 is the deduced amino acid sequence of a soybean
.beta.-amyrin synthase derived from the nucleotide sequence of SEQ
ID NO:3.
[0029] SEQ ID NO:5 is the nucleotide sequence comprising the
soybean cDNA insert in clone sah1c.pk002.n23 encoding a soybean
oxidosqualene cyclase.
[0030] SEQ ID NO:6 is the deduced amino acid sequence of a soybean
oxidosqualene cyclase derived from the nucleotide sequence of SEQ
ID NO:5.
[0031] SEQ ID NO:7 is the nucleotide sequence comprising the wheat
cDNA insert in clone wdk1c.pk010.o10 encoding a wheat oxidosqualene
cyclase.
[0032] SEQ ID NO:8 is the deduced amino acid sequence of a wheat
oxidosqualene cyclase derived from the nucleotide sequence of SEQ
ID NO:7.
[0033] SEQ ID NO:9 is the nucleotide sequence of an oligonucleotide
primer used to amplify the cDNA insert from clone
src3c.pk0024.m11.
[0034] SEQ ID NO:10 is the nucleotide sequence of an
oligonucleotide primer used to amplify the cDNA insert from clone
sah1c.pk002.n23.
[0035] SEQ ID NO:11 is the nucleotide sequence of an
oligonucleotide primer used for PCR amplification of oxidosqualene
cyclase sequences from clones src3c.pk0024.m11 and
sah1c.pk002.n23.
[0036] SEQ ID NO:12 is the amino acid sequence of a Panax ginseng
.beta.-amyrin synthase having NCBI General Identifier No.
3721856.
[0037] SEQ ID NO:13 is the amino acid sequence of a Panax ginseng
.beta.-amyrin synthase having NCBI General Identifier No.
3688600.
[0038] The Sequence Listing contains the one letter code for
nucleotide sequence characters and the three letter codes for amino
acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Research 13:3021-3030 (1985) and in the
Biochemical Journal 219 (No. 2):345-373 (1984) which are herein
incorporated by reference. The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37 C.F.R. .sctn.1.822.
DETAILED DESCRIPTION OF THE INVENTION
[0039] In the context of this disclosure, a number of terms shall
be utilized. The terms "polynucleotide/isolated polynucleotide" and
"nucleic acid fragment"/"isolated nucleic acid fragment" are used
interchangeably herein. These terms encompass nucleotide sequences
and the like. A polynucleotide may be a polymer of RNA or DNA that
is single- or double-stranded, that optionally contains synthetic,
non-natural or altered nucleotide bases. A polynucleotide in the
form of a polymer of DNA may be comprised of one or more segments
of cDNA, genomic DNA, synthetic DNA or mixtures thereof. An
isolated polynucleotide of the present invention may include at
least one of 60 contiguous nucleotides, preferably at least one of
40 contiguous nucleotides, most preferably one of at least 30
contiguous nucleotides derived from a nucleotide sequence selected
from the group consisting of SEQ ID NOs:1, 3, 5, and 7, or the
complement of such sequences.
[0040] The term "isolated" polynucleotide is one that has been
substantially separated or purified away from other nucleic acid
sequences in the cell of the organism in which the nucleic acid
naturally occurs, i.e., other chromosomal and extrachromosomal DNA
and RNA, by conventional nucleic acid purification methods. The
term also embraces recombinant polynucleotides and chemically
synthesized polynucleotides.
[0041] The present invention is directed to isolated
polynucleotides and chimeric genes encoding oxidosqualene cyclase
enzymes. While not intending to be bound by any theory or theories
of operation, it is believed that these enzymes are membrane bound.
Oxidosqualene cyclases include and are not limited to .beta.-amyrin
synthase, squalene monooxygenase, cycloartenol synthase and the
like. Triterpene synthesis is catalyzed by oxidosqualene cyclases.
Triterpenes, also known as triterpenoids, include and are not
limited to sapinogenins and sterols. The sapinogenin,
.beta.-amyrin, is produced by the action of .beta.-amyrin synthase
on 2,3-oxidosqualene, for example.
[0042] As used herein, "substantially similar" refers to nucleic
acid sequences wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, that do not
affect the functional properties of the polypeptide encoded by the
nucleic acid sequence. "Substantially similar" also refers to
polynucleotides wherein changes in one or more nucleotide bases
does not affect the ability of the nucleic acid sequence to mediate
alteration of gene expression by antisense or co-suppression
technology among others. "Substantially similar" also refers to
modifications of the nucleic acid fragments of the instant
invention such as deletion or insertion of one or more nucleotides
that do not substantially affect the functional properties of the
resulting transcript vis-a-vis the ability to mediate gene
silencing or alteration of the functional properties of the
resulting polpeptide. It is therefore understood that the invention
encompasses more than the specific exemplary sequences.
[0043] Substantially similar nucleic acid fragments may be selected
by screening nucleic acid fragments representing subfragments or
modifications of the nucleic acid fragments of the instant
invention, wherein one or more nucleotides are substituted, deleted
and/or inserted, for their ability to affect the level of the
polypeptide encoded by the unmodified nucleic acid fragment in a
plant or plant cell. For example, a substantially similar nucleic
acid fragment representing at least one of 30 contiguous
nucleotides derived from the instant nucleic acid fragment can be
constructed and introduced into a plant or plant cell. The level of
the polypeptide encoded by the unmodified nucleic acid fragment
present in a plant or plant cell exposed to the substantially
similar nucleic fragment can then be compared to the level of the
polypeptide in a plant or plant cell that is not exposed to the
substantially similar nucleic acid fragment.
[0044] For example, it is well known in the art that antisense
suppression and co-suppression of gene expression may be
accomplished using nucleic acid fragments representing less than
the entire coding region of a gene, and by nucleic acid fragments
that do not share 100% sequence identity with the gene to be
suppressed. Moreover, alterations in a nucleic acid sequence which
result in the production of a chemically equivalent amino acid at a
given site, but do not effect the functional properties of the
encoded polypeptide, are well known in the art. Thus, a codon for
the amino acid alanine, a hydrophobic amino acid, may be
substituted by a codon encoding another less hydrophobic residue,
such as glycine, or a more hydrophobic residue such as valine,
leucine, or isoleucine. Similarly, changes which result in
substitution of one negatively charged residue for another, such as
aspartic acid for glutamic acid, or one positively charged residue
for another, such as lysine for arginine, can also be expected to
produce a functionally equivalent product. Nucleotide changes which
result in alteration of the N-terminal and C-terminal portions of
the polypeptide molecule would also not be expected to alter the
activity of the polypeptide. Each of the proposed modifications is
well within the routine skill in the art, as is determination of
retention of biological activity of the encoded products.
Consequently, an isolated polynucleotide comprising a nucleotide
sequence of at least one of 60 (preferably at least one of 40, most
preferably at least one of 30) contiguous nucleotides derived from
a nucleotide sequence selected from the group consisting of SEQ ID
NOs:1, 3, 5, and 7 and the complement of such nucleotide sequences
may be used in methods of selecting an isolated polynucleotide that
affects the expression of an oxidosqualene cyclase polypeptide in a
host cell. A method of selecting an isolated polynucleotide that
affects the level of expression of an oxidosqualene cyclase
polypeptide in a host cell (eukaryotic, such as plant or yeast for
example, or prokaryotic, such as bacterial for example) or virus
may comprise the steps of: constructing an isolated polynucleotide
of the present invention or an isolated chimeric gene of the
present invention; introducing the isolated polynucleotide or the
isolated chimeric gene into a host cell; measuring the level a
polypeptide in the host cell containing the isolated
polynucleotide; and comparing the level of a polypeptide in the
host cell containing the isolated polynucleotide with the level of
a polypeptide in a host cell that does not contain the isolated
polynucleotide.
[0045] Moreover, substantially similar nucleic acid fragments may
also be characterized by their ability to hybridize. Estimates of
such homology are provided by either DNA-DNA or DNA-RNA
hybridization under conditions of stringency as is well understood
by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic
Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions
can be adjusted to screen for moderately similar fragments, such as
homologous sequences from distantly related organisms, to highly
similar fragments, such as genes that duplicate functional enzymes
from closely related organisms. Post-hybridization washes determine
stringency conditions. One set of preferred conditions uses a
series of washes starting with 6.times.SSC, 0.5% SDS at room
temperature for 15 min, then repeated with 2.times.SSC, 0.5% SDS at
45.degree. C. for 30 min, and then repeated twice with
0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30 min. A more
preferred set of stringent conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times.SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of highly
stringent conditions uses two final washes in 0.1.times.SSC, 0.1%
SDS at 65.degree. C.
[0046] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent identity of the
amino acid sequences that they encode to the amino acid sequences
disclosed herein, as determined by algorithms commonly employed by
those skilled in this art. Suitable nucleic acid fragments
(isolated polynucleotides of the present invention) encode
polypeptides that are at least about 70% identical, preferably at
least about 80% identical to the amino acid sequences reported
herein. Preferred nucleic acid fragments encode amino acid
sequences that are about 85% identical to the amino acid sequences
reported herein. More preferred nucleic acid fragments encode amino
acid sequences that are at least about 90% identical to the amino
acid sequences reported herein. Most preferred are nucleic acid
fragments that encode amino acid sequences that are at least about
95% identical to the amino acid sequences reported herein. Suitable
nucleic acid fragments not only have the above homologies but
typically encode a polypeptide having at least 50 amino acids,
preferably at least 100 amino acids, more preferably at least 150
amino acids, still more preferably at least 200 amino acids, and
most preferably at least 250 amino acids. Sequence alignments and
percent identity calculations were performed using the Megalign
program of the LASERGENE bioinformatics computing suite (DNASTAR
Inc., Madison, Wis.). Multiple alignment of the sequences was
performed using the Clustal method of alignment (Higgins and Sharp
(1989) CABIOS. 5:151-153) with the default parameters (GAP
PENALTY=10, GAP LENGTH PENALTYL=10). Default parameters for
pairwise alignments using the Clustal method were KTUPLE 1, GAP
PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
[0047] A "substantial portion" of an amino acid or nucleotide
sequence comprises an amino acid or a nucleotide sequence that is
sufficient to afford putative identification of the protein or gene
that the amino acid or nucleotide sequence comprises. Amino acid
and nucleotide sequences can be evaluated either manually by one
skilled in the art, or by using computer-based sequence comparison
and identification tools that employ algorithms such as BLAST
(Basic Local Alignment Search Tool; Altschul et al., (1993) J. Mol.
Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In
general, a sequence of ten or more contiguous amino acids or thirty
or more contiguous nucleotides is necessary in order to putatively
identify a polypeptide or nucleic acid sequence as homologous to a
known protein or gene. Moreover, with respect to nucleotide
sequences, gene specific oligonucleotide probes comprising 30 or
more contiguous nucleotides may be used in sequence-dependent
methods of gene identification (e.g., Southern hybridization) and
isolation (e.g., in situ hybridization of bacterial colonies or
bacteriophage plaques). In addition, short oligonucleotides of 12
or more nucleotides may be used as amplification primers in PCR in
order to obtain a particular nucleic acid fragment comprising the
primers. Accordingly, a "substantial portion" of a nucleotide
sequence comprises a nucleotide sequence that will afford specific
identification and/or isolation of a nucleic acid fragment
comprising the sequence. The instant specification teaches amino
acid and nucleotide sequences encoding polypeptides that comprise
one or more particular plant proteins. The skilled artisan, having
the benefit of the sequences as reported herein, may now use all or
a substantial portion of the disclosed sequences for purposes known
to those skilled in this art. Accordingly, the instant invention
comprises the complete sequences as reported in the accompanying
Sequence Listing, as well as substantial portions of those
sequences as defined above.
[0048] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid fragment comprising a
nucleotide sequence that encodes all or a substantial portion of
the amino acid sequence encoding the oxidosqualene cyclase proteins
as set forth in SEQ ID NOs:2, 4, 6, and 8. The skilled artisan is
well aware of the "codon-bias" exhibited by a specific host cell in
usage of nucleotide codons to specify a given amino acid.
Therefore, when synthesizing a polynucleotide for improved
expression of a specific gene in a host cell, it is desirable to
design the polynucleotide such that its frequency of codon usage
approaches the frequency of preferred codon usage of the host
cell.
[0049] "Synthetic nucleic acid fragments" can be assembled from
oligonucleotide building blocks that are chemically synthesized
using procedures known to those skilled in the art. These building
blocks are ligated and annealed to form larger nucleic acid
fragments which may then be enzymatically assembled to construct
the entire desired nucleic acid fragment. "Chemically synthesized",
as related to nucleic acid fragment, means that the component
nucleotides were assembled in vitro. Manual chemical synthesis of
nucleic acid fragments may be accomplished using well established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the nucleic acid fragments can be tailored for optimal gene
expression based on optimization of nucleotide sequence to reflect
the codon bias of the host cell. The skilled artisan appreciates
the likelihood of successful gene expression if codon usage is
biased towards those codons favored by the host. Determination of
preferred codons can be based on a survey of genes derived from the
host cell where sequence information is available.
[0050] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences upstream (5'
non-coding sequences), within, and downstream (3' non-coding
sequences) the coding sequence. "Native gene" refers to a gene as
found in nature with its own regulatory sequences. "Chimeric or
heterologous" "gene or polynucleotide" refers any gene or
polynucleotide that is not native to a plant. A chimeric or
heterologous gene comprises regulatory and coding sequences that
are not found together in nature. Accordingly, a chimeric gene may
comprise regulatory sequences and coding sequences that are derived
from different sources, or regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different than that found in nature. "Endogenous gene" refers to a
native gene in its natural location in the genome of an organism. A
"foreign" gene refers to a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes. A "transgene" is a gene
that has been introduced into the genome by a transformation
procedure.
[0051] "Coding sequence" refers to a nucleotide sequence that codes
for a specific amino acid sequence. "Regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, and polyadenylation recognition sequences.
[0052] "Promoter" refers to a polynucleotide sequence capable of
controlling the expression of a coding sequence or functional RNA.
In general, a coding sequence is located 3' to a promoter sequence.
The promoter sequence consists of proximal and more distal upstream
elements; the latter elements often referred to as enhancers.
Accordingly, an "enhancer" is a nucleotide sequence, which can
stimulate promoter activity, and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or be composed of different elements
derived from different promoters found in nature, or even comprise
synthetic nucleotide segments. It is understood by those skilled in
the art that different promoters may direct the expression of a
gene in different tissues or cell types, or at different stages of
development, or in response to different environmental conditions.
Promoters which cause a gene to be expressed in most cell types at
most times are commonly referred to as "constitutive promoters".
New promoters of various types useful in plant cells are constantly
being discovered; numerous examples may be found in the compilation
by Okamuro and Goldberg, (1989) Biochemistry of Plants 15:1-82. It
is further recognized that since in most cases the exact boundaries
of regulatory sequences have not been completely defined, nucleic
acid fragments of different lengths may have identical promoter
activity.
[0053] The "translation leader sequence" refers to a polynucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner and Foster (1995) Mol. Biotechnol.
3:225-236).
[0054] The "3' non-coding sequences" refer to DNA sequences located
downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.
[0055] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from posttranscriptional processing of the primary
transcript and is referred to as the mature RNA. "Messenger RNA
(mRNA)" refers to the RNA that is without introns and that can be
translated into protein by the cell. "cDNA" refers to a DNA that is
complementary to and derived from an mRNA. The cDNA can be
single-stranded or converted into the double stranded form using,
for example, the klenow fragment of DNA polymerase I. "Sense" RNA
refers to RNA transcript that includes the mRNA and so can be
translated into a polypeptide by the cell. "Antisense RNA" refers
to an RNA transcript that is complementary to all or part of a
target primary transcript or mRNA and that blocks the expression of
a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by
reference). The complementarity of an antisense RNA may be with any
part of the specific gene transcript, i.e., at the 5' non-coding
sequence, 3' non-coding sequence, introns, or the coding sequence.
"Functional RNA" refers to sense RNA, antisense RNA, ribozyme RNA,
or other RNA that may not be translated but yet has an effect on
cellular processes.
[0056] The term "operably linked" refers to the association of
nucleic acid sequences on a single polynucleotide so that the
function of one is affected by the other. For example, a promoter
is operably linked with a coding sequence when it is capable of
affecting the expression of that coding sequence (i.e., that the
coding sequence is under the transcriptional control of the
promoter). Coding sequences can be operably linked to regulatory
sequences in sense or antisense orientation.
[0057] The term "recombinant" means, for example, that a
recombinant nucleic acid sequence is made by an artificial
combination of two otherwise separated segments of sequence, e.g.,
by chemical synthesis or by the manipulation of isolated segments
of nucleic acids by genetic engineering techniques.
[0058] The term "expression", as used herein refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from a polynucleotide of the invention. Expression may
also refer to translation of mRNA into a polypeptide. "Antisense
inhibition" refers to the production of antisense RNA transcripts
capable of suppressing the expression of the target protein.
"Overexpression" refers to the production of a gene product in
transgenic organisms that exceeds levels of production in normal or
non-transformed organisms. "Co-suppression" refers to the
production of sense RNA transcripts capable of suppressing the
expression of identical or substantially similar foreign or
endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by
reference).
[0059] "Altered levels" or "altered expression" refers to the
production of gene product(s) in transgenic organisms in amounts or
proportions that differ from that of normal or non-transformed
organisms.
[0060] "Mature" protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or pro-peptides present
in the primary translation product have been removed. "Precursor"
protein refers to the primary product of translation of mRNA; i.e.,
with pre- and propeptides still present. Pre- and pro-peptides may
be but are not limited to intracellular localization signals.
[0061] A "signal peptide" is an amino acid sequence which is
translated in conjunction with a protein and directs the protein to
the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant
Mol. Biol. 42:21-53). If the protein is to be directed to a
vacuole, a vacuolar targeting signal (supra) can further be added,
or if to the endoplasmic reticulum, an endoplasmic reticulum
retention signal (supra) may be added. If the protein is to be
directed to the nucleus, any signal peptide present should be
removed and instead a nuclear localization signal included Raikhel
(1992) Plant Phys. 100:1627-1632). A "chloroplast transit peptide"
is an amino acid sequence which is translated in conjunction with a
protein and directs the protein to the chloroplast or other plastid
types present in the cell in which the protein is made.
"Chloroplast transit sequence" refers to a nucleotide sequence that
encodes a chloroplast transit peptide.
[0062] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. Examples of methods of plant transformation include
Agrobacterium-mediated transformation (De Blaere et al. (1987)
Meth. Enzymol. 143:277) and particle-accelerated or "gene gun"
transformation technology (Klein et al. (1987) Nature (London)
327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by
reference).
[0063] Expression of a chimeric oxidosqualene cyclase, for example,
results in the production of a level of the encoded oxidosqualene
cyclase protein in a transformed host cell that is altered as
compared to the level produced in an untransformed host cell. Also,
a transgenic plant, or plant part, comprising a polynucleotide of
the present invention, such as for example, SEQ BD NOS: 1, 3, 5,
and 7, under the control of a heterologous promoter results in
plants having altered levels of triterpenes. Plants may be selected
from the group consisting of monocots and dicots. Monocots include
and are not limited to corn, rice, wheat, barley, palm, and the
like. Dicots include and are not limited to Arabidopsis, soybean,
oilseed Brassica, peanut, sunflower, safflower, cotton, tobacco,
tomato, potato, cocoa, and the like. Plant parts include and are
not limited to seeds and grains, for example.
[0064] Thus, isolated polynucleotides of the present invention can
be incorporated into recombinant constructs capable of introduction
into and replication in a host cell. Such a construct can be a
vector that includes a replication system and sequences that are
capable of transcription and translation of a polypeptide-encoding
sequence in a given host cell. A number of vectors suitable for
stable transfection of plant cells or for the establishment of
transgenic plants have been described in, e.g., Pouwels et al.,
Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach
and Weissbach, Methods for Plant Molecular Biology, Academic Press,
1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer
Academic Publishers, 1990. Typically, plant expression vectors
include, for example, one or more cloned plant genes under the
transcriptional control of 5' and 3' regulatory sequences and a
dominant selectable marker. Such plant expression vectors also can
contain a promoter regulatory region (e.g., a regulatory region
controlling inducible or constitutive, environmentally- or
developmentally-regulated, or cell- or tissue-specific expression),
a transcription initiation start site, a ribosome binding site, an
RNA processing signal, a transcription termination site, and/or a
polyadenylation signal.
[0065] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, 1989
(hereinafter "Sambrook").
[0066] "PCR" or "polymerase chain reaction" is a technique for the
synthesis of large quantities of specific DNA segments. It consists
of a series of repetitive cycles (Perkin Elmer Cetus Instruements,
Norwark, Conn.). Typically, the double-stranded DNA is heat
denatured, the two primers complementary to the 3' boundaries of
the target segments are annealed at low temperature and then
extended at an intermediate temperature. One set of these three
consecutive steps is referred to as a cycle.
[0067] Nucleic acid fragments encoding oxidosqualene cyclases have
been isolated and identified by comparison of random plant cDNA
sequences to public databases containing nucleotide and protein
sequences using the BLAST algorithms well known to those skilled in
the art. Furthermore, their expression has been functionally
demonstrated in yeast. Included herein are nucleotide and amino
acid sequences with similarities to oxidosqualene cyclase.
.beta.-amyrin synthase converts 2,3-oxidosqualene to .beta.-amyrin,
which is a required step in the synthesis of saponins in soybean
seeds. Elimination of saponins may therefore lead to improved
flavor.
[0068] The nucleic acid fragments of the instant invention may be
used to isolate cDNAs and genes encoding homologous proteins from
the same or other plant species. Isolation of homologous genes
using sequence-dependent protocols is well known in the art.
Examples of sequence-dependent protocols include, but are not
limited to, methods of nucleic acid hybridization, and methods of
DNA and RNA amplification as exemplified by various uses of nucleic
acid amplification technologies (e.g., polymerase chain reaction,
ligase chain reaction).
[0069] For example, genes encoding other oxidosqualene cyclases,
either as cDNAs or genomic DNAs, could be isolated directly by
using all or a portion of the instant nucleic acid fragments as DNA
hybridization probes to screen libraries from any desired plant
employing methodology well known to those skilled in the art.
Specific oligonucleotide probes based upon the instant nucleic acid
sequences can be designed and synthesized by methods known in the
art (Sambrook). Moreover, the entire sequences can be used directly
to synthesize DNA probes by methods known to the skilled artisan
such as random primer DNA labeling, nick translation, or
end-labeling techniques, or RNA probes using available in vitro
transcription systems. In addition, specific primers can be
designed and used to amplify a part or all of the instant
sequences. The resulting amplification products can be labeled
directly during amplification reactions or labeled after
amplification reactions, and used as probes to isolate full-length
cDNA or genomic fragments under conditions of appropriate
stringency.
[0070] In addition, two short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols to
amplify longer nucleic acid fragments encoding homologous genes
from DNA or RNA. The polymerase chain reaction may also be
performed on a library of cloned nucleic acid fragments wherein the
sequence of one primer is derived from the instant nucleic acid
fragments, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding plant genes. Alternatively, the second
primer sequence may be based upon sequences derived from the
cloning vector. For example, the skilled artisan can follow the
RACE protocol (Frohman et al., (1988) Proc. Natl. Acad. Sci. USA
85:8998-9002) to generate cDNAs by using PCR to amplify copies of
the region between a single point in the transcript and the 3' or
5' end. Primers oriented in the 3' and 5' directions can be
designed from the instant sequences. Using commercially available
3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments
can be isolated (Ohara et al., (1989) Proc. Natl. Acad. Sci. USA
86:5673-5677; Loh et al., (1989) Science 243:217-220). Products
generated by the 3' and 5' RACE procedures can be combined to
generate full-length cDNAs (Frohman and Martin (1989) Techniques
1:165).
[0071] Availability of the instant nucleotide and deduced amino
acid sequences facilitates immunological screening of cDNA
expression libraries. Synthetic peptides representing portions of
the instant amino acid sequences may be synthesized. These peptides
can be used to immunize animals to produce polyclonal or monoclonal
antibodies with specificity for peptides or proteins comprising the
amino acid sequences. These antibodies can be then be used to
screen cDNA expression libraries to isolate full-length cDNA clones
of interest (Lerner (1984) Adv. Immunol. 36:1-34; Sambrook).
[0072] The nucleic acid fragments of the instant invention may be
used to create transgenic plants in which the disclosed
oxidosqualene cyclase is present at higher or lower levels than
normal or in cell types or developmental stages in which it is not
normally found. This would have the effect of altering the relative
sterol composition in those cells. These changes in the plant seed
may be useful to improve the seed nutritional value, and in the
plant leaf may aid in insect tolerance.
[0073] Overexpression of oxidosqualene cyclase proteins of the
instant invention may be accomplished by first constructing a
chimeric gene in which the coding region is operably linked to a
promoter capable of directing expression of a gene in the desired
tissues at the desired stage of development. The chimeric gene may
comprise promoter sequences and translation leader sequences
derived from the same genes. 3' Non-coding sequences encoding
transcription termination signals may also be provided. The instant
chimeric gene may also comprise one or more introns in order to
facilitate gene expression.
[0074] Plasmid vectors comprising the isolated polynucleotide (or
chimeric gene) may be constructed. The choice of plasmid vector is
dependent upon the method that will be used to transform host
cells. The skilled artisan is well aware of the genetic elements
that must be present on the plasmid vector in order to successfully
transform, select and propagate host cells containing the chimeric
gene. The skilled artisan will also recognize that different
independent transformation events will result in different levels
and patterns of expression (Jones et al., (1985) EMBO J.
4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics
218:78-86), and thus that multiple events must be screened in order
to obtain lines displaying the desired expression level and
pattern. Such screening may be accomplished by Southern analysis of
DNA, Northern analysis of mRNA expression, Western analysis of
protein expression, or phenotypic analysis.
[0075] For some applications it may be useful to direct the instant
polypeptide to different cellular compartments, or to facilitate
its secretion from the cell. It is thus envisioned that the
chimeric gene described above may be further supplemented by
altering the coding sequence to encode a triterpenoid with
appropriate intracellular targeting sequences such as transit
sequences (Keegstra (1989) Cell 56:247-253), signal sequences or
sequences encoding endoplasmic reticulum localization (Chrispeels
(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear
localization signals (Raikhel (1992) Plant Phys. 100: 1627-1632)
with or without removing targeting sequences that are already
present. While the references cited give examples of each of these,
the list is not exhaustive and more targeting signals of utility
may be discovered in the future.
[0076] It may also be desirable to reduce or eliminate expression
of genes encoding oxidosqualene cyclase in plants for some
applications. In order to accomplish this, a chimeric gene designed
for co-suppression of the instant enzymes can be constructed by
linking a gene or gene fragment encoding an oxidosqualene cyclase
to plant promoter sequences. Alternatively, a chimeric gene
designed to express antisense RNA for all or part of the instant
nucleic acid fragment can be constructed by linking the gene or
gene fragment in reverse orientation to plant promoter sequences.
Either the co-suppression or antisense chimeric genes could be
introduced into plants via transformation wherein expression of the
corresponding endogenous genes are reduced or eliminated.
[0077] The instant oxidosqualene cyclase (or portions thereof) may
be produced in heterologous host cells, particularly in the cells
of microbial hosts, and can be used to prepare antibodies to these
proteins by methods well known to those skilled in the art. The
antibodies are useful for detecting oxidosqualene cyclase in situ
in cells or in vitro in cell extracts. Preferred heterologous host
cells for production of the instant oxidosqualene cyclase are
microbial hosts. Microbial expression systems and expression
vectors containing regulatory sequences that direct high level
expression of foreign proteins are well known to those skilled in
the art. Any of these could be used to construct a chimeric gene
for production of the instant oxidosqualene cyclase. This chimeric
gene could then be introduced into appropriate microorganisms via
transformation to provide high level expression of the encoded
enzymes involved in squalene metabolism. An example of a vector for
high level expression of the instant oxidosqualene cyclase in a
bacterial host is provided (Example 7).
[0078] All or a substantial portion of the nucleic acid fragments
of the instant invention may also be used as probes for genetically
and physically mapping the genes that they are a part of, and as
markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. For example, the instant nucleic acid fragments may be
used as restriction fragment length polymorphism (FP) markers.
Southern blots (Sambrook) of restriction-digested plant genomic DNA
may be probed with the nucleic acid fragments of the instant
invention. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander
et al., (1987) Genomics 1:174-181) in order to construct a genetic
map. In addition, the nucleic acid fragments of the instant
invention may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the instant nucleic acid sequence in the
genetic map previously obtained using this population (Botstein et
al., (1980) Am. J. Hum. Genet. 32:314-331).
[0079] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bernatzky and Tanksley (1986)
Plant Mol. Biol. Reporter 4(1):37-41. Numerous publications
describe genetic mapping of specific cDNA clones using the
methodology outlined above or variations thereof. For example, F2
intercross populations, backcross populations, randomly mated
populations, near isogenic lines, and other sets of individuals may
be used for mapping. Such methodologies are well known to those
skilled in the art.
[0080] Nucleic acid probes derived from the instant nucleic acid
sequences may also be used for physical mapping (i.e., placement of
sequences on physical maps; see Hoheisel et al. In: Nonmammalian
Genomic Analysis: A Practical Guide, Academic press 1996, pp.
319-346, and references cited therein).
[0081] In another embodiment, nucleic acid probes derived from the
instant nucleic acid sequences may be used in direct fluorescence
in situ hybridization (FISH) mapping (Trask (1991) Trends Genet.
7:149-154). Although current methods of FISH mapping favor use of
large clones (several to several hundred KB; see Laan et al. (1995)
Genome Res. 5:13-20), improvements in sensitivity may allow
performance of FISH mapping using shorter probes.
[0082] A variety of nucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the instant
nucleic acid sequences. Examples include allele-specific
amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96),
polymorphism of PCR-amplified fragments (CAPS; Sheffield et al.
(1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy
Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For
these methods, the sequence of a nucleic acid fragment is used to
design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such
primers is well known to those skilled in the art. In methods
employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0083] Loss of function mutant phenotypes may be identified for the
instant cDNA clones either by targeted gene disruption protocols or
by identifying specific mutants for these genes contained in a
maize population carrying mutations in all possible genes
(Ballinger and Benzer (1989) Proc. Natl. Acad. Sci. USA
86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci. USA
92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter
approach may be accomplished in two ways. First, short segments of
the instant nucleic acid fragments may be used in polymerase chain
reaction protocols in conjunction with a mutation tag sequence
primer on DNAs prepared from a population of plants in which
Mutator transposons or some other mutation-causing DNA element has
been introduced (see Bensen, supra). The amplification of a
specific DNA fragment with these primers indicates the insertion of
the mutation tag element in or near the plant gene encoding the
oxidosqualene cyclase. Alternatively, the instant nucleic acid
fragment may be used as a hybridization probe against PCR
amplification products generated from the mutation population using
the mutation tag sequence primer in conjunction with an arbitrary
genomic site primer, such as that for a restriction enzyme
site-anchored synthetic adaptor. With either method, a plant
containing a mutation in the endogenous gene encoding an
oxidosqualene cyclase can be identified and obtained. This mutant
plant can then be used to determine or confirm the natural function
of the oxidosqualene cyclase gene product.
[0084] While not intending to be bound by any theory or theories of
operation, it is believed by those of skill in the art that altered
levels of triterpenes have different effects. Foods originating
from plants having an increased level of triterpenes are thought to
have a cholesterol lowering effect while decreased triterpenes are
believed to result in better tasting foods. Accordingly, plants
grown with altered levels of oxidosqualene cyclases may contribute
to nutritious and/or better flavored foods.
[0085] In another embodiment, the present invention is directed to
a variety of plant or vegetable protein products. A variety of
processed vegetable protein products are produced from soybean.
These are useful in and as food products including and not limited
to human foods as well as animal feed products. These range from
minimally processed, soy beans and soy nuts such as toasted soy
nuts, defatted items such as soybean meal, grits, and flours to
more highly processed items such as soy protein concentrates and
soy protein isolates. In other soy protein products, such as
full-fat soy flour, the oil is not extracted. In addition to these
processed products, there are also a number of specialty products
based on traditional Oriental processes, which utilize the entire
bean as the starting material, also known as the "whole bean
method." Examples of additional products include milk (liquid and
powder) including and not limited to soy milk, soy sauce, nuts
including soy nuts, tofu, natto, miso, tempeh, and yuba.
[0086] Examples of use of soy protein products in human foods
include soy milk, applying soy protein concentrates and soy protein
isolates in nutritional beverage, emulsified meat and whole muscle
meat applications, textured soy protein in meat analogue, and soy
protein isolate in infant formula. Facilities and methods to
produce protein concentrates and isolates from soybeans are
available across the world. To the extent that they are retained in
these processed soy fractions and the foods prepared from them, the
saponin content of the starting beans influences the flavor of the
food.
[0087] The present invention is further defined in the following
Examples, in which all parts and percentages are by weight and
degrees are Celsius, unless otherwise stated. Examples 1-4 are
actual, Examples 5-7 are prophetic. It should be understood that
these Examples, while indicating preferred embodiments of the
invention, are given by way of illustration only. From the above
discussion and these Examples, one skilled in the art can ascertain
the essential characteristics of this invention, and without
departing from the spirit and scope thereof, can make various
changes and modifications of the invention to adapt it to various
usages and conditions. Thus, various modifications of the invention
in addition to those shown and described herein will be apparent to
those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
[0088] The disclosure of each reference set forth herein is
incorporated by reference in its entirety.
EXAMPLES
Example 1
Composition of cDNA Libraries: Isolation and Sequencing of cDNA
Clones
[0089] cDNA libraries representing mRNAs from various soybean and
wheat tissues were prepared. The characteristics of the libraries
are described below. TABLE-US-00001 TABLE 2 cDNA Libraries from
Soybean and Wheat Library Tissue Clone sdp3c Soybean (Glycine max
L.) developing sdp3c.pk020.o10 pods 8-9 mm src3c Soybean (Glycine
max L., Bell) 8 day old src3c.pk024.m11 root inoculated with eggs
of Cyst Nematode (Race 14) for 4 days sah1c Soybean (Glycine max
L., 9151) sprayed sah1c.pk002.n23 with Authority Herbicide wdk1c
Wheat (Triticum aestivum L.) developing wdk1c.pk010.o10 kernel, 3
days after anthesis
[0090] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid
vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries
are converted into plasmid libraries according to the protocol
provided by Stratagene. Upon conversion, cDNA inserts will be
contained in the plasmid vector pBluescript. In addition, the cDNAs
may be introduced directly into precut Bluescript II SK(+) vectors
(Stratagene) using T4 DNA ligase (New England Biolabs), followed by
transfection into DH10B cells according to the manufacturer's
protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid
vectors, plasmid DNAs are prepared from randomly picked bacterial
colonies containing recombinant pBluescript plasmids, or the insert
cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al. (1991) Science
252:1651-1656). The resulting ESTs are analyzed using a Perkin
Elmer Model 377 fluorescent sequencer.
Example 2
Identification of cDNA Clones
[0091] cDNA clones encoding enzymes involved in squalene metabolism
were identified by conducting BLAST (Basic Local Alignment Search
Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences
contained in the BLAST "nr" database (comprising all non-redundant
GenBank CDS translations, sequences derived from the 3-dimensional
structure Brookhaven Protein Data Bank, the last major release of
the SWISS-PROT protein sequence database, EMBL, and DDBJ
databases). The cDNA sequences obtained in Example 1 were analyzed
for similarity to all publicly available DNA sequences contained in
the "nr" database using the BLASTN algorithm provided by the
National Center for Biotechnology Information (NCBI). The DNA
sequences were translated in all reading frames and compared for
similarity to all publicly available protein sequences contained in
the "nr" database using the BLASTX algorithm (Gish and States
(1993) Nat. Genet. 3:266-272) provided by the NCBI. For
convenience, the P-value (probability) of observing a match of a
cDNA sequence to a sequence contained in the searched databases
merely by chance as calculated by BLAST are reported herein as
"pLog" values, which represent the negative of the logarithm of the
reported P-value. Accordingly, the greater the pLog value, the
greater the likelihood that the cDNA sequence and the BLAST "hit"
represent homologous proteins.
Example 3
Characterization of cDNA Clones Encoding Oxidosqualene Cyclases
[0092] The BLASTX search using the EST sequences from clones
sdp3c.pk020.o10, src3c.pk024.m11, sah1c.pk002.n23, wdk1c.pk010.o10
revealed similarity of the proteins encoded by the cDNAs to
.beta.-amyrin synthases from Panax ginseng (NCBI General Identifier
Nos. 3721856 and 3688600) and to the proteins encoded by the contig
to cycloartenol synthase from Arabidopsis thaliana (NCBI General
Identifier No. 3779033). The BLAST results for each of these ESTs
are shown in Table 3: TABLE-US-00002 TABLE 3 BLAST Results for
Clones Encoding Polypeptides Homologous to .beta.-amyrin synthase
Clone NCBI General Identifier No. BLAST pLog Score sdp3c.pk020.o10
3688600 0.02 src3c.pk024.m11 3721856 12.77 sah1c.pk002.n23 3721856
6.09 wdk1c.pk010.o10 3779033 18.96
[0093] A 550 base pair soybean EST having NCBI General Identifier
No. 5606831 has been identified as "similar to .beta.-amyrin
synthase," according to the NCBI entrez. Based on Clustal
alignment, this sequence is 62.0% identical to SEQ ID NO:1, 62.9%
to SEQ ID NO:3, 64.2% to SEQ ID NO:5, and 38.4% to SEQ ID NO:7. The
longest contiguous stretch of identity is 18 nucleotides. This 18
nucleotide stretch corresponds to nucleotides 2372 through 2390
from sah1c.pk002.n23 and nucleotides 120 through 138 from NCBI
General Identifier No. 5606831.
[0094] The sequence of the entire cDNA insert from clone
sdp3c.pk020.o10 is shown in SEQ ID NO:1; the deduced amino acid
sequence of this cDNA is shown in SEQ ID NO:2. The sequence of the
entire cDNA insert from clone src3c.pk024.m11 is shown in SEQ ID
NO:3; the deduced amino acid sequence of this cDNA is shown in SEQ
ID NO:4. The sequence of the entire cDNA insert from clone
sah1c.pk002.n23 is shown in SEQ ID NO:5; the deduced amino acid
sequence of this cDNA is shown in SEQ ID NO:6. The sequence of the
entire cDNA insert from clone wdk1c.pk010.o10 is shown in SEQ ID
NO:7; the deduced amino acid sequence of this cDNA is shown in SEQ
ID NO:8.
[0095] FIG. 1 presents an alignment of the amino acid sequences set
forth in SEQ ID NOs:2, 4, 6, and 8 and the Panax ginseng sequences
(SEQ ID NO:12; NCBI General Identifier No. 3721856, and SEQ ID
NO:13; NCBI General Identifier No. 3688600). The data in Table 4
represents a calculation of the percent identity of the amino acid
sequences set forth in SEQ ID NOs:2, 4 6, and 8 and the Panax
ginseng sequences (SEQ ID NOs:12 and 13). TABLE-US-00003 TABLE 4
Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to .beta.-amyrin synthase Percent Identity to SEQ ID NO.
3721856 3688600 2 75.2 76.2 4 78.4 79.0 6 71.1 70.9 8 51.2 50.9
[0096] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode entire or almost entire soybean and wheat
oxidosqualene cyclases. These sequences are among the first monocot
and soybean sequences known to encode oxidosqualene cyclases.
Example 4
Demonstration of Functional Expression of .beta.-Amyrin Synthase in
Yeast
[0097] The inserts in cDNA clones src3c.pk024.m11 and
sah1c.pk002.n23 were identified as candidate .beta.-amyrin synthase
genes by a BLAST search against the NCBI database. The 5' sequence
of these inserts was determined to be related to either of the
.beta.-amyrin synthases from Panax ginseng, the complete coding
sequence of which may be found as DDBJ Accession Nos. A-B014057 and
AB009030 having NCBI General Identifier Nos. 3721856 and 3688600.
.beta.-amyrin synthase catalyzes the cyclization of
2,3-oxidosqualene to .beta.-amyrin. In order to confirm the
identity of the polypeptides encoded by the inserts in cDNA clones,
src3c.pk024.m11 and sah1c.pk002.n23 as .beta.-amyrin synthase, the
polypeptides encoded by these inserts were evaluated for their
ability to catalyze the formation of .beta.-amyrin.
[0098] The ability of the cDNA inserts in clones src3c.pk024.m11
and sah1c.pk002.n23 to encode .beta.-amyrin synthase was evaluated
by expression of the encoded polypeptides in a yeast (Saccharomyces
cerivisae) strain YPH (Stratagene) and examining the membrane
components. Plasmid DNA (200 ng) from cDNA clone src3c.pk024.m11
was used as template for PCR using primers set forth in SEQ ID NO:9
and SEQ ID NO:11. Plasmid DNA (200 ng) from cDNA clone
sah1c.pk002.n23 was used as template for PCR using primers set
forth in SEQ ID NO:10 and SEQ ID NO:11. The 24 nucleotides at the
5' terminus of both primers set forth as SEQ ID NO:9 and SEQ ID
NO:10 are homologous to the modified pRS315 plasmid described
below. The 22 nucleotides at the 3' terminus of the primer set
forth in SEQ ID NO:9 is derived from the start of transcription of
the protein encoded by clone src3c.pk0024.m11. The 22 nucleotides
at the 3' terminus of the primer set forth in SEQ ID NO:10 is
derived from the start of transcription of the protein encoded by
clone sah1c.pk002.n23. The sequence of the primer set forth as SEQ
ID NO:11 is homologous to vector sequences. TABLE-US-00004 [SEQ ID
NO:9] 5'-TCAAGGAGAAAAAACCCCGGATCCATGTGGAGGCTGAAGAT AGCAG-3' [SEQ ID
NO:10] 5'-TCAAGGAGAAAAAACCCCGGATCCATGTGGAGGTTAAAGATA GCAG-3' [SEQ
ID NO:11] 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGCG-3'
[0099] Amplification was performed using the GC melt kit (Clontech)
with a 1 M final concentration of GC melt reagent. Amplification
took place in a Perkin Elmer 9700 thermocycler for 30 cycles as
follows: 94.degree. C. for 30 seconds, 60.degree. C. for 30
seconds, and 72.degree. C. for 2 minutes. The amplified insert was
then incubated with a modified pRS315 plasmid (NCBI General
Identifier No. 984798; Sikorski, R. S. and Hieter, P. (1989)
Genetics 122:19-27) that had been digested with Not I and Spe I.
Plasmid pRS315 had been previously modified by the insertion of a
bidirectional gal 1/10 promoter between the Xho I and Hind III
sites. The plasmid was then transformed into the YPH yeast strain
using standard procedures where the insert recombines through gap
repair to form the desired transformed yeast strain (Hua, S. B. et
al. (1997) Plasmid 38:91-96.). The resulting transformed yeast
strains were named Yeast Strains .beta.-amyrin X and .beta.-amyrin
Y.
[0100] Yeast cells were prepared according to a modification of the
methods of Pompon et al. (Pompon, D. et al. (1996) Meth. Enz.
272:51-64). Briefly, a yeast colony was grown overnight (to
saturation) in SG (-Leucine) medium at 30.degree. C. with good
aeration. A 1:50 dilution of this culture was made into 500 mL of
YPGE medium with adenine supplementation and allowed to grow at
30.degree. C. with good aeration to an OD.sub.600 of 1.6 (24-30 h).
Fifty mL of 20% galactose was added, and the culture was allowed to
grow overnight at 30.degree. C. The cells were recovered by
centrifugation at 5,500 rpm for five minutes in a Sorvall GS-3
rotor. The cell pellet was resuspended in 500 mL of 0.1 M potassium
phosphate buffer (H 7.0) and then allow to grow at 30.degree. C.
for another 24 hours.
[0101] The cells were recovered by centrifugation as described
above and the presence of .beta.-amyrin was determined by HPLC/mass
spectrometry.
[0102] The cDNA insert in clones src3c.pk024.m11 and
sah1c.pk002.n23 resulted in decectable levels of .beta.-amyrin. The
results were not repeatable using the cDNA insert in clone
sah1c.pk002.n23. This example was repeated in a different yeast
background (overexpressing HMG-CoA reductase) using the cDNA
inserts from clones src3c.pk024.m11, sah1c.pk002.n23,
sdp3c.pk020.o10 and wdk1c.pk010.o10. Detectable levels of
.beta.-amyrin resulted using the cDNA insert from
src3c.pk024.m11.
Example 5
Expression of Chimeric Genes in Monocot Cells
[0103] A chimeric gene comprising a cDNA encoding .beta.-amyrin
synthase in sense orientation with respect to the maize 27 kD zein
promoter that is located 5' to the cDNA fragment, and the 10 kD
zein 3' end that is located 3' to the cDNA fragment, can be
constructed. The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites (Nco I or Sma I) can be
incorporated into the oligonucleotides to provide proper
orientation of the DNA fragment when inserted into the digested
vector pML103 as described below. Amplification is then performed
in a standard PCR reaction. The amplified DNA is then digested with
restriction enzymes Nco I and Sma I and fractionated on an agarose
gel. The appropriate band can be isolated from the gel and combined
with a 4.9 kb Nco I-Sma I fragment of the plasmid pML103. Plasmid
pML103 has been deposited under the terms of the Budapest Treaty at
ATCC (American Type Culture Collection, 10801 University Blvd.,
Manassas, Va. 20110-2209), and bears accession number ATCC 97366.
The DNA segment from pML103 contains a 1.05 kb Sal I-Nco I promoter
fragment of the maize 27 kD zein gene and a 0.96 kb Sma I-Sal I
fragment from the 3' end of the maize 10 kD zein gene in the vector
pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at
15.degree. C. overnight, essentially as described (Sambrook). The
ligated DNA may then be used to transform E. coli XL1-Blue
(Epicurian Coli XL-1 Blue.TM.; Stratagene). Bacterial transformants
can be screened by restriction enzyme digestion of plasmid DNA and
limited nucleotide sequence analysis using the dideoxy chain
termination method (Sequenase.TM. DNA Sequencing Kit; U.S.
Biochemical). The resulting plasmid construct would comprise a
chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD
zein promoter, a cDNA encoding .beta.-amyrin synthase, and the 10
kD zein 3' region.
[0104] The chimeric gene described above can then be introduced
into corn cells by the following procedure. Immature corn embryos
can be dissected from developing caryopses derived from crosses of
the inbred corn lines H99 and LH132. The embryos are isolated 10 to
11 days after pollination when they are 1.0 to 1.5 mm long. The
embryos are then placed with the axis-side facing down and in
contact with agarose-solidified N6 medium (Chu et al. (1975) Sci.
Sin. Peking 18:659-668). The embryos are kept in the dark at
27.degree. C. Friable embryogenic callus consisting of
undifferentiated masses of cells with somatic proembryoids and
embryoids borne on suspensor structures proliferates from the
scutellum of these immature embryos. The embryogenic callus
isolated from the primary explant can be cultured on N6 medium and
sub-cultured on this medium every 2 to 3 weeks.
[0105] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst
Ag, Frankfurt, Germany) may be used in transformation experiments
in order to provide for a selectable marker. This plasmid contains
the Pat gene (see European Patent Publication 0 242,236) which
encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT
confers resistance to herbicidal glutamine synthetase inhibitors
such as phosphinothricin. The pat gene in p35S/Ac is under the
control of the 35S promoter from Cauliflower Mosaic Virus (Odell et
al. (1985) Nature 313:810-812) and the 3' region of the nopaline
synthase gene from the T-DNA of the Ti plasmid of Agrobacterium
tumefaciens.
[0106] The particle bombardment method (Klein et al. (1987) Nature
327:70-73) may be used to transfer genes to the callus culture
cells. According to this method, gold particles (1 .mu.M in
diameter) are coated with DNA using the following technique. Ten
.mu.g of plasmid DNAs are added to 50 .mu.L of a suspension of gold
particles (60 mg per mL). Calcium chloride (50 .mu.L of a 2.5 M
solution) and spermidine free base (20 .mu.L of a 1.0 M solution)
are added to the particles. The suspension is vortexed during the
addition of these solutions. After 10 minutes, the tubes are
briefly centrifuged (5 sec at 15,000 rpm) and the supernatant
removed. The particles are resuspended in 200 .mu.L of absolute
ethanol, centrifuged again and the supernatant removed. The ethanol
rinse is performed again and the particles resuspended in a final
volume of 30 .mu.L of ethanol. An aliquot (5 mL) of the DNA-coated
gold particles can be placed in the center of a Kapton.TM. flying
disc (Bio-Rad Labs). The particles are then accelerated into the
corn tissue with a Biolistic.TM. PDS-1000/He (Bio-Rad Instruments,
Hercules Calif.), using a helium pressure of 1000 psi, a gap
distance of 0.5 cm and a flying distance of 1.0 cm.
[0107] For bombardment the embryogenic tissue is placed on filter
paper over agarose-solidified N6 medium. The tissue is arranged as
a thin lawn and covered a circular area of about 5 cm in diameter.
The petri dish containing the tissue can be placed in the chamber
of the PDS-1000/He approximately 8 cm from the stopping screen. The
air in the chamber is then evacuated to a vacuum of 28 inches of
Hg. The macrocarrier is accelerated with a helium shock wave using
a rupture membrane that bursts when the He pressure in the shock
tube reaches 1000 psi.
[0108] Seven days after bombardment the tissue can be transferred
to N6 medium that contains glufosinate (2 mg per liter) and lacks
casein or proline. The tissue continues to grow slowly on this
medium. After an additional 2 weeks the tissue can be transferred
to fresh N6 medium containing gluphosinate. After 6 weeks, areas of
about 1 cm in diameter of actively growing callus can be identified
on some of the plates containing the glufosinate-supplemented
medium. These calluses may continue to grow when sub-cultured on
the selective medium.
[0109] Plants can be regenerated from the transgenic callus by
first transferring clusters of tissue to N6 medium supplemented
with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be
transferred to regeneration medium (Fromm et al., (1990)
Bio/Technology 8:833-839).
Example 6
Expression of Chimeric Genes in Dicot Cells
[0110] A seed-specific expression cassette composed of the promoter
and transcription terminator from the gene encoding the D subunit
of the seed storage protein phaseolin from the bean Phaseolus
vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-91238) can be
used for expression of the instant enzymes .beta.-amyrin synthase
in transformed soybean. The phaseolin cassette includes about 500
nucleotides upstream (5' from the translation initiation codon and
about 1650 nucleotides downstream (3') from the translation stop
codon of phaseolin. Between the 5' and 3' regions are the unique
restriction endonuclease sites Nco I (which includes the ATG
translation initiation codon), Sma L Kpn I and Xba I. The entire
cassette is flanked by Hind III sites.
[0111] The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites can be incorporated into the
oligonucleotides to provide proper orientation of the DNA fragment
when inserted into the expression vector. Amplification is then
performed as described above, and the isolated fragment is inserted
into a pUC18 vector carrying the seed expression cassette.
[0112] Soybean embryos may then be transformed with the expression
vector comprising sequences encoding .beta.-amyrin synthase. To
induce somatic embryos, cotyledons, 3-5 mm in length dissected from
surface sterilized, immature seeds of the soybean cultivar A2872,
can be cultured in the light or dark at 26.degree. C. on an
appropriate agar medium for 6-10 weeks. Somatic embryos which
produce secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos which multiplied as early, globular staged embryos,
the suspensions are maintained as described below.
[0113] Soybean embryogenic suspension cultures can maintained in 35
mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
florescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 mL of liquid medium.
[0114] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Kline et al.
(1987) Nature (London) 327:70, U.S. Pat. No. 4,945,050). A DuPont
Biolistic.TM. PDS1000/HE instrument (helium retrofit) can be used
for these transformations.
[0115] A selectable marker gene which can be used to facilitate
soybean transformation is a chimeric gene composed of the 35S
promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) and the
3' region of the nopaline synthase gene from the T-DNA of the Ti
plasmid of Agrobacterium tumefaciens. The seed expression cassette
comprising the phaseolin 5' region, the fragment encoding the
enzyme involved in squalene metabolism and the phaseolin 3' region
can be isolated as a restriction fragment. This fragment can then
be inserted into a unique restriction site of the vector carrying
the marker gene.
[0116] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order: 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.l
spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.L 70% ethanol and
resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
.mu.L of the DNA-coated gold particles are then loaded on each
macro carrier disk.
[0117] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0118] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post
bombardment with fresh media containing 50 mg/mL hygromycin. This
selective media can be refreshed weekly. Seven to eight weeks post
bombardment, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated green tissue
is removed and inoculated into individual flasks to generate new,
clonally propagated, transformed embryogenic suspension cultures.
Each new line may be treated as an independent transformation
event. These suspensions can then be subcultured and maintained as
clusters of immature embryos or regenerated into whole plants by
maturation and germination of individual somatic embryos.
Example 7
Expression of Chimeric Genes in Microbial Cells
[0119] The cDNAs encoding the instant enzyme .beta.-amyrin synthase
can be inserted into the T7 E. coli expression vector pBT430. This
vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene
56:125-135) which employs the bacteriophage T7 RNA polymerase/T7
promoter system. Plasmid pBT430 may be constructed by first
destroying the EcoR I and Hind III sites in pET-3a at their
original positions. An oligonucleotide adaptor containing EcoR I
and Hind III sites may be inserted at the BamH I site of pET-3&
This creates pET-3 aM with additional unique cloning sites for
insertion of genes into the expression vector. Then, the Nde I site
at the position of translation initiation is then converted to an
Nco I site using oligonucleotide-directed mutagenesis. The DNA
sequence of pET-3aM in this region, 5'-CATATGG, is converted to
5'-CCCATGG in pBT430.
[0120] Plasmid DNA containing a cDNA may be appropriately digested
to release a nucleic acid fragment encoding the protein. This
fragment may then be purified on a 1% NuSieve GTG.TM. low melting
agarose gel (FMC). Buffer and agarose contain 10 .mu.g/ml ethidium
bromide for visualization of the DNA fragment. The fragment can
then be purified from the agarose gel by digestion with GELase.TM.
(Epicentre Technologies) according to the manufacturer's
instructions, ethanol precipitated, dried and resuspended in 20
.mu.L of water. Appropriate oligonucleotide adapters may be ligated
to the fragment using T4 DNA ligase (New England Biolabs, Beverly,
Mass.). The fragment containing the ligated adapters can be
purified from the excess adapters using low melting agarose as
described above. The vector pBT430 is digested, dephosphorylated
with alkaline phosphatase (NEB) and deproteinized with
phenol/chloroform as described above. The prepared vector pBT430
and fragment can then be ligated at 16.degree. C. for 15 hours
followed by transformation into DH5 electrocompetent cells (GIBCO
BRL). Transformants can be selected on agar plates containing LB
media and 100 .mu.g/mL ampicillin. Transformants containing the
gene encoding the enzyme involved in squalene metabolism may then
be screened for the correct orientation with respect to the T7
promoter by restriction enzyme analysis.
[0121] For high level expression, a plasmid clone with the cDNA
insert in the correct orientation relative to the T7 promoter can
be transformed into E. coli strain BL21(DE3) (Studier et al. (1986)
J. Mol. Biol. 189:113-130). Cultures are grown in LB medium
containing ampicillin (100 mg/L) at 25.degree. C. At an optical
density at 600 nm of approximately 1, IPTG
(isopropylthio-p-galactoside, the inducer) can be added to a final
concentration of 0.4 mM and incubation can be continued for 3 h at
25.degree.. Cells are then harvested by centrifugation and
re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1
mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of
1 mm glass beads can be added and the mixture sonicated 3 times for
about 5 seconds each time with a microprobe sonicator. The mixture
is centrifuged and the protein concentration of the supernatant
determined. One .mu.g of protein from the soluble fraction of the
culture can be separated by SDS-polyacrylamide gel electrophoresis.
Gels can be observed for protein bands migrating at the expected
molecular weight.
[0122] Various modifications of the invention in addition to those
shown and described herein will be apparent to those skilled in the
art from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
[0123] The disclosure of each reference set forth above is
incorporated herein by reference in its entirety.
Sequence CWU 1
1
13 1 2731 DNA Glycine max 1 atagattcga ggacagattt taaaagcaac
ctaagttttg tatttacaaa actgagcttc 60 cagaaggaaa aaaaaaatca
atttgctaaa gttattttag gataactaac taagcatgtc 120 aagaaatcga
gtttattctc taaaaatatt ttcaacataa gaaaaacaat actttttgaa 180
tggttagtaa aacatgtctt taatcagttg cttcgtattt tcatgcagtt aggtgcatca
240 attgactgtg gcattatagc atattaagag aaagcaacaa aatgtggagg
ctgaagatag 300 cagatggtgg aaaagaccca tacattttca gcacaaacaa
ttttgtagga agacagacat 360 gggagtttga tcctgaggca ggaactccag
aggaacgagc tcaagttgaa gcagctcgtc 420 aaaattttta taacaatcgc
ttcaaggtca aggcatgtgg tgatctcctt tggcgttttc 480 agattctgag
agaaaaaaac ttcaaacaat caatacctag tgtgaagata gaagatggag 540
aagaaataac atacgaaaaa gtcataagca cgttgagaag agccgcacac cacctatcag
600 cattgcagac cagtgatggc cattggcctg cacaaattgc aggtcctctg
ttttttctgc 660 ctcctttggt tttttgtatg tacattacag ggcatcttga
tttggtattc ccagaagagt 720 atcgaaaaga gattctccgt tacatatact
atcaccagaa tgaagatgga ggatggggac 780 tacacataga gggtcatagc
accatgtttg gtactacact aaactatata tgtatgcgaa 840 ttcttggaga
agggcctaat ggaggtcatg aaaatgcatg tgctagagga aaaaagtgga 900
ttcatgatca tggtggtgta acacacatcc cttcatgggg gaaaacttgg ctttcgatac
960 ttggtgtatt tgattggtgt ggaagcaacc caatgccccc agagttttgg
attattcctt 1020 cgtttcttcc tatgcatcca gctaaaatgt ggtgttattg
tcgattggta tatatgccta 1080 tgtcttatat gggaaaaggg tttgtgggtc
caatcacacc actcatctta caattgagag 1140 aagagctctt tactcagcct
tatgaaaaag ttaattggaa aaaagtgcgt catcaatgtg 1200 caaaggaaga
tctttattat cctcattctt tgatacaaga cctagtatgg gacagtctat 1260
acatgtttac tgagccacta cttacttgtt ggcctttcaa caaactaatt agagaaaagg
1320 cccttcaagt aacaatgaac catattcatt acgaagatga gaatagtcga
tacataacta 1380 ttgggtgtgt ggaaaaggtt ctatgtatgc ttgcttgttg
ggttgaagat ccacagggag 1440 atgcctttaa gaagcatctt gcaagggtct
cagattactt atgggtttct gaagatggaa 1500 tgaccatgca aagttttggt
agtcaagaat gggatgctgg ttttgcagtt caagctttgc 1560 ttgcaactaa
gctaattgac gaaattggcc attcacttgc aaaagggcat gatttcatca 1620
agaagtctca ggtgagagac aacccttcag gagattttaa gagtatgtat cgtcatatta
1680 ctaaagggtc ttggacattc tcagatcaag accatggatg gcaagtttct
gattgcactg 1740 cagaaggttt aaagtgttgt cttcttctat caaagttgtc
accagagatt gtgggagaaa 1800 aagtgaaacc tgaaagattt tatgattcag
tcaatatctt gttgtcactt cagagtaaaa 1860 aaggtggtat agcagcatgg
gaaccaatag gagctcaaga atggttggaa ttactcaatc 1920 ccactgaatt
ttttgaggac attgtaattg agcatgaata tgttgagtgc actggatctg 1980
caattcaagc tttagttttg ttccagaagc tatatccaga gcataggaag acagagatta
2040 agaatttcat tgtcaatgca gttcaattcc ttgaagatac acaaacaacc
aatggttcat 2100 ggtatggatg ttggggagtt tgctacactt atggctcttg
gtttgcactt ggtggtctag 2160 cagctgccgg taagacttac actaattgta
atgccattcg caaggctgtg aaatttctac 2220 ttacaacaca gagagaggat
ggtgggtggg gagagagtta tctttcaagc ccaaaaaaga 2280 tatacatacc
tcttgaagga agtcgatcaa acgttgtaca aacagcatgg gctcttatgg 2340
gtctaattta tgctggacag tcagagagag accttactcc tcttcatcga gctgcaaagt
2400 tgctcattaa ttcccagttg gaagaaggtg attggcccca acaggaaatc
actggagtat 2460 tcttgaaaaa ctgcatgatg cattacccaa tgtatagaaa
tatttttcca atgtgggctc 2520 tagctgaata tcgtaggcga gttccattgc
catccactga aggttaattt tgaaaaggtg 2580 tgtgcataaa agggcaaaga
catatcaatg aggaatgggg caagaagcac ccattgtctc 2640 ttacttggac
tctattgatc tgtaattttg aagcttgctt tttattataa aataaaaaat 2700
tatttacgca caaaaaaaaa aaaaaaaaaa a 2731 2 761 PRT Glycine max 2 Met
Trp Arg Leu Lys Ile Ala Asp Gly Gly Lys Asp Pro Tyr Ile Phe 1 5 10
15 Ser Thr Asn Asn Phe Val Gly Arg Gln Thr Trp Glu Phe Asp Pro Glu
20 25 30 Ala Gly Thr Pro Glu Glu Arg Ala Gln Val Glu Ala Ala Arg
Gln Asn 35 40 45 Phe Tyr Asn Asn Arg Phe Lys Val Lys Ala Cys Gly
Asp Leu Leu Trp 50 55 60 Arg Phe Gln Ile Leu Arg Glu Lys Asn Phe
Lys Gln Ser Ile Pro Ser 65 70 75 80 Val Lys Ile Glu Asp Gly Glu Glu
Ile Thr Tyr Glu Lys Val Ile Ser 85 90 95 Thr Leu Arg Arg Ala Ala
His His Leu Ser Ala Leu Gln Thr Ser Asp 100 105 110 Gly His Trp Pro
Ala Gln Ile Ala Gly Pro Leu Phe Phe Leu Pro Pro 115 120 125 Leu Val
Phe Cys Met Tyr Ile Thr Gly His Leu Asp Leu Val Phe Pro 130 135 140
Glu Glu Tyr Arg Lys Glu Ile Leu Arg Tyr Ile Tyr Tyr His Gln Asn 145
150 155 160 Glu Asp Gly Gly Trp Gly Leu His Ile Glu Gly His Ser Thr
Met Phe 165 170 175 Gly Thr Thr Leu Asn Tyr Ile Cys Met Arg Ile Leu
Gly Glu Gly Pro 180 185 190 Asn Gly Gly His Glu Asn Ala Cys Ala Arg
Gly Lys Lys Trp Ile His 195 200 205 Asp His Gly Gly Val Thr His Ile
Pro Ser Trp Gly Lys Thr Trp Leu 210 215 220 Ser Ile Leu Gly Val Phe
Asp Trp Cys Gly Ser Asn Pro Met Pro Pro 225 230 235 240 Glu Phe Trp
Ile Ile Pro Ser Phe Leu Pro Met His Pro Ala Lys Met 245 250 255 Trp
Cys Tyr Cys Arg Leu Val Tyr Met Pro Met Ser Tyr Met Gly Lys 260 265
270 Gly Phe Val Gly Pro Ile Thr Pro Leu Ile Leu Gln Leu Arg Glu Glu
275 280 285 Leu Phe Thr Gln Pro Tyr Glu Lys Val Asn Trp Lys Lys Val
Arg His 290 295 300 Gln Cys Ala Lys Glu Asp Leu Tyr Tyr Pro His Ser
Leu Ile Gln Asp 305 310 315 320 Leu Val Trp Asp Ser Leu Tyr Met Phe
Thr Glu Pro Leu Leu Thr Cys 325 330 335 Trp Pro Phe Asn Lys Leu Ile
Arg Glu Lys Ala Leu Gln Val Thr Met 340 345 350 Asn His Ile His Tyr
Glu Asp Glu Asn Ser Arg Tyr Ile Thr Ile Gly 355 360 365 Cys Val Glu
Lys Val Leu Cys Met Leu Ala Cys Trp Val Glu Asp Pro 370 375 380 Gln
Gly Asp Ala Phe Lys Lys His Leu Ala Arg Val Ser Asp Tyr Leu 385 390
395 400 Trp Val Ser Glu Asp Gly Met Thr Met Gln Ser Phe Gly Ser Gln
Glu 405 410 415 Trp Asp Ala Gly Phe Ala Val Gln Ala Leu Leu Ala Thr
Lys Leu Ile 420 425 430 Asp Glu Ile Gly His Ser Leu Ala Lys Gly His
Asp Phe Ile Lys Lys 435 440 445 Ser Gln Val Arg Asp Asn Pro Ser Gly
Asp Phe Lys Ser Met Tyr Arg 450 455 460 His Ile Thr Lys Gly Ser Trp
Thr Phe Ser Asp Gln Asp His Gly Trp 465 470 475 480 Gln Val Ser Asp
Cys Thr Ala Glu Gly Leu Lys Cys Cys Leu Leu Leu 485 490 495 Ser Lys
Leu Ser Pro Glu Ile Val Gly Glu Lys Val Lys Pro Glu Arg 500 505 510
Phe Tyr Asp Ser Val Asn Ile Leu Leu Ser Leu Gln Ser Lys Lys Gly 515
520 525 Gly Ile Ala Ala Trp Glu Pro Ile Gly Ala Gln Glu Trp Leu Glu
Leu 530 535 540 Leu Asn Pro Thr Glu Phe Phe Glu Asp Ile Val Ile Glu
His Glu Tyr 545 550 555 560 Val Glu Cys Thr Gly Ser Ala Ile Gln Ala
Leu Val Leu Phe Gln Lys 565 570 575 Leu Tyr Pro Glu His Arg Lys Thr
Glu Ile Lys Asn Phe Ile Val Asn 580 585 590 Ala Val Gln Phe Leu Glu
Asp Thr Gln Thr Thr Asn Gly Ser Trp Tyr 595 600 605 Gly Cys Trp Gly
Val Cys Tyr Thr Tyr Gly Ser Trp Phe Ala Leu Gly 610 615 620 Gly Leu
Ala Ala Ala Gly Lys Thr Tyr Thr Asn Cys Asn Ala Ile Arg 625 630 635
640 Lys Ala Val Lys Phe Leu Leu Thr Thr Gln Arg Glu Asp Gly Gly Trp
645 650 655 Gly Glu Ser Tyr Leu Ser Ser Pro Lys Lys Ile Tyr Ile Pro
Leu Glu 660 665 670 Gly Ser Arg Ser Asn Val Val Gln Thr Ala Trp Ala
Leu Met Gly Leu 675 680 685 Ile Tyr Ala Gly Gln Ser Glu Arg Asp Leu
Thr Pro Leu His Arg Ala 690 695 700 Ala Lys Leu Leu Ile Asn Ser Gln
Leu Glu Glu Gly Asp Trp Pro Gln 705 710 715 720 Gln Glu Ile Thr Gly
Val Phe Leu Lys Asn Cys Met Met His Tyr Pro 725 730 735 Met Tyr Arg
Asn Ile Phe Pro Met Trp Ala Leu Ala Glu Tyr Arg Arg 740 745 750 Arg
Val Pro Leu Pro Ser Thr Glu Gly 755 760 3 2478 DNA Glycine max 3
ggtttgtttg gtgtgagtga atagggatca gggatgtgga ggctgaagat agcagatgga
60 ggaaatgatc catacatatt cagcacaaac aatttcgttg ggaggcagac
atgggagttt 120 gatcctgaag caggcagtcc agaggaacgg gcccaggttg
aagcagctcg tcagcatttc 180 taccacaacc gcttcaaggt caagccctgc
gctgacctcc tttggcgttt tcaggttctc 240 agagaaaata acttcaaaca
aacaattcct cgtgtgacta tagaagatgg agaggaaatc 300 acataccaaa
aagtcacaag cgccgtcaga aggggcgcac accaccttgc ggcactgcag 360
acctctgatg gccattggcc tgctcaaatt gcaggtcctc tcttctttct tcctcccttg
420 gttttttgta tgtatattac aggaaatctt gaatcagtat ttccagaaga
acatcgcaaa 480 gaaattcttc gttacacata ttatcaccag aatgaagacg
gaggatgggg actacacata 540 gagggtcata gcactatgtt ttgtactgca
ctgaactata tatgcatgcg aatgcttgga 600 gaaggaccta atggaggtca
tgacaatgct tgtgctagag caagaaagtg gattcgagat 660 catggtggtg
taacacatat accttcatgg ggaaaaactt ggctttcgat actcggtgta 720
tttgattggt gcggaagcaa cccaatgccc ccagagtttt ggatccttcc atcttttctt
780 cctatgcatc cagctaagat gtggtgttac tgtcgattgg tatacatgcc
tatgtcttac 840 ttatatggga agaggtttgt gggtccaatc acaccactca
tcttacaatt aagagaagag 900 ttgtttactc aaccttatga aaaagttaat
tggaagaaag cgcgtcacca atgtgcaaag 960 gaagatcttt actatcccca
tcctttgata caagacctaa tatgggatag tttatacata 1020 ttcactgaac
cgctacttac tcgttggcct ttcaacaagt tgattagaga aaaggccctt 1080
caagtaacta tgaaacatat tcattatgaa gatgagacta gtcgatacat aaccattggt
1140 tgtgtggaaa aggttttatg tatgcttgct tgttgggtgg aagatccaaa
cggagatgct 1200 ttcaagaagc atcttgcaag ggtcccagat tacttatggg
tttctgaaga tggaatgacc 1260 atgcagagtt ttggtagcca agaatgggat
gctggctttg ctgttcaagc tttgcttgcc 1320 actaacataa ttgaagaaat
tggtcctacg tttgcaaaag gacatgattt catcaagaag 1380 tctcaggtga
aggataatcc ttttggagat tttaaaagta tgcatcgtca tatttctaaa 1440
gggtcttgga cattctctga tcaagaccat ggatggcaag tttctgattg cactgcagaa
1500 ggtttaaagt gttgtctact tctatcaatg ttgccaccag agattgtggg
agaaaagatg 1560 gaacctgaaa gattatacga ttcagtcaat gtcttgttgt
cgcttcagag taaaaaaggt 1620 ggtttagcag catgggagcc tgcaggagct
caagagtggt tagaattact caatcccaca 1680 gaattttttg cggacattgt
agttgaacat gaatatgttg agtgcactgg atctgcaatc 1740 caagctttag
ttttgttcaa gaagctatat ccaggacata ggaagaaaga gatagaaaat 1800
ttcattacca atgcagttcg attccttgaa gatacacaaa cagctgatgg ttcatggtat
1860 ggaaattggg gagtttgctt cacttatggc tcttggtttg cacttggagg
tctagcagct 1920 gctggtaaga cttacaccaa ttgtgctgcc attcgcaaag
ccgttaaatt tctacttaca 1980 acacaaagag aggacggtgg atggggagag
agttatcttt caagcccaaa aaagatatat 2040 gtacctctag aaggaagccg
atcaaatgtt gtacatacag catgggctct tatgggacta 2100 attcatgctg
gacaggcgga tagagacccc atgcctcttc accgtgctgc aaagttgctc 2160
attaattctc agttggaaga gggtgattgg ccccaacagg aaatcacggg agtattcatg
2220 aaaaattgca tgttgcatta tccaatgtac agagatattt atccaatgtg
ggctctagct 2280 gaatatcgaa ggcgggttcc attgccttcc actgaagttt
aatttagaat ggtttgagca 2340 cgaaaaggca aaggcatttt cattaagatt
gaggcaaata agttgtgtgt aatcaagctt 2400 aatcaatttt ttcatattcc
tatgtttatt tcctacatat attggtagaa aaattatttc 2460 aaaaaaaaaa
aaaaaaaa 2478 4 762 PRT Glycine max 4 Met Trp Arg Leu Lys Ile Ala
Asp Gly Gly Asn Asp Pro Tyr Ile Phe 1 5 10 15 Ser Thr Asn Asn Phe
Val Gly Arg Gln Thr Trp Glu Phe Asp Pro Glu 20 25 30 Ala Gly Ser
Pro Glu Glu Arg Ala Gln Val Glu Ala Ala Arg Gln His 35 40 45 Phe
Tyr His Asn Arg Phe Lys Val Lys Pro Cys Ala Asp Leu Leu Trp 50 55
60 Arg Phe Gln Val Leu Arg Glu Asn Asn Phe Lys Gln Thr Ile Pro Arg
65 70 75 80 Val Thr Ile Glu Asp Gly Glu Glu Ile Thr Tyr Gln Lys Val
Thr Ser 85 90 95 Ala Val Arg Arg Gly Ala His His Leu Ala Ala Leu
Gln Thr Ser Asp 100 105 110 Gly His Trp Pro Ala Gln Ile Ala Gly Pro
Leu Phe Phe Leu Pro Pro 115 120 125 Leu Val Phe Cys Met Tyr Ile Thr
Gly Asn Leu Glu Ser Val Phe Pro 130 135 140 Glu Glu His Arg Lys Glu
Ile Leu Arg Tyr Thr Tyr Tyr His Gln Asn 145 150 155 160 Glu Asp Gly
Gly Trp Gly Leu His Ile Glu Gly His Ser Thr Met Phe 165 170 175 Cys
Thr Ala Leu Asn Tyr Ile Cys Met Arg Met Leu Gly Glu Gly Pro 180 185
190 Asn Gly Gly His Asp Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile Arg
195 200 205 Asp His Gly Gly Val Thr His Ile Pro Ser Trp Gly Lys Thr
Trp Leu 210 215 220 Ser Ile Leu Gly Val Phe Asp Trp Cys Gly Ser Asn
Pro Met Pro Pro 225 230 235 240 Glu Phe Trp Ile Leu Pro Ser Phe Leu
Pro Met His Pro Ala Lys Met 245 250 255 Trp Cys Tyr Cys Arg Leu Val
Tyr Met Pro Met Ser Tyr Leu Tyr Gly 260 265 270 Lys Arg Phe Val Gly
Pro Ile Thr Pro Leu Ile Leu Gln Leu Arg Glu 275 280 285 Glu Leu Phe
Thr Gln Pro Tyr Glu Lys Val Asn Trp Lys Lys Ala Arg 290 295 300 His
Gln Cys Ala Lys Glu Asp Leu Tyr Tyr Pro His Pro Leu Ile Gln 305 310
315 320 Asp Leu Ile Trp Asp Ser Leu Tyr Ile Phe Thr Glu Pro Leu Leu
Thr 325 330 335 Arg Trp Pro Phe Asn Lys Leu Ile Arg Glu Lys Ala Leu
Gln Val Thr 340 345 350 Met Lys His Ile His Tyr Glu Asp Glu Thr Ser
Arg Tyr Ile Thr Ile 355 360 365 Gly Cys Val Glu Lys Val Leu Cys Met
Leu Ala Cys Trp Val Glu Asp 370 375 380 Pro Asn Gly Asp Ala Phe Lys
Lys His Leu Ala Arg Val Pro Asp Tyr 385 390 395 400 Leu Trp Val Ser
Glu Asp Gly Met Thr Met Gln Ser Phe Gly Ser Gln 405 410 415 Glu Trp
Asp Ala Gly Phe Ala Val Gln Ala Leu Leu Ala Thr Asn Ile 420 425 430
Ile Glu Glu Ile Gly Pro Thr Phe Ala Lys Gly His Asp Phe Ile Lys 435
440 445 Lys Ser Gln Val Lys Asp Asn Pro Phe Gly Asp Phe Lys Ser Met
His 450 455 460 Arg His Ile Ser Lys Gly Ser Trp Thr Phe Ser Asp Gln
Asp His Gly 465 470 475 480 Trp Gln Val Ser Asp Cys Thr Ala Glu Gly
Leu Lys Cys Cys Leu Leu 485 490 495 Leu Ser Met Leu Pro Pro Glu Ile
Val Gly Glu Lys Met Glu Pro Glu 500 505 510 Arg Leu Tyr Asp Ser Val
Asn Val Leu Leu Ser Leu Gln Ser Lys Lys 515 520 525 Gly Gly Leu Ala
Ala Trp Glu Pro Ala Gly Ala Gln Glu Trp Leu Glu 530 535 540 Leu Leu
Asn Pro Thr Glu Phe Phe Ala Asp Ile Val Val Glu His Glu 545 550 555
560 Tyr Val Glu Cys Thr Gly Ser Ala Ile Gln Ala Leu Val Leu Phe Lys
565 570 575 Lys Leu Tyr Pro Gly His Arg Lys Lys Glu Ile Glu Asn Phe
Ile Thr 580 585 590 Asn Ala Val Arg Phe Leu Glu Asp Thr Gln Thr Ala
Asp Gly Ser Trp 595 600 605 Tyr Gly Asn Trp Gly Val Cys Phe Thr Tyr
Gly Ser Trp Phe Ala Leu 610 615 620 Gly Gly Leu Ala Ala Ala Gly Lys
Thr Tyr Thr Asn Cys Ala Ala Ile 625 630 635 640 Arg Lys Ala Val Lys
Phe Leu Leu Thr Thr Gln Arg Glu Asp Gly Gly 645 650 655 Trp Gly Glu
Ser Tyr Leu Ser Ser Pro Lys Lys Ile Tyr Val Pro Leu 660 665 670 Glu
Gly Ser Arg Ser Asn Val Val His Thr Ala Trp Ala Leu Met Gly 675 680
685 Leu Ile His Ala Gly Gln Ala Asp Arg Asp Pro Met Pro Leu His Arg
690 695 700 Ala Ala Lys Leu Leu Ile Asn Ser Gln Leu Glu Glu Gly Asp
Trp Pro 705 710 715 720 Gln Gln Glu Ile Thr Gly Val Phe Met Lys Asn
Cys Met Leu His Tyr 725 730 735 Pro Met Tyr Arg Asp Ile Tyr Pro Met
Trp Ala Leu Ala Glu Tyr Arg 740 745 750 Arg Arg Val Pro Leu Pro Ser
Thr Glu Val 755 760 5 2766 DNA Glycine max 5 ttcatctccc acgcttcact
ttctccctcc ccctccctct ccctctccct ctccccaccc 60 cgagacctca
ccctcccctc cttctccctt tcgccaccac aacgcccaac gtccacataa 120
gctagatgag atcaatctga agcaaatggt tataatttca aaattttaag agtggaggac
180 ctgtgttgtg cacgttagag tgaatcgttc aagattaatc cttaacaacc
tgaccaccag 240 gaacaaccag ctatcatttt acattgaact agaaattcat
ttagaagatc aaagacaaaa 300
ttttccgatt aaaacgtact taaattgaag aggggttgtt ggcattgtgc accaaaaagg
360 aaaaaaaatg tggaggttaa agatagcaga tggagggaat gatccctata
tatttagcac 420 aaataatttt gtggggaggc aaacatggga gtttgattct
gaggcaggta ccgctgagga 480 acgagctcaa attgaagcag ctcgtcaaaa
cttttatgaa aatcgcttca tggtcaaggc 540 ttgtggtgat cgactttggc
ggtttcagat tttgagggaa aataatttca aacaaacaat 600 aagtggcgta
aagatagaag atgatgagaa aattacatgc gagaaaatta ggagcaccat 660
gaagagggcc actcattacc tatcgtcact acagactagt gatggtcatt ggcctgctca
720 tcttggaggt tccctctttt ttactccacc gttggtcatt tgtttatata
ttacaggaca 780 tattgattct atattttcag aagagtatcg taaagagatt
cttcgttaca tatattacca 840 ccagaacaaa gatggaggtt ggggactaca
catagaaggt cacagtatca tgttttgcac 900 tacactcaat tatatatgca
tgcgaattct tggagaagga cctaatggag gtcataacaa 960 tgcttgtgct
aaagcaagaa agtggattca tgatcatggt ggtgcaacac atataccttc 1020
atgggggaaa ttttggcttt cggtacttgg tatagttgat tggtgtggaa gcaacccaat
1080 gccgcctgaa ttttggatcc ttccttcttt tctccctatg catccgggta
aaatgtggtg 1140 ttattgtcgg ttggtataca tgcccatgtc ttatttgtat
gggaagaaat ttacgggtcc 1200 aatcacaccg ttagttgtaa atttgagaga
agaacttttt attcaacctt atgatgaaaa 1260 tagttggaag aaagcacgtc
ataaatgtgc aaatgaagat ctttactatc cccatcattg 1320 gatacaagat
ctattatggg atagtttgta tgtattcacc gagcctcttc taaattgttg 1380
gcctttcaac aagttggtta gagaaaaggc acttcaagta acaatgaaac atattcatta
1440 tgaagacgaa aatagtcggt atattgccat cgggtgtgtg gaaaaggttc
tatgtatgct 1500 tgcttgttgg gttgaagatc caaatggaga tgctttcaag
aagcatcttg caaggatccc 1560 agattattta tgggtttctg aagatggaat
gaccatgcag ggtattggta ctcaatcatg 1620 ggatgttggt ttcattgttc
aagctttact tgctactaac cttatagatg attttggacc 1680 tacaattgca
aaagctcacg atttcatcaa gaaatctcag gtaagagaaa atccttcggg 1740
agattttaag agtatgtatc gtcacatttg taaaggctca tggacccttg ccgatagaga
1800 tcatgcatgg caagtttctg ataccactgc agaatgtttg aagtgttgtc
tacttttatc 1860 agtgctgcca caagatattg tgggagaaaa aatggaactt
gaaaagttac atgattcaat 1920 caatttgata ctgtcacttc agagtaaaaa
tggaggtatg actgcgtggg agcccgcagg 1980 agcttataaa tggttggaac
tactcaatcc tacggaattt tttgctgaca tagtagttga 2040 gcacgaatat
cttgaatgca ctgcatcagc aattcaggtt ttagtgttgt tcaaaaagct 2100
ttaccctgag catagaaagg aagagataga gaacttcatt gctaaagcag taacattcat
2160 tgaagataca caattagaga atggttcttg gtatgggaat tgggcagttt
gtttcactta 2220 cagctcttgg tttgcacttg gaggtctagt tgctgctggc
aagacttaca caaattgtgt 2280 tactattcgt aaagctgtga aatttctact
caaaatacaa aataaggacg gtgggtgggg 2340 agagagttat ctttcttgcc
caaggaagat gtacgtacct cttgaaggaa gtcgatcaaa 2400 tgttgtacaa
acatcatggg ctctaatggc tctaattcat gctgagcagg ctgagagaga 2460
tccaactccc cttcatcatg cagcaaagtt actcattaat tctcagttag aagatggcga
2520 ttggccccaa caagaaactc ttggagtata cttgagaaat tgcttggttc
attactcatt 2580 ctatagaaat atttttccaa tgtgggcttt ggctgaatac
cgcacaaatg ttttattgcc 2640 ttcctttact atttaagttg aaaaattgtg
agctcaaaaa gataatgtca taccaataaa 2700 agtctagaaa aaaaaaagtt
ggtaatgaag tttaataggc ttattcataa aaaaaaaaaa 2760 aaaaaa 2766 6 762
PRT Glycine max 6 Met Trp Arg Leu Lys Ile Ala Asp Gly Gly Asn Asp
Pro Tyr Ile Phe 1 5 10 15 Ser Thr Asn Asn Phe Val Gly Arg Gln Thr
Trp Glu Phe Asp Ser Glu 20 25 30 Ala Gly Thr Ala Glu Glu Arg Ala
Gln Ile Glu Ala Ala Arg Gln Asn 35 40 45 Phe Tyr Glu Asn Arg Phe
Met Val Lys Ala Cys Gly Asp Arg Leu Trp 50 55 60 Arg Phe Gln Ile
Leu Arg Glu Asn Asn Phe Lys Gln Thr Ile Ser Gly 65 70 75 80 Val Lys
Ile Glu Asp Asp Glu Lys Ile Thr Cys Glu Lys Ile Arg Ser 85 90 95
Thr Met Lys Arg Ala Thr His Tyr Leu Ser Ser Leu Gln Thr Ser Asp 100
105 110 Gly His Trp Pro Ala His Leu Gly Gly Ser Leu Phe Phe Thr Pro
Pro 115 120 125 Leu Val Ile Cys Leu Tyr Ile Thr Gly His Ile Asp Ser
Ile Phe Ser 130 135 140 Glu Glu Tyr Arg Lys Glu Ile Leu Arg Tyr Ile
Tyr Tyr His Gln Asn 145 150 155 160 Lys Asp Gly Gly Trp Gly Leu His
Ile Glu Gly His Ser Ile Met Phe 165 170 175 Cys Thr Thr Leu Asn Tyr
Ile Cys Met Arg Ile Leu Gly Glu Gly Pro 180 185 190 Asn Gly Gly His
Asn Asn Ala Cys Ala Lys Ala Arg Lys Trp Ile His 195 200 205 Asp His
Gly Gly Ala Thr His Ile Pro Ser Trp Gly Lys Phe Trp Leu 210 215 220
Ser Val Leu Gly Ile Val Asp Trp Cys Gly Ser Asn Pro Met Pro Pro 225
230 235 240 Glu Phe Trp Ile Leu Pro Ser Phe Leu Pro Met His Pro Gly
Lys Met 245 250 255 Trp Cys Tyr Cys Arg Leu Val Tyr Met Pro Met Ser
Tyr Leu Tyr Gly 260 265 270 Lys Lys Phe Thr Gly Pro Ile Thr Pro Leu
Val Val Asn Leu Arg Glu 275 280 285 Glu Leu Phe Ile Gln Pro Tyr Asp
Glu Asn Ser Trp Lys Lys Ala Arg 290 295 300 His Lys Cys Ala Asn Glu
Asp Leu Tyr Tyr Pro His His Trp Ile Gln 305 310 315 320 Asp Leu Leu
Trp Asp Ser Leu Tyr Val Phe Thr Glu Pro Leu Leu Asn 325 330 335 Cys
Trp Pro Phe Asn Lys Leu Val Arg Glu Lys Ala Leu Gln Val Thr 340 345
350 Met Lys His Ile His Tyr Glu Asp Glu Asn Ser Arg Tyr Ile Ala Ile
355 360 365 Gly Cys Val Glu Lys Val Leu Cys Met Leu Ala Cys Trp Val
Glu Asp 370 375 380 Pro Asn Gly Asp Ala Phe Lys Lys His Leu Ala Arg
Ile Pro Asp Tyr 385 390 395 400 Leu Trp Val Ser Glu Asp Gly Met Thr
Met Gln Gly Ile Gly Thr Gln 405 410 415 Ser Trp Asp Val Gly Phe Ile
Val Gln Ala Leu Leu Ala Thr Asn Leu 420 425 430 Ile Asp Asp Phe Gly
Pro Thr Ile Ala Lys Ala His Asp Phe Ile Lys 435 440 445 Lys Ser Gln
Val Arg Glu Asn Pro Ser Gly Asp Phe Lys Ser Met Tyr 450 455 460 Arg
His Ile Cys Lys Gly Ser Trp Thr Leu Ala Asp Arg Asp His Ala 465 470
475 480 Trp Gln Val Ser Asp Thr Thr Ala Glu Cys Leu Lys Cys Cys Leu
Leu 485 490 495 Leu Ser Val Leu Pro Gln Asp Ile Val Gly Glu Lys Met
Glu Leu Glu 500 505 510 Lys Leu His Asp Ser Ile Asn Leu Ile Leu Ser
Leu Gln Ser Lys Asn 515 520 525 Gly Gly Met Thr Ala Trp Glu Pro Ala
Gly Ala Tyr Lys Trp Leu Glu 530 535 540 Leu Leu Asn Pro Thr Glu Phe
Phe Ala Asp Ile Val Val Glu His Glu 545 550 555 560 Tyr Leu Glu Cys
Thr Ala Ser Ala Ile Gln Val Leu Val Leu Phe Lys 565 570 575 Lys Leu
Tyr Pro Glu His Arg Lys Glu Glu Ile Glu Asn Phe Ile Ala 580 585 590
Lys Ala Val Thr Phe Ile Glu Asp Thr Gln Leu Glu Asn Gly Ser Trp 595
600 605 Tyr Gly Asn Trp Ala Val Cys Phe Thr Tyr Ser Ser Trp Phe Ala
Leu 610 615 620 Gly Gly Leu Val Ala Ala Gly Lys Thr Tyr Thr Asn Cys
Val Thr Ile 625 630 635 640 Arg Lys Ala Val Lys Phe Leu Leu Lys Ile
Gln Asn Lys Asp Gly Gly 645 650 655 Trp Gly Glu Ser Tyr Leu Ser Cys
Pro Arg Lys Met Tyr Val Pro Leu 660 665 670 Glu Gly Ser Arg Ser Asn
Val Val Gln Thr Ser Trp Ala Leu Met ala 675 680 685 Leu Ile His Ala
Glu Gln Ala Glu Arg Asp Pro Thr Pro Leu His His 690 695 700 Ala Ala
Lys Leu Leu Ile Asn Ser Gln Leu Glu Asp Gly Asp Trp Pro 705 710 715
720 Gln Gln Glu Thr Leu Gly Val Tyr Leu Arg Asn Cys Leu Val His Tyr
725 730 735 Ser Phe Tyr Arg Asn Ile Phe Pro Met Trp Ala Leu Ala Glu
Tyr Arg 740 745 750 Thr Asn Val Leu Leu Pro Ser Phe Thr Ile 755 760
7 2538 DNA Triticum aestivum 7 gtttggggtc agcgatagtg atcgggcatt
tcgtgagcta gctgagctaa ccacctgttc 60 agagagagat gtggaggctc
aaggtgtccg agggtggcgg cccgtggctg cggtcggtga 120 acaacttcct
cggcagggca gtgtgggagt tcgaccccga ctacggcaca ccggaggagc 180
gcgccgaggt gaagagggtg cgccgggagt tcaccgaccg ccgtttcgag aaaaaggagt
240 cgcaggatct tctcatgcgc atgcagtatg caaaagaaaa gcatcttcag
gtggaccttc 300 cagccatcaa gcttgcagac agtgcacaag tcacagaaga
gactttacta acatcattga 360 ggcgatgcct tagccaacat tctgctctac
aagcacacga tgggcattgg gctggggact 420 tcagtggaat tttgttcatt
atgcccatct tgatatttgc tctacatgtt actggatcac 480 tcaatactgt
cctatcaaca gaacatcgat gtgagatttg tcgctatatt tacaaccatc 540
agaatgaaga tggtggttgg ggcacgcaag tgttgggtcc gagcaccatg tttggatcat
600 gcttaaacta tgttacctta aggcttcttg gcgaggtgga aaatgatgcc
ttaaccaagg 660 gacgtgcttg gattctattg cgtggaagtg caactgcaat
accacaatgg ggaaagatat 720 ggctctcggt ggttggttta tatgaatggt
ctggaaataa ttcgatcatt cctgagttat 780 ggcttgtccc gtattttctt
ccgattcatc caggacgatt ctggtgcttt tgccggttgg 840 tttatatgcc
aatgtcttat ctttacggca aaaagtttgt tggcccaatt acaccaacaa 900
tagtggcaat aagagaggag ctctatagtg tatcatacag cgagattgat tggaacaaag
960 cacgtgatac ttgtgctaag gaagaccttc gctatccacg gtcgttgctg
caaaatgtta 1020 tttggacttg ccttaataaa tttgtagaac cagtgttgaa
ttgttggcca atcaataagt 1080 tgagagatac agcgctgaag aacctcatga
aacatataca ttatgaagac gaaagcacta 1140 aatacattgg cgtatgtccg
attaacaagg cactagatat gatttgttgt tggagcgagg 1200 atccaaattc
agatgcactg aagttgcatc ttccaaggat ctatgactat ttatggcttg 1260
cagaagatgg catgaaagca caggtttatg atggttgtca aagctgggag cttgctttta
1320 ttgttcaagc atattgctcg acagaccttg ttaatgagtt tggtccaaca
cttcggaaag 1380 cccatgagtt cattaaaagt tcacaggttc ttgagaacca
tcctaacagt gaaacttatt 1440 accgccatag gtcaaaaggt tcatggacac
tttcaacagc ggataatggg tggtctgtat 1500 cagattgtac tgcagaagca
cttaaggcat tgttgttgtt gtcgaagatc tctcctaatc 1560 ttgtggggga
tcccgtaaaa ggagaaaggt tgcatgatgc agtcgattgc ttactttctt 1620
ttatgaataa agatggcaca ttttctacat atgagtgtaa gagaactaca tctctattag
1680 aggttctcaa cccttctgaa agttttctga acattattgt cgactatcca
tctgtcgaat 1740 gtacatcatc tgtgcttcag gccctaatta tgttcaaaga
gctttaccct gggtaccgca 1800 aagaagagat aggaaaatgt attaaaaatg
cttccaagtt cattgaggac aagcaacgaa 1860 aggatggctc atggtttggc
acttggggta tatgtttcac ttatgggacg ttctttggtg 1920 taaaaggatt
gattgcttct ggaagaactt acgaaaatag ttcttccata aggaaagcat 1980
gcaattttct gttgtcaaag caactaagta caggtggatg gggagagtct tatctttcta
2040 gtgaaactga ggcttatgtg gaggccacta gtcctcatgc agtgaacact
gcttgggcaa 2100 tgttggcttt aatttatgct gggcaggttg aacgagatcc
tactccacta tatcatgctg 2160 caaaagagtt gatcaatatg caactagaga
caggagagtt tccccagcaa gaacacgttg 2220 gatgcttcaa ctgctccata
tactttaatt acggcaacta tcgcaactta taccccattt 2280 gggctcttgg
ggagtttcgt cgtcgactgc ttgcgaagaa ctgaaactga tgacgatgat 2340
atgtcgcttc actgctctta ggtttaggtg tggtcgtgcc tgtgacgaaa aggatgacct
2400 tagccaaact atattatata tgtgtgtgta acacatactg caataacact
tacaaccaaa 2460 gtgactaatg caaacataat agcgcctgtt tggtttgtaa
aaaaaaaaaa aaaaaaaaaa 2520 aaaaaaaaaa aaaaaaaa 2538 8 751 PRT
Triticum aestivum 8 Met Trp Arg Leu Lys Val Ser Glu Gly Gly Gly Pro
Trp Leu Arg Ser 1 5 10 15 Val Asn Asn Phe Leu Gly Arg Ala Val Trp
Glu Phe Asp Pro Asp Tyr 20 25 30 Gly Thr Pro Glu Glu Arg Ala Glu
Val Lys Arg Val Arg Arg Glu Phe 35 40 45 Thr Asp Arg Arg Phe Glu
Lys Lys Glu Ser Gln Asp Leu Leu Met Arg 50 55 60 Met Gln Tyr Ala
Lys Glu Lys His Leu Gln Val Asp Leu Pro Ala Ile 65 70 75 80 Lys Leu
Ala Asp Ser Ala Gln Val Thr Glu Glu Thr Leu Leu Thr Ser 85 90 95
Leu Arg Arg Cys Leu Ser Gln His Ser Ala Leu Gln Ala His Asp Gly 100
105 110 His Trp Ala Gly Asp Phe Ser Gly Ile Leu Phe Ile Met Pro Ile
Leu 115 120 125 Ile Phe Ala Leu His Val Thr Gly Ser Leu Asn Thr Val
Leu Ser Thr 130 135 140 Glu His Arg Cys Glu Ile Cys Arg Tyr Ile Tyr
Asn His Gln Asn Glu 145 150 155 160 Asp Gly Gly Trp Gly Thr Gln Val
Leu Gly Pro Ser Thr Met Phe Gly 165 170 175 Ser Cys Leu Asn Tyr Val
Thr Leu Arg Leu Leu Gly Glu Val Glu Asn 180 185 190 Asp Ala Leu Thr
Lys Gly Arg Ala Trp Ile Leu Leu Arg Gly Ser Ala 195 200 205 Thr Ala
Ile Pro Gln Trp Gly Lys Ile Trp Leu Ser Val Val Gly Leu 210 215 220
Tyr Glu Trp Ser Gly Asn Asn Ser Ile Ile Pro Glu Leu Trp Leu Val 225
230 235 240 Pro Tyr Phe Leu Pro Ile His Pro Gly Arg Phe Trp Cys Phe
Cys Arg 245 250 255 Leu Val Tyr Met Pro Met Ser Tyr Leu Tyr Gly Lys
Lys Phe Val Gly 260 265 270 Pro Ile Thr Pro Thr Ile Val Ala Ile Arg
Glu Glu Leu Tyr Ser Val 275 280 285 Ser Tyr Ser Glu Ile Asp Trp Asn
Lys Ala Arg Asp Thr Cys Ala Lys 290 295 300 Glu Asp Leu Arg Tyr Pro
Arg Ser Leu Leu Gln Asn Val Ile Trp Thr 305 310 315 320 Cys Leu Asn
Lys Phe Val Glu Pro Val Leu Asn Cys Trp Pro Ile Asn 325 330 335 Lys
Leu Arg Asp Thr Ala Leu Lys Asn Leu Met Lys His Ile His Tyr 340 345
350 Glu Asp Glu Ser Thr Lys Tyr Ile Gly Val Cys Pro Ile Asn Lys Ala
355 360 365 Leu Asp Met Ile Cys Cys Trp Ser Glu Asp Pro Asn Ser Asp
Ala Leu 370 375 380 Lys Leu His Leu Pro Arg Ile Tyr Asp Tyr Leu Trp
Leu Ala Glu Asp 385 390 395 400 Gly Met Lys Ala Gln Val Tyr Asp Gly
Cys Gln Ser Trp Glu Leu Ala 405 410 415 Phe Ile Val Gln Ala Tyr Cys
Ser Thr Asp Leu Val Asn Glu Phe Gly 420 425 430 Pro Thr Leu Arg Lys
Ala His Glu Phe Ile Lys Ser Ser Gln Val Leu 435 440 445 Glu Asn His
Pro Asn Ser Glu Thr Tyr Tyr Arg His Arg Ser Lys Gly 450 455 460 Ser
Trp Thr Leu Ser Thr Ala Asp Asn Gly Trp Ser Val Ser Asp Cys 465 470
475 480 Thr Ala Glu Ala Leu Lys Ala Leu Leu Leu Leu Ser Lys Ile Ser
Pro 485 490 495 Asn Leu Val Gly Asp Pro Val Lys Gly Glu Arg Leu His
Asp Ala Val 500 505 510 Asp Cys Leu Leu Ser Phe Met Asn Lys Asp Gly
Thr Phe Ser Thr Tyr 515 520 525 Glu Cys Lys Arg Thr Thr Ser Leu Leu
Glu Val Leu Asn Pro Ser Glu 530 535 540 Ser Phe Leu Asn Ile Ile Val
Asp Tyr Pro Ser Val Glu Cys Thr Ser 545 550 555 560 Ser Val Leu Gln
Ala Leu Ile Met Phe Lys Glu Leu Tyr Pro Gly Tyr 565 570 575 Arg Lys
Glu Glu Ile Gly Lys Cys Ile Lys Asn Ala Ser Lys Phe Ile 580 585 590
Glu Asp Lys Gln Arg Lys Asp Gly Ser Trp Phe Gly Thr Trp Gly Ile 595
600 605 Cys Phe Thr Tyr Gly Thr Phe Phe Gly Val Lys Gly Leu Ile Ala
Ser 610 615 620 Gly Arg Thr Tyr Glu Asn Ser Ser Ser Ile Arg Lys Ala
Cys Asn Phe 625 630 635 640 Leu Leu Ser Lys Gln Leu Ser Thr Gly Gly
Trp Gly Glu Ser Tyr Leu 645 650 655 Ser Ser Glu Thr Glu Ala Tyr Val
Glu Ala Thr Ser Pro His Ala Val 660 665 670 Asn Thr Ala Trp Ala Met
Leu Ala Leu Ile Tyr Ala Gly Gln Val Glu 675 680 685 Arg Asp Pro Thr
Pro Leu Tyr His Ala Ala Lys Glu Leu Ile Asn Met 690 695 700 Gln Leu
Glu Thr Gly Glu Phe Pro Gln Gln Glu His Val Gly Cys Phe 705 710 715
720 Asn Cys Ser Ile Tyr Phe Asn Tyr Gly Asn Tyr Arg Asn Leu Tyr Pro
725 730 735 Ile Trp Ala Leu Gly Glu Phe Arg Arg Arg Leu Leu Ala Lys
Asn 740 745 750 9 46 DNA Artificial Sequence Description of
Artificial SequencePCR primer 9 tcaaggagaa aaaaccccgg atccatgtgg
aggctgaaga tagcag 46 10 46 DNA Artificial Sequence Description of
Artificial SequencePCR primer 10 tcaaggagaa aaaaccccgg atccatgtgg
aggttaaaga tagcag 46 11 35 DNA Artificial Sequence Description of
Artificial SequencePCR primer 11 ggccagtgaa ttgtaatacg actcactata
gggcg 35 12 761 PRT Panax ginseng 12 Met Trp Arg Leu Met Thr Ala
Lys Gly Gly Asn Asp Pro Tyr Leu Tyr 1 5 10 15 Ser Thr Asn Asn Phe
Ile Gly Arg Gln Thr Trp Glu Phe Asp Pro Asp
20 25 30 Tyr Gly Thr Pro Ala Glu Arg Ala Glu Val Glu Glu Ala Arg
Leu His 35 40 45 Phe Trp Asn Asn Arg Tyr Gln Val Lys Pro Ser Ser
Asp Val Leu Trp 50 55 60 Arg Met Gln Phe Leu Lys Glu Lys Asn Phe
Lys Gln Ile Ile Pro Gln 65 70 75 80 Val Lys Val Glu Asp Gly Glu Glu
Ile Thr Tyr Glu Ala Ala Thr Thr 85 90 95 Thr Leu Arg Arg Ala Val
His Tyr Phe Ser Ala Leu Gln Ala Asp Asp 100 105 110 Gly His Trp Pro
Ala Glu Asn Ala Gly Pro Leu Phe Phe Leu Pro Pro 115 120 125 Leu Val
Met Cys Leu Tyr Ile Thr Gly His Leu Asn Thr Val Phe Pro 130 135 140
Ala Glu His Arg Ile Glu Ile Leu Arg Tyr Ile Tyr Cys His Gln Asn 145
150 155 160 Asp Asp Gly Gly Trp Gly Leu His Ile Glu Gly His Ser Thr
Met Phe 165 170 175 Cys Thr Ala Leu Ser Tyr Ile Cys Met Arg Ile Leu
Gly Glu Gly Arg 180 185 190 Asp Gly Gly Glu Asn Asn Ala Cys Ala Arg
Ala Arg Lys Trp Ile Leu 195 200 205 Asp His Gly Ser Val Thr Ala Ile
Pro Ser Trp Gly Lys Thr Trp Leu 210 215 220 Ser Ile Leu Gly Leu Phe
Asp Trp Ser Gly Ser Asn Pro Met Pro Pro 225 230 235 240 Glu Phe Trp
Ile Leu Pro Pro Phe Leu Pro Met His Pro Ala Lys Met 245 250 255 Trp
Cys Tyr Cys Arg Met Val Tyr Met Pro Met Ser Tyr Leu Tyr Gly 260 265
270 Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Leu Gln Leu Arg Glu
275 280 285 Glu Leu Tyr Ala Gln Ala Tyr Asp Glu Ile Asn Trp Arg Lys
Val Arg 290 295 300 His Asn Cys Ala Lys Glu Asp Leu Tyr Tyr Pro His
Pro Leu Ile Gln 305 310 315 320 Asp Leu Met Trp Asp Ser Leu Tyr Ile
Phe Thr Glu Pro Phe Leu Thr 325 330 335 Arg Trp Pro Phe Asn Lys Leu
Arg Glu Lys Ala Leu Gln Thr Thr Met 340 345 350 Lys His Ile His Tyr
Glu Asp Glu Asn Ser Arg Tyr Ile Thr Ile Gly 355 360 365 Cys Val Glu
Lys Val Leu Cys Met Leu Ala Cys Trp Val Glu Asp Pro 370 375 380 Asn
Gly Asp Tyr Phe Lys Gln His Leu Ala Arg Ile Pro Asp Tyr Ile 385 390
395 400 Trp Val Ala Glu Asp Gly Met Lys Met Gln Ser Phe Gly Ser Gln
Glu 405 410 415 Trp Asp Thr Gly Phe Ala Ile Gln Ala Leu Leu Ala Ser
Asp Leu Ile 420 425 430 Asp Glu Ile Arg Pro Thr Leu Met Lys Gly His
Asp Phe Ile Lys Lys 435 440 445 Ser Gln Val Lys Glu Asn Pro Ser Gly
Asp Phe Lys Ser Met His Arg 450 455 460 His Ile Ser Lys Gly Ser Trp
Thr Phe Ser Asp Gln Asp His Gly Trp 465 470 475 480 Gln Val Ser Asp
Cys Thr Ala Glu Ala Leu Lys Cys Cys Leu Leu Phe 485 490 495 Ser Arg
Met Pro Thr Glu Ile Val Gly Asp Lys Met Glu Asp Asn Gln 500 505 510
Leu Phe Asp Ala Val Asn Met Leu Leu Ser Leu Gln Ser Lys Asn Gly 515
520 525 Gly Leu Ala Ala Trp Glu Pro Ala Gly Ser Ser Glu Trp Leu Glu
Leu 530 535 540 Leu Asn Pro Thr Glu Phe Phe Glu Asp Ile Val Ile Glu
His Glu Tyr 545 550 555 560 Val Glu Cys Thr Ser Ser Ala Ile Gln Ala
Met Val Met Phe Lys Lys 565 570 575 Leu Tyr Pro Gly His Arg Lys Lys
Glu Ile Glu Val Ser Ile Thr Asn 580 585 590 Ala Val Gln Tyr Leu Glu
Asp Ile Gln Met Pro Asp Gly Ser Trp Tyr 595 600 605 Gly Asn Trp Gly
Val Cys Phe Thr Tyr Gly Thr Trp Phe Ala Met Gly 610 615 620 Gly Leu
Thr Ala Ala Gly Lys Thr Tyr Asn Asn Cys Gln Thr Leu His 625 630 635
640 Lys Ala Val Asp Phe Leu Ile Lys Ser Gln Arg Ser Asp Gly Gly Trp
645 650 655 Gly Glu Ser Tyr Leu Ser Cys Pro Asn Lys Glu Tyr Thr Pro
Leu Glu 660 665 670 Gly Asn Arg Ser Asn Leu Val His Thr Ser Trp Ala
Met Met Gly Leu 675 680 685 Ile His Ser Arg Gln Ala Glu Arg Asp Pro
Thr Pro Leu His Arg Ala 690 695 700 Ala Lys Leu Leu Ile Asn Ser Gln
Met Glu Ser Gly Asp Phe Pro Gln 705 710 715 720 Gln Glu Ile Thr Gly
Val Phe Met Lys Asn Cys Met Leu His Tyr Ala 725 730 735 Ala Ser Arg
Asn Ile Tyr Pro Leu Trp Ala Leu Ala Glu Tyr Arg Lys 740 745 750 Asn
Val Arg Leu Pro Ser Lys Ser Val 755 760 13 763 PRT Panax ginseng 13
Met Trp Lys Leu Lys Ile Ala Glu Gly Asn Lys Asn Asp Pro Tyr Leu 1 5
10 15 Tyr Ser Thr Asn Asn Phe Val Gly Arg Gln Thr Trp Glu Phe Asp
Pro 20 25 30 Asp Tyr Val Ala Ser Pro Gly Glu Leu Glu Glu Val Glu
Gln Val Arg 35 40 45 Arg Gln Phe Trp Asp Asn Arg Tyr Gln Val Lys
Pro Ser Gly Asp Leu 50 55 60 Leu Trp Arg Met Gln Phe Leu Arg Glu
Lys Asn Phe Arg Gln Thr Ile 65 70 75 80 Pro Gln Val Lys Val Gly Asp
Asp Glu Ala Val Thr Tyr Glu Ala Ala 85 90 95 Thr Thr Thr Leu Arg
Arg Ala Val His Phe Phe Ser Ala Leu Gln Ala 100 105 110 Ser Asp Gly
His Trp Pro Ala Glu Asn Ser Gly Pro Leu Phe Phe Leu 115 120 125 Pro
Pro Leu Val Met Cys Val Tyr Ile Thr Gly His Leu Asp Thr Val 130 135
140 Phe Pro Ala Glu His Arg Lys Glu Ile Leu Arg Tyr Ile Tyr Cys His
145 150 155 160 Gln Asn Glu Asp Gly Gly Trp Gly Leu His Ile Glu Gly
His Ser Thr 165 170 175 Met Phe Cys Thr Thr Leu Ser Tyr Ile Cys Met
Arg Ile Leu Gly Glu 180 185 190 Gly Pro Asp Gly Gly Val Asn Asn Ala
Cys Ala Arg Gly Arg Lys Trp 195 200 205 Ile Leu Asp His Gly Ser Val
Thr Ala Ile Pro Ser Trp Gly Lys Thr 210 215 220 Trp Leu Ser Ile Leu
Gly Val Tyr Glu Trp Ile Gly Ser Asn Pro Met 225 230 235 240 Pro Pro
Glu Phe Trp Ile Leu Pro Ser Phe Leu Pro Met His Pro Ala 245 250 255
Lys Met Trp Cys Tyr Cys Arg Met Val Tyr Met Pro Met Ser Tyr Leu 260
265 270 Tyr Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Leu Gln
Leu 275 280 285 Arg Glu Glu Leu Tyr Gly Gln Pro Tyr Asn Glu Ile Asn
Trp Arg Lys 290 295 300 Thr Arg Arg Val Cys Ala Lys Glu Asp Ile Tyr
Tyr Pro His Pro Leu 305 310 315 320 Ile Gln Asp Leu Leu Trp Asp Ser
Leu Tyr Val Leu Thr Glu Pro Leu 325 330 335 Leu Thr Arg Trp Pro Phe
Asn Lys Leu Arg Glu Lys Ala Leu Gln Thr 340 345 350 Thr Met Lys His
Ile His Tyr Glu Asp Glu Asn Ser Arg Tyr Ile Thr 355 360 365 Ile Gly
Cys Val Glu Lys Val Leu Cys Met Leu Val Cys Trp Val Glu 370 375 380
Asp Pro Asn Gly Asp Tyr Phe Arg Lys His Leu Ala Arg Ile Pro Asp 385
390 395 400 Tyr Ile Trp Val Ala Glu Asp Gly Met Lys Met Gln Ser Phe
Gly Ser 405 410 415 Gln Glu Trp Asp Thr Gly Phe Ser Ile Gln Ala Leu
Leu Asp Ser Asp 420 425 430 Leu Thr His Glu Ile Gly Pro Thr Leu Met
Lys Gly His Asp Phe Ile 435 440 445 Lys Lys Ser Gln Val Lys Asp Asn
Pro Ser Gly Asp Phe Lys Ser Met 450 455 460 Tyr Arg His Ile Ser Lys
Gly Ser Trp Thr Phe Ser Asp Gln Asp His 465 470 475 480 Gly Trp Gln
Val Ser Asp Cys Thr Ala Glu Gly Leu Lys Cys Cys Leu 485 490 495 Ile
Phe Ser Thr Met Pro Glu Glu Ile Val Gly Lys Lys Ile Lys Pro 500 505
510 Glu Arg Leu Tyr Asp Ser Val Asn Val Leu Leu Ser Leu Gln Arg Lys
515 520 525 Asn Gly Gly Leu Ser Ala Trp Glu Pro Ala Gly Ala Gln Glu
Trp Leu 530 535 540 Glu Leu Leu Asn Pro Thr Glu Phe Phe Ala Asp Ile
Val Ile Glu His 545 550 555 560 Glu Tyr Val Glu Cys Thr Ser Ser Ala
Ile Gln Ala Leu Val Leu Phe 565 570 575 Lys Lys Leu Tyr Pro Gly His
Arg Lys Lys Glu Ile Asp Asn Phe Ile 580 585 590 Thr Asn Ala Val Arg
Tyr Leu Glu Asp Thr Gln Met Pro Asp Gly Ser 595 600 605 Trp Tyr Gly
Asn Trp Gly Val Cys Phe Thr Tyr Gly Ser Trp Phe Ala 610 615 620 Leu
Gly Gly Leu Ala Ala Ala Gly Lys Thr Tyr Tyr Asn Cys Ala Ala 625 630
635 640 Val Arg Lys Ala Val Glu Phe Leu Leu Lys Ser Gln Met Asp Asp
Gly 645 650 655 Gly Trp Gly Glu Ser Tyr Leu Ser Cys Pro Lys Lys Val
Tyr Val Pro 660 665 670 Leu Glu Gly Asn Arg Ser Asn Leu Val His Thr
Gly Trp Ala Leu Met 675 680 685 Gly Leu Ile His Ser Glu Gln Ala Glu
Arg Asp Pro Thr Pro Leu His 690 695 700 Arg Ala Ala Lys Leu Leu Ile
Asn Ser Gln Met Glu Asp Gly Asp Phe 705 710 715 720 Pro Gln Gln Glu
Ile Ser Gly Val Phe Met Lys Asn Cys Met Leu His 725 730 735 Tyr Ala
Ala Tyr Arg Asn Ile Tyr Pro Leu Trp Ala Leu Ala Glu Tyr 740 745 750
Arg Arg Arg Val Pro Leu Pro Ser Leu Gly Thr 755 760
* * * * *
References