U.S. patent application number 14/843038 was filed with the patent office on 2015-12-17 for use of dimerization domain component stacks to modulate plant architecture.
The applicant listed for this patent is PIONEER HI BRED INTERNATIONAL INC. Invention is credited to Shai Lawit, Dwight Tomes.
Application Number | 20150361442 14/843038 |
Document ID | / |
Family ID | 42668195 |
Filed Date | 2015-12-17 |
United States Patent
Application |
20150361442 |
Kind Code |
A1 |
Lawit; Shai ; et
al. |
December 17, 2015 |
USE OF DIMERIZATION DOMAIN COMPONENT STACKS TO MODULATE PLANT
ARCHITECTURE
Abstract
This invention provides means for altering the harvest index of
crop plants by modulating the expression of transgenic genes using
dimerization domain and component stacks, thereby modulating plant
architecture. The transgene/dimerization domain stacks are provided
in a single transformation vector unit and are used to modulate
plant growth, yield, and harvest index in plants.
Inventors: |
Lawit; Shai; (Urbandale,
IA) ; Tomes; Dwight; (Grimes, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIONEER HI BRED INTERNATIONAL INC |
Johnston |
IA |
US |
|
|
Family ID: |
42668195 |
Appl. No.: |
14/843038 |
Filed: |
September 2, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12837553 |
Jul 16, 2010 |
9175301 |
|
|
14843038 |
|
|
|
|
61286061 |
Dec 14, 2009 |
|
|
|
61228195 |
Jul 24, 2009 |
|
|
|
Current U.S.
Class: |
800/287 ;
435/161; 435/252.1; 435/252.31; 435/252.33; 435/254.2; 435/254.21;
435/254.23; 435/320.1; 435/325; 435/348; 435/352; 435/358; 435/369;
435/419; 536/23.6; 568/840; 800/290; 800/298; 800/306; 800/312;
800/314; 800/320; 800/320.1; 800/320.2; 800/320.3; 800/322 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8255 20130101; C12N 15/8261 20130101; C12N 15/827 20130101;
C12N 15/8297 20130101; C07C 31/08 20130101; C12N 15/8242 20130101;
C12P 7/06 20130101; C12N 15/8269 20130101; Y02A 40/146 20180101;
C12N 15/8293 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415; C07C 31/08 20060101
C07C031/08; C12P 7/06 20060101 C12P007/06 |
Claims
1. An isolated nucleic acid encoding a dimerization domain, the
dimerization domain comprising a consensus amino acid sequence of
SEQ ID NO: 41 or a sequence that is 90% identical to SEQ ID NO:
41.
2. The nucleic acid of claim 1 encoding a dimerization domain, the
dimerization domain consisting essentially of the amino acid
sequence of SEQ ID NO: 19 or 21 or a sequence that is 90% identical
to SEQ ID NO: 19 or 21.
3. The isolated nucleic acid of claim 1 comprising a polynucleotide
sequence of SEQ ID NO: 9.
4. The isolated nucleic acid of claim 1, wherein the dimerization
domain binds to a native maize D8 protein or D9 protein to produce
a nonfunctional D8 or D9 dimer.
5. A recombinant expression cassette, comprising the polynucleotide
of claim 3, wherein the polynucleotide is operably linked to a
promoter.
6. A host cell comprising the expression cassette of claim 5.
7. A transgenic plant comprising the recombinant expression
cassette of claim 5.
8. The transgenic plant of claim 7, wherein said plant is a
monocot.
9. The transgenic plant of claim 7, wherein said plant is a
dicot.
10. The transgenic plant of claim 7, wherein said plant is selected
from the group consisting of: maize, soybean, sunflower, sorghum,
canola, wheat, alfalfa, cotton, rice, barley, millet, peanut, sugar
cane, grass, turfgrass and cocoa.
11. A transgenic seed from the transgenic plant of claim 7.
12. A method of modulating harvest index in a transgenic plant, the
method comprising expressing a recombinant polynucleotide encoding
a dimerization domain of a dwarf gene.
13. The method of claim 12, wherein the dwarf gene is D8 from
maize.
14. The method of claim 12, wherein the polynucleotide comprises
the nucleic acid sequence of claim 3 operably linked to a
promoter.
15. The method of claim 12, wherein the plant is selected from the
group consisting of: maize, soybean, sunflower, sorghum, canola,
wheat, alfalfa, cotton, rice, barley, millet, peanut, sugar cane,
grass, turfgrass and cocoa.
16. The method of claim 12, wherein the dimerization domain forms a
non-functional dimer of endogenous maize D8 or D9 protein.
17. A method of modulating plant tissue growth with a dimerization
domain in a plant, comprising expressing a recombinant expression
cassette comprising the polynucleotide of claim 3 operably linked
to a promoter.
18. The method of claim 17, wherein plant tissue growth is due to
reduced inhibition by endogenous gibberellic acid.
19. The method of claim 17, wherein the plant is selected from the
group consisting of: maize, soybean, sorghum, canola, wheat,
alfalfa, cotton, rice, barley, millet, peanut, sugar cane, grass,
turfgrass and cocoa.
20. A product derived from the method of processing of transgenic
plant component expressing an isolated polynucleotide encoding a
dimerization domain, the method comprising: a. growing a plant that
expresses a polynucleotide having at least 90% sequence identity to
the full length sequence of SEQ ID NO: 9, operably linked to a
promoter; and b. processing the plant component to obtain a
product.
21. The product of claim 20, wherein the plant component is a
seed.
22. A product according to claim 20, wherein the polynucleotide
further encodes a polypeptide selected of SEQ ID NO: 19.
23. A product according to claim 20, which is a constituent of
ethanol.
24. The method of claim 12, wherein the plant has improved canopy
shape.
25. The method of claim 12, wherein the plant has increased
photosynthetic capacity in leaf tissue.
26. The method of claim 12, wherein the plant has improved stalk
strength.
27. The method of claim 12, wherein the plant has improved plant
standibility.
28. The method of claim 12, wherein the plant has altered vascular
bundle structure or number.
29. The method of claim 12, wherein the plant has increased root
biomass.
30. The method of claim 12, wherein the plant has enhanced root
growth.
31. The method of claim 12, wherein the plant has modulated shoot
development.
32. The method of claim 12, wherein the plant has modulated leaf
development.
33. The method of claim 12, wherein the plant has improved silage
quality and digestibility.
34. The method of claim 14, wherein the promoter is selected from
the group consisting of a leaf specific promoter, vascular element
preferred promoter and a root specific promoter.
Description
CROSS REFERENCE
[0001] This utility application is a continuation of, and claims
the benefit of co-pending U.S. non provisional application Ser. No.
12/837,553, filed 16 Jul. 2010, and further claims the benefit U.S.
Provisional Application Ser. Nos. 61/228,195 and 61/286,061, filed
Jul. 24, 2009 and Dec. 14, 2009 respectively, which are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to the field of molecular
biology.
BACKGROUND OF THE INVENTION
[0003] Harvest index, ratio of grain to total above ground biomass,
has remained nearly constant around 50% in maize over the past 100
years (Sinclair, (1998) Crop Science 38:638-643; Tollenaar and Wu,
(1999) "Crop Science 39:1597-1604). Thus, the quadrupling of grain
yield over the last 50-60 years has resulted from an increase in
total biomass production per unit land area, which has been
accomplished by increased planting density (Duvick and Cassman,
(1999) Crop Science 39:1622-1630). Selection for higher grain yield
under increasing planting densities has led to a significant
architectural change in plant structure that of relatively erect
and narrow leaves to minimize shading. An undesirable consequence
of higher density planting (or higher plant populations) has been
the increased frequency of stalk and root lodging. The relationship
between planting density and biomass production deviates
significantly from linearity as the optimal density is approached
for maximal biomass yield per unit land area. This is reflected in
a proportionately greater reduction in the individual plant
biomass, which manifests in the form of weaker stalks and hence
increased lodging. In addition, approximately 20% of total biomass
at maturity stays in the form of roots in the soil, contributing to
its organic matter content (Amos and Walters, 2006). Since both
stalk and root lodging are agronomic characteristics affecting
harvest index, dwarf type plants could have potential advantages in
yield stability.
[0004] Dwarf plants have had a major impact on agriculture. Dwarf
varieties of wheat (and other small grain cereals) are widely used
in North America due to both reduced potential for lodging and
response to more intensive management and yield stability and
potentially higher yields. There are other benefits that may be
realized from the higher harvest index of dwarf crop plants
including reductions in the amounts of pesticides and fertilizers
required, higher planting densities and reduced labor costs. Dwarf
plants provide ease in harvesting, simplified management of crops
and potential reductions in water and nutrient use.
[0005] In view of the current trends of both increasing human
population and the decreasing land area suitable for agriculture,
increasing agricultural productivity is, and will continue to be, a
challenge of paramount importance. Dwarf crop plants are important
components of our agricultural production system. Increased usage
of dwarf crop plants may help to meet the agricultural production
demands of the future.
[0006] Genes that increase stalk strength, i.e., Cellulose
Synthase, are responsible for cellulose production in crop plants,
can be modified to increase size and strength of various plant
tissues. Cellulose in a unit length of the maize stalk was found to
be the best indicator of mechanical strength (Appenzeller, et al.,
(2004) Cellulose 11:287-299; Ching, et al., (2006)). Increasing
cellulose concentration in the stalk dry matter could lead to
improving stalk mechanical strength and increasing biomass which in
turn increases yield and potentially harvest index. Improvements in
plant strength (biomass) and growth of specific plant tissues
(organs) provides plants with greater biomass and increased harvest
index.
[0007] Flowering time determines maturity, an important agronomic
trait. Genes that control the transition from vegetative to
reproductive growth are essential for manipulation of flowering
time. In maize, flowering genes provide opportunities for enhanced
crop yield, adaptation of germplasm to different climatic zones and
synchronous flowering for hybrid seed production. The development
of inbred lines having modified flowering facilitates the movement
of elite germplasm across maturity zones. In addition, additional
opportunities exist to increase the rate of grain fill and/or grain
dry down to complement changes in the onset of flowering.
[0008] The combined controlled expression of plant architecture
genes, flowering time genes and dwarfing gene components within
transformed plants would not only increase the yield potential and
harvest index of crop plants but would also improve the agronomic
characteristics that simplify management practices and increase the
adaptation of crop species into new geographic areas.
[0009] This invention provides means for altering the harvest index
of crop plants by modulating the expression of transgenes using
multiple stacked plant genes and dwarf gene components, thereby
modulating plant architecture. A component of Dwarf gene D8, the
dimerization domain (DD), a leucine-zipper dimerization domain (SEQ
ID NO: 9) is overexpressed as a dominant negative transgene. The
transgene/dimerization domain component stacks are provided in a
single transformation vector unit and are used to modulate specific
plant organs of a plant that can increase growth, yield and harvest
index in plants. The expression in specific plant tissues, such as
roots, ears or tassels can lead to elongation of the specific plant
organs.
[0010] These stacked units could be used to enhance crop plant
performance and value in several areas including: 1) plant
standability (composed of stalk and root lodging), harvest index
and yield potential; 2) modification of specific plant organ size;
3) plant dry matter as a feedstock for ethanol or for other
renewable bioproducts and 4) silage.
BRIEF SUMMARY OF THE INVENTION
[0011] Compositions and methods for controlling plant growth and
dimerization domain component stack formation for increasing yield
in a plant are provided. The compositions include dimerization
domain component stacks from maize. Compositions of the invention
comprise amino acid sequences and nucleotide sequences selected
from SEQ ID NOS: 1-22 as well as variants and fragments
thereof.
[0012] Polynucleotides encoding the dimerization domain component
stacks are provided in DNA constructs for expression in a plant of
interest. Expression cassettes, plants, plant cells, plant parts
and seeds comprising the sequences of the invention are further
provided. In specific embodiments, the polynucleotide is operably
linked to a constitutive promoter.
[0013] Methods for modulating the level of a dimerization domain
component stack sequence in a plant or plant part are provided. The
methods comprise introducing into a plant or plant part a
heterologous polynucleotide comprising a dimerization domain
component stack sequence of the invention. The level of a
dimerization domain component stack polypeptide can be increased or
decreased. Such method can be used to increase the yield in plants;
in one embodiment, the method is used to increase grain yield in
cereals.
[0014] The plant hormone GA is active in various growth processes,
specifically the elongation of stem and root during plant growth.
The D8 (and D9) genes of maize encode for transcriptional
regulators that act as inhibitors of the giberellic acid signal
transduction pathway, and consist of a DELLA and GRAS domain. The
GA receptor interacts with DELLA proteins in the presence of GA,
which leads to poly-ubiquitination of the DELLA protein.
Poly-ubiquination signals for protein degradation by the 26S
proteasome. The degradation of the DELLA proteins removes their
inhibition of the GA growth response. In general, the rate of
degradation of the D8/D9 proteins appears to correlate with plant
size (i.e. slower degradation results in less response to GA, less
elongation and a greater height reduction). Deletions and specific
mutations in the DELLA domain of D8 are responsible for the
dwarfing phenotype because of the altered degradation kinetics of
these proteins.
[0015] The D8 (and D9) proteins are thought to function in-vivo as
a dimer, whose catabolism regulates plant elongation. Dimers of
strong dwarf genes such as D8 are less sensitive to degradation
while moderate dwarf genes such as D8MPL are relatively more
sensitive to degradation. The native wild type gene d8 is sensitive
to degradation and a tall or normal height is observed. A specific
leucine-zipper domain of the D8 protein, ZM-D8 243-331, is involved
in the formation of the dimers. An altered dimerization domain
protein is formed by over expression of the ZM-D8 243-331 protein.
These truncated protein fragments compete for binding to the
leucine-zipper domain of full length D8 and D9. This competitive
binding leads to the formation of defective dimers having a
full-length protein::truncated protein. The resultant
non-functional dimer lacks the capacity to inhibit the GA response,
and when present in a plant or plant organ increases elongation.
Further, tissue specific expression using promoters for specific
plant organs such as roots, ears or tassels are expected to have
increased size (length) compared to dwarf plants. Specifically, a
dwarf plant type could have roots that are similar in size to wild
type or normal statured plants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1: Root Growth as measured in Mini Rhizitrons in
Johnston Iowa in 2006 with Hybrid 33A14, PHP24843, PHP26998 and
PHP26998
[0017] FIG. 2: D8-MPL Stack Average Harvest Index by construct,
based on late season plant dry weight.
[0018] FIG. 3: D8-MPL Stack Yield Comparison at 24K
[0019] FIG. 4: Diagram describing selective architecture
modification of Zm-D8 243-331, a dominant negative transgene,
overexpression of DD, leading to non-functional dimers.
Non-functional Dimers (DN) increase elongation when expressed in
tissues such as roots, ears or tassels.
[0020] FIG. 5: Alignment of DD domains across various species,
Glycine max (SEQ ID NOS: 24, 26, 28 30, Arabidopsis thaliana (SEQ
ID NOS: 32, 34, 36, 38 and 40), Zea mays (SEQ ID NO: 19), showing
conserved regions and consensus sequence (SEQ ID NO: 41).
DETAILED DESCRIPTION OF THE INVENTION
[0021] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Unless
mentioned otherwise, the techniques employed or contemplated herein
are standard methodologies well known to one of ordinary skill in
the art. The materials, methods and examples are illustrative only
and not limiting. The following is presented by way of illustration
and is not intended to limit the scope of the invention.
[0022] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0023] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
[0024] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of botany,
microbiology, tissue culture, molecular biology, chemistry,
biochemistry and recombinant DNA technology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Langenheim and Thimann, BOTANY: PLANT
BIOLOGY AND ITS RELATION TO HUMAN AFFAIRS, John Wiley (1982); CELL
CULTURE AND SOMATIC CELL GENETICS OF PLANTS, vol. 1, Vasil, ed.
(1984); Stanier, et al., THE MICROBIAL WORLD, 5.sup.th ed.,
Prentice-Hall (1986); Dhringra and Sinclair, BASIC PLANT PATHOLOGY
METHODS, CRC Press (1985); Maniatis, et al., MOLECULAR CLONING: A
LABORATORY MANUAL (1982); DNA CLONING, vols. I and II, Glover, ed.
(1985); OLIGONUCLEOTIDE SYNTHESIS, Gait, ed. (1984); NUCLEIC ACID
HYBRIDIZATION, Hames and Higgins, eds. (1984) and the series
METHODS IN ENZYMOLOGY, Colowick and Kaplan, eds, Academic Press,
Inc., San Diego, Calif.
[0025] Units, prefixes and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxy orientation,
respectively. Numeric ranges are inclusive of the numbers defining
the range. Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes. The terms defined below are more
fully defined by reference to the specification as a whole.
[0026] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0027] By "microbe" is meant any microorganism (including both
eukaryotic and prokaryotic microorganisms), such as fungi, yeast,
bacteria, actinomycetes, algae and protozoa, as well as other
unicellular structures.
[0028] By "amplified" is meant the construction of multiple copies
of a nucleic acid sequence or multiple copies complementary to the
nucleic acid sequence using at least one of the nucleic acid
sequences as a template. Amplification systems include the
polymerase chain reaction (PCR) system, ligase chain reaction (LCR)
system, nucleic acid sequence based amplification (NASBA, Cangene,
Mississauga, Ontario), 0-Beta Replicase systems,
transcription-based amplification system (TAS), and strand
displacement amplification (SDA). See, e.g., DIAGNOSTIC MOLECULAR
MICROBIOLOGY: PRINCIPLES AND APPLICATIONS, Persing, et al., eds.,
American Society for Microbiology, Washington, D.C. (1993). The
product of amplification is termed an amplicon.
[0029] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refer to
those nucleic acids that encode identical or conservatively
modified variants of the amino acid sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations" and represent one species
of conservatively modified variation. Every nucleic acid sequence
herein that encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of ordinary skill will
recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine; one exception is
Micrococcus rubens, for which GTG is the methionine codon
(Ishizuka, et al., (1993) J. Gen. Microbiol. 139:425-32) can be
modified to yield a functionally identical molecule. Accordingly,
each silent variation of a nucleic acid, which encodes a
polypeptide of the present invention, is implicit in each described
polypeptide sequence and incorporated herein by reference.
[0030] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" when
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Thus, any number of amino acid
residues selected from the group of integers consisting of from 1
to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7 or 10
alterations can be made. Conservatively modified variants typically
provide similar biological activity as the unmodified polypeptide
sequence from which they are derived. For example, substrate
specificity, enzyme activity, or ligand/receptor binding is
generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, preferably
60-90% of the native protein for it's native substrate.
Conservative substitution tables providing functionally similar
amino acids are well known in the art.
[0031] The following six groups each contain amino acids that are
conservative substitutions for one another:
[0032] 1) Alanine (A), Serine (S), Threonine (T);
[0033] 2) Aspartic acid (D), Glutamic acid (E);
[0034] 3) Asparagine (N), Glutamine (Q);
[0035] 4) Arginine (R), Lysine (K);
[0036] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V)
and
[0037] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton, PROTEINS, W.H. Freeman and Co. (1984).
[0038] As used herein in the context of nucleic acids in general,
"consisting essentially of" means the inclusion of additional
sequences to an object polynucleotide where the additional
sequences do not selectively hybridize, under stringent
hybridization conditions, to the same cDNA as the polynucleotide
and where the hybridization conditions include a wash step in
0.1.times.SSC and 0.1% sodium dodecyl sulfate at 65.degree. C.
[0039] The term "consisting essentially of" or "consists
essentially of" in the context of a nucleic acid sequence encoding
a dimerization domain or the amino acid sequence of the
dimerization domain, generally refers to a recombinant dimerization
domain sequence and any other sequence that does not materially
alter the basic binding property of the dimerization domain
fragment, for example, to form a defective dimer with the target
protein. For example, the ZM-D8 243-331 is a portion of the D8
protein that corresponds to a dimerization domain region. In an
embodiment, this domain fragment may contain other sequences both
to the amino and/or carboxy-terminus as long as the additional
sequences do not materially alter the basic binding characteristics
of the dimerization domain fragment with the target protein that
results in reduced inhibition by giberrellic acid (GA) hormone. For
example, a full-length D8 amino acid sequence is not suitable as it
will result in the formation of a functional dimer that blocks GA
response.
[0040] By "encoding" or "encoded," with respect to a specified
nucleic acid, is meant comprising the information for translation
into the specified protein. A nucleic acid encoding a protein may
comprise non-translated sequences (e.g., introns) within translated
regions of the nucleic acid, or may lack such intervening
non-translated sequences (e.g., as in cDNA). The information by
which a protein is encoded is specified by the use of codons.
Typically, the amino acid sequence is encoded by the nucleic acid
using the "universal" genetic code. However, variants of the
universal code, such as is present in some plant, animal and fungal
mitochondria, the bacterium Mycoplasma capricolum (Yamao, et al.,
(1985) Proc. Natl. Acad. Sci. USA 82:2306-2309) or the ciliate
Macronucleus, may be used when the nucleic acid is expressed using
these organisms.
[0041] When the nucleic acid is prepared or altered synthetically,
advantage can be taken of known codon preferences of the intended
host where the nucleic acid is to be expressed. For example,
although nucleic acid sequences of the present invention may be
expressed in both monocotyledonous and dicotyledonous plant
species, sequences can be modified to account for the specific
codon preferences and GC content preferences of monocotyledonous
plants or dicotyledonous plants as these preferences have been
shown to differ (Murray, et al., (1989) Nucleic Acids Res.
17:477-98 and herein incorporated by reference). Thus, the maize
preferred codon for a particular amino acid might be derived from
known gene sequences from maize. Maize codon usage for 28 genes
from maize plants is listed in Table 4 of Murray, et al.,
supra.
[0042] As used herein, "heterologous" in reference to a nucleic
acid is a nucleic acid that originates from a foreign species, or,
if from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a
heterologous structural gene is from a species different from that
from which the structural gene was derived or, if from the same
species, one or both are substantially modified from their original
form. A heterologous protein may originate from a foreign species
or, if from the same species, is substantially modified from its
original form by deliberate human intervention.
[0043] By "host cell" is meant a cell, which contains a vector and
supports the replication and/or expression of the expression
vector. Host cells may be prokaryotic cells such as E. coli, or
eukaryotic cells such as yeast, insect, plant, amphibian or
mammalian cells. Preferably, host cells are monocotyledonous or
dicotyledonous plant cells, including but not limited to maize,
sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola,
barley, millet and tomato. A particularly preferred
monocotyledonous host cell is a maize host cell.
[0044] The term "hybridization complex" includes reference to a
duplex nucleic acid structure formed by two single-stranded nucleic
acid sequences selectively hybridized with each other.
[0045] The term "introduced" in the context of inserting a nucleic
acid into a cell, means "transfection" or "transformation" or
"transduction" and includes reference to the incorporation of a
nucleic acid into a eukaryotic or prokaryotic cell where the
nucleic acid may be incorporated into the genome of the cell (e.g.,
chromosome, plasmid, plastid or mitochondrial DNA), converted into
an autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0046] The terms "isolated" refers to material, such as a nucleic
acid or a protein, which is substantially or essentially free from
components which normally accompany or interact with it as found in
its naturally occurring environment. The isolated material
optionally comprises material not found with the material in its
natural environment. Nucleic acids, which are "isolated", as
defined herein, are also referred to as "heterologous" nucleic
acids. Unless otherwise stated, the term "dimerization domain
component stack nucleic acid" means a nucleic acid comprising a
polynucleotide ("dimerization domain component stack
polynucleotide") encoding a dimerization domain component stack
polypeptide.
[0047] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues having the essential nature of natural nucleotides
in that they hybridize to single-stranded nucleic acids in a manner
similar to naturally occurring nucleotides (e.g., peptide nucleic
acids).
[0048] By "nucleic acid library" is meant a collection of isolated
DNA or RNA molecules, which comprise and substantially represent
the entire transcribed fraction of a genome of a specified
organism. Construction of exemplary nucleic acid libraries, such as
genomic and cDNA libraries, is taught in standard molecular biology
references such as Berger and Kimmel, GUIDE TO MOLECULAR CLONING
TECHNIQUES, from the series METHODS IN ENZYMOLOGY, vol. 152,
Academic Press, Inc., San Diego, Calif. (1987); Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2.sup.nd ed., vols. 1-3
(1989) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, et al.,
eds, Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc. (1994
Supplement).
[0049] As used herein "operably linked" includes reference to a
functional linkage between a first sequence, such as a promoter and
a second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA sequence corresponding to the
second sequence. Generally, operably linked means that the nucleic
acid sequences being linked are contiguous and, where necessary to
join two protein coding regions, contiguous and in the same reading
frame.
[0050] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and
plant cells and progeny of same. Plant cell, as used herein
includes, without limitation, seeds suspension cultures, embryos,
meristematic regions, callus tissue, leaves, roots, shoots,
gametophytes, sporophytes, pollen and microspores. The class of
plants, which can be used in the methods of the invention, is
generally as broad as the class of higher plants amenable to
transformation techniques, including both monocotyledonous and
dicotyledonous plants including species from the genera: Cucurbita,
Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis,
Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot,
Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum,
Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,
Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus,
Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium,
Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis,
Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena,
Hordeum, Secale, Allium and Triticum. Also included are grass
plants from the Poaceae family including but not limited to the
genera: Poa, Agrostis, Lolium, Festuca, Zoysia, Cynodon,
Stenotaphrum, Paspalum, Eremochloa, Axonopus, Buchloe, Bouteloua,
including Bluegrass, Bentgrass, Ryegrasses, Fescues, Zoysiagrass,
Bermudagrass, St. Augustine grass, Bahiagrass, Centipedegrass,
Carpetgrass, Buffalograss and Gramagrass. A particularly preferred
plant is Zea mays.
[0051] As used herein, "yield" includes reference to bushels per
acre of a grain crop at harvest, as adjusted for grain moisture
(15% typically). Grain moisture is measured in the grain at
harvest. The adjusted test weight of grain is determined to be the
weight in pounds per bushel, adjusted for grain moisture level at
harvest.
[0052] As used herein, "polynucleotide" includes reference to a
deoxyribopolynucleotide, ribopolynucleotide or analogs thereof that
have the essential nature of a natural ribonucleotide in that they
hybridize, under stringent hybridization conditions, to
substantially the same nucleotide sequence as naturally occurring
nucleotides and/or allow translation into the same amino acid(s) as
the naturally occurring nucleotide(s). A polynucleotide can be
full-length or a subsequence of a native or heterologous structural
or regulatory gene. Unless otherwise indicated, the term includes
reference to the specified sequence as well as the complementary
sequence thereof. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons are "polynucleotides" as that term
is intended herein. Moreover, DNAs or RNAs comprising unusual
bases, such as inosine, or modified bases, such as tritylated
bases, to name just two examples, are polynucleotides as the term
is used herein. It will be appreciated that a great variety of
modifications have been made to DNA and RNA that serve many useful
purposes known to those of skill in the art. The term
polynucleotide as it is employed herein embraces such chemically,
enzymatically or metabolically modified forms of polynucleotides,
as well as the chemical forms of DNA and RNA characteristic of
viruses and cells, including inter alia, simple and complex
cells.
[0053] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0054] As used herein "promoter" includes reference to a region of
DNA upstream from the start of transcription and involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription. A "plant promoter" is a promoter capable of
initiating transcription in plant cells. Exemplary plant promoters
include, but are not limited to, those that are obtained from
plants, plant viruses and bacteria which comprise genes expressed
in plant cells such Agrobacterium or Rhizobium. Examples are
promoters that preferentially initiate transcription in certain
tissues, such as leaves, roots, seeds, fibres, xylem vessels,
tracheids or sclerenchyma. Such promoters are referred to as
"tissue preferred." A "cell type" specific promoter primarily
drives expression in certain cell types in one or more organs, for
example, vascular cells in roots or leaves. An "inducible" or
"regulatable" promoter is a promoter, which is under environmental
control. Examples of environmental conditions that may effect
transcription by inducible promoters include anaerobic conditions
or the presence of light. Another type of promoter is a
developmentally regulated promoter, for example, a promoter that
drives expression during pollen development. Tissue preferred, cell
type specific, developmentally regulated and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter, which is active under most
environmental conditions.
[0055] The term "dimerization domain component stack polypeptide"
refers to one or more amino acid sequences that include the
dimerization domain region of interest and another polypeptide
sequence that is not the same parent sequence from which the
dimerization domain sequence was derived. The term is also
inclusive of fragments, variants, homologs, alleles or precursors
(e.g., preproproteins or proproteins) thereof. A "dimerization
domain component stack protein" comprises a dimerization domain
component stack polypeptide. Unless otherwise stated, the term
"dimerization domain component stack nucleic acid" means a nucleic
acid comprising a polynucleotide ("dimerization domain component
stack polynucleotide") encoding a dimerization domain component
stack polypeptide.
[0056] As used herein "recombinant" includes reference to a cell or
vector, that has been modified by the introduction of a
heterologous nucleic acid or that the cell is derived from a cell
so modified. Thus, for example, recombinant cells express genes
that are not found in identical form within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all as a result of deliberate human intervention. The term
"recombinant" as used herein does not encompass the alteration of
the cell or vector by naturally occurring events (e.g., spontaneous
mutation, natural transformation/transduction/transposition) such
as those occurring without deliberate human intervention. The term
"recombinant polypeptide" or "recombinant nucleic acid" refers to
the peptide and nucleic acid sequences that have been modified such
that they do not exist in nature in their present form.
[0057] As used herein, a "recombinant expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically,
with a series of specified nucleic acid elements, which permit
transcription of a particular nucleic acid in a target cell. The
recombinant expression cassette can be incorporated into a plasmid,
chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid
fragment. Typically, the recombinant expression cassette portion of
an expression vector includes, among other sequences, a nucleic
acid to be transcribed and a promoter.
[0058] The term "residue" or "amino acid residue" or "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide or peptide (collectively
"protein"). The amino acid may be a naturally occurring amino acid
and, unless otherwise limited, may encompass known analogs of
natural amino acids that can function in a similar manner as
naturally occurring amino acids.
[0059] The term "selectively hybridizes" includes reference to
hybridization, under stringent hybridization conditions, of a
nucleic acid sequence to a specified nucleic acid target sequence
to a detectably greater degree (e.g., at least 2-fold over
background) than its hybridization to non-target nucleic acid
sequences and to the substantial exclusion of non-target nucleic
acids. Selectively hybridizing sequences typically have about at
least 40% sequence identity, preferably 60-90% sequence identity
and most preferably 100% sequence identity (i.e., complementary)
with each other.
[0060] The terms "stringent conditions" or "stringent hybridization
conditions" include reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than other sequences (e.g., at least 2-fold over
background).
[0061] Stringent conditions are sequence-dependent and will be
different in different circumstances. By controlling the stringency
of the hybridization and/or washing conditions, target sequences
can be identified which can be up to 100% complementary to the
probe (homologous probing). Alternatively, stringency conditions
can be adjusted to allow some mismatching in sequences so that
lower degrees of similarity are detected (heterologous probing).
Optimally, the probe is approximately 500 nucleotides in length,
but can vary greatly in length from less than 500 nucleotides to
equal to the entire length of the target sequence.
[0062] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide or Denhardt's. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at
37.degree. C. and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.
and a wash in 0.1.times.SSC at 60 to 65.degree. C. Specificity is
typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash
solution. For DNA-DNA hybrids, the T.sub.m can be approximated from
the equation of Meinkoth and Wahl, (1984) Anal. Biochem.
138:267-84: T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61
(% form)-500/L; where M is the molarity of monovalent cations, % GC
is the percentage of guanosine and cytosine nucleotides in the DNA,
% form is the percentage of formamide in the hybridization solution
and L is the length of the hybrid in base pairs. The T.sub.m is the
temperature (under defined ionic strength and pH) at which 50% of a
complementary target sequence hybridizes to a perfectly matched
probe. T.sub.m is reduced by about 1.degree. C. for each 1% of
mismatching; thus, T.sub.m, hybridization and/or wash conditions
can be adjusted to hybridize to sequences of the desired identity.
For example, if sequences with .gtoreq.90% identity are sought, the
T.sub.m can be decreased 10.degree. C. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence and its
complement at a defined ionic strength and pH. However, severely
stringent conditions can utilize a hybridization and/or wash at 1,
2, 3 or 4.degree. C. lower than the thermal melting point
(T.sub.m); moderately stringent conditions can utilize a
hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C. lower than
the thermal melting point (T.sub.m); low stringency conditions can
utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or
20.degree. C. lower than the thermal melting point (T.sub.m). Using
the equation, hybridization and wash compositions, and desired
T.sub.m, those of ordinary skill will understand that variations in
the stringency of hybridization and/or wash solutions are
inherently described. If the desired degree of mismatching results
in a T.sub.m of less than 45.degree. C. (aqueous solution) or
32.degree. C. (formamide solution) it is preferred to increase the
SSC concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR
BIOLOGY--HYBRIDIZATION WITH NUCLEIC ACID PROBES, part I, chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," Elsevier, New York (1993) and CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, chapter 2, Ausubel, et al., eds,
Greene Publishing and Wiley-Interscience, New York (1995). Unless
otherwise stated, in the present application high stringency is
defined as hybridization in 4.times.SSC, 5.times.Denhardt's (5 g
Ficoll, 5 g polyvinypyrrolidone, 5 g bovine serum albumin in 500 ml
of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na
phosphate at 65.degree. C. and a wash in 0.1.times.SSC, 0.1% SDS at
65.degree. C.
[0063] As used herein, "transgenic plant" includes reference to a
plant, which comprises within its genome a heterologous
polynucleotide. Generally, the heterologous polynucleotide is
stably integrated within the genome such that the polynucleotide is
passed on to successive generations. The heterologous
polynucleotide may be integrated into the genome alone or as part
of a recombinant expression cassette. "Transgenic" is used herein
to include any cell, cell line, callus, tissue, plant part or
plant, the genotype of which has been altered by the presence of
heterologous nucleic acid including those transgenics initially so
altered as well as those created by sexual crosses or asexual
propagation from the initial transgenic. The term "transgenic" as
used herein does not encompass the alteration of the genome
(chromosomal or extra-chromosomal) by conventional plant breeding
methods or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition or spontaneous mutation.
[0064] As used herein, "vector" includes reference to a nucleic
acid used in transfection of a host cell and into which can be
inserted a polynucleotide. Vectors are often replicons. Expression
vectors permit transcription of a nucleic acid inserted
therein.
[0065] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides
or polypeptides: (a) "reference sequence," (b) "comparison window,"
(c) "sequence identity," (d) "percentage of sequence identity" and
(e) "substantial identity."
[0066] As used herein, "reference sequence" is a defined sequence
used as a basis for sequence comparison. A reference sequence may
be a subset or the entirety of a specified sequence; for example,
as a segment of a full-length cDNA or gene sequence or the complete
cDNA or gene sequence.
[0067] As used herein, "comparison window" means includes reference
to a contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence may be compared to a reference
sequence and wherein the portion of the polynucleotide sequence in
the comparison window may comprise additions or deletions (i.e.,
gaps) compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Generally, the comparison window is at least 20 contiguous
nucleotides in length, and optionally can be 30, 40, 50, 100 or
longer. Those of skill in the art understand that to avoid a high
similarity to a reference sequence due to inclusion of gaps in the
polynucleotide sequence a gap penalty is typically introduced and
is subtracted from the number of matches.
[0068] Methods of alignment of nucleotide and amino acid sequences
for comparison are well known in the art. The local homology
algorithm (BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math
2:482, may conduct optimal alignment of sequences for comparison;
by the homology alignment algorithm (GAP) of Needleman and Wunsch,
(1970) J. Mol. Biol. 48:443-53; by the search for similarity method
(Tfasta and Fasta) of Pearson and Lipman, (1988) Proc. Natl. Acad.
Sci. USA 85:2444; by computerized implementations of these
algorithms, including, but not limited to: CLUSTAL in the PC/Gene
program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT,
BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software
Package.RTM., Version 8 (available from Genetics Computer Group
(GCG.RTM. programs (Accelrys, Inc., San Diego, Calif.)). The
CLUSTAL program is well described by Higgins and Sharp, (1988) Gene
73:237-44; Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et
al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992)
Computer Applications in the Biosciences 8:155-65 and Pearson, et
al., (1994) Meth. Mol. Biol. 24:307-31. The preferred program to
use for optimal global alignment of multiple sequences is PileUp
(Feng and Doolittle, (1987) J. Mol. Evol., 25:351-60 which is
similar to the method described by Higgins and Sharp, (1989) CABIOS
5:151-53 and hereby incorporated by reference). The BLAST family of
programs which can be used for database similarity searches
includes: BLASTN for nucleotide query sequences against nucleotide
database sequences; BLASTX for nucleotide query sequences against
protein database sequences; BLASTP for protein query sequences
against protein database sequences; TBLASTN for protein query
sequences against nucleotide database sequences and TBLASTX for
nucleotide query sequences against nucleotide database sequences.
See, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Chapter 19, Ausubel,
et al., eds., Greene Publishing and Wiley-Interscience, New York
(1995).
[0069] GAP uses the algorithm of Needleman and Wunsch, supra, to
find the alignment of two complete sequences that maximizes the
number of matches and minimizes the number of gaps. GAP considers
all possible alignments and gap positions and creates the alignment
with the largest number of matched bases and the fewest gaps. It
allows for the provision of a gap creation penalty and a gap
extension penalty in units of matched bases. GAP must make a profit
of gap creation penalty number of matches for each gap it inserts.
If a gap extension penalty greater than zero is chosen, GAP must,
in addition, make a profit for each gap inserted of the length of
the gap times the gap extension penalty. Default gap creation
penalty values and gap extension penalty values in Version 10 of
the Wisconsin Genetics Software Package.RTM. are 8 and 2,
respectively. The gap creation and gap extension penalties can be
expressed as an integer selected from the group of integers
consisting of from 0 to 100. Thus, for example, the gap creation
and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 30, 40, 50 or greater.
[0070] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the Wisconsin Genetics Software Package.RTM. is
BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci.
USA 89:10915).
[0071] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using the BLAST 2.0
suite of programs using default parameters (Altschul, et al.,
(1997) Nucleic Acids Res. 25:3389-402).
[0072] As those of ordinary skill in the art will understand, BLAST
searches assume that proteins can be modeled as random sequences.
However, many real proteins comprise regions of nonrandom
sequences, which may be homopolymeric tracts, short-period repeats
or regions enriched in one or more amino acids. Such low-complexity
regions may be aligned between unrelated proteins even though other
regions of the protein are entirely dissimilar. A number of
low-complexity filter programs can be employed to reduce such
low-complexity alignments. For example, the SEG (Wooten and
Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie and
States, (1993) Comput. Chem. 17:191-201) low-complexity filters can
be employed alone or in combination.
[0073] As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences includes
reference to the residues in the two sequences, which are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences, which differ by such conservative substitutions, are
said to have "sequence similarity" or "similarity." Means for
making this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., according to the algorithm of
Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17,
e.g., as implemented in the program PC/GENE (Intelligenetics,
Mountain View, Calif., USA).
[0074] As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0075] The term "substantial identity" of polynucleotide sequences
means that a polynucleotide comprises a sequence that has between
50-100% sequence identity, preferably at least 50% sequence
identity, preferably at least 60% sequence identity, preferably at
least 70%, more preferably at least 80%, more preferably at least
90% and most preferably at least 95%, compared to a reference
sequence using one of the alignment programs described using
standard parameters. One of skill will recognize that these values
can be appropriately adjusted to determine corresponding identity
of proteins encoded by two nucleotide sequences by taking into
account codon degeneracy, amino acid similarity, reading frame
positioning and the like. Substantial identity of amino acid
sequences for these purposes normally means sequence identity of
between 55-100%, preferably at least 55%, preferably at least 60%,
more preferably at least 70%, 80%, 90% and most preferably at least
95%.
[0076] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. The degeneracy of the genetic code
allows for many amino acids substitutions that lead to variety in
the nucleotide sequence that code for the same amino acid, hence it
is possible that the DNA sequence could code for the same
polypeptide but not hybridize to each other under stringent
conditions. This may occur, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code. One indication that two nucleic acid sequences are
substantially identical is that the polypeptide, which the first
nucleic acid encodes, is immunologically cross reactive with the
polypeptide encoded by the second nucleic acid.
[0077] The terms "substantial identity" in the context of a peptide
indicates that a peptide comprises a sequence with between 55-100%
sequence identity to a reference sequence preferably at least 55%
sequence identity, preferably 60% preferably 70%, more preferably
80%, most preferably at least 90% or 95% sequence identity to the
reference sequence over a specified comparison window. Preferably,
optimal alignment is conducted using the homology alignment
algorithm of Needleman and Wunsch, supra. An indication that two
peptide sequences are substantially identical is that one peptide
is immunologically reactive with antibodies raised against the
second peptide. Thus, a peptide is substantially identical to a
second peptide, for example, where the two peptides differ only by
a conservative substitution. In addition, a peptide can be
substantially identical to a second peptide when they differ by a
non-conservative change if the epitope that the antibody recognizes
is substantially identical. Peptides, which are "substantially
similar" share sequences as, noted above except that residue
positions, which are not identical, may differ by conservative
amino acid changes.
[0078] The invention discloses dimerization domain polynucleotides
and polypeptides. The novel nucleotides and proteins of the
invention have an expression pattern which indicates that they
alter cell wall formation and thus play an important role in plant
development. The polynucleotides are expressed in various plant
tissues. The polynucleotides and polypeptides thus provide an
opportunity to manipulate plant development to alter seed and
vegetative tissue development, timing or composition. This may be
used to create a sterile plant, a seedless plant or a plant with
altered endosperm composition.
Nucleic Acids
[0079] The present invention provides, inter alia, isolated nucleic
acids of RNA, DNA and analogs and/or chimeras thereof, comprising a
dimerization domain polynucleotide.
[0080] The present invention also includes polynucleotides
optimized for expression in different organisms. For example, for
expression of the polynucleotide in a maize plant, the sequence can
be altered to account for specific codon preferences and to alter
GC content as according to Murray, et al, supra. Maize codon usage
for 28 genes from maize plants is listed in Table 4 of Murray, et
al., supra.
[0081] The dimerization domain nucleic acids include isolated
dimerization domain polynucleotides which are inclusive of: [0082]
(a) a polynucleotide encoding a dimerization domain polypeptide and
conservatively modified and polymorphic variants thereof; [0083]
(b) a polynucleotide having at least 70% sequence identity with
polynucleotides of (a) or (b); [0084] (c) complementary sequences
of polynucleotides of (a) or (b).
[0085] The following table, Table 1, lists the specific identities
of the sequences disclosed herein.
TABLE-US-00001 TABLE 1 SEQ ID NO: Identity SEQ ID NO: 1 MS-S2A
promoter SEQ ID NO: 2 ZmCesA10 polynucleotide SEQ ID NO: 3 Pin II
terminator SEQ ID NO: 4 F3.7 promoter SEQ ID NO: 5 ZmCesA4
polynucleotide SEQ ID NO: 6 ZmD8 polynucleotide SEQ ID NO: 7 ZmNAS2
promoter SEQ ID NO: 8 ZmNAS2 5'UTR SEQ ID NO: 9 ZmD8 Dimerization
Domain polynucletide (start and stop codons are artificial
appendages to the 243-331 coding sequence) SEQ ID NO: 10 NOS
terminator SEQ ID NO: 11 ZmFTM1 polynucleotide SEQ ID NO: 12 GmGAl1
polynucleotide SEQ ID NO: 13 ZRP2.47 promoter SEQ ID NO: 14 ADH1
intron SEQ ID NO: 15 ZmRootMet2 promoter SEQ ID NO: 16 ZmCesA10
polypeptide SEQ ID NO: 17 ZmCesA4 polypeptide SEQ ID NO: 18 ZmD8
polypeptide SEQ ID NO: 19 ZmD8 243-331 Dimerization Domain
polypeptide (ATG start codon is artificial and leads to an
N-terminal methionine added to the 243-331 amino acids). SEQ ID NO:
20 ZmFTM1 polypeptide SEQ ID NO: 21 GmGAl1 Dimerization Domain
polypeptide SEQ ID NO: 22 GmGAl1 polypeptide SEQ ID NO: 23 Gm
05g27190.1 SEQ ID NO: 24 Gm 05g27190.1 Dimerization Domain SEQ ID
NO: 25 Gm 08g10140.1 SEQ ID NO: 26 Gm 08g10140.1 Dimerization
Domain SEQ ID NO: 27 Gm 11g33720.1 SEQ ID NO: 28 Gm 11g33720.1
Dimerization Domain SEQ ID NO: 29 Gm 18g04500.1 SEQ ID NO: 30 Gm
18g04500.1 Dimerization Domain SEQ ID NO: 31 At GAl SEQ ID NO: 32
At GAl Dimerization Domain SEQ ID NO: 33 At RGA SEQ ID NO: 34 At
RGA Dimerization Domain SEQ ID NO: 35 At RGL1 SEQ ID NO: 36 At RGL1
Dimerization Domain SEQ ID NO: 37 At RGL2 SEQ ID NO: 38 At RGL2
Dimerization Domain SEQ ID NO: 39 At RGL3 SEQ ID NO: 40 At RGL3
Dimerization Domain SEQ ID NO: 41 Consensus Dimerization Domain SEQ
ID NO: 42 Primer SEQ ID NO: 43 Primer
Construction of Nucleic Acids
[0086] The isolated nucleic acids of the present invention can be
made using (a) standard recombinant methods, (b) synthetic
techniques, or combinations thereof. In some embodiments, the
polynucleotides of the present invention will be cloned, amplified
or otherwise constructed from a fungus or bacteria.
[0087] The nucleic acids may conveniently comprise sequences in
addition to a polynucleotide of the present invention. For example,
a multi-cloning site comprising one or more endonuclease
restriction sites may be inserted into the nucleic acid to aid in
isolation of the polynucleotide. Also, translatable sequences may
be inserted to aid in the isolation of the translated
polynucleotide of the present invention. For example, a
hexa-histidine marker sequence provides a convenient means to
purify the proteins of the present invention. The nucleic acid of
the present invention--excluding the polynucleotide sequence--is
optionally a vector, adapter or linker for cloning and/or
expression of a polynucleotide of the present invention. Additional
sequences may be added to such cloning and/or expression sequences
to optimize their function in cloning and/or expression, to aid in
isolation of the polynucleotide or to improve the introduction of
the polynucleotide into a cell. Typically, the length of a nucleic
acid of the present invention less the length of its polynucleotide
of the present invention is less than 20 kilobase pairs, often less
than 15 kb and frequently less than 10 kb. Use of cloning vectors,
expression vectors, adapters, and linkers is well known in the art.
Exemplary nucleic acids include such vectors as: M13, lambda ZAP
Express, lambda ZAP II, lambda gt10, lambda gt11, pBK-CMV, pBK-RSV,
pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4,
pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/-, pSG5, pBK,
pCR-Script, pET, pSPUTK, p3'SS, pGEM, pSK+/-, pGEX, pSPORTI and II,
pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pOG44,
pOG45, pFRT.beta.GAL, pNEO.beta.GAL, pRS403, pRS404, pRS405,
pRS406, pRS413, pRS414, pRS415, pRS416, lambda MOSSlox and lambda
MOSElox. Optional vectors for the present invention, include but
are not limited to, lambda ZAP II and pGEX. For a description of
various nucleic acids see, e.g., Stratagene Cloning Systems,
Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life
Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).
Synthetic Methods for Constructing Nucleic Acids
[0088] The isolated nucleic acids of the present invention can also
be prepared by direct chemical synthesis by methods such as the
phosphotriester method of Narang, et al., (1979) Meth. Enzymol.
68:90-9; the phosphodiester method of Brown, et al., (1979) Meth.
Enzymol. 68:109-51; the diethylphosphoramidite method of Beaucage,
et al., (1981) Tetra. Letts. 22(20):1859-62; the solid phase
phosphoramidite triester method described by Beaucage, et al.,
supra, e.g., using an automated synthesizer, e.g., as described in
Needham-VanDevanter, et al., (1984) Nucleic Acids Res. 12:6159-68
and the solid support method of U.S. Pat. No. 4,458,066. Chemical
synthesis generally produces a single stranded oligonucleotide.
This may be converted into double stranded DNA by hybridization
with a complementary sequence or by polymerization with a DNA
polymerase using the single strand as a template. One of skill will
recognize that while chemical synthesis of DNA is limited to
sequences of about 100 bases, longer sequences may be obtained by
the ligation of shorter sequences.
UTRs and Codon Preference
[0089] In general, translational efficiency has been found to be
regulated by specific sequence elements in the 5' non-coding or
untranslated region (5' UTR) of the RNA. Positive sequence motifs
include translational initiation consensus sequences (Kozak, (1987)
Nucleic Acids Res. 15:8125) and the 5<G> 7 methyl GpppG RNA
cap structure (Drummond, et al., (1985) Nucleic Acids Res.
13:7375). Negative elements include stable intramolecular 5' UTR
stem-loop structures (Muesing, et al., (1987) Cell 48:691) and AUG
sequences or short open reading frames preceded by an appropriate
AUG in the 5' UTR (Kozak, supra, Rao, et al., (1988) Mol. and Cell.
Biol. 8:284). Accordingly, the present invention provides 5' and/or
3' UTR regions for modulation of translation of heterologous coding
sequences.
[0090] Further, the polypeptide-encoding segments of the
polynucleotides of the present invention can be modified to alter
codon usage. Altered codon usage can be employed to alter
translational efficiency and/or to optimize the coding sequence for
expression in a desired host or to optimize the codon usage in a
heterologous sequence for expression in maize. Codon usage in the
coding regions of the polynucleotides of the present invention can
be analyzed statistically using commercially available software
packages such as "Codon Preference" available from the University
of Wisconsin Genetics Computer Group. See, Devereaux, et al.,
(1984) Nucleic Acids Res. 12:387-395; or MacVector 4.1 (Eastman
Kodak Co., New Haven, Conn.). Thus, the present invention provides
a codon usage frequency characteristic of the coding region of at
least one of the polynucleotides of the present invention. The
number of polynucleotides (3 nucleotides per amino acid) that can
be used to determine a codon usage frequency can be any integer
from 3 to the number of polynucleotides of the present invention as
provided herein. Optionally, the polynucleotides will be
full-length sequences. An exemplary number of sequences for
statistical analysis can be at least 1, 5, 10, 20, 50 or 100.
Sequence Shuffling
[0091] The present invention provides methods for sequence
shuffling using polynucleotides of the present invention, and
compositions resulting therefrom. Sequence shuffling is described
in PCT publication number 96/19256. See also, Zhang, et al., (1997)
Proc. Natl. Acad. Sci. USA 94:4504-9 and Zhao, et al., (1998)
Nature Biotech 16:258-61. Generally, sequence shuffling provides a
means for generating libraries of polynucleotides having a desired
characteristic, which can be selected or screened for. Libraries of
recombinant polynucleotides are generated from a population of
related sequence polynucleotides, which comprise sequence regions,
which have substantial sequence identity and can be homologously
recombined in vitro or in vivo. The population of
sequence-recombined polynucleotides comprises a subpopulation of
polynucleotides which possess desired or advantageous
characteristics and which can be selected by a suitable selection
or screening method. The characteristics can be any property or
attribute capable of being selected for or detected in a screening
system, and may include properties of: an encoded protein, a
transcriptional element, a sequence controlling transcription, RNA
processing, RNA stability, chromatin conformation, translation or
other expression property of a gene or transgene, a replicative
element, a protein-binding element, or the like, such as any
feature which confers a selectable or detectable property. In some
embodiments, the selected characteristic will be an altered K.sub.m
and/or K.sub.a, over the wild-type protein as provided herein. In
other embodiments, a protein or polynucleotide generated from
sequence shuffling will have a ligand binding affinity greater than
the non-shuffled wild-type polynucleotide. In yet other
embodiments, a protein or polynucleotide generated from sequence
shuffling will have an altered pH optimum as compared to the
non-shuffled wild-type polynucleotide. The increase in such
properties can be at least 110%, 120%, 130%, 140% or greater than
150% of the wild-type value.
Recombinant Expression Cassettes
[0092] The present invention further provides recombinant
expression cassettes comprising a nucleic acid of the present
invention. A nucleic acid sequence coding for the desired
polynucleotide of the present invention, for example a cDNA or a
genomic sequence encoding a polypeptide long enough to code for an
active protein of the present invention, can be used to construct a
recombinant expression cassette which can be introduced into the
desired host cell. A recombinant expression cassette will typically
comprise a polynucleotide of the present invention operably linked
to transcriptional initiation regulatory sequences which will
direct the transcription of the polynucleotide in the intended host
cell, such as tissues of a transformed plant.
[0093] For example, plant expression vectors may include (1) a
cloned plant gene under the transcriptional control of 5' and 3'
regulatory sequences and (2) a dominant selectable marker. Such
plant expression vectors may also contain, if desired, a promoter
regulatory region (e.g., one conferring inducible or constitutive,
environmentally- or developmentally-regulated or cell- or
tissue-specific/selective expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site and/or a polyadenylation signal.
[0094] A plant promoter fragment can be employed which will direct
expression of a polynucleotide of the present invention in all
tissues of a regenerated plant. Such promoters are referred to
herein as "constitutive" promoters and are active under most
environmental conditions and states of development or cell
differentiation. Examples of constitutive promoters include the 1'-
or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the
Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S.
Pat. No. 5,683,439), the Nos promoter, the rubisco promoter, the
GRP1-8 promoter, the 35S promoter from cauliflower mosaic virus
(CaMV), as described in Odell, et al., (1985) Nature 313:810-2;
rice actin (McElroy, et al., (1990) Plant Cell 163-171); ubiquitin
(Christensen, et al., (1992) Plant Mol. Biol. 12:619-632 and
Christensen, et al., (1992) Plant Mol. Biol. 18:675-89); pEMU
(Last, et al., (1991) Theor. Appl. Genet. 81:581-8); MAS (Velten,
et al., (1984) EMBO J. 3:2723-30) and maize H3 histone (Lepetit, et
al., (1992) Mol. Gen. Genet. 231:276-85 and Atanassvoa, et al.,
(1992) Plant Journal 2(3):291-300); ALS promoter, as described in
PCT Application Number WO 96/30530; GOS2 (U.S. Pat. No. 6,504,083)
and other transcription initiation regions from various plant genes
known to those of skill. For the present invention ubiquitin is the
preferred promoter for expression in monocot plants.
[0095] Alternatively, the plant promoter can direct expression of a
polynucleotide of the present invention in a specific tissue or may
be otherwise under more precise environmental or developmental
control. Such promoters are referred to here as "inducible"
promoters (Rab17, RAD29). Environmental conditions that may effect
transcription by inducible promoters include pathogen attack,
anaerobic conditions or the presence of light. Examples of
inducible promoters are the Adh1 promoter, which is inducible by
hypoxia or cold stress, the Hsp70 promoter, which is inducible by
heat stress and the PPDK promoter, which is inducible by light.
[0096] Examples of promoters under developmental control include
promoters that initiate transcription only, or preferentially, in
certain tissues, such as leaves, roots, fruit, seeds or flowers.
The operation of a promoter may also vary depending on its location
in the genome. Thus, an inducible promoter may become fully or
partially constitutive in certain locations.
[0097] If polypeptide expression is desired, it is generally
desirable to include a polyadenylation region at the 3'-end of a
polynucleotide coding region. The polyadenylation region can be
derived from a variety of plant genes, or from T-DNA. The 3' end
sequence to be added can be derived from, for example, the nopaline
synthase or octopine synthase genes, or alternatively from another
plant gene, or less preferably from any other eukaryotic gene.
Examples of such regulatory elements include, but are not limited
to, 3' termination and/or polyadenylation regions such as those of
the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan,
et al., (1983) Nucleic Acids Res. 12:369-85); the potato proteinase
inhibitor II (PINII) gene (Keil, et al., (1986) Nucleic Acids Res.
14:5641-50 and An, et al., (1989) Plant Cell 1:115-22) and the CaMV
19S gene (Mogen, et al., (1990) Plant Cell 2:1261-72).
[0098] An intron sequence can be added to the 5' untranslated
region or the coding sequence of the partial coding sequence to
increase the amount of the mature message that accumulates in the
cytosol. Inclusion of a spliceable intron in the transcription unit
in both plant and animal expression constructs has been shown to
increase gene expression at both the mRNA and protein levels up to
1000-fold (Buchman and Berg, (1988) Mol. Cell Biol. 8:4395-4405;
Callis, et al., (1987) Genes Dev. 1:1183-200). Such intron
enhancement of gene expression is typically greatest when placed
near the 5' end of the transcription unit. Use of maize introns
Adh1-S intron 1, 2 and 6, the Bronze-1 intron are known in the art.
See generally, THE MAIZE HANDBOOK, Chapter 116, Freeling and
Walbot, eds., Springer, New York (1994).
[0099] Plant signal sequences, including, but not limited to,
signal-peptide encoding DNA/RNA sequences which target proteins to
the extracellular matrix of the plant cell (Dratewka-Kos, et al.,
(1989) J. Biol. Chem. 264:4896-900), such as the Nicotiana
plumbaginifolia extension gene (DeLoose, et al., (1991) Gene
99:95-100); signal peptides which target proteins to the vacuole,
such as the sweet potato sporamin gene (Matsuka, et al., (1991)
Proc. Natl. Acad. Sci. USA 88:834) and the barley lectin gene
(Wilkins, et al., (1990) Plant Cell, 2:301-13); signal peptides
which cause proteins to be secreted, such as that of PRIb (Lind, et
al., (1992) Plant Mol. Biol. 18:47-53) or the barley alpha amylase
(BAA) (Rahmatullah, et al., (1989) Plant Mol. Biol. 12:119, and
hereby incorporated by reference) or signal peptides which target
proteins to the plastids such as that of rapeseed enoyl-Acp
reductase (Verwaert, et al., (1994) Plant Mol. Biol. 26:189-202)
are useful in the invention. The barley alpha amylase signal
sequence fused to the dimerization domain component stack
polynucleotide is the preferred construct for expression in maize
for the present invention.
[0100] The vector comprising the sequences from a polynucleotide of
the present disclosure will typically comprise a marker gene, which
confers a selectable phenotype on plant cells. Usually, the
selectable marker gene will encode antibiotic resistance, with
suitable genes including genes coding for resistance to the
antibiotic spectinomycin (e.g., the aada gene), the streptomycin
phosphotransferase (SPT) gene coding for streptomycin resistance,
the neomycin phosphotransferase (NPTII) gene encoding kanamycin or
geneticin resistance, the hygromycin phosphotransferase (HPT) gene
coding for hygromycin resistance, genes coding for resistance to
herbicides which act to inhibit the action of acetolactate synthase
(ALS), in particular the sulfonylurea-type herbicides (e.g., the
acetolactate synthase (ALS) gene containing mutations leading to
such resistance in particular the S4 and/or Hra mutations), genes
coding for resistance to herbicides which act to inhibit action of
glutamine synthase, such as phosphinothricin or basta (e.g., the
bar gene) or other such genes known in the art. The bar gene
encodes resistance to the herbicide basta and the ALS gene encodes
resistance to the herbicide chlorsulfuron.
[0101] Typical vectors useful for expression of genes in higher
plants are well known in the art and include vectors derived from
the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens
described by Rogers, et al., (1987) Meth. Enzymol. 153:253-77.
These vectors are plant integrating vectors in that on
transformation, the vectors integrate a portion of vector DNA into
the genome of the host plant. Exemplary A. tumefaciens vectors
useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al.,
(1987) Gene 61:1-11 and Berger, et al., (1989) Proc. Natl. Acad.
Sci. USA, 86:8402-6. Another useful vector herein is plasmid
pBI101.2 that is available from CLONTECH Laboratories, Inc. (Palo
Alto, Calif.).
Expression of Proteins in Host Cells
[0102] Using the nucleic acids of the present invention, one may
express a protein of the present invention in a recombinantly
engineered cell such as bacteria, yeast, insect, mammalian or
preferably plant cells. The cells produce the protein in a
non-natural condition (e.g., in quantity, composition, location
and/or time), because they have been genetically altered through
human intervention to do so.
[0103] It is expected that those of skill in the art are
knowledgeable in the numerous expression systems available for
expression of a nucleic acid encoding a protein of the present
invention. No attempt to describe in detail the various methods
known for the expression of proteins in prokaryotes or eukaryotes
will be made.
[0104] In brief summary, the expression of isolated nucleic acids
encoding a protein of the present invention will typically be
achieved by operably linking, for example, the DNA or cDNA to a
promoter (which is either constitutive or inducible), followed by
incorporation into an expression vector. The vectors can be
suitable for replication and integration in either prokaryotes or
eukaryotes. Typical expression vectors contain transcription and
translation terminators, initiation sequences and promoters useful
for regulation of the expression of the DNA encoding a protein of
the present invention. To obtain high level expression of a cloned
gene, it is desirable to construct expression vectors which
contain, at the minimum, a strong promoter, such as ubiquitin, to
direct transcription, a ribosome binding site for translational
initiation and a transcription/translation terminator. Constitutive
promoters are classified as providing for a range of constitutive
expression. Thus, some are weak constitutive promoters and others
are strong constitutive promoters. Generally, by "weak promoter" is
intended a promoter that drives expression of a coding sequence at
a low level. By "low level" is intended at levels of about 1/10,000
transcripts to about 1/100,000 transcripts to about 1/500,000
transcripts. Conversely, a "strong promoter" drives expression of a
coding sequence at a "high level" or about 1/10 transcripts to
about 1/100 transcripts to about 1/1,000 transcripts.
[0105] One of skill would recognize that modifications could be
made to a protein of the present invention without diminishing its
biological activity. Some modifications may be made to facilitate
the cloning, expression or incorporation of the targeting molecule
into a fusion protein. Such modifications are well known to those
of skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site or additional
amino acids (e.g., poly His) placed on either terminus to create
conveniently located restriction sites or termination codons or
purification sequences.
Expression in Prokaryotes
[0106] Prokaryotic cells may be used as hosts for expression.
Prokaryotes most frequently are represented by various strains of
E. coli; however, other microbial strains may also be used.
Commonly used prokaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding site
sequences, include such commonly used promoters as the beta
lactamase (penicillinase) and lactose (lac) promoter systems
(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp)
promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057)
and the lambda derived P L promoter and N-gene ribosome binding
site (Shimatake, et al., (1981) Nature 292:128). The inclusion of
selection markers in DNA vectors transfected in E. coli is also
useful. Examples of such markers include genes specifying
resistance to ampicillin, tetracycline or chloramphenicol.
[0107] The vector is selected to allow introduction of the gene of
interest into the appropriate host cell. Bacterial vectors are
typically of plasmid or phage origin. Appropriate bacterial cells
are infected with phage vector particles or transfected with naked
phage vector DNA. If a plasmid vector is used, the bacterial cells
are transfected with the plasmid vector DNA. Expression systems for
expressing a protein of the present invention are available using
Bacillus sp. and Salmonella (Palva, et al., (1983) Gene 22:229-35;
Mosbach, et al., (1983) Nature 302:543-5). The pGEX-4T-1 plasmid
vector from Pharmacia is the preferred E. coli expression vector
for the present invention.
Expression in Eukaryotes
[0108] A variety of eukaryotic expression systems such as yeast,
insect cell lines, plant and mammalian cells, are known to those of
skill in the art. As explained briefly below, the present invention
can be expressed in these eukaryotic systems. In some embodiments,
transformed/transfected plant cells, as discussed infra, are
employed as expression systems for production of the proteins of
the instant invention.
[0109] Synthesis of heterologous proteins in yeast is well known.
Sherman, et al., (1982) METHODS IN YEAST GENETICS, Cold Spring
Harbor Laboratory is a well recognized work describing the various
methods available to produce the protein in yeast. Two widely
utilized yeasts for production of eukaryotic proteins are
Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains and
protocols for expression in Saccharomyces and Pichia are known in
the art and available from commercial suppliers (e.g., Invitrogen).
Suitable vectors usually have expression control sequences, such as
promoters, including 3-phosphoglycerate kinase or alcohol oxidase,
and an origin of replication, termination sequences and the like as
desired.
[0110] A protein of the present invention, once expressed, can be
isolated from yeast by lysing the cells and applying standard
protein isolation techniques to the lysates or the pellets. The
monitoring of the purification process can be accomplished by using
Western blot techniques or radioimmunoassay of other standard
immunoassay techniques.
[0111] The sequences encoding proteins of the present invention can
also be ligated to various expression vectors for use in
transfecting cell cultures of, for instance, mammalian, insect or
plant origin. Mammalian cell systems often will be in the form of
monolayers of cells although mammalian cell suspensions may also be
used. A number of suitable host cell lines capable of expressing
intact proteins have been developed in the art, and include the
HEK293, BHK21 and CHO cell lines. Expression vectors for these
cells can include expression control sequences, such as an origin
of replication, a promoter (e.g., the CMV promoter, a HSV tk
promoter or pgk (phosphoglycerate kinase) promoter), an enhancer
(Queen, et al., (1986) Immunol. Rev. 89:49) and necessary
processing information sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly
A addition site) and transcriptional terminator sequences. Other
animal cells useful for production of proteins of the present
invention are available, for instance, from the American Type
Culture Collection Catalogue of Cell Lines and Hybridomas (7.sup.th
ed., 1992).
[0112] Appropriate vectors for expressing proteins of the present
invention in insect cells are usually derived from the SF9
baculovirus. Suitable insect cell lines include mosquito larvae,
silkworm, armyworm, moth and Drosophila cell lines such as a
Schneider cell line (see, e.g., Schneider, (1987) J. Embryol. Exp.
Morphol. 27:353-65).
[0113] As with yeast, when higher animal or plant host cells are
employed, polyadenlyation or transcription terminator sequences are
typically incorporated into the vector. An example of a terminator
sequence is the polyadenlyation sequence from the bovine growth
hormone gene. Sequences for accurate splicing of the transcript may
also be included. An example of a splicing sequence is the VP1
intron from SV40 (Sprague, et al., (1983) J. Virol. 45:773-81).
Additionally, gene sequences to control replication in the host
cell may be incorporated into the vector such as those found in
bovine papilloma virus type-vectors (Saveria-Campo, "Bovine
Papilloma Virus DNA a Eukaryotic Cloning Vector," in DNA CLONING: A
PRACTICAL APPROACH, vol. II, Glover, ed., IRL Press, Arlington,
Va., pp. 213-38 (1985)).
[0114] In addition, the gene for dimerization domain placed in the
appropriate plant expression vector can be used to transform plant
cells. The polypeptide can then be isolated from plant callus or
the transformed cells can be used to regenerate transgenic plants.
Such transgenic plants can be harvested and the appropriate tissues
(seed or leaves, for example) can be subjected to large scale
protein extraction and purification techniques.
Plant Transformation Methods
[0115] Numerous methods for introducing foreign genes into plants
are known and can be used to insert a dimerization domain
polynucleotide into a plant host, including biological and physical
plant transformation protocols. See, e.g., Miki, et al., "Procedure
for Introducing Foreign DNA into Plants," in METHODS IN PLANT
MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick and Thompson, eds., CRC
Press, Inc., Boca Raton, pp. 67-88 (1993). The methods chosen vary
with the host plant, and include chemical transfection methods such
as calcium phosphate, microorganism-mediated gene transfer such as
Agrobacterium (Horsch, et al., (1985) Science 227:1229-31),
electroporation, micro-injection and biolistic bombardment.
[0116] Expression cassettes and vectors and in vitro culture
methods for plant cell or tissue transformation and regeneration of
plants are known and available. See, e.g., Gruber, et al., "Vectors
for Plant Transformation," in METHODS IN PLANT MOLECULAR BIOLOGY
AND BIOTECHNOLOGY, supra, pp. 89-119.
[0117] The isolated polynucleotides or polypeptides may be
introduced into the plant by one or more techniques typically used
for direct delivery into cells. Such protocols may vary depending
on the type of organism, cell, plant or plant cell, i.e., monocot
or dicot, targeted for gene modification. Suitable methods of
transforming plant cells include microinjection (Crossway, et al.,
(1986) Biotechniques 4:320-334; and U.S. Pat. No. 6,300,543),
electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA
83:5602-5606), direct gene transfer (Paszkowski, et al., (1984)
EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for
example, Sanford, et al., U.S. Pat. No. 4,945,050; WO 91/10725 and
McCabe, et al., (1988) Biotechnology 6:923-926). Also see, Tomes,
et al., Direct DNA Transfer into Intact Plant Cells Via
Microprojectile Bombardment pp. 197-213 in Plant Cell, Tissue and
Organ Culture, Fundamental Methods eds. Gamborg and Phillips,
Springer-Verlag Berlin Heidelberg New York, 1995; U.S. Pat. No.
5,736,369 (meristem); Weissinger, et al., (1988) Ann. Rev. Genet.
22:421-477; Sanford, et al., (1987) Particulate Science and
Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol.
87:671-674 (soybean); Datta, et al., (1990) Biotechnology 8:736-740
(rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA
85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563
(maize); WO 91/10725 (maize); Klein, et al., (1988) Plant Physiol.
91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839
and Gordon-Kamm, et al., (1990) Plant Cell 2:603-618 (maize);
Hooydaas-Van Slogteren and Hooykaas, (1984) Nature (London)
311:763-764; Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA
84:5345-5349 (Liliaceae); De Wet, et al., (1985) In The
Experimental Manipulation of Ovule Tissues, ed. G. P. Chapman, et
al., pp. 197-209; Longman, N.Y. (pollen); Kaeppler, et al., (1990)
Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992) Theor.
Appl. Genet. 84:560-566 (whisker-mediated transformation); U.S.
Pat. No. 5,693,512 (sonication); D'Halluin, et al., (1992) Plant
Cell 4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell
Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany
75:407-413 (rice); Osjoda, et al., (1996) Nature Biotech.
14:745-750; Agrobacterium mediated maize transformation (U.S. Pat.
No. 5,981,840); silicon carbide whisker methods (Frame, et al.,
(1994) Plant J. 6:941-948); laser methods (Guo, et al., (1995)
Physiologia Plantarum 93:19-24); sonication methods (Bao, et al.,
(1997) Ultrasound in Medicine & Biology 23:953-959; Finer and
Finer, (2000) Lett Appl Microbiol. 30:406-10; Amoah, et al., (2001)
J Exp Bot 52:1135-42); polyethylene glycol methods (Krens, et al.,
(1982) Nature 296:72-77); protoplasts of monocot and dicot cells
can be transformed using electroporation (Fromm, et al., (1985)
Proc. Natl. Acad. Sci. USA 82:5824-5828) and microinjection
(Crossway, et al., (1986) Mol. Gen. Genet. 202:179-185), all of
which are herein incorporated by reference.
Agrobacterium-Mediated Transformation
[0118] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium. A. tumefaciens and A.
rhizogenes are plant pathogenic soil bacteria, which genetically
transform plant cells. The Ti and Ri plasmids of A. tumefaciens and
A. rhizogenes, respectively, carry genes responsible for genetic
transformation of plants. See, e.g., Kado, (1991) Crit. Rev. Plant
Sci. 10:1. Descriptions of the Agrobacterium vector systems and
methods for Agrobacterium-mediated gene transfer are provided in
Gruber, et al., supra; Miki, et al., supra and Moloney, et al.,
(1989) Plant Cell Reports 8:238.
[0119] Similarly, the gene can be inserted into the T-DNA region of
a Ti or Ri plasmid derived from A. tumefaciens or A. rhizogenes,
respectively. Thus, expression cassettes can be constructed as
above, using these plasmids. Many control sequences are known which
when coupled to a heterologous coding sequence and transformed into
a host organism show fidelity in gene expression with respect to
tissue/organ specificity of the original coding sequence. See,
e.g., Benfey and Chua, (1989) Science 244:174-81. Particularly
suitable control sequences for use in these plasmids are promoters
for constitutive leaf-specific expression of the gene in the
various target plants. Other useful control sequences include a
promoter and terminator from the nopaline synthase gene (NOS). The
NOS promoter and terminator are present in the plasmid pARC2,
available from the American Type Culture Collection and designated
ATCC 67238. If such a system is used, the virulence (vir) gene from
either the Ti or Ri plasmid must also be present, either along with
the T-DNA portion or via a binary system where the vir gene is
present on a separate vector. Such systems, vectors for use
therein, and methods of transforming plant cells are described in
U.S. Pat. No. 4,658,082; US Patent Application Serial Number
913,914, filed Oct. 1, 1986, as referenced in U.S. Pat. No.
5,262,306, issued Nov. 16, 1993 and Simpson, et al., (1986) Plant
Mol. Biol. 6:403-15 (also referenced in the '306 patent), all
incorporated by reference in their entirety.
[0120] Once constructed, these plasmids can be placed into A.
rhizogenes or A. tumefaciens and these vectors used to transform
cells of plant species, which are ordinarily susceptible to
Fusarium or Alternaria infection. Several other transgenic plants
are also contemplated by the present invention including but not
limited to soybean, corn, sorghum, alfalfa, rice, clover, cabbage,
banana, coffee, celery, tobacco, cowpea, cotton, melon and pepper.
The selection of either A. tumefaciens or A. rhizogenes will depend
on the plant being transformed thereby. In general A. tumefaciens
is the preferred organism for transformation. Most dicotyledonous
plants, some gymnosperms and a few monocotyledonous plants (e.g.,
certain members of the Liliales and Arales) are susceptible to
infection with A. tumefaciens. A. rhizogenes also has a wide host
range, embracing most dicots and some gymnosperms, which includes
members of the Leguminosae, Compositae and Chenopodiaceae. Monocot
plants can now be transformed with some success. EP Application
Number 604 662 A1 discloses a method for transforming monocots
using Agrobacterium. EP Application Number 672 752 A1 discloses a
method for transforming monocots with Agrobacterium using the
scutellum of immature embryos. Ishida, et al., discuss a method for
transforming maize by exposing immature embryos to A. tumefaciens
(Nature Biotechnology 14:745-50 (1996)).
[0121] Once transformed, these cells can be used to regenerate
transgenic plants. For example, whole plants can be infected with
these vectors by wounding the plant and then introducing the vector
into the wound site. Any part of the plant can be wounded,
including leaves, stems and roots. Alternatively, plant tissue, in
the form of an explant, such as cotyledonary tissue or leaf disks,
can be inoculated with these vectors, and cultured under
conditions, which promote plant regeneration. Roots or shoots
transformed by inoculation of plant tissue with A. rhizogenes or A.
tumefaciens, containing the gene coding for the fumonisin
degradation enzyme, can be used as a source of plant tissue to
regenerate fumonisin-resistant transgenic plants, either via
somatic embryogenesis or organogenesis. Examples of such methods
for regenerating plant tissue are disclosed in Shahin, (1985)
Theor. Appl. Genet. 69:235-40; U.S. Pat. No. 4,658,082; Simpson, et
al., supra and US Patent Application Serial Numbers 913,913 and
913,914, both filed Oct. 1, 1986, as referenced in U.S. Pat. No.
5,262,306, issued Nov. 16, 1993, the entire disclosures therein
incorporated herein by reference.
Direct Gene Transfer
[0122] Despite the fact that the host range for
Agrobacterium-mediated transformation is broad, some major cereal
crop species and gymnosperms have generally been recalcitrant to
this mode of gene transfer, even though some success has recently
been achieved in rice (Hiei, et al., (1994) The Plant Journal
6:271-82). Several methods of plant transformation, collectively
referred to as direct gene transfer, have been developed as an
alternative to Agrobacterium-mediated transformation.
[0123] A generally applicable method of plant transformation is
microprojectile-mediated transformation, where DNA is carried on
the surface of microprojectiles measuring about 1 to 4 .mu.m. The
expression vector is introduced into plant tissues with a biolistic
device that accelerates the microprojectiles to speeds of 300 to
600 m/s which is sufficient to penetrate the plant cell walls and
membranes (Sanford, et al., (1987) Part. Sci. Technol. 5:27;
Sanford, (1988) Trends Biotech 6:299; Sanford, (1990) Physiol.
Plant 79:206 and Klein, et al., (1992) Biotechnology 10:268).
[0124] Another method for physical delivery of DNA to plants is
sonication of target cells as described in Zang, et al., (1991)
BioTechnology 9:996. Alternatively, liposome or spheroplast fusions
have been used to introduce expression vectors into plants. See,
e.g., Deshayes, et al., (1985) EMBO J. 4:2731 and Christou, et al.,
(1987) Proc. Natl. Acad. Sci. USA 84:3962. Direct uptake of DNA
into protoplasts using CaCl.sub.2 precipitation, polyvinyl alcohol,
or poly-L-ornithine has also been reported. See, e.g., Hain, et
al., (1985) Mol. Gen. Genet. 199:161 and Draper, et al., (1982)
Plant Cell Physiol. 23:451.
[0125] Electroporation of protoplasts and whole cells and tissues
has also been described. See, e.g., Donn, et al., (1990) in
Abstracts of the VIlth Intl. Congress on Plant Cell and Tissue
Culture IAPTC, A2-38, p. 53; D'Halluin, et al., (1992) Plant Cell
4:1495-505 and Spencer, et al., (1994) Plant Mol. Biol.
24:51-61.
Increasing the Activity and/or Level of a Dimerization Domain
Polypeptide
[0126] Methods are provided to increase the activity and/or level
of the dimerization domain polypeptide. An increase in the level
and/or activity of the dimerization domain polypeptide can be
achieved by providing to the plant a dimerization domain
polypeptide. The dimerization domain polypeptide can be provided by
introducing the amino acid sequence encoding the dimerization
domain polypeptide into the plant, introducing into the plant a
nucleotide sequence encoding a dimerization domain polypeptide or
alternatively by selecting for different variants of the genomic
locus encoding the dimerization domain polypeptide of the
invention.
[0127] As discussed elsewhere herein, many methods are known in the
art for providing a polypeptide to a plant including, but not
limited to, direct introduction of the polypeptide into the plant,
introducing into the plant (transiently or stably) a polynucleotide
construct encoding a polypeptide having dimerization domain
component stack which directs plant development activity. It is
also recognized that the methods of the invention may employ a
polynucleotide that is not capable of directing, in the transformed
plant, the expression of a protein or an RNA. Thus, the level
and/or activity of a dimerization domain polypeptide may be
increased by altering the gene encoding the dimerization domain
polypeptide or its promoter. See, e.g., Kmiec, U.S. Pat. No.
5,565,350; Zarling, et al., PCT/US93/03868. Therefore mutagenized
plants that carry mutations in dimerization domain genes, where the
mutations increase expression of the dimerization domain gene or
increase the plant growth and/or dimerization domain activity of
the encoded dimerization domain polypeptide are provided.
Reducing the Activity and/or Level of a Dimerization Domain
Polypeptide
[0128] Methods are provided to reduce or eliminate the activity of
a dimerization domain polypeptide of the invention by transforming
a plant cell with an expression cassette that expresses a
polynucleotide that inhibits the expression of the dimerization
domain polypeptide. The polynucleotide may inhibit the expression
of the dimerization domain polypeptide directly, by preventing
translation of the dimerization domain messenger RNA, or
indirectly, by encoding a polypeptide that inhibits the
transcription or translation of a dimerization domain gene encoding
a dimerization domain polypeptide. Methods for inhibiting or
eliminating the expression of a gene in a plant are well known in
the art, and any such method may be used in the present invention
to inhibit the expression of a dimerization domain polypeptide.
[0129] The expression of a target polypeptide is inhibited if the
protein level of the polypeptide is less than 70% of the protein
level of the polypeptide in a plant that has not been genetically
modified or mutagenized to inhibit the expression of that
dimerization domain polypeptide. In particular embodiments of the
invention, the protein level of the dimerization domain polypeptide
in a modified plant according to the invention is less than 60%,
less than 50%, less than 40%, less than 30%, less than 20%, less
than 10%, less than 5% or less than 2% of the protein level of the
same dimerization domain polypeptide in a plant that is not a
mutant or that has not been genetically modified to inhibit the
expression of that dimerization domain polypeptide. The expression
level of the dimerization domain polypeptide may be measured
directly, for example, by assaying for the level of dimerization
domain polypeptide expressed in the plant cell or plant, or
indirectly, for example, by measuring the plant growth and/or
dimerization domain activity of the dimerization domain polypeptide
in the plant cell or plant, or by measuring the biomass in the
plant. Methods for performing such assays are described elsewhere
herein.
[0130] In other embodiments of the invention, the activity of the
dimerization domain polypeptides is reduced or eliminated by
transforming a plant cell with an expression cassette comprising a
polynucleotide encoding a polypeptide that inhibits the activity of
a dimerization domain polypeptide. The plant growth and/or
dimerization domain activity of a dimerization domain component
stack polypeptide is inhibited according to the present invention
if the plant growth and/or dimerization domain activity of the
dimerization domain component stack polypeptide is less than 70% of
the plant growth and/or dimerization domain activity of the same
dimerization domain polypeptide in a plant that has not been
modified to inhibit the plant growth and/or dimerization domain
activity of that dimerization domain component stack polypeptide.
In particular embodiments of the invention, the plant growth and/or
dimerization domain activity of the dimerization domain polypeptide
in a modified plant according to the invention is less than 60%,
less than 50%, less than 40%, less than 30%, less than 20%, less
than 10% or less than 5% of the plant growth and/or dimerization
domain activity of the same dimerization domain polypeptide in a
plant that that has not been modified to inhibit the expression of
that dimerization domain polypeptide. The plant growth and/or
dimerization domain activity of a dimerization domain polypeptide
is "eliminated" according to the invention when it is not
detectable by the assay methods described elsewhere herein. Methods
of determining the plant growth and/or dimerization domain activity
of a dimerization domain polypeptide are described elsewhere
herein.
[0131] In other embodiments, the activity of a dimerization domain
componet stack polypeptide may be reduced or eliminated by
disrupting the gene encoding the dimerization domain polypeptide.
The invention encompasses mutagenized plants that carry mutations
in dimerization domain genes, where the mutations reduce expression
of the dimerization domain gene or inhibit the plant growth and/or
dimerization domain activity of the encoded dimerization domain
polypeptide.
[0132] Thus, many methods may be used to reduce or eliminate the
activity of a dimerization domain polypeptide. In addition, more
than one method may be used to reduce the activity of a single
dimerization domain polypeptide. Non-limiting examples of methods
of reducing or eliminating the expression of dimerization domain
polypeptides are given below.
[0133] 1. Polynucleotide-Based Methods:
[0134] In some embodiments of the present invention, a plant is
transformed with an expression cassette that is capable of
expressing a polynucleotide that inhibits the expression of a
dimerization domain polypeptide of the invention. The term
"expression" as used herein refers to the biosynthesis of a gene
product, including the transcription and/or translation of said
gene product. For example, for the purposes of the present
invention, an expression cassette capable of expressing a
polynucleotide that inhibits the expression of at least one
dimerization domain polypeptide is an expression cassette capable
of producing an RNA molecule that inhibits the transcription and/or
translation of at least one dimerization domain polypeptide of the
invention. The "expression" or "production" of a protein or
polypeptide from a DNA molecule refers to the transcription and
translation of the coding sequence to produce the protein or
polypeptide, while the "expression" or "production" of a protein or
polypeptide from an RNA molecule refers to the translation of the
RNA coding sequence to produce the protein or polypeptide.
[0135] Examples of polynucleotides that inhibit the expression of a
dimerization domain polypeptide are given below.
[0136] i. Sense Suppression/Cosuppression
[0137] In some embodiments of the invention, inhibition of the
expression of a dimerization domain polypeptide may be obtained by
sense suppression or cosuppression. For cosuppression, an
expression cassette is designed to express an RNA molecule
corresponding to all or part of a messenger RNA encoding a
dimerization domain polypeptide in the "sense" orientation. Over
expression of the RNA molecule can result in reduced expression of
the native gene. Accordingly, multiple plant lines transformed with
the cosuppression expression cassette are screened to identify
those that show the greatest inhibition of dimerization domain
polypeptide expression.
[0138] The polynucleotide used for cosuppression may correspond to
all or part of the sequence encoding the dimerization domain
polypeptide, all or part of the 5' and/or 3' untranslated region of
a dimerization domain polypeptide transcript or all or part of both
the coding sequence and the untranslated regions of a transcript
encoding a dimerization domain polypeptide. In some embodiments
where the polynucleotide comprises all or part of the coding region
for the dimerization domain polypeptide, the expression cassette is
designed to eliminate the start codon of the polynucleotide so that
no protein product will be translated.
[0139] Cosuppression may be used to inhibit the expression of plant
genes to produce plants having undetectable protein levels for the
proteins encoded by these genes. See, for example, Broin, et al.,
(2002) Plant Cell 14:1417-1432. Cosuppression may also be used to
inhibit the expression of multiple proteins in the same plant. See,
for example, U.S. Pat. No. 5,942,657. Methods for using
cosuppression to inhibit the expression of endogenous genes in
plants are described in Flavell, et al., (1994) Proc. Natl. Acad.
Sci. USA 91:3490-3496; Jorgensen, et al., (1996) Plant Mol. Biol.
31:957-973; Johansen and Carrington, (2001) Plant Physiol.
126:930-938; Broin, et al., (2002) Plant Cell 14:1417-1432;
Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Yu, et
al., (2003) Phytochemistry 63:753-763 and U.S. Pat. Nos. 5,034,323,
5,283,184 and 5,942,657, each of which is herein incorporated by
reference. The efficiency of cosuppression may be increased by
including a poly-dT region in the expression cassette at a position
3' to the sense sequence and 5' of the polyadenylation signal. See,
US Patent Application Publication Number 2002/0048814, herein
incorporated by reference. Typically, such a nucleotide sequence
has substantial sequence identity to the sequence of the transcript
of the endogenous gene, optimally greater than about 65% sequence
identity, more optimally greater than about 85% sequence identity,
most optimally greater than about 95% sequence identity. See, U.S.
Pat. Nos. 5,283,184 and 5,034,323, herein incorporated by
reference.
[0140] ii. Antisense Suppression
[0141] In some embodiments of the invention, inhibition of the
expression of the dimerization domain polypeptide may be obtained
by antisense suppression. For antisense suppression, the expression
cassette is designed to express an RNA molecule complementary to
all or part of a messenger RNA encoding the dimerization domain
polypeptide. Over expression of the antisense RNA molecule can
result in reduced expression of the native gene. Accordingly,
multiple plant lines transformed with the antisense suppression
expression cassette are screened to identify those that show the
greatest inhibition of dimerization domain polypeptide
expression.
[0142] The polynucleotide for use in antisense suppression may
correspond to all or part of the complement of the sequence
encoding the dimerization domain polypeptide, all or part of the
complement of the 5' and/or 3' untranslated region of the
dimerization domain transcript or all or part of the complement of
both the coding sequence and the untranslated regions of a
transcript encoding the dimerization domain polypeptide. In
addition, the antisense polynucleotide may be fully complementary
(i.e., 100% identical to the complement of the target sequence) or
partially complementary (i.e., less than 100% identical to the
complement of the target sequence) to the target sequence.
Antisense suppression may be used to inhibit the expression of
multiple proteins in the same plant. See, for example, U.S. Pat.
No. 5,942,657. Furthermore, portions of the antisense nucleotides
may be used to disrupt the expression of the target gene.
Generally, sequences of at least 50 nucleotides, 100 nucleotides,
200 nucleotides, 300, 400, 450, 500, 550 or greater may be used.
Methods for using antisense suppression to inhibit the expression
of endogenous genes in plants are described, for example, in Liu,
et al., (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos.
5,759,829 and 5,942,657, each of which is herein incorporated by
reference. Efficiency of antisense suppression may be increased by
including a poly-dT region in the expression cassette at a position
3' to the antisense sequence and 5' of the polyadenylation signal.
See, US Patent Application Publication Number 2002/0048814, herein
incorporated by reference.
[0143] iii. Double-Stranded RNA Interference
[0144] In some embodiments of the invention, inhibition of the
expression of a dimerization domain polypeptide may be obtained by
double-stranded RNA (dsRNA) interference. For dsRNA interference, a
sense RNA molecule like that described above for cosuppression and
an antisense RNA molecule that is fully or partially complementary
to the sense RNA molecule are expressed in the same cell, resulting
in inhibition of the expression of the corresponding endogenous
messenger RNA.
[0145] Expression of the sense and antisense molecules can be
accomplished by designing the expression cassette to comprise both
a sense sequence and an antisense sequence. Alternatively, separate
expression cassettes may be used for the sense and antisense
sequences. Multiple plant lines transformed with the dsRNA
interference expression cassette or expression cassettes are then
screened to identify plant lines that show the greatest inhibition
of dimerization domain polypeptide expression. Methods for using
dsRNA interference to inhibit the expression of endogenous plant
genes are described in Waterhouse, et al., (1998) Proc. Natl. Acad.
Sci. USA 95:13959-13964, Liu, et al., (2002) Plant Physiol.
129:1732-1743 and WO 99/49029, WO 99/53050, WO 99/61631 and WO
00/49035, each of which is herein incorporated by reference.
[0146] iv. Hairpin RNA Interference and Intron-Containing Hairpin
RNA Interference
[0147] In some embodiments of the invention, inhibition of the
expression of one or a dimerization domain polypeptide may be
obtained by hairpin RNA (hpRNA) interference or intron-containing
hairpin RNA (ihpRNA) interference. These methods are highly
efficient at inhibiting the expression of endogenous genes. See,
Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38 and the
references cited therein.
[0148] For hpRNA interference, the expression cassette is designed
to express an RNA molecule that hybridizes with itself to form a
hairpin structure that comprises a single-stranded loop region and
a base-paired stem. The base-paired stem region comprises a sense
sequence corresponding to all or part of the endogenous messenger
RNA encoding the gene whose expression is to be inhibited and an
antisense sequence that is fully or partially complementary to the
sense sequence. Thus, the base-paired stem region of the molecule
generally determines the specificity of the RNA interference. hpRNA
molecules are highly efficient at inhibiting the expression of
endogenous genes and the RNA interference they induce is inherited
by subsequent generations of plants. See, for example, Chuang and
Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;
Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731 and
Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods
for using hpRNA interference to inhibit or silence the expression
of genes are described, for example, in Chuang and Meyerowitz,
(2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et
al., (2002) Plant Physiol. 129:1723-1731; Waterhouse and Helliwell,
(2003) Nat. Rev. Genet. 4:29-38; Pandolfini, et al., BMC
Biotechnology 3:7 and US Patent Application Publication Number
2003/0175965, each of which is herein incorporated by reference. A
transient assay for the efficiency of hpRNA constructs to silence
gene expression in vivo has been described by Panstruga, et al.,
(2003) Mol. Biol. Rep. 30:135-140, herein incorporated by
reference.
[0149] For ihpRNA, the interfering molecules have the same general
structure as for hpRNA, but the RNA molecule additionally comprises
an intron that is capable of being spliced in the cell in which the
ihpRNA is expressed. The use of an intron minimizes the size of the
loop in the hairpin RNA molecule following splicing, and this
increases the efficiency of interference. See, for example, Smith,
et al., (2000) Nature 407:319-320. In fact, Smith, et al., show
100% suppression of endogenous gene expression using
ihpRNA-mediated interference. Methods for using ihpRNA interference
to inhibit the expression of endogenous plant genes are described,
for example, in Smith, et al., (2000) Nature 407:319-320; Wesley,
et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001)
Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003)
Nat. Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods
30:289-295 and US Patent Application Publication Number
2003/0180945, each of which is herein incorporated by
reference.
[0150] The expression cassette for hpRNA interference may also be
designed such that the sense sequence and the antisense sequence do
not correspond to an endogenous RNA. In this embodiment, the sense
and antisense sequence flank a loop sequence that comprises a
nucleotide sequence corresponding to all or part of the endogenous
messenger RNA of the target gene. Thus, it is the loop region that
determines the specificity of the RNA interference. See, for
example, WO 02/00904, herein incorporated by reference.
[0151] v. Amplicon-Mediated Interference
[0152] Amplicon expression cassettes comprise a plant virus-derived
sequence that contains all or part of the target gene but generally
not all of the genes of the native virus. The viral sequences
present in the transcription product of the expression cassette
allow the transcription product to direct its own replication. The
transcripts produced by the amplicon may be either sense or
antisense relative to the target sequence (i.e., the messenger RNA
for the dimerization domain polypeptide). Methods of using
amplicons to inhibit the expression of endogenous plant genes are
described, for example, in Angell and Baulcombe, (1997) EMBO J.
16:3675-3684, Angell and Baulcombe, (1999) Plant J. 20:357-362 and
U.S. Pat. No. 6,646,805, each of which is herein incorporated by
reference.
[0153] vi. Ribozymes
[0154] In some embodiments, the polynucleotide expressed by the
expression cassette of the invention is catalytic RNA or has
ribozyme activity specific for the messenger RNA of the
dimerization domain polypeptide. Thus, the polynucleotide causes
the degradation of the endogenous messenger RNA, resulting in
reduced expression of the dimerization domain polypeptide. This
method is described, for example, in U.S. Pat. No. 4,987,071,
herein incorporated by reference.
[0155] vii. Small Interfering RNA or Micro RNA
[0156] In some embodiments of the invention, inhibition of the
expression of a dimerization domain polypeptide may be obtained by
RNA interference by expression of a gene encoding a micro RNA
(miRNA). miRNAs are regulatory agents consisting of about 22
ribonucleotides. miRNA are highly efficient at inhibiting the
expression of endogenous genes. See, for example, Javier, et al.,
(2003) Nature 425:257-263, herein incorporated by reference.
[0157] For miRNA interference, the expression cassette is designed
to express an RNA molecule that is modeled on an endogenous miRNA
gene. The miRNA gene encodes an RNA that forms a hairpin structure
containing a 22-nucleotide sequence that is complementary to
another endogenous gene (target sequence). For suppression of
dimerization domain expression, the 22-nucleotide sequence is
selected from a dimerization domain transcript sequence and
contains 22 nucleotide of said dimerization domain sequence in
sense orientation and 21 nucleotides of a corresponding antisense
sequence that is complementary to the sense sequence. miRNA
molecules are highly efficient at inhibiting the expression of
endogenous genes, and the RNA interference they induce is inherited
by subsequent generations of plants.
[0158] 2. Polypeptide-Based Inhibition of Gene Expression
[0159] In one embodiment, the polynucleotide encodes a zinc finger
protein that binds to a gene encoding a dimerization domain
polypeptide, resulting in reduced expression of the gene. In
particular embodiments, the zinc finger protein binds to a
regulatory region of a dimerization domain gene. In other
embodiments, the zinc finger protein binds to a messenger RNA
encoding a dimerization domain polypeptide and prevents its
translation. Methods of selecting sites for targeting by zinc
finger proteins have been described, for example, in U.S. Pat. No.
6,453,242 and methods for using zinc finger proteins to inhibit the
expression of genes in plants are described, for example, in US
Patent Application Publication Number 2003/0037355, each of which
is herein incorporated by reference.
[0160] 3. Polypeptide-Based Inhibition of Protein Activity
[0161] In some embodiments of the invention, the polynucleotide
encodes an antibody that binds to at least one dimerization domain
polypeptide and reduces the dimerization domain activity of the
dimerization domain polypeptide. In another embodiment, the binding
of the antibody results in increased turnover of the
antibody-dimerization domain complex by cellular quality control
mechanisms. The expression of antibodies in plant cells and the
inhibition of molecular pathways by expression and binding of
antibodies to proteins in plant cells are well known in the art.
See, for example, Conrad and Sonnewald, (2003) Nature Biotech.
21:35-36, incorporated herein by reference.
[0162] 4. Gene Disruption
[0163] In some embodiments of the present invention, the activity
of a dimerization domain polypeptide is reduced or eliminated by
disrupting the gene encoding the dimerization domain polypeptide.
The gene encoding the dimerization domain polypeptide may be
disrupted by any method known in the art. For example, in one
embodiment, the gene is disrupted by transposon tagging. In another
embodiment, the gene is disrupted by mutagenizing plants using
random or targeted mutagenesis and selecting for plants that have
reduced dimerization domain activity.
[0164] i. Transposon Tagging
[0165] In one embodiment of the invention, transposon tagging is
used to reduce or eliminate the dimerization domain activity of one
or more dimerization domain polypeptide. Transposon tagging
comprises inserting a transposon within an endogenous dimerization
domain gene to reduce or eliminate expression of the dimerization
domain polypeptide. "dimerization domain gene" is intended to mean
the gene that encodes a dimerization domain polypeptide according
to the invention.
[0166] In this embodiment, the expression of one or more
dimerization domain polypeptide is reduced or eliminated by
inserting a transposon within a regulatory region or coding region
of the gene encoding the dimerization domain polypeptide. A
transposon that is within an exon, intron, 5' or 3' untranslated
sequence, a promoter or any other regulatory sequence of a
dimerization domain gene may be used to reduce or eliminate the
expression and/or activity of the encoded dimerization domain
polypeptide.
[0167] Methods for the transposon tagging of specific genes in
plants are well known in the art. See, for example, Maes, et al.,
(1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti, (1999) FEMS
Microbiol. Lett. 179:53-59; Meissner, et al., (2000) Plant J.
22:265-274; Phogat, et al., (2000) J. Biosci. 25:57-63; Walbot,
(2000) Curr. Opin. Plant Biol. 2:103-107; Gai, et al., (2000)
Nucleic Acids Res. 28:94-96; Fitzmaurice, et al., (1999) Genetics
153:1919-1928). In addition, the TUSC process for selecting Mu
insertions in selected genes has been described in Bensen, et al.,
(1995) Plant Cell 7:75-84; Mena, et al., (1996) Science
274:1537-1540 and U.S. Pat. No. 5,962,764, each of which is herein
incorporated by reference.
[0168] ii. Mutant Plants with Reduced Activity
[0169] Additional methods for decreasing or eliminating the
expression of endogenous genes in plants are also known in the art
and can be similarly applied to the instant invention. These
methods include other forms of mutagenesis, such as ethyl
methanesulfonate-induced mutagenesis, deletion mutagenesis and fast
neutron deletion mutagenesis used in a reverse genetics sense (with
PCR) to identify plant lines in which the endogenous gene has been
deleted. For examples of these methods see, Ohshima, et al., (1998)
Virology 243:472-481; Okubara, et al., (1994) Genetics 137:867-874
and Quesada, et al., (2000) Genetics 154:421-436, each of which is
herein incorporated by reference. In addition, a fast and
automatable method for screening for chemically induced mutations,
TILLING (Targeting Induced Local Lesions In Genomes), using
denaturing HPLC or selective endonuclease digestion of selected PCR
products is also applicable to the instant invention. See,
McCallum, et al., (2000) Nat. Biotechnol. 18:455-457, herein
incorporated by reference.
[0170] Mutations that impact gene expression or that interfere with
the function (dimerization domain activity) of the encoded protein
are well known in the art. Insertional mutations in gene exons
usually result in null-mutants. Mutations in conserved residues are
particularly effective in inhibiting the dimerization domain
activity of the encoded protein. Conserved residues of plant
dimerization domain polypeptides suitable for mutagenesis with the
goal to eliminate dimerization domain activity have been described.
Such mutants can be isolated according to well-known procedures and
mutations in different dimerization domain loci can be stacked by
genetic crossing. See, for example, Gruis, et al., (2002) Plant
Cell 14:2863-2882.
[0171] In another embodiment of this invention, dominant mutants
can be used to trigger RNA silencing due to gene inversion and
recombination of a duplicated gene locus. See, for example, Kusaba,
et al., (2003) Plant Cell 15:1455-1467.
[0172] The invention encompasses additional methods for reducing or
eliminating the activity of one or more dimerization domain
polypeptide. Examples of other methods for altering or mutating a
genomic nucleotide sequence in a plant are known in the art and
include, but are not limited to, the use of RNA:DNA vectors,
RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex
oligonucleotides, self-complementary RNA:DNA oligonucleotides and
recombinogenic oligonucleobases. Such vectors and methods of use
are known in the art. See, for example, U.S. Pat. Nos. 5,565,350;
5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, each of
which are herein incorporated by reference. See also, WO 98/49350,
WO 99/07865, WO 99/25821 and Beetham, et al., (1999) Proc. Natl.
Acad. Sci. USA 96:8774-8778, each of which is herein incorporated
by reference.
[0173] iii. Modulating Plant Growth and/or Dimerization Domain
Component Stack Activity
[0174] In specific methods, the level and/or activity of a
dimerization domain gene in a plant is increased by increasing the
level or activity of the dimerization domain polypeptide in the
plant. Methods for increasing the level and/or activity of
dimerization domain polypeptides in a plant are discussed elsewhere
herein. Briefly, such methods comprise providing a dimerization
domain polypeptide of the invention to a plant and thereby
increasing the level and/or activity of the dimerization domain
polypeptide. In other embodiments, a dimerization domain nucleotide
sequence encoding a dimerization domain polypeptide can be provided
by introducing into the plant a polynucleotide comprising a
dimerization domain nucleotide sequence of the invention,
expressing the dimerization domain sequence, increasing the
activity of the dimerization domain polypeptide and thereby
increasing the dimerization domain activity and therefore the
tissue growth in the plant or plant part. In other embodiments, the
dimerization domain nucleotide construct introduced into the plant
is stably incorporated into the genome of the plant.
[0175] In other methods, the number of cells and biomass of a plant
tissue is increased by increasing the level and/or activity of the
dimerization domain polypeptide in the plant. Such methods are
disclosed in detail elsewhere herein. In one such method, a
dimerization domain nucleotide sequence is introduced into the
plant and expression of said dimerization domain nucleotide
sequence decreases the activity of the dimerization domain
polypeptide and thereby increasing the plant growth and/or
dimerization domain in the plant or plant part. In other
embodiments, the dimerization domain nucleotide construct
introduced into the plant is stably incorporated into the genome of
the plant.
[0176] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate the level/activity of a
plant growth and/or dimerization domain polynucleotide and
polypeptide in the plant. Exemplary promoters for this embodiment
have been disclosed elsewhere herein.
[0177] Accordingly, the present invention further provides plants
having a modified plant growth and/or dimerization domain when
compared to the plant growth and/or dimerization domain of a
control plant tissue. In one embodiment, the plant of the invention
has an increased level/activity of the dimerization domain
polypeptide of the invention and thus has increased plant growth
and/or dimerization domain in the plant tissue. In other
embodiments, the plant of the invention has a reduced or eliminated
level of the dimerization domain polypeptide of the invention and
thus has decreased plant growth and/or dimerization domain in the
plant tissue. In other embodiments, such plants have stably
incorporated into their genome a nucleic acid molecule comprising a
dimerization domain nucleotide sequence of the invention operably
linked to a promoter that drives expression in the plant cell.
[0178] iv. Modulating Root Development
[0179] Methods for modulating root development in a plant are
provided. By "modulating root development" is intended any
alteration in the development of the plant root when compared to a
control plant. Such alterations in root development include, but
are not limited to, alterations in the growth rate of the primary
root, the fresh root weight, the extent of lateral and adventitious
root formation, the vasculature system, meristem development or
radial expansion. In particular, the most desirable outcome would
be a root with a stronger vasculature that improves the
standability of the plant and thus reduces root lodging as well as
being less susceptible to pests.
[0180] Methods for modulating root development in a plant are
provided. The methods comprise modulating the level and/or activity
of the dimerization domain polypeptide in the plant. In one method,
a dimerization domain sequence of the invention is provided to the
plant. In another method, the dimerization domain nucleotide
sequence is provided by introducing into the plant a polynucleotide
comprising a dimerization domain nucleotide sequence of the
invention, expressing the dimerization domain sequence and thereby
modifying root development. In still other methods, the
dimerization domain nucleotide construct introduced into the plant
is stably incorporated into the genome of the plant.
[0181] In other methods, root development is modulated by altering
the level or activity of the dimerization domain polypeptide in the
plant. An increase in dimerization domain activity can result in at
least one or more of the following alterations to root development,
including, but not limited to, larger root meristems, increased in
root growth, enhanced radial expansion, an enhanced vasculature
system, increased root branching, more adventitious roots and/or an
increase in fresh root weight when compared to a control plant.
[0182] As used herein, "root growth" encompasses all aspects of
growth of the different parts that make up the root system at
different stages of its development in both monocotyledonous and
dicotyledonous plants. It is to be understood that enhanced root
growth can result from enhanced growth of one or more of its parts
including the primary root, lateral roots, adventitious roots,
etc.
[0183] Methods of measuring such developmental alterations in the
root system are known in the art. See, for example, US Patent
Application Number 2003/0074698 and Werner, et al., (2001) PNAS
18:10487-10492, both of which are herein incorporated by
reference.
[0184] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate root development in the
plant. Exemplary promoters for this embodiment include constitutive
promoters and root-preferred promoters. Exemplary root-preferred
promoters have been disclosed elsewhere herein.
[0185] Stimulating root growth and increasing root mass by
increasing the activity and/or level of the dimerization domain
polypeptide also finds use in improving the standability of a
plant. The term "resistance to lodging" or "standability" refers to
the ability of a plant to fix itself to the soil. For plants with
an erect or semi-erect growth habit, this term also refers to the
ability to maintain an upright position under adverse
(environmental) conditions. This trait relates to the size, depth
and morphology of the root system. In addition, stimulating root
growth and increasing root mass by increasing the level and/or
activity of the dimerization domain polypeptide also finds use in
promoting in vitro propagation of explants.
[0186] Furthermore, higher root biomass production due to an
increased level and/or activity of dimerization domain activity has
a direct effect on the yield and an indirect effect of production
of compounds produced by root cells or transgenic root cells or
cell cultures of said transgenic root cells. One example of an
interesting compound produced in root cultures is shikonin, the
yield of which can be advantageously enhanced by said methods.
[0187] Accordingly, the present invention further provides plants
having modulated root development when compared to the root
development of a control plant. In some embodiments, the plant of
the invention has an increased level/activity of the dimerization
domain polypeptide of the invention and has enhanced root growth
and/or root biomass. In other embodiments, such plants have stably
incorporated into their genome a nucleic acid molecule comprising a
dimerization domain nucleotide sequence of the invention operably
linked to a promoter that drives expression in the plant cell.
[0188] v. Modulating Shoot and Leaf Development
[0189] Methods are also provided for modulating shoot and leaf
development in a plant. By "modulating shoot and/or leaf
development" is intended any alteration in the development of the
plant shoot and/or leaf. Such alterations in shoot and/or leaf
development include, but are not limited to, alterations in shoot
meristem development, in leaf number, leaf size, leaf and stem
vasculature, internode length and leaf senescence. As used herein,
"leaf development" and "shoot development" encompasses all aspects
of growth of the different parts that make up the leaf system and
the shoot system, respectively, at different stages of their
development, both in monocotyledonous and dicotyledonous plants.
Methods for measuring such developmental alterations in the shoot
and leaf system are known in the art. See, for example, Werner, et
al., (2001) PNAS 98:10487-10492 and US Patent Application
Publication Number 2003/0074698, each of which is herein
incorporated by reference.
[0190] The method for modulating shoot and/or leaf development in a
plant comprises modulating the activity and/or level of a
dimerization domain polypeptide of the invention. In one
embodiment, a dimerization domain sequence of the invention is
provided. In other embodiments, the dimerization domain nucleotide
sequence can be provided by introducing into the plant a
polynucleotide comprising a dimerization domain nucleotide sequence
of the invention, expressing the dimerization domain sequence and
thereby modifying shoot and/or leaf development. In other
embodiments, the dimerization domain nucleotide construct
introduced into the plant is stably incorporated into the genome of
the plant.
[0191] In specific embodiments, shoot or leaf development is
modulated by decreasing the level and/or activity of the
dimerization domain polypeptide in the plant. An decrease in
dimerization domain activity can result in at least one or more of
the following alterations in shoot and/or leaf development,
including, but not limited to, reduced leaf number, reduced leaf
surface, reduced vascular, shorter internodes and stunted growth
and retarded leaf senescence when compared to a control plant.
[0192] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate shoot and leaf development
of the plant. Exemplary promoters for this embodiment include
constitutive promoters, shoot-preferred promoters, shoot
meristem-preferred promoters and leaf-preferred promoters.
Exemplary promoters have been disclosed elsewhere herein.
[0193] Decreasing dimerization domain activity and/or level in a
plant results in shorter internodes and stunted growth. Thus, the
methods of the invention find use in producing dwarf plants. In
addition, as discussed above, modulation of dimerization domain
activity in the plant modulates both root and shoot growth. Thus,
the present invention further provides methods for altering the
root/shoot ratio. Shoot or leaf development can further be
modulated by decreasing the level and/or activity of the
dimerization domain polypeptide in the plant.
[0194] Accordingly, the present invention further provides plants
having modulated shoot and/or leaf development when compared to a
control plant. In some embodiments, the plant of the invention has
an increased level/activity of the dimerization domain polypeptide
of the invention, altering the shoot and/or leaf development. Such
alterations include, but are not limited to, increased leaf number,
increased leaf surface, increased vascularity, longer internodes
and increased plant stature, as well as alterations in leaf
senescence, as compared to a control plant. In other embodiments,
the plant of the invention has a decreased level/activity of the
dimerization domain polypeptide of the invention.
[0195] vi Modulating Reproductive Tissue Development
[0196] Methods for modulating reproductive tissue development are
provided. In one embodiment, methods are provided to modulate
floral development in a plant. By "modulating floral development"
is intended any alteration in a structure of a plant's reproductive
tissue as compared to a control plant in which the activity or
level of the dimerization domain polypeptide has not been
modulated. "Modulating floral development" further includes any
alteration in the timing of the development of a plant's
reproductive tissue (i.e., a delayed or an accelerated timing of
floral development) when compared to a control plant in which the
activity or level of the dimerization domain polypeptide has not
been modulated. Macroscopic alterations may include changes in
size, shape, number or location of reproductive tissues, the
developmental time period that these structures form or the ability
to maintain or proceed through the flowering process in times of
environmental stress. Microscopic alterations may include changes
to the types or shapes of cells that make up the reproductive
tissues.
[0197] The method for modulating floral development in a plant
comprises modulating dimerization domain activity in a plant. In
one method, a dimerization domain sequence of the invention is
provided. A dimerization domain nucleotide sequence can be provided
by introducing into the plant a polynucleotide comprising a
dimerization domain nucleotide sequence of the invention,
expressing the dimerization domain sequence and thereby modifying
floral development. In other embodiments, the dimerization domain
nucleotide construct introduced into the plant is stably
incorporated into the genome of the plant.
[0198] In specific methods, floral development is modulated by
decreasing the level or activity of the dimerization domain
polypeptide in the plant. A decrease in dimerization domain
activity can result in at least one or more of the following
alterations in floral development, including, but not limited to,
retarded flowering, reduced number of flowers, partial male
sterility and reduced seed set when compared to a control plant.
Inducing delayed flowering or inhibiting flowering can be used to
enhance yield in forage crops such as alfalfa. Methods for
measuring such developmental alterations in floral development are
known in the art. See, for example, Mouradov, et al., (2002) The
Plant Cell S111-S130, herein incorporated by reference.
[0199] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate floral development of the
plant. Exemplary promoters for this embodiment include constitutive
promoters, inducible promoters, shoot-preferred promoters and
inflorescence-preferred promoters.
[0200] In other methods, floral development is modulated by
increasing the level and/or activity of the dimerization domain
sequence of the invention. Such methods can comprise introducing a
dimerization domain nucleotide sequence into the plant and
increasing the activity of the dimerization domain polypeptide. In
other methods, the dimerization domain nucleotide construct
introduced into the plant is stably incorporated into the genome of
the plant. Increasing expression of the dimerization domain
sequence of the invention can modulate floral development during
periods of stress. Such methods are described elsewhere herein.
Accordingly, the present invention further provides plants having
modulated floral development when compared to the floral
development of a control plant. Compositions include plants having
an increased level/activity of the dimerization domain polypeptide
of the invention and having an altered floral development.
Compositions also include plants having an increased level/activity
of the dimerization domain polypeptide of the invention wherein the
plant maintains or proceeds through the flowering process in times
of stress.
[0201] Methods are also provided for the use of the dimerization
domain of the invention to increase seed size and/or weight. The
method comprises increasing the activity of the dimerization domain
in a plant or plant part, such as the seed. An increase in seed
size and/or weight comprises an increased size or weight of the
seed and/or an increase in the size or weight of one or more seed
part including, for example, the embryo, endosperm, seed coat,
aleurone or cotyledon.
[0202] As discussed above, one of skill will recognize the
appropriate promoter to use to increase seed size and/or seed
weight. Exemplary promoters of this embodiment include constitutive
promoters, inducible promoters, seed-preferred promoters,
embryo-preferred promoters and endosperm-preferred promoters.
[0203] The method for decreasing seed size and/or seed weight in a
plant comprises decreasing dimerization domain activity in the
plant. In one embodiment, the dimerization domain nucleotide
sequence can be provided by introducing into the plant a
polynucleotide comprising a dimerization domain nucleotide sequence
of the invention, expressing the dimerization domain sequence and
thereby increasing seed weight and/or size. In other embodiments,
the dimerization domain nucleotide construct introduced into the
plant is stably incorporated into the genome of the plant.
[0204] It is further recognized that increasing seed size and/or
weight can also be accompanied by an increase in the speed of
growth of seedlings or an increase in early vigor. As used herein,
the term "early vigor" refers to the ability of a plant to grow
rapidly during early development and relates to the successful
establishment, after germination, of a well-developed root system
and a well-developed photosynthetic apparatus. In addition, an
increase in seed size and/or weight can also result in an increase
in plant yield when compared to a control.
[0205] Accordingly, the present invention further provides plants
having an increased seed weight and/or seed size when compared to a
control plant. In other embodiments, plants having an increased
vigor and plant yield are also provided. In some embodiments, the
plant of the invention has an increased level/activity of the
dimerization domain polypeptide of the invention and has an
increased seed weight and/or seed size. In other embodiments, such
plants have stably incorporated into their genome a nucleic acid
molecule comprising a dimerization domain nucleotide sequence of
the invention operably linked to a promoter that drives expression
in the plant cell.
[0206] vii. Method of Use for Dimerization Domain Promoter
Polynucleotides
[0207] The polynucleotides comprising the dimerization domain
promoters disclosed in the present invention, as well as variants
and fragments thereof, are useful in the genetic manipulation of
any host cell, preferably plant cell, when assembled with a DNA
construct such that the promoter sequence is operably linked to a
nucleotide sequence comprising a polynucleotide of interest. In
this manner, the dimerization domain promoter polynucleotides of
the invention are provided in expression cassettes along with a
polynucleotide sequence of interest for expression in the host cell
of interest. The dimerization domain promoter sequences of the
invention are expressed in a variety of tissues containing cells
that have dimerization domain and thus the promoter sequences can
find use in regulating the temporal and/or the spatial expression
of polynucleotides of interest particularly in the dimerization
domain containing cells.
[0208] Synthetic hybrid promoter regions are known in the art. Such
regions comprise upstream promoter elements of one polynucleotide
operably linked to the promoter element of another polynucleotide.
In an embodiment of the invention, heterologous sequence expression
is controlled by a synthetic hybrid promoter comprising the
dimerization domain promoter sequences of the invention or a
variant or fragment thereof, operably linked to upstream promoter
element(s) from a heterologous promoter. Upstream promoter elements
that are involved in the plant defense system have been identified
and may be used to generate a synthetic promoter. See, for example,
Rushton, et al., (1998) Curr. Opin. Plant Biol. 1:311-315.
Alternatively, a synthetic dimerization domain promoter sequence
may comprise duplications of the upstream promoter elements found
within the dimerization domain promoter sequences.
[0209] It is recognized that the promoter sequence of the invention
may be used with its native dimerization domain coding sequences. A
DNA construct comprising the dimerization domain promoter operably
linked with its native dimerization domain gene may be used to
transform any plant of interest to bring about a desired phenotypic
change, such as modulating cell number, modulating root, shoot,
leaf, floral and embryo development, stress tolerance and any other
phenotype described elsewhere herein.
[0210] The promoter nucleotide sequences and methods disclosed
herein are useful in regulating expression of any heterologous
nucleotide sequence in a host plant in order to vary the phenotype
of a plant. Various changes in phenotype are of interest including
modifying the fatty acid composition in a plant, altering the amino
acid content of a plant, altering a plant's pathogen defense
mechanism, and the like. These results can be achieved by providing
expression of heterologous products or increased expression of
endogenous products in plants. Alternatively, the results can be
achieved by providing for a reduction of expression of one or more
endogenous products, particularly enzymes or cofactors in the
plant. These changes result in a change in phenotype of the
transformed plant.
[0211] Genes of interest are reflective of the commercial markets
and interests of those involved in the development of the crop.
Crops and markets of interest change, and as developing nations
open up world markets, new crops and technologies will emerge also.
In addition, as our understanding of agronomic traits and
characteristics such as yield and heterosis increase, the choice of
genes for transformation will change accordingly. General
categories of genes of interest include, for example, those genes
involved in information, such as zinc fingers, those involved in
communication, such as kinases and those involved in housekeeping,
such as heat shock proteins. More specific categories of
transgenes, for example, include genes encoding important traits
for agronomics, insect resistance, disease resistance, herbicide
resistance, sterility, grain characteristics and commercial
products. Genes of interest include, generally, those involved in
oil, starch, carbohydrate or nutrient metabolism as well as those
affecting kernel size, sucrose loading and the like.
[0212] In certain embodiments the nucleic acid sequences of the
present invention can be used in combination ("stacked") with other
polynucleotide sequences of interest in order to create plants with
a desired phenotype. The combinations generated can include
multiple copies of any one or more of the polynucleotides of
interest. The polynucleotides of the present invention may be
stacked with any gene or combination of genes to produce plants
with a variety of desired trait combinations, including but not
limited to traits desirable for animal feed such as high oil genes
(e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g.,
hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802 and
5,703,409); barley high lysine (Williamson, et al., (1987) Eur. J.
Biochem. 165:99-106 and WO 98/20122) and high methionine proteins
(Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et
al., (1988) Gene 71:359 and Musumura, et al., (1989) Plant Mol.
Biol. 12:123)); increased digestibility (e.g., modified storage
proteins (U.S. patent application Ser. No. 10/053,410, filed Nov.
7, 2001) and thioredoxins (U.S. patent application Ser. No.
10/005,429, filed Dec. 3, 2001)), the disclosures of which are
herein incorporated by reference. The polynucleotides of the
present invention can also be stacked with traits desirable for
insect, disease or herbicide resistance (e.g., Bacillus
thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450;
5,737,514; 5,723,756; 5,593,881; Geiser, et al., (1986) Gene
48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol.
24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931);
avirulence and disease resistance genes (Jones, et al., (1994)
Science 266:789; Martin, et al., (1993) Science 262:1432;
Mindrinos, et al., (1994) Cell 78:1089); acetolactate synthase
(ALS) mutants that lead to herbicide resistance such as the S4
and/or Hra mutations; inhibitors of glutamine synthase such as
phosphinothricin or basta (e.g., bar gene); and glyphosate
resistance (EPSPS gene)) and traits desirable for processing or
process products such as high oil (e.g., U.S. Pat. No. 6,232,529);
modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.
5,952,544; WO 94/11516)); modified starches (e.g., ADPG
pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE) and starch debranching enzymes (SDBE)) and
polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;
beta-ketothiolase, polyhydroxybutyrate synthase and acetoacetyl-CoA
reductase (Schubert, et al., (1988) J. Bacteriol. 170:5837-5847)
facilitate expression of polyhydroxyalkanoates (PHAs)), the
disclosures of which are herein incorporated by reference. One
could also combine the polynucleotides of the present invention
with polynucleotides affecting agronomic traits such as male
sterility (e.g., see, U.S. Pat. No. 5,583,210), stalk strength,
flowering time or transformation technology traits such as cell
cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364;
WO 99/25821), the disclosures of which are herein incorporated by
reference.
[0213] In one embodiment, sequences of interest improve plant
growth and/or crop yields. For example, sequences of interest
include agronomically important genes that result in improved
primary or lateral root systems. Such genes include, but are not
limited to, nutrient/water transporters and growth induces.
Examples of such genes, include but are not limited to, maize
plasma membrane H.sup.+-ATPase (MHA2) (Frias, et al., (1996) Plant
Cell 8:1533-44); AKT1, a component of the potassium uptake
apparatus in Arabidopsis, (Spalding, et al., (1999) J Gen Physiol
113:909-18); RML genes which activate cell division cycle in the
root apical cells (Cheng, et al., (1995) Plant Physiol 108:881);
maize glutamine synthetase genes (Sukanya, et al., (1994) Plant Mol
Biol 26:1935-46) and hemoglobin (Duff, et al., (1997) J. Biol. Chem
27:16749-16752, Arredondo-Peter, et al., (1997) Plant Physiol.
115:1259-1266; Arredondo-Peter, et al., (1997) Plant Physiol
114:493-500 and references sited therein). The sequence of interest
may also be useful in expressing antisense nucleotide sequences of
genes that that negatively affects root development.
[0214] Additional, agronomically important traits such as oil,
starch and protein content can be genetically altered in addition
to using traditional breeding methods. Modifications include
increasing content of oleic acid, saturated and unsaturated oils,
increasing levels of lysine and sulfur, providing essential amino
acids and also modification of starch. Hordothionin protein
modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801,
5,885,802 and 5,990,389, herein incorporated by reference. Another
example is lysine and/or sulfur rich seed protein encoded by the
soybean 2S albumin described in U.S. Pat. No. 5,850,016 and the
chymotrypsin inhibitor from barley, described in Williamson, et
al., (1987) Eur. J. Biochem. 165:99-106, the disclosures of which
are herein incorporated by reference.
[0215] Derivatives of the coding sequences can be made by
site-directed mutagenesis to increase the level of preselected
amino acids in the encoded polypeptide. For example, the gene
encoding the barley high lysine polypeptide (BHL) is derived from
barley chymotrypsin inhibitor, U.S. patent application Ser. No.
08/740,682, filed Nov. 1, 1996 and WO 98/20133, the disclosures of
which are herein incorporated by reference. Other proteins include
methionine-rich plant proteins such as from sunflower seed (Lilley,
et al., (1989) Proceedings of the World Congress on Vegetable
Protein Utilization in Human Foods and Animal Feedstuffs, ed.
Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.
497-502; herein incorporated by reference); corn (Pedersen, et al.,
(1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene
71:359, both of which are herein incorporated by reference) and
rice (Musumura, et al., (1989) Plant Mol. Biol. 12:123, herein
incorporated by reference). Other agronomically important genes
encode latex, Floury 2, growth factors, seed storage factors and
transcription factors.
[0216] Insect resistance genes may encode resistance to pests that
have great yield drag such as rootworm, cutworm, European Corn
Borer and the like. Such genes include, for example, Bacillus
thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892;
5,747,450; 5,736,514; 5,723,756; 5,593,881 and Geiser, et al.,
(1986) Gene 48:109), and the like.
[0217] Genes encoding disease resistance traits include
detoxification genes, such as against fumonosin (U.S. Pat. No.
5,792,931); avirulence (avr) and disease resistance (R) genes
(Jones, et al., (1994) Science 266:789; Martin, et al., (1993)
Science 262:1432 and Mindrinos, et al., (1994) Cell 78:1089), and
the like.
[0218] Herbicide resistance traits may include genes coding for
resistance to herbicides that act to inhibit the action of
acetolactate synthase (ALS), in particular the sulfonylurea-type
herbicides (e.g., the acetolactate synthase (ALS) gene containing
mutations leading to such resistance, in particular the S4 and/or
Hra mutations), genes coding for resistance to herbicides that
inhibit enol-pyruvylshikimate phosphate synthase (EPSPS), e.g.,
glyphosate acetyl transferase (GAT), genes coding for resistance to
herbicides that act to inhibit action of glutamine synthase, such
as phosphinothricin or basta (e.g., the bar gene), a combination
thereof or other such genes known in the art. The bar gene encodes
resistance to the herbicide basta, the nptII gene encodes
resistance to the antibiotics kanamycin and geneticin and the
ALS-gene mutants encode resistance to the herbicide
chlorsulfuron.
[0219] Sterility genes can also be encoded in an expression
cassette and provide an alternative to physical detasseling.
Examples of genes used in such ways include male tissue-preferred
genes and genes with male sterility phenotypes such as QM,
described in U.S. Pat. No. 5,583,210. Other genes include kinases
and those encoding compounds toxic to either male or female
gametophytic development.
[0220] The quality of grain is reflected in traits such as levels
and types of oils, saturated and unsaturated, quality and quantity
of essential amino acids and levels of cellulose. In corn, modified
hordothionin proteins are described in U.S. Pat. Nos. 5,703,049,
5,885,801, 5,885,802 and 5,990,389.
[0221] Commercial traits can also be encoded on a gene or genes
that could increase for example, starch for ethanol production, or
provide expression of proteins. Another important commercial use of
transformed plants is the production of polymers and bioplastics
such as described in U.S. Pat. No. 5,602,321. Genes such as
13-Ketothiolase, PHBase (polyhydroxyburyrate synthase) and
acetoacetyl-CoA reductase (see, Schubert, et al., (1988) J.
Bacteriol. 170:5837-5847) facilitate expression of
polyhyroxyalkanoates (PHAs).
[0222] Exogenous products include plant enzymes and products as
well as those from other sources including procaryotes and other
eukaryotes. Such products include enzymes, cofactors, hormones and
the like. The level of proteins, particularly modified proteins
having improved amino acid distribution to improve the nutrient
value of the plant, can be increased. This is achieved by the
expression of such proteins having enhanced amino acid content.
[0223] This invention can be better understood by reference to the
following non-limiting examples. It will be appreciated by those
skilled in the art that other embodiments of the invention may be
practiced without departing from the spirit and the scope of the
invention as herein disclosed and claimed.
Examples
Example 1
Yield and Harvest Index Tests--RT810ZBS_T (Intro-EF09B/GR1B5)
[0224] PHP26963 (S2a:D8MPL+S2a:CesA10)--10 evts [0225] PHP26998
(S2a:D8mpl+Nas2:DD+S2a:CesA10)--8 evts [0226] PHP24843
(S2a:D8MPL+NAS2:DD)--4 evts [0227] Construct Nulls (3 events/null)
[0228] WT (Intro-EF09BZTZ/GR1B5) 2 Densities [0229] 36,000 PPA (JH,
MR)--Yield (5 reps) [0230] 48,000 PPA (JH, MR)--Yield (5 reps),
Harvest Index (3 reps)
[0231] Three constructs were tested at Johnston (JH) and Marion
(MR) Iowa at two densities, 36,000 plants per acre (PPA) and 48,000
PPA in 20'' row width. The genes tested consisted of the dwarf
mutant D8mpl and the additional genes (stacks) DD (dimerization
domain of the D8 gene) or the Ces A10 gene. The constructs are
shown below in which the transgenic events are shown with their
plasmid identification and nulls (segregating non transgenic sibs)
are shown with their plasmid designation and the letter n.
TABLE-US-00002 TABLE 2 D8mpl + DD php24843 E7216.51.1.1 php24843n
E7216.49.1.5 D8 + CesA php26963 E7216.49.2.1 php26963 E7216.49.2.2
php26963 E7216.49.3.1 php26963n CN D8 + DD + CesA php26998
E7216.50.1.1 php26998 E7216.50.1.3 php26998n CN
[0232] A higher plant population density was chosen to determine if
the dwarf and dwarf stack transgenic plants behaved the same or
differently than the construct null sibs (no transgenes with normal
height) for yield and harvest index. In general, corn shows a
decline in yield in populations above the optimum economic yield
such that yield levels decline. Harvest index in corn has been
relatively stable, from 45 to 50% as defined by ear dry
matter/total above ground dry matter. Increases in biomass and
harvest index are the major determinants of yield, thus a positive
change in either attribute could lead to higher potential
yield.
[0233] The yield comparison for selected events of the different
constructs at the Johnston and Marion locations was performed. In
general, the reduction in yield levels due to high plant population
was more pronounced in the null sibs compared to the respective
transgenic stack constructs. In some instances, the transgenic
treatments showed an unexpected and increased yield response,
particularly in the Marion, Iowa location. Such an observation
indicates that further breeding with a variety of different
germplasm sources in addition to those used with these transgene
stacks or additional optimization of the agronomic factors such as
row width, fertilization practices or optimized plant population
for the dwarf phenotype would further improve yield potential.
[0234] Harvest index of the entries at the higher population
density of 48,000 PPA was measured. Generally, the harvest index of
the null sibs were just between 0.5 and 0.52 while most of the
transgenic stacks had a harvest index in the range of 0.54 to 0.58.
The increase in harvest index could be expected to make better use
of available soil moisture and nutrients since a greater proportion
of the dry matter produced is in the form of grain.
Example 2
Yield and Harvest Index Tests--(Intro-EF09B/HG11)
[0235] Topcrosses were made from PHP26963, PHP26998 and PHP24843 T0
plants onto HG11 females. This produced a background genotype
similar to commercial hybrid 33A14 which could be used as a
reference. These plants were then grown in Johnston observation
plots. A small planting of PHP17881 hybrids were also included.
[0236] PHP26963 (S2a:D8MPL+S2a:CesA10)--2 evts, 6 rows [0237]
PHP26998 (S2a:D8mpl+Nas2:DD+S2a:CesA10)--2 evts, 6 rows [0238]
PHP24843 (S2a:D8MPL+NAS2:DD)--2 evts, 8 rows [0239] PHP17881
(S2a:D8MPL) [0240] WT (EF09B/HG11-33A14)--11 rows
[0241] Minirhizotron tubes were inserted in the soil near these
plants to allow for imaging of roots that intersected the tubes.
This allowed for a direct measurement of root length of the NAS2:DD
stack constructs (FIGS. 2-5). The DD stacks had longer root systems
at earlier time points and appeared to be colonizing the soil more
rapidly than the non DD counterparts. The later time point showed
the non-DD constructs having similar root lengths to the DD stacks
at more shallow depths, but not yet fully colonizing the lowest
depth of soil measured. The surface area of these plants increased
proportionately with the length, indicating that there is no
sacrifice of root width. Plant height and yields were consistent
with previous observations of S2a:D8MPL constructs (FIG. 4 and
Table 3--Heights from field experiments).
TABLE-US-00003 TABLE 3 Average Height Standard Deviation (m) (m)
33A14 2.90 0.06 PHP17881 (D8 MPL) 1.99 0.28 PHP24843 (D8/DD) 1.96
0.06 PHP26963 (D8/CES) 2.10 0.09 PHP26998(D8/DD/CES) 1.90 0.10
Example 3
Greenhouse Grown Transgenic Stacks
[0242] Three constructs were tested in the introEF09B background at
the T0 generation to determine the agronomic characteristics of the
D8 dimerization domain stacks and for preparation for field
testing. Each stacked construct (PHP24843, PHP26963 and PHP26998)
utilized the S2A PRO:D8MPL gene, NAS2 PRO:D8 243-331 and/or S2A
PRO:ZM-CES A10. [0243] Genes Tested (Intro EF09B) [0244] S2a:D8MPL
(Vascular Element Preferred Promoter:moderate dwarfing Gene) [0245]
Nas2:DD (Root Preferred Promoter: Leucine Zipper Dimerization
Domain) [0246] S2a:CesA10 (Vascular Element Preferred
Promoter:Cellulose Synthase Gene in Stalk Tissue. [0247] Gene
Combinations ("Stacks") of Two and Three Genes [0248]
PHP24843--NAS2 PRO:D8 243-331/S2A PRO:D8MPL Stack (13 events)
[0249] PHP26963--S2A PRO:ZM-CES A10/S2A PRO:D8MPL Stack (15 events)
[0250] PHP26998--NAS2 PRO:D8 243-331/S2A PRO:ZM-CES A10/S2A
PRO:D8MPL (14 events)
[0251] Morphometric analyses were performed on the mature T0 plants
from this experiment (FIG. 6). The NAS2 PRO:D8 243-331 gene
increased leaf width and area in this experiment. The S2A
PRO:ZM-CES A10 gene increased leaf angle, decreased leaf length and
increased seed number.
Example 4
Greenhouse Grown Transgenic Stacks
[0252] Five constructs were tested in GS3.times.GF3 at the T0
generation to determine the effectiveness of the D8 dimerization
domain for reversing dwarfing of the maize root system. Each
stacked construct (PHP24843, PHP24844 and PHP24861) utilized a
different root preferred promoter to drive expression the D8
243-331 coding sequence. [0253] Genes Tested (GS3.times.GF#) [0254]
S2a:D8MPL (Vascular Element Preferred Promoter:moderate dwarfing
Gene) [0255] Nas2:DD (Root Preferred Promoter: Leucine Zipper
Dimerization Domain) [0256] ZRP2.47 PRO:D8 243-331 (Root Preferred
Promoter: Leucine Zipper Dimerization Domain) [0257] ROOTMET2
PRO:D8 243-331 (Root Preferred Promoter: Leucine Zipper
Dimerization Domain) [0258] ROOTMET2 PRO:GUSINT (Root Preferred
Promoter: .beta.-glucuronidase reporter gene) [0259] Gene
Combinations ("Stacks") of Two Genes [0260] PHP24843--NAS2 PRO:D8
243-331/S2A PRO:D8MPL Stack (25 events) [0261] PHP24844--ZRP2.47
PRO:D8 243-331/S2A PRO:D8MPL Stack (23 events) [0262]
PHP24861--ROOTMET2 PRO:D8 243-331/S2A PRO:D8MPL Stack (22 events)
[0263] PHP17881--S2A PRO:D8MPL (Dwarf Control) (14 events) [0264]
PHP23206--ROOTMET2 PRO:GUSINT (Full Size Control) (14 events)
[0265] Morphometric analyses were performed on the mature T0 plants
from this experiment (FIG. 7). The findings were that root weight
was not significantly altered. The expected root change is in root
length, which could not be measured due to root bound growth in
greenhouse pots. Each construct with the S2A PRO:D8MPL gene
displayed a reduced stature with plant height reduced by
.about.25-35%. Stalk weight was lower in the dimerization domain
constructs than in the S2A PRO:D8MPL alone, which was in-turn lower
than the full size control. Leaf weight and seed number were
reduced in PHP24844, PHP24861 and PHP17881 compared to the full
size control; however, PHP24843 (NAS2 PRO:D8 243-331/S2A PRO:D8MPL
Stack) retained leaf weight and seed numbers equal to those of the
full size control. Seed number is a component of yield and stalk
weight is a component of biomass, indicating that PHP24843 may
increase harvest index.
Example 5
Greenhouse Grown D8 Dimerization Domain Transgenics
[0266] Four constructs were tested in GS3.times.GF3 at the T0
generation to determine the effects of the D8 dimerization domain
when expressed in roots in a non-stacked configuration. Each
stacked construct (PHP24711, PHP24712 and PHP24713) utilized a
different root preferred promoter to drive expression the D8
243-331 coding sequence. [0267] Genes Tested (GS3.times.GF#) [0268]
Nas2:DD (Root Preferred Promoter: Leucine Zipper Dimerization
Domain) [0269] ZRP2.47 PRO:D8 243-331 (Root Preferred Promoter:
Leucine Zipper Dimerization Domain) [0270] ROOTMET2 PRO:D8 243-331
(Root Preferred Promoter: Leucine Zipper Dimerization Domain)
[0271] Gene Constructs [0272] PHP24711--ZRP2.47 PRO:D8 243-331 (25
events) [0273] PHP24712--ROOTMET2 PRO:D8 243-331 (25 events) [0274]
PHP24713--NAS2 PRO:D8 243-331 (25 events) [0275] PHP24715--S2A
PRO:AC-GFP1 (Full Size Control) (25 events)
[0276] Morphometric analyses were performed on the mature T0 plants
from this experiment (FIG. 8). Stalk weight and seed number were
increased in the ROOTMET2 PRO:D8 243-331 and NAS2 PRO:D8 243-331
constructs.
Example 6
Two Location, 3 Construct, Yield and Harvest Index Trial
[0277] Yield and harvest index comparisons were made with three
different constructs in genotype "Intro EF09B/GR1B5" compared to
their respective construct nulls. The data is described in Table 4.
The yield and harvest index was measured in replicated experiments
(5) at 48,000 PPA seeded in 20'' rows in Johnston and Marion Iowa
using a randomized complete block design. Generally, the semi-dwarf
plant height was approximately 60-70% of the construct nulls (CN)
with each construct. The phP29693 and phP26998 plant height was
about 65% (about 12'' taller) than phP24843. Compared to the
construct nulls, yield in Johnston was near equal to the construct
null with the events shown in this table. Harvest index was
significantly higher in the Johnston location. At the Marion
location, several constructs/events were higher in yield than their
respective construct nulls. Harvest index was numerically higher
and in most cases significantly higher than their respective
construct nulls. The semi-dwarf transgenics had a better yield
response at high populations compared to the construct nulls when
grown at a lower population of 36,000 ppa. The `stacked`
combinations of D8mpl+DD was not significantly lower than the
construct null for yield but had higher harvest index at both
locations. The combination in construct phP26963 had higher yield
potential in Johnston and Marion with higher harvest index. The
combination of D8mpl+DD+CesA 10 had similar or equal yield and
higher harvest index in Johnston while the triple gene stack in the
Marion, Iowa location shows lower yields but higher harvest index.
Although there were some individual plots that showed root and
stalk lodging, neither location had significant differences between
the transgenic and their construct nulls.
TABLE-US-00004 TABLE 4 % JH % MR JH of HI MR of HI Gene PHP ID #
yield null (%) yield null (%) 0.57 0.56 D8mpl/ control E7216.51.1.1
168 95% (110) 152 98% (103) DD CN 177 0.51 156 0.54 0.58 0.54 D8/
26963 E7216.49.1.5 163 92 (114) 141 105% (111) CesA 0.56 0.58
E7216.49.2.1 172 97% (111) 147 110% (112) 0.56 0.53 E7216.49.2.2
176 100% (111) 152 113% (104) 0.53 0.55 E7216.49.3.1 176 99% (104)
151 113% (108) CN 177 0.51 134 0.51 0.56 0.58 D8/DD/ 26998
E7216.50.1.1 158 93% (109) 133 90% (113) CesA 0.57 0.54
E7216.50.1.3 168 99% (110) 133 90% (104) CN 169 0.51 149 0.52 HI =
Harvest Index JH = Johnston MR = Marion
Example 7
Transformation and Regeneration of Transgenic Plants
[0278] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing the Zm dimerization domain
sequence operably linked to the drought-inducible promoter RAB17
promoter (Vilardell, et al., (1990) Plant Mol Biol 14:423-432) and
the selectable marker gene PAT, which confers resistance to the
herbicide Bialaphos. Alternatively, the selectable marker gene is
provided on a separate plasmid. Transformation is performed as
follows. Media recipes follow below.
[0279] Preparation of Target Tissue:
[0280] The ears are husked and surface sterilized in 30%
Clorox.RTM. bleach plus 0.5% Micro detergent for 20 minutes and
rinsed two times with sterile water. The immature embryos are
excised and placed embryo axis side down (scutellum side up), 25
embryos per plate, on 560Y medium for 4 hours and then aligned
within the 2.5-cm target zone in preparation for bombardment.
[0281] Preparation of DNA:
[0282] A plasmid vector comprising the dimerization domain sequence
operably linked to an ubiquitin promoter is made. This plasmid DNA
plus plasmid DNA containing a PAT selectable marker is precipitated
onto 1.1 .mu.m (average diameter) tungsten pellets using a
CaCl.sub.2 precipitation procedure as follows:
[0283] 100 .mu.l prepared tungsten particles in water
[0284] 10 .mu.l (1 .mu.g) DNA in Tris EDTA buffer (1 .mu.g total
DNA)
[0285] 100 .mu.l 2.5M CaCl.sub.2
[0286] 10 .mu.l 0.1 M spermidine
[0287] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 ml 100%
ethanol and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.l spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
[0288] Particle Gun Treatment:
[0289] The sample plates are bombarded at level #4 in particle gun
#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
[0290] Subsequent Treatment:
[0291] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288J medium to initiate plant
regeneration. Following somatic embryo maturation (2-4 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5'' pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
for increased drought tolerance. Assays to measure improved drought
tolerance are routine in the art and include, for example,
increased kernel-earring capacity yields under drought conditions
when compared to control maize plants under identical environmental
conditions. Alternatively, the transformed plants can be monitored
for a modulation in meristem development (i.e., a decrease in
spikelet formation on the ear). See, for example, Bruce, et al.,
(2002) Journal of Experimental Botany 53:1-13.
[0292] Bombardment and Culture Media:
[0293] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts
(SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000.times.
SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l
2,4-D and 2.88 g/l L-proline (brought to volume with D-I H.sub.2O
following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite.RTM.
(added after bringing to volume with D-I H.sub.2O) and 8.5 mg/l
silver nitrate (added after sterilizing the medium and cooling to
room temperature). Selection medium (560R) comprises 4.0 g/l N6
basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times. SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose
and 2.0 mg/l 2,4-D (brought to volume with D-I H.sub.2O following
adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite.RTM. (added after
bringing to volume with D-I H.sub.2O) and 0.85 mg/l silver nitrate
and 3.0 mg/l bialaphos (both added after sterilizing the medium and
cooling to room temperature).
[0294] Plant regeneration medium (288J) comprises 4.3 g/l MS salts
(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and
0.40 g/l glycine brought to volume with polished D-I H.sub.2O)
(Murashige and Skoog, (1962) Physiol. Plant. 15:473), 100 mg/l
myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose and 1.0 ml/l of 0.1
mM abscisic acid (brought to volume with polished D-I H.sub.2O
after adjusting to pH 5.6); 3.0 g/l Gelrite.RTM. (added after
bringing to volume with D-I H.sub.2O) and 1.0 mg/l indoleacetic
acid and 3.0 mg/l bialaphos (added after sterilizing the medium and
cooling to 60.degree. C.). Hormone-free medium (272V) comprises 4.3
g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution
(0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l
pyridoxine HCL and 0.40 g/l glycine brought to volume with polished
D-I H.sub.2O), 0.1 g/l myo-inositol and 40.0 g/l sucrose (brought
to volume with polished D-I H.sub.2O after adjusting pH to 5.6) and
6 g/l Bacto.TM.-agar (added after bringing to volume with polished
D-I H.sub.2O), sterilized and cooled to 60.degree. C.
Example 8
Agrobacterium-Mediated Transformation
[0295] For Agrobacterium-mediated transformation of maize with an
antisense sequence of the Zmdimerization domain sequence of the
present invention, preferably the method of Zhao is employed (U.S.
Pat. No. 5,981,840 and PCT Patent Publication WO98/32326, the
contents of which are hereby incorporated by reference). Briefly,
immature embryos are isolated from maize and the embryos contacted
with a suspension of Agrobacterium, where the bacteria are capable
of transferring the dimerization domain sequence to at least one
cell of at least one of the immature embryos (step 1: the infection
step). In this step the immature embryos are preferably immersed in
an Agrobacterium suspension for the initiation of inoculation. The
embryos are co-cultured for a time with the Agrobacterium (step 2:
the co-cultivation step). Preferably the immature embryos are
cultured on solid medium following the infection step. Following
this co-cultivation period an optional "resting" step is
contemplated. In this resting step, the embryos are incubated in
the presence of at least one antibiotic known to inhibit the growth
of Agrobacterium without the addition of a selective agent for
plant transformants (step 3: resting step). Preferably the immature
embryos are cultured on solid medium with antibiotic, but without a
selecting agent, for elimination of Agrobacterium and for a resting
phase for the infected cells. Next, inoculated embryos are cultured
on medium containing a selective agent and growing transformed
callus is recovered (step 4: the selection step). Preferably, the
immature embryos are cultured on solid medium with a selective
agent resulting in the selective growth of transformed cells. The
callus is then regenerated into plants (step 5: the regeneration
step) and preferably calli grown on selective medium are cultured
on solid medium to regenerate the plants. Plants are monitored and
scored for a modulation in meristem development. For instance,
alterations of size and appearance of the shoot and floral
meristems and/or increased yields of leaves, flowers and/or fruits
are monitored.
Example 9
Sugar Cane Transformation
[0296] This protocol describes routine conditions for production of
transgenic sugarcane lines. The same conditions are close to
optimal for number of transiently expressing cells following
bombardment into embryogenic sugarcane callus. See also, Bower, et
al., (1996). Molec Breed 2:239-249; Birch and Bower, (1994).
Principles of gene transfer using particle bombardment. In Particle
Bombardment Technology for Gene Transfer, Yang and Christou, eds
(New York: Oxford University Press), pp. 3-37 and Santosa, et al.,
(2004), Molecular Biotechnology 28:113-119, incorporated herein by
reference.
Sugarcane Transformation Protocol:
1. Subculture Callus on MSC3, 4 Days Prior to Bombardment:
[0297] (a) Use actively growing embryogenic callus (predominantly
globular pro-embryoids rather than more advanced stages of
differentiation) for bombardment and through the subsequent
selection period. [0298] (b) Divide callus into pieces around 5 mm
in diameter at the time of subculture and use forceps to make a
small crater in the agar surface for each transferred callus piece.
[0299] (c) Incubate at 28.degree. C. in the dark, in deep (25 mm)
Petri dishes with micropore tape seals for gas exchange. 2. Place
embryogenic callus pieces in a circle (.about.2.5 cm diameter), on
MSC3Osm medium. Incubate for 4 hours prior to bombardment. 3.
Sterilize 0.7 .mu.m diameter tungsten (Grade M-10, Bio-Rad
#165-2266) in absolute ethanol. Vortex the suspension, then pellet
the tungsten in a microfuge for .about.30 seconds. Draw off the
supernatant and resuspend the particles at the same concentration
in sterile H.sub.20. Repeat the washing step with sterile H.sub.20
twice and thoroughly resuspend particles before transferring 50
.mu.l aliquots into microfuge tubes. 4. Add the precipitation mix
components:
TABLE-US-00005 [0299] Component (stock solution) Volume to add
Final conc in mix Tungsten (100 .mu.g/.mu.l in H.sub.20) 50 .mu.l
38.5 .mu.g/.mu.l DNA (1 .mu.g/.mu.l) 10 .mu.l 0.38 .mu.g/.mu.l
CaCl.sub.2 (2.5M in H20) 50 .mu.l 963 mM Spermidine free base (0.1M
in H.sub.20) 20 .mu.l 15 mM
5. Allow the mixture to stand on ice for 5 min. During this time,
complete steps 6-8 below. 6. Disinfect the inside of the `gene gun`
target chamber by swabbing with ethanol and allow it to dry. 7.
Adjust the outlet pressure at the helium cylinder to the desired
bombardment pressure. 8. Adjust the solenoid timer to 0.05 seconds.
Pass enough helium to remove air from the supply line (2-3 pulses).
9. After 5 min on ice, remove (and discard) 100 .mu.l of
supernatant from the settled precipitation mix. 10. Thoroughly
disperse the particles in the remaining solution. 11. Immediately
place 4 .mu.l of the dispersed tungsten-DNA preparation in the
center of the support screen in a 13 mm plastic syringe filter
holder. 12. Attach the filter holder to the helium outlet in the
target chamber. 13. Replace the lid over the target tissue with a
sterile protective screen. Place the sample into the target
chamber, centered 16.5 cm under the particle source and close the
door. 14. Open the valve to the vacuum source. When chamber vacuum
reaches 28'' of mercury, press the button to apply the accelerating
gas pulse, which discharges the particles into the target chamber.
15. Close the valve to the vacuum source. Allow air to return
slowly into the target chamber through a sterilizing filter. Open
the door, cover the sample with a sterile lid and remove the sample
dish from the chamber. 16. Repeat steps 10-15 for consecutive
target plates using the same precipitation mix, filter and screen.
17. Approximately 4 hours after bombardment, transfer the callus
pieces from MSC3Osm to MSC3. 18. Two days after shooting, transfer
the callus onto selection medium. During this transfer, divide the
callus into pieces .about.5 mm in diameter, with each piece being
kept separate throughout the selection process. 19. Subculture
callus pieces at 2-3 week intervals. 20. When callus pieces grow to
.about.5 to 10 mm in diameter (typically 8 to 12 weeks after
bombardment) transfer onto regeneration medium at 28.degree. C. in
the light. 21. When regenerated shoots are 30-60 mm high with
several well-developed roots, transfer them into potting mix with
the usual precautions against mechanical damage, pathogen attack
and desiccation until plantlets are established in the
greenhouse.
Example 10
Soybean Embryo Transformation
[0300] Soybean embryos are bombarded with a plasmid containing a
dimerization domain sequence operably linked to an ubiquitin
promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm
in length dissected from surface-sterilized, immature seeds of the
soybean cultivar A2872, are cultured in the light or dark at
26.degree. C. on an appropriate agar medium for six to ten weeks.
Somatic embryos producing secondary embryos are then excised and
placed into a suitable liquid medium. After repeated selection for
clusters of somatic embryos that multiplied as early,
globular-staged embryos, the suspensions are maintained as
described below.
[0301] Soybean embryogenic suspension cultures can be 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.
[0302] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein, et
al., (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0303] A selectable marker gene that can be used to facilitate
soybean transformation is a transgene 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 expression cassette
comprising a dimerization domain sense sequence operably linked to
the ubiquitin promoter 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.
[0304] 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
microliters of the DNA-coated gold particles are then loaded on
each macro carrier disk.
[0305] 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.
[0306] 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 11
Sunflower Meristem Tissue Transformation
[0307] Sunflower meristem tissues are transformed with an
expression cassette containing a dimerization domain sequence
operably linked to a ubiquitin promoter as follows (see also, EP
Patent Number 0 486233, herein incorporated by reference and
Malone-Schoneberg, et al., (1994) Plant Science 103:199-207).
Mature sunflower seed (Helianthus annuus L.) are dehulled using a
single wheat-head thresher. Seeds are surface sterilized for 30
minutes in a 20% Clorox.RTM. bleach solution with the addition of
two drops of Tween.RTM. 20 per 50 ml of solution. The seeds are
rinsed twice with sterile distilled water.
[0308] Split embryonic axis explants are prepared by a modification
of procedures described by Schrammeijer, et al., (Schrammeijer, et
al., (1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in
distilled water for 60 minutes following the surface sterilization
procedure. The cotyledons of each seed are then broken off,
producing a clean fracture at the plane of the embryonic axis.
Following excision of the root tip, the explants are bisected
longitudinally between the primordial leaves. The two halves are
placed, cut surface up, on GBA medium consisting of Murashige and
Skoog mineral elements (Murashige, et al., (1962) Physiol. Plant.,
15:473-497), Shepard's vitamin additions (Shepard (1980) in
Emergent Techniques for the Genetic Improvement of Crops
(University of Minnesota Press, St. Paul, Minn.), 40 mg/l adenine
sulfate, 30 g/l sucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25
mg/l indole-3-acetic acid (IAA), 0.1 mg/l gibberellic acid
(GA.sub.3), pH 5.6 and 8 g/l Phytagar.
[0309] The explants are subjected to microprojectile bombardment
prior to Agrobacterium treatment (Bidney, et al., (1992) Plant Mol.
Biol. 18:301-313). Thirty to forty explants are placed in a circle
at the center of a 60.times.20 mm plate for this treatment.
Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are
resuspended in 25 ml of sterile TE buffer (10 mM Tris HCl, 1 mM
EDTA, pH 8.0) and 1.5 ml aliquots are used per bombardment. Each
plate is bombarded twice through a 150 mm nytex screen placed 2 cm
above the samples in a PDS 1000.RTM. particle acceleration
device.
[0310] Disarmed Agrobacterium tumefaciens strain EHA105 is used in
all transformation experiments. A binary plasmid vector comprising
the expression cassette that contains the dimerization domain gene
operably linked to the ubiquitin promoter is introduced into
Agrobacterium strain EHA105 via freeze-thawing as described by
Holsters, et al., (1978) Mol. Gen. Genet. 163:181-187. This plasmid
further comprises a kanamycin selectable marker gene (i.e, nptII).
Bacteria for plant transformation experiments are grown overnight
(28.degree. C. and 100 RPM continuous agitation) in liquid YEP
medium (10 gm/l yeast extract, 10 gm/l Bacto.RTM. peptone, and 5
gm/l NaCl, pH 7.0) with the appropriate antibiotics required for
bacterial strain and binary plasmid maintenance. The suspension is
used when it reaches an OD.sub.600 of about 0.4 to 0.8. The
Agrobacterium cells are pelleted and resuspended at a final
OD.sub.600 of 0.5 in an inoculation medium comprised of 12.5 mM MES
pH 5.7, 1 gm/l NH.sub.4Cl, and 0.3 gm/l MgSO.sub.4.
[0311] Freshly bombarded explants are placed in an Agrobacterium
suspension, mixed, and left undisturbed for 30 minutes. The
explants are then transferred to GBA medium and co-cultivated, cut
surface down, at 26.degree. C. and 18-hour days. After three days
of co-cultivation, the explants are transferred to 374B (GBA medium
lacking growth regulators and a reduced sucrose level of 1%)
supplemented with 250 mg/l cefotaxime and 50 mg/l kanamycin
sulfate. The explants are cultured for two to five weeks on
selection and then transferred to fresh 374B medium lacking
kanamycin for one to two weeks of continued development. Explants
with differentiating, antibiotic-resistant areas of growth that
have not produced shoots suitable for excision are transferred to
GBA medium containing 250 mg/l cefotaxime for a second 3-day
phytohormone treatment. Leaf samples from green,
kanamycin-resistant shoots are assayed for the presence of NPTII by
ELISA and for the presence of transgene expression by assaying for
a modulation in meristem development (i.e., an alteration of size
and appearance of shoot and floral meristems).
[0312] NPTII-positive shoots are grafted to Pioneer.RTM. hybrid
6440 in vitro-grown sunflower seedling rootstock. Surface
sterilized seeds are germinated in 48-0 medium (half-strength
Murashige and Skoog salts, 0.5% sucrose, 0.3% Gelrite.RTM., pH 5.6)
and grown under conditions described for explant culture. The upper
portion of the seedling is removed, a 1 cm vertical slice is made
in the hypocotyl and the transformed shoot inserted into the cut.
The entire area is wrapped with Parafilm.RTM. to secure the shoot.
Grafted plants can be transferred to soil following one week of in
vitro culture. Grafts in soil are maintained under high humidity
conditions followed by a slow acclimatization to the greenhouse
environment. Transformed sectors of T.sub.0 plants (parental
generation) maturing in the greenhouse are identified by NPTII
ELISA and/or by dimerization domain activity analysis of leaf
extracts while transgenic seeds harvested from NPTII-positive
T.sub.0 plants are identified by dimerization domain activity
analysis of small portions of dry seed cotyledon.
[0313] An alternative sunflower transformation protocol allows the
recovery of transgenic progeny without the use of chemical
selection pressure. Seeds are dehulled and surface-sterilized for
20 minutes in a 20% Clorox.RTM. bleach solution with the addition
of two to three drops of Tween.RTM. 20 per 100 ml of solution, then
rinsed three times with distilled water. Sterilized seeds are
imbibed in the dark at 26.degree. C. for 20 hours on filter paper
moistened with water. The cotyledons and root radical are removed
and the meristem explants are cultured on 374E (GBA medium
consisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate,
3% sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8%
Phytagar at pH 5.6) for 24 hours under the dark. The primary leaves
are removed to expose the apical meristem, around 40 explants are
placed with the apical dome facing upward in a 2 cm circle in the
center of 374M (GBA medium with 1.2% Phytagar) and then cultured on
the medium for 24 hours in the dark.
[0314] Approximately 18.8 mg of 1.8 .mu.m tungsten particles are
resuspended in 150 .mu.l absolute ethanol. After sonication, 8
.mu.l of it is dropped on the center of the surface of
macrocarrier. Each plate is bombarded twice with 650 psi rupture
discs in the first shelf at 26 mm of Hg helium gun vacuum.
[0315] The plasmid of interest is introduced into Agrobacterium
tumefaciens strain EHA105 via freeze thawing as described
previously. The pellet of overnight-grown bacteria at 28.degree. C.
in a liquid YEP medium (10 g/l yeast extract, 10 g/l Bacto.RTM.
peptone and 5 g/l NaCl, pH 7.0) in the presence of 50 .mu.g/l
kanamycin is resuspended in an inoculation medium (12.5 mM 2-mM
2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH.sub.4CI and 0.3
g/I MgSO.sub.4 at pH 5.7) to reach a final concentration of 4.0 at
OD.sub.600. Particle-bombarded explants are transferred to GBA
medium (374E) and a droplet of bacteria suspension is placed
directly onto the top of the meristem. The explants are
co-cultivated on the medium for 4 days, after which the explants
are transferred to 374C medium (GBA with 1% sucrose and no BAP,
IAA, GA3 and supplemented with 250 .mu.g/ml cefotaxime). The
plantlets are cultured on the medium for about two weeks under
16-hour day and 26.degree. C. incubation conditions.
[0316] Explants (around 2 cm long) from two weeks of culture in
374C medium are screened for a modulation in meristem development
(i.e., an alteration of size and appearance of shoot and floral
meristems). After positive (i.e., a change in dimerization domain
expression) explants are identified, those shoots that fail to
exhibit an alteration in dimerization domain activity are discarded
and every positive explant is subdivided into nodal explants. One
nodal explant contains at least one potential node. The nodal
segments are cultured on GBA medium for three to four days to
promote the formation of auxiliary buds from each node. Then they
are transferred to 374C medium and allowed to develop for an
additional four weeks. Developing buds are separated and cultured
for an additional four weeks on 374C medium. Pooled leaf samples
from each newly recovered shoot are screened again by the
appropriate protein activity assay. At this time, the positive
shoots recovered from a single node will generally have been
enriched in the transgenic sector detected in the initial assay
prior to nodal culture.
[0317] Recovered shoots positive for altered dimerization domain
expression are grafted to Pioneer hybrid 6440 in vitro-grown
sunflower seedling rootstock. The rootstocks are prepared in the
following manner. Seeds are dehulled and surface-sterilized for 20
minutes in a 20% Clorox.RTM. bleach solution with the addition of
two to three drops of Tween.RTM. 20 per 100 ml of solution, and are
rinsed three times with distilled water. The sterilized seeds are
germinated on the filter moistened with water for three days, then
they are transferred into 48 medium (half-strength MS salt, 0.5%
sucrose, 0.3% Gelrite.RTM. pH 5.0) and grown at 26.degree. C. under
the dark for three days, then incubated at 16-hour-day culture
conditions. The upper portion of selected seedling is removed, a
vertical slice is made in each hypocotyl and a transformed shoot is
inserted into 8V-Cut. The cut area is wrapped with Parafilm.RTM..
After one week of culture on the medium, grafted plants are
transferred to soil. In the first two weeks, they are maintained
under high humidity conditions to acclimatize to a greenhouse
environment.
Example 12
Agrobacterium Mediated Grass Transformation
[0318] Grass plants may be transformed by following the
Agrobacterium mediated transformation of Luo, et al., (2004) Plant
Cell Rep (2004) 22:645-652.
Materials and Methods
Plant Material
[0319] A commercial cultivar of creeping bentgrass (Agrostis
stolonifera L., cv. Penn-A-4) supplied by Turf-Seed (Hubbard, Ore.)
can be used. Seeds are stored at 4.degree. C. until used.
Bacterial Strains and Plasmids
[0320] Agrobacterium strains containing one of 3 vectors are used.
One vector includes a pUbi-gus/Act1-hyg construct consisting of the
maize ubiquitin (ubi) promoter driving an intron-containing
b-glucuronidase (GUS) reporter gene and the rice actin 1 promoter
driving a hygromycin (hyg) resistance gene. The other two
pTAP-arts/35S-bar and pTAP-barnase/Ubi-bar constructs are vectors
containing a rice tapetum-specific promoter driving either a rice
tapetum-specific antisense gene, rts (Lee, et al., (1996) Int Rice
Res Newsl 21:2-3) or a ribonuclease gene, barnase (Hartley, (1988)
J Mol Biol 202:913-915), linked to the cauliflower mosaic virus 35S
promoter (CaMV 35S) or the rice ubi promoter (Huq, et al, (1997)
Plant Physiol 113:305) driving the bar gene for herbicide
resistance as the selectable marker.
Induction of Embryogenic Callus and Agrobacterium-Mediated
Transformation
[0321] Mature seeds are dehusked with sand paper and surface
sterilized in 10% (v/v) Clorox.RTM. bleach (6% sodium hypochlorite)
plus 0.2% (v/v) Tween.RTM. 20 (Polysorbate 20) with vigorous
shaking for 90 min. Following rinsing five times in sterile
distilled water, the seeds are placed onto callus-induction medium
containing MS basal salts and vitamins (Murashige and Skoog, (1962)
Physiol Plant 15:473-497), 30 g/l sucrose, 500 mg/l casein
hydrolysate, 6.6 mg/l 3,6-dichloro-o-anisic acid (dicamba), 0.5
mg/l 6-benzylaminopurine (BAP) and 2 g/l Phytagel. The pH of the
medium is adjusted to 5.7 before autoclaving at 120.degree. C. for
20 min. The culture plates containing prepared seed explants are
kept in the dark at room temperature for 6 weeks. Embryogenic calli
are visually selected and subcultured on fresh callus-induction
medium in the dark at room temperature for 1 week before
co-cultivation.
Transformation
[0322] The transformation process is divided into five sequential
steps: agro-infection, co-cultivation, antibiotic treatment,
selection and plant regeneration. One day prior to agro-infection,
the embryogenic callus is divided into 1- to 2-mm pieces and placed
on callus-induction medium containing 100 .mu.M acetosyringone. A
10-ml aliquot of Agrobacterium suspension (OD=1.0 at 660 nm) is
then applied to each piece of callus, followed by 3 days of
co-cultivation in the dark at 25.degree. C. For the antibiotic
treatment step, the callus is then transferred and cultured for 2
weeks on callus-induction medium plus 125 mg/l cefotaxime and 250
mg/l carbenicillin to suppress bacterial growth. Subsequently, for
selection, the callus is moved to callus-induction medium
containing 250 mg/l cefotaxime and 10 mg/l phosphinothricin (PPT)
or 200 mg/l hygromycin for 8 weeks. Antibiotic treatment and the
entire selection process is performed at room temperature in the
dark. The subculture interval during selection is typically 3
weeks. For plant regeneration, the PPT- or hygromycin-resistant
proliferating callus is first moved to regeneration medium (MS
basal medium, 30 g/l sucrose, 100 mg/l myo-inositol, 1 mg/l BAP and
2 WI Phytagel) supplemented with cefotaxime. PPT or hygromycin.
These calli are kept in the dark at room temperature for 1 week and
then moved into the light for 2-3 weeks to develop shoots. Small
shoots are then separated and transferred to hormone-free
regeneration medium containing PPT or hygromycin and cefotaxime to
promote root growth while maintaining selection pressure and
suppressing any remaining Agrobacterium cells. Plantlets with
well-developed roots (3-5 weeks) are then transferred to soil and
grown either in the greenhouse or in the field.
Staining for GUS Activity
[0323] GUS activity in transformed callus is assayed by
histochemical staining with 1 mM
5-bromo-4-chloro-3-indolyl-b-d-glucuronic acid (X-Gluc, Biosynth,
Staad, Switzerland) as described in Jefferson, (1987) Plant Mol
Biol Rep 5:387-405. The hygromycin-resistant callus surviving from
selection was incubated at 37 C overnight in 100 .mu.l of reaction
buffer containing X-Gluc. GUS expression is then documented by
photography.
Vernalization and Out-Crossing of Transgenic Plants
[0324] Transgenic plants are maintained out of doors in a
containment nursery (3-6 months) until the winter solstice in
December. The vernalized plants are then transferred to the
greenhouse and kept at 25.degree. C. under a 16/8 h [day/light
(artificial light)] photoperiod and surrounded by non-transgenic
wild-type plants that physically isolated them from other pollen
sources. The plants will initiate flowering 3-4 weeks after being
moved back into the greenhouse. They are out-crossed with the
pollen from the surrounding wild-type plants. The seeds collected
from each individual transgenic plant are germinated in soil at
25.degree. C. and Ti plants are grown in the greenhouse for further
analysis.
Seed Testing
[0325] Test of the Transgenic Plants and their Progeny for
Resistance to PPT
[0326] Transgenic plants and their progeny are evaluated for
tolerance to glufosinate (PPT) indicating functional expression of
the bar gene. The seedlings are sprayed twice at concentrations of
1-10% (v/v) Finale.COPYRGT. (AgrEvo USA, Montvale, N.J.) containing
11% glufosinate as the active ingredient. Resistant and sensitive
seedlings are clearly distinguishable 1 week after the application
of Finale.COPYRGT. in all the sprayings.
Statistical Analysis
[0327] Transformation efficiency for a given experiment is
estimated by the number of PPT-resistant events recovered per 100
embryogenic calli infected and regeneration efficiency is
determined using the number of regenerated events per 100 events
attempted. The mean transformation and regeneration efficiencies
are determined based on the data obtained from multiple independent
experiments. A Chi-square test can be used to determine whether the
segregation ratios observed among Ti progeny for the inheritance of
the bar gene as a single locus fit the expected 1:1 ratio when
out-crossed with pollen from untransformed wild-type plants.
DNA Extraction and Analysis
[0328] Genomic DNA is extracted from approximately 0.5-2 g of fresh
leaves essentially as described by Luo, et al., (1995) Mol Breed
1:51-63. Ten micrograms of DNA is digested with HindIII or BamHI
according to the supplier's instructions (New England Biolabs,
Beverly, Mass.). Fragments are size-separated through a 1.0% (w/v)
agarose gel and blotted onto a Hybond-N+ membrane (Amersham
Biosciences, Piscataway, N.J.). The bar gene, isolated by
restriction digestion from pTAP-arts/35S-bar, is used as a probe
for Southern blot analysis. The DNA fragment is radiolabeled using
a Random Priming Labeling kit (Amersham Biosciences) and the
Southern blots are processed as described by Sambrook, et al.,
(1989) Molecular cloning: a laboratory manual, 2nd edn, Cold Spring
Harbor Laboratory Press, New York.
Polymerase Chain Reaction
[0329] The two primers designed to amplify the bar gene are as
follows: 5-GTCTGCACCATCGTCAACC-3' (SEQ ID NO: 42), corresponding to
the proximity of the 5' end of the bar gene and
5'-GAAGTCCAGCTGCCAGAAACC-3' (SEQ ID NO: 43), corresponding to the
3' end of the bar coding region. The amplification of the bar gene
using this pair of primers should result in a product of 0.44 kb.
The reaction mixtures (25 .mu.l total volume) consist of 50 mM KCl,
10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 0.1% (w/v) Triton X-100, 200
.mu.M each of dATP, dCTP, dGTP and dTTP, 0.5 .mu.M of each primer,
0.2 .mu.g of template DNA and 1 U Taq DNA polymerase (QIAGEN,
Valencia, Calif.). Amplification is performed in a Stratagene
Robocycler Gradient 96 thermal cycler (La Jolla, Calif.) programmed
for 25 cycles of 1 min at 94.degree. C. (denaturation), 2 min at
55.degree. C. (hybridization), 3 min at 72.degree. C. (elongation)
and a final elongation step at 72.degree. C. for 10 min. PCR
products are separated on a 1.5% (w/v) agarose gel and detected by
staining with ethidium bromide.
Example 13
Plant Characterization Analysis--Greenhouse
[0330] Greenhouse experiments were performed with two constructs
plus a comparative control. All had 35s::BAR as the selectable
marker. Php37407 contained S2A PRO::D8 MPL+F3.7 PRO::CESA4+FTM1
PRO::DD+NAS2 PRO::DD. Php39175 contained S2A PRO::D8 MPL S89T
(ALT4). For each construct, 10 events were planted. An equal number
of positive and negative plants (4 per week.times.4 weeks) were
expected. Due to greenhouse growth conditions and subsequent extra
plantings, the outcome of samples was:
For php37407: 28 positive plants from 10 events and 24 negative
siblings from 9 events. For php39175: 25 positive plants from 8
events and 29 negative siblings from 10 events.
[0331] Observations were performed on each of the plants, and
measurements recorded. Data collected included: Plant Height, Leaf
Width and Leaf Length (leaves -2, +2, +4 from ear node), Central
Tassel Spike Measurement (absolute value and normalized to height),
Anther Exertion Length, Tassel Score (1-9: 1 being very small with
no branches, 5 average size with approximately 6 branches, 9 very
large with 20 or more branches), Pollen Score (1-5: low to high, a
measurement of collected pollen, each unit equivalent to 0.7'' of
collected pollen in a 0.25'' wide apparatus) and Leaf Count.
[0332] Final analysis of the plants showed that the stacked
php37407 construct containing the dimerization domain had
moderating effects on the dwarf gene's phenotype exhibited in
php39175 plants. Plant height for php37407 increased 8.8% as
compared with php39175. The increases in the leaf width and the
reductions in leaf length with the dwarf gene in php39175 were also
moderated with the stack in php37407. In the php37407 plants, leaf
width was reduced 6% in the leaf two nodes below the ear, 4.2% in
the leaf two nodes above the ear and 5.4% in the leaf four nodes
above the ear. The leaf lengths in the php37407 plants were
increased 2.7% and 2.8% respectively for the nodes two below and
two above the ear and showed no difference for the leaf four nodes
above the ear. Additionally, the absolute length of the central
tassel spike was increased by 3% in the php37407 samples as
compared with the php39175 samples. The tassel length as a
percentage of height was reduced by 6.4% in php37407, moderating
the dwarf gene's effect to increase the relative tassel length
compared to the vegetative plant height. Furthermore, on a
representative subset of samples, the exerted anther length
including the filament plus anther in php37407 plants was increased
by 9.9% as compared with the php39175 anthers. In addition, the
tassel score index (1-9) taken by the greenhouse in the stacked
php37407 plants showed an increase of 9.5%. The pollen score index
(1-5) on a representative subset of samples showed no difference
between the samples. Leaf counts were also similar between php37407
and php39175 and found one node greater per plant as compared with
their negative siblings. Overall, the stack of genes in the
php37407 plants displayed a moderating effect of the dwarf gene
phenotype in the php39175 samples. Moderating the effect of the
dwarf gene included, but was not limited to: increased plant height
and tassel size, leaf length and anther exerted.
Example 14
Arabidopsis Dimerization Domain Study
[0333] Arabidopsis plants ecotype Columbia were transformed with a
construct containing a constitutive promoter or tissue-preferred
promoter driving expression of the dimerization domain (DD). Plants
were transformed using Agrobacterium-mediated transformation method
and positive transformants were selected by resistance to an
herbicide. Transgenic Arabidopsis plants were grown in
nutrient-rich soil under greenhouse conditions. Seeds were
collected to determine improvements in yield or yield-related
traits between transgenic plants and control plants. Control plants
are positive transformants that contain the vector backbone without
the promoter and dimerization domain. Effective transgenic events
are those that show an increase in yield or seed weight under
normal growing conditions.
[0334] Transgenic plants containing a putative leaf-preferred
promoter driving expression of the dimerization domain showed an
approximately 22% increase in seed weight over control plants.
Example 15
Root Growth Analysis
[0335] Seed segregating for transgene heterozygote and wild type
are planted in Custom 200C pot filled with Turface MVP then watered
with nutrient solution containing 1 mM KNO3 or 4 mM KNO3 as
nitrogen source along with a full complement of other
nutrients:
TABLE-US-00006 Nutrient 1 mM KNO.sub.3 4 mM KNO.sub.3 10x Micron
utrients 400 ml 400 ml KH.sub.2PO.sub.4 136.02 Mwt 272 g 272 g
MgSO.sub.4 120.36 Mwt 963 g 963 g KNO.sub.3 fertilizer grade 400 g
1200 g KCl 74.55 Mwt 596 g -- *CaCl.sub.2 147.01 Mwt 588 g 588 g
Sprint 330 335 g 335 g / 100 l / 100 l
Add 84 ml H.sub.2SO.sub.4 to reduce pH. Optimum pH is 5-5.5. Add
200 ul of the nutrient solution to 3 ml tap water and check the pH,
it should be 5-5.8. If distilled water is used the pH will have to
be raised with 10M KOH instead of decreased. *If using tap water
with Ca.sup.++ concentration in the 0.5-0.7 mM level reduce this
amount to 235 g. If comparing 6 mM growth to any other nutrient mix
maintain the CaCl.sub.2 level at 588 g/100 l.
TABLE-US-00007 10x Micronutrients Stock solution mg/liter 15 mM
H.sub.3BO.sub.3 1852 mg 5 mM MnCl.sub.2.cndot.4H.sub.2O 1980 mg 5
mM ZnSO.sub.4.cndot.7 H.sub.2O 2874 mg 0.5 mM
CuSO.sub.4.cndot.5H.sub.2O 250 mg 0.5 mM
H.sub.2MoO.sub.4.cndot.H.sub.2O 242 mg
[0336] After 3 weeks of growth in these media SPAD meter
measurements are made by averaging at least 5 readings taken from
the base of the youngest most fully expanded leaf. Plants are
removed from the pots, the Turface washed from the roots and
separated into shoots and roots. These samples are dried
(70.degree. C. for 72 hr) and dried roots are weighed separately
from the shoots. The dried shoots are ground to a fine powder and
total N determined using a sample of the ground tissue. From these
parameters greenness (SPAD), total plant weight, shoot weight, root
weight, root/shoot ratio, shoot nitrogen concentration and total N
are calculated for low and high N fertility grown plants.
[0337] Plants have a higher root/shoot ratio when grown in lower
nitrogen fertility. Agronomic conditions for growing maize have
higher soil nitrate conditions when the plants are the smallest.
Higher soil nitrate conditions favor lower root/shoot ratios which
does not favor extensive soil exploration by roots. These
transgenes that increase the root/shoot ratio under high or low
nitrogen fertility would likely explore a greater portion of the
soil early during growth and maximize plant growth. Root/shoot
ratios would be higher in higher N fertilities.
[0338] The use of a root preferred promoter such as NAS2 and the
dimerization domain (DD) enhances root growth early in development.
The changes in root growth can be detected at the tissue culture
stages of plant regeneration following transformation with this
gene specifically by the appearance of more roots and larger
diameter roots in test tubes prepared for rooting (ref. Zhao).
Transgenic seed expressing NAS2:DD would be expected to have an
enhanced early root growth phenotype similar to that observed in
tissue culture experiments. The expected phenotype in the assay
mentioned above would be expected to produce a higher root dry
weight at the end of the growth period of three weeks. An altered
root growth (higher) would be especially desirable under higher N
conditions because of a greater soil exploration capacity in
transgenic versus non transgenic plants.
Example 16
The Use of DD (Dimerization Domain) Components with Moderate
Dwarfing Genes (D8MPL) to Improve Creeping Bentgrass (Agrostis
stolonifera L.) for Turf Grass Applications
[0339] The semi-dwarf characteristics of S2a:D8MPL in corn could be
used to improve turf grass species such as creeping bentgrass
(Agrostis stolonifera L). Specifically, a more compact leaf with
increased width and reduced length is desirable and the dark green
leaf color observed in corn would be especially desirable in turf
grass. In addition to the reduced leaf length with S2a:D8MPL, roots
may also have shorter length compared to non transgenic creeping
bent grass. The use of the DD dominant negative transgene with a
root preferred promoter such as NAS2 could be combined in a
transgene stack to selectively increase the root growth relative to
a more compact leaf phenotype desired in the leaves. The compact
leaf structure would also have advantages in terms of reduced
maintenance (mowing) with similar or reduced amounts of added
fertilizer. Furthermore, the use of the DD with a root preferred or
specific promoter would increase the relative root length and root
density compared to the expectation of smaller roots with a dwarf
shoot/leaf phenotype. Increasing root length and density,
especially earlier in plant development, would aide establishment
and could also moderate irrigation requirements for establishment
and maintenance of commercial turf grass plantings. Similar
advantages are anticipated for non-commercial home use of
transgenic Agrostis species--ease in establishment because of
strong root formation from seedlings and more efficient maintenance
in terms of less mowing and irrigation to maintain a desirable
turfgrass (i.e., dark green) appearance above ground and deeper
more vigorous roots to support leaf growth and turf quality
maintenance.
Example 17
Variants of Dimerization Domain Sequences
[0340] A. Variant Nucleotide Sequences of Dimerization Domain that
do not Alter the Encoded Amino Acid Sequence
[0341] The dimerization domain nucleotide sequences are used to
generate variant nucleotide sequences having the nucleotide
sequence of the open reading frame with about 70%, 75%, 80%, 85%,
90% and 95% nucleotide sequence identity when compared to the
starting unaltered ORF nucleotide sequence of the corresponding SEQ
ID NO. These functional variants are generated using a standard
codon table. While the nucleotide sequence of the variants are
altered, the amino acid sequence encoded by the open reading frames
do not change. These variants are associated with the ability of
the dimerization domain to form defective dimmers thereby
preventing the inhibitory response to GA.
[0342] B. Variant Amino Acid Sequences of Dimerization Domain
Polypeptides
[0343] Variant amino acid sequences of the dimerization domain
polypeptides are generated. In this example, one amino acid is
altered. Specifically, the open reading frames are reviewed to
determine the appropriate amino acid alteration. The selection of
the amino acid to change is made by consulting the protein
alignment (with the other orthologs and other gene family members
from various species). An amino acid is selected that is deemed not
to be under high selection pressure (not highly conserved) and
which is rather easily substituted by an amino acid with similar
chemical characteristics (i.e., similar functional side-chain).
Using a protein alignment, an appropriate amino acid can be
changed. Once the targeted amino acid is identified, the procedure
outlined in the following section C is followed. Variants having
about 70%, 75%, 80%, 85%, 90% and 95% nucleic acid sequence
identity are generated using this method.
[0344] C. Additional Variant Amino Acid Sequences of Dimerization
Domain Polypeptides
[0345] In this example, artificial protein sequences are created
having 80%, 85%, 90% and 95% identity relative to the reference
protein sequence. This latter effort requires identifying conserved
and variable regions from an alignment and then the judicious
application of an amino acid substitutions table. These parts will
be discussed in more detail below.
[0346] Largely, the determination of which amino acid sequences are
altered is made based on the conserved regions among dimerization
domain protein or among the other dimerization domain polypeptides.
It is recognized that conservative substitutions can be made in the
conserved regions below without altering function. In addition, one
of skill will understand that functional variants of the
dimerization domain sequence of the invention can have minor
non-conserved amino acid alterations in the conserved domain.
[0347] Artificial protein sequences are then created that are
different from the original in the intervals of 80-85%, 85-90%,
90-95% and 95-100% identity. Midpoints of these intervals are
targeted, with liberal latitude of plus or minus 1%, for example.
The amino acids substitutions will be effected by a custom Perl
script. The substitution table is provided below in Table 5.
TABLE-US-00008 TABLE 5 Substitution Table Strongly Similar and Rank
of Optimal Order to Amino Acid Substitution Change Comment I L, V 1
50:50 substitution L I, V 2 50:50 substitution V I, L 3 50:50
substitution A G 4 G A 5 D E 6 E D 7 W Y 8 Y W 9 S T 10 T S 11 K R
12 R K 13 N Q 14 Q N 15 F Y 16 M L 17 First methionine cannot
change H Na No good substitutes C Na No good substitutes P Na No
good substitutes
[0348] First, any conserved amino acids in the protein that should
not be changed is identified and "marked off" for insulation from
the substitution. The start methionine will of course be added to
this list automatically. Next, the changes are made.
[0349] H, C and P are not changed in any circumstance. The changes
will occur with isoleucine first, sweeping N-terminal to
C-terminal. Then leucine, and so on down the list until the desired
target it reached. Interim number substitutions can be made so as
not to cause reversal of changes. The list is ordered 1-17, so
start with as many isoleucine changes as needed before leucine, and
so on down to methionine. Clearly many amino acids will in this
manner not need to be changed. L, I and V will involve a 50:50
substitution of the two alternate optimal substitutions.
[0350] The variant amino acid sequences are written as output. Perl
script is used to calculate the percent identities. Using this
procedure, variants of the dimerization domain polypeptides are
generating having about 80%, 85%, 90% and 95% amino acid identity
to the starting unaltered ORF nucleotide sequence of SEQ ID NO:
9.
[0351] All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated by reference.
[0352] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the invention.
Sequence CWU 1
1
431879DNAMedicago sativa 1aattcccatg atcttctctc cttcatcaat
ggatgccatg tttcataaca ataacaccaa 60atgtttgatg agctaccaac aattgcgcaa
agactatggc taagctcgag ctcgctcgct 120acaagttgtt gactttcaaa
tacaagtttg tttttggaac accaaatatt ctacatgatc 180tttcactaag
ttgcgcacca ctatcaaaag attatctagg ccattattca agtaaagagt
240gaacacgtct aagacccaca accacaccaa atagaatacg catacatgca
acatattgtg 300caagaagtat ccaactggac tcccatgtat tctaaaacta
ttttcgtaga gttaaagtta 360tgacaaactt atcaaataaa aatttgaacg
ctggaccaaa actttcatct ttcaaatcca 420ccatcgtcta tcctcataaa
ttgttttgat tataacacat ctacgtaaat catttgtttt 480gaacaatact
aatttaattt tattaagtca aataacctgc ttagaaaata atccctccac
540ctcatttaac aatttcttgt caaacacaca ccaagaaaaa aattaatgaa
agagaaaaga 600aatgaaaagg acatggagtt gaatactagc aaaattgatt
gaaggaagat tcacaattga 660aattgaaacc atttaattta ttttcgggtc
cataataata aattggtaag aataaaaacc 720cgatcaagtc cggtacagta
caattccact ccaccaactc cttacttaaa cccctattta 780tacccactct
catcctcact cttccttcac ctctcacact ctcttctctc tctcaaaacc
840ctcacacaaa cgctgcgttt agtgtaagaa attcaatcc 87923237DNAZea mays
2atggacgccg gctcggtcac cggtggcctc gccgcgggct cgcacatgcg ggacgagctg
60catgtcatgc gcgcccgcga ggagccgaac gccaaggtcc ggagcgccga cgtgaagacg
120tgccgcgtgt gcgccgacga ggtcgggacg cgggaggacg ggcagccctt
cgtggcgtgc 180gccgagtgcg gcttccccgt ctgccggccc tgctacgagt
acgagcgcag cgagggcacg 240cagtgctgcc cgcagtgcaa cacccgctac
aagcgccaga aagggtgccc gagggtggaa 300ggggacgagg aggagggccc
ggagatggac gacttcgagg acgagttccc cgccaagagc 360cccaagaagc
ctcacgagcc tgtcgcgttc gacgtctact cggagaacgg cgagcacccg
420gcgcagaaat ggcggacggg tggccagacg ctgtcgtcct tcaccggaag
cgtcgccggg 480aaggacctgg aggcggagag ggagatggag gggagcatgg
agtggaagga ccggatcgac 540aagtggaaga ccaagcagga gaagaggggc
aagctcaacc acgacgacag cgacgacgac 600gacgacaaga acgaagacga
gtacatgctg cttgccgagg cccgacagcc gctgtggcgc 660aaggttccga
tcccgtcgag catgatcaac ccgtaccgca tcgtcatcgt gctccgcctg
720gtggtgctct gcttcttcct caagttccgg atcacgacgc ccgccacgga
cgccgtgcct 780ctgtggctgg cgtccgtcat ctgcgagctc tggttcgcct
tctcctggat cctggaccag 840ctgccaaagt gggcgccggt gacgcgggag
acgtacctgg accgcctggc gctgcggtac 900gaccgtgagg gcgaggcgtg
ccggctgtcc cccatcgact tcttcgtcag cacggtggac 960ccgctcaagg
agccgcccat catcaccgcc aacaccgtgc tgtccatcct cgccgtcgac
1020taccccgtgg accgcgtcag ctgctacgtc tccgacgacg gcgcgtccat
gctgctcttc 1080gacgcgctgt ccgagaccgc cgagttcgcg cgccgctggg
tgcccttctg caagaagttc 1140gccgtggagc cgcgcgcccc ggagttctac
ttctcgcaga agatcgacta cctcaaggac 1200aaggtgcagc cgacgttcgt
caaggagcgc cgcgccatga agagggagta cgaggagttc 1260aaggtgcgca
tcaacgcgct ggtggccaag gcgcagaaga agcccgagga ggggtgggtc
1320atgcaggacg gcacgccgtg gcccgggaac aacacgcgcg accacccggg
tatgatccag 1380gtctacctcg gcaaccaggg cgcgctggac gtggagggcc
acgagctgcc gcgcctcgtc 1440tacgtgtccc gtgagaagcg ccccgggtac
aaccaccaca agaaggcggg cgccatgaac 1500gcgctggtgc gcgtctccgc
cgtgctcacc aacgcgccct tcatcctcaa cctcgactgc 1560gaccactacg
tcaacaacag caaggccgtg cgcgaggcca tgtgcttcct catggacccg
1620cagctgggga agaagctctg ctacgtccag ttcccgcagc gcttcgatgg
catcgatcgc 1680cacgaccgat acgccaaccg caacgtcgtc ttcttcgaca
tcaacatgaa ggggctggac 1740ggcatccagg gcccggtgta cgtcggcacg
gggtgcgtgt tcaaccgcca ggcgctgtac 1800ggctacgacc cgccgcggcc
cgagaagcgg cccaagatga cgtgcgactg ctggccgtcg 1860tggtgctgct
gctgctgctg cttcggcggc ggcaagcgcg gcaaggcgcg caaggacaag
1920aagggcgacg gcggcgagga gccgcgccgg ggcctgctcg gcttctacag
gaagcggagc 1980aagaaggaca agctcggcgg cgggtcggtg gccggcagca
agaagggcgg cgggctgtac 2040aagaagcacc agcgcgcgtt cgagctggag
gagatcgagg aggggctgga ggggtacgac 2100gagctggagc gctcctcgct
catgtcgcag aagagcttcg agaagcggtt cggccagtcg 2160cccgtgttca
tcgcctccac gctcgtcgag gacggcggcc tgccgcaggg cgccgccgcc
2220gaccccgccg cgctcatcaa ggaggccatc cacgtcatca gctgcggata
cgaggagaag 2280accgagtggg gcaaggagat tgggtggatc tatgggtcgg
tgacagagga tatcctgacg 2340gggttcaaga tgcactgccg ggggtggaag
tccgtgtact gcacgccgac acggccggcg 2400ttcaaggggt cggcgcccat
caacttgtct gatcgtctcc accaggtgct gcgctgggcg 2460ctggggtccg
tggagatctt catgagccgc cactgcccgc tccggtacgc ctacggcggc
2520cggctcaagt ggctggagcg cttcgcctac accaacacca tcgtgtaccc
cttcacctcc 2580atcccgctcc tcgcctactg caccatcccc gccgtctgcc
tgctcaccgg caagttcatc 2640attcccacgc tgaacaacct cgccagcatc
tggttcatcg cgctcttcct gtccatcatc 2700gcgacgagcg tcctggagct
gcggtggagc ggggtgagca tcgaggactg gtggcgcaac 2760gagcagttct
gggtcatcgg cggcgtgtcc gcgcatctct tcgccgtgtt ccagggcttc
2820ctcaaggttc tgggcggcgt ggacaccagc ttcaccgtca cctccaaggc
ggccggcgac 2880gaggccgacg ccttcgggga cctctacctc ttcaagtgga
ccaccctgct ggtgcccccc 2940accacgctca tcatcatcaa catggtgggc
atcgtggccg gcgtgtccga cgccgtcaac 3000aacggctacg gctcctgggg
cccgctcttc ggcaagctct tcttctcctt ctgggtcatc 3060gtccacctct
acccgttcct caaggggctc atggggaggc agaaccggac gcccaccatc
3120gtcgtgctct ggtccatcct cctcgcctcc atcttctcgc tcgtctgggt
caggatcgac 3180ccgtttatcc cgaaggccaa gggccccatc ctcaagccat
gcggagtcga gtgctga 32373319DNAZea mays 3caacctagac ttgtccatct
tctggattgg ccaacttaat taatgtatga aataaaagga 60tgcacacata gtgacatgct
aatcactata atgtgggcat caaagttgtg tgttatgtgt 120aattactagt
tatctgaata aaagagaaag agatcatcca tatttcttat cctaaatgaa
180tgtcacgtgt ctttataatt ctttgatgaa ccagatgcat ttcattaacc
aaatccatat 240acatataaat attaatcata tataattaat atcaattggg
ttagcaaaac aaatctagtc 300taggtgtgtt ttgcgaatt 3194748DNAZea mays
4catggtggca cagaatcgag ttgatgttgt agctggcggc tagggtttga agtggagaag
60aggtccggct ggtggcatcc tatcgtctat tgagggttgg gtccggtggc atcatacttg
120atgacaattg aaagtaattt taatcaactt gtcatgagta gtgagtcttt
tataaaaaat 180aagctgaaat aagcaccctt tgatgagctt ataggattat
cataatctca aatgctaaat 240tatataattt tattagataa gttgcttgtt
tgtttcccca ctagcttatt tacattggat 300tatataatct acataaatta
taatctcaaa caaaaagtcc ttaatcagag atcagcgagg 360tctcacgagt
gagaaggcga gagcttgtcc aaacgagcat tttcgggcgt gtgaacaccc
420atttcagcaa agccgtcgtt gtccagttca gcgaagcgca ttctgcggct
ttggcgtgac 480ccattctgct agctcagcac tgagaatacg cgtccgctgc
agcgttggcg tacaggccgg 540actacattag ccaacgcgta tcggcagtgg
caaacctctt cgcttctaac tccgctgggc 600caccagcttt gaccgccgcc
tcccttcccc tccgctactg ctcctcccca ccccactccc 660ccgcaggagc
ggcggcggcg gcggcgaggt cgtaccccac atcggcgagc ggcggcggca
720ccgccggagg caaaggcaag tctagaac 74853234DNAZea mays 5atggagggcg
acgcggacgg cgtgaagtcg gggaggcgcg gtggcggaca ggtgtgccag 60atctgcggcg
acggcgtggg caccacggcg gagggggacg tcttcgccgc ctgcgacgtc
120tgcgggtttc cggtgtgccg cccctgctac gagtacgagc gcaaggacgg
cacgcaggcg 180tgcccccagt gcaagaccaa gtacaagcgc cacaagggga
gcccggcgat ccgtggggag 240gaaggagacg acactgatgc cgatagcgac
ttcaattacc ttgcatctgg caatgaggac 300cagaagcaga agattgccga
cagaatgcgc agctggcgca tgaacgttgg gggcagcggg 360gatgttggtc
gccccaagta tgacagtggc gagatcgggc ttaccaagta tgacagtggc
420gagattcctc ggggatacat cccatcagtc actaacagcc agatctcagg
agaaatccct 480ggtgcttccc ctgaccatca tatgatgtcc ccaactggga
acattggcaa gcgtgctcca 540tttccctatg tgaaccattc gccaaatccg
tcaagggagt tctctggtag cattgggaat 600gttgcctgga aagagagggt
tgatggctgg aaaatgaagc aggacaaggg gacgattccc 660atgacgaatg
gcacaagcat tgctccctct gagggtcggg gtgttggtga tattgatgca
720tcaactgatt acaacatgga agatgcctta ttgaacgacg aaactcgaca
gcctctatct 780aggaaagttc cacttccttc ctccaggata aatccataca
ggatggtcat tgtgctgcga 840ttgattgttc taagcatctt cttgcactac
cgtatcacaa atcctgtgcg caatgcatac 900ccattatggc ttctatctgt
tatatgtgag atctggtttg ctctttcgtg gatattggat 960cagttcccta
agtggtttcc aatcaaccgg gagacgtacc ttgataggct ggcattaagg
1020tatgaccggg aaggtgagcc atctcagttg gctgctgttg acattttcgt
cagtacagtc 1080gacccaatga aggagcctcc tcttgtcact gccaataccg
tgctatccat tcttgctgtg 1140gattaccctg tggataaggt ctcttgctat
gtatctgatg atggagctgc gatgctgaca 1200tttgatgcac tagctgagac
ttcagagttt gctagaaaat gggtaccatt tgttaagaag 1260tacaacattg
aacctagagc tcctgaatgg tacttctccc agaaaattga ttacttgaag
1320gacaaagtgc acccttcatt tgttaaagac cgccgggcca tgaagagaga
atatgaagaa 1380ttcaaagtta gggtaaatgg ccttgttgct aaggcacaga
aagttcctga ggaaggatgg 1440atcatgcaag atggcacacc atggccagga
aacaataccm gggaccatcc tggaatgatt 1500caggttttcc ttggtcacag
tggtggcctt gatactgagg gcaatgagct accccgtttg 1560gtctatgttt
ctcgtgaaaa gcgtcctgga ttccagcatc acaagaaagc tggtgccatg
1620aatgctcttg ttcgtgtctc agctgtgctt accaatggac aatacatgtt
gaatcttgat 1680tgtgatcact acattaacaa cagtaaggct ctcagggaag
ctatgtgctt ccttatggac 1740cctaacctag gaaggagtgt ctgctacgtc
cagtttcccc agagattcga tggcattgac 1800aggaatgatc gatatgccaa
caggaacacc gtgtttttcg atattaactt gagaggtctt 1860gatggcatcc
aaggaccagt ttatgtcgga actggctgtg ttttcaaccg aacagctcta
1920tatggttatg agcccccaat taagcagaag aagggtggtt tcttgtcatc
actatgtggc 1980ggtaggaaga aggcaagcaa atcaaagaag ggctcggaca
agaagaagtc gcagaagcat 2040gtggacagtt ctgtgccagt attcaacctt
gaagatatag aggagggagt tgaaggcgct 2100ggatttgacg acgagaaatc
acttcttatg tctcaaatga gcctggagaa gagatttggc 2160cagtccgcag
cgtttgttgc ctccactctg atggagtatg gtggtgttcc tcagtccgca
2220actccggagt ctcttctgaa agaagctatc catgttataa gctgtggcta
tgaggacaag 2280actgaatggg gaactgagat cgggtggatc tacggttctg
tgacagaaga cattctcacc 2340ggattcaaga tgcacgcgcg aggctggcgg
tcgatctact gcatgcccaa gcggccagct 2400ttcaaggggt ctgcccccat
caatctttcg gaccgtctga accaggtgct ccggtgggct 2460cttgggtccg
tggagatcct cttcagccgg cactgccccc tgtggtacgg ctacggaggg
2520cggctcaagt tcctggagag attcgcgtac atcaacacca ccatctaccc
gctcacgtcc 2580atcccgcttc tcatctactg catcctgccc gccatctgtc
tgctcaccgg aaagttcatc 2640attccagaga tcagcaactt cgccagcatc
tggttcatct ccctcttcat ctcgatcttc 2700gccacgggca tcctggagat
gaggtggagc ggggtgggca tcgacgagtg gtggaggaac 2760gagcagttct
gggtgatcgg gggcatctcc gcgcacctct tcgccgtgtt ccagggcctg
2820ctcaaggtgc tggccggcat cgacaccaac ttcaccgtca cctccaaggc
ctcggacgag 2880gacggcgact tcgcggagct gtacatgttc aagtggacga
cgctcctgat cccgcccacc 2940accatcctga tcatcaacct ggtcggcgtc
gtcgccggca tctcctacgc catcaacagc 3000ggataccagt cgtggggccc
gctcttcggc aagctcttct tcgccttctg ggtcatcgtc 3060cacctgtacc
cgttcctcaa gggcctcatg ggcaggcaga accgcacccc gaccatcgtc
3120gtcgtctggg ccatcctgct ggcgtccatc ttctccttgc tgtgggttcg
catcgacccc 3180ttcaccaccc gcgtcactgg cccggatacc cagacgtgtg
gcatcaactg ctag 323461578DNAZea mays 6atgctgtccg agctcaacgc
gcccccagcg ccgctcccgc ccgcgacgcc ggccccaagg 60ctcgcgtcca catcgtccac
cgtcacaagt ggcgccgccg ccggtgctgg ctacttcgat 120ctcccgcccg
ccgtggactc gtccagcagt acctacgctc tgaagccgat cccctcgccg
180gtggcggcgc cgtcggccga cccgtccacg gactcggcgc gggagcccaa
gcgaatgagg 240actggcggcg gcagcacgtc ctcctcctct tcctcgtcgt
catccatgga tggcggtcgc 300actaggagct ccgtggtcga agctgcgccg
ccggcgacgc aagcatccgc agcggccaac 360gggcccgcgg tgccggtggt
ggtggtggac acgcaggagg ccgggatccg gctcgtgcac 420gcgctgctgg
cgtgcgcgga ggccgtgcag caggagaact tctctgcggc ggaggcgctg
480gtcaagcaga tccccatgct ggcctcgtcg cagggcggtg ccatgcgcaa
ggtcgccgcc 540tacttcggcg aggcgcttgc ccgccgcgtg tatcgcttcc
gcccaccacc ggacagctcc 600ctcctcgacg ccgccttcgc cgacctctta
cacgcgcact tctacgagtc ctgcccctac 660ctgaagttcg cccacttcac
cgcgaaccag gccatcctcg aggccttcgc cggctgccgc 720cgcgtccacg
tcgtcgactt cggcatcaag caggggatgc agtggccggc tcttctccag
780gccctcgccc tccgccctgg cggccccccg tcgttccggc tcaccggcgt
cgggccgccg 840cagcccgacg agaccgacgc cttgcagcag gtgggctgga
aacttgccca gttcgcgcac 900actatccgcg tggacttcca gtaccgtggc
ctcgtcgcgg ccacgctcgc cgacctggag 960ccgttcatgc tgcaaccgga
gggcgatgac acggatgacg agcccgaggt gatcgccgtg 1020aactccgtgt
tcgagctgca ccggcttctt gcgcagcccg gtgcactcga gaaggtcctg
1080ggcacggtgc gcgcggtgcg gccgaggatc gtgaccgtgg tcgagcagga
ggccaaccac 1140aactccggca cgttcctcga ccgcttcacc gagtcgctgc
actactactc caccatgttc 1200gattctctcg agggcgccgg cgccggctcc
ggccagtcca ccgacgcctc cccggccgcg 1260gccggcggca cggaccaggt
catgtcggag gtgtacctcg gccggcagat ctgcaacgtg 1320gtggcgtgcg
agggcgcgga gcgcacggaa cgccacgaga cgctggggca gtggcgcagc
1380cgcctcggcg gctccgggtt cgcgcccgtg cacctgggct ccaatgccta
caagcaggcg 1440agcacgctgc tggcgctctt cgccggcggc gacgggtaca
gggtggagga gaaggacggg 1500tgcctgaccc tggggtggca tacgcgcccg
ctcatcgcca cctcggcgtg gcgcgtcgcc 1560gccgccgccg ctccgtga
15787825DNAZea mays 7aaatccttac agaattgctg tagtttcata gtgctagatg
tggacagcaa agcgccgctg 60tatgcttctg cttttctttt ttggtgtgtg tagccacatc
ctttgttcct gcccggcgcc 120atcccacttg gttgtttttt tttatgattg
aaagccttca tgcttcctcg gtcaatcacc 180ggtgcgcact gggagcatcg
ccggaaaaaa aattcttcgg ctaagagtaa cttctttctc 240cttttcttct
ctgatctcgc gagcagtgct gataacgtgt tgtaatctac ttagcggtaa
300cgagattgag agagacaaaa tgacagaact attgtcttta ttgcagagtg
tcatgtattt 360atacagggga tacaaagtct cccaaggggt gtgtcccttg
ggagtaactg ccagttgatc 420acaggacaat attttgtaac aaaacgtaca
catcgtcaaa atagcgaggc atgaaactgg 480ccttggccat ggacgcgtga
agcgcgccat gcgttggata tgtggtcaat aagtatatac 540aatacaatgt
ttaacagagc tgatagtact gctttggcac atttttgtcc acgcttcatg
600agagataaaa cacctgcacg taaattcaca tgctgcactg aaggcccgat
cactgaggag 660cgaactgccg taactccctt ctatatatac ccccagtccc
tgtttcagtt ttcgtcaagc 720tagcagcacc aagttgtcga tcacttgcct
gctcttgagc tcgattaagc tatcatcagc 780tacagcatcc gatcccaaac
tgcaactgta gcagcgacaa ctgcc 8258162DNAZea mays 8actgccgtaa
ctcccttcta tatatacccc cagtccctgt ttcagttttc gtcaagctag 60cagcaccaag
ttgtcgatca cttgcctgct cttgagctcg attaagctat catcagctac
120agcatccgat cccaaactgc aactgtagca gcgacaactg cc 1629273DNAZea
mays 9atgctcgtgc acgcgctgct ggcgtgcgcg gaggccgtgc agcaggagaa
cttctctgcg 60gcggaggcgc tggtcaagca gatccccatg ctggcctcgt cgcagggcgg
tgccatgcgc 120aaggtcgccg cctacttcgg cgaggcgctt gcccgccgcg
tgtatcgctt ccgcccgcca 180ccggacagct ccctcctcga cgccgccttc
gccgacctct tgcacgcgca cttctacgag 240tcctgcccct acctgaagtt
cgcccacttc tag 27310277DNAZea mays 10ctaaagaagg agtgcgtcga
agcagatcgt tcaaacattt ggcaataaag tttcttaaga 60ttgaatcctg ttgccggtct
tgcgatgatt atcatataat ttctgttgaa ttacgttaag 120catgtaataa
ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga
180gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc
aaactaggat 240aaattatcgc gcgcggtgtc atctatgtta ctagatc
27711738DNAZea mays 11atggggcgcg ggaaggtgca gctgaagcgg atcgagaaca
agatcaaccg ccaggtgaca 60ttctccaagc gccgctcggg gctactcaag aaggcgcacg
agatctccgt gctctgcgac 120gccgaggtcg cgctcatcat cttctccacc
aagggcaagc tctacgagta ctctaccgat 180tcatgtatgg acaaaattct
tgaacggtat gagcgctact cctatgcaga aaaggttctc 240atttccgcag
aatatgaaac tcagggcaat tggtgccatg aatatagaaa actaaaggcg
300aaggtcgaga caatacagaa atgtcaaaag cacctcatgg gagaggatct
tgaaactttg 360aatctcaaag agcttcagca actagagcag cagctggaga
gttcactgaa acatatcaga 420acaaggaaga gccagcttat ggtcgagtca
atttcagcgc tccaacggaa ggagaagtca 480ctgcaggagg agaacaaggt
tctgcagaag gagctcgcgg agaagcagaa agaccagcgg 540cagcaagtgc
aacgggacca aactcaacag cagaccagtt cgtcttccac gtccttcatg
600ttaagggaag ctgccccaac aacaaatgtc agcatcttcc ctgtggcagc
aggcgggagg 660gtggtggaag gggcagcagc gcagccgcag gctcgcgttg
gactgccacc atggatgctt 720agccatctga gctgctga 738121554DNAGlycine
max 12atgaagaggg aacgcgagca gcttggttcc atcgcaggga cctcaagctg
cggttattca 60agcggaaaat cgaatctttg ggaggaagaa ggaggcatgg acgagcttct
tgcggtggtg 120ggttacaagg ttaggtcatc ggacatggcg gaagtggcgc
agaagcttga gcgtctcgaa 180gaagccatgg gaaatgtcca agatgacctc
ccggagattt caaacgacgt cgttcattac 240aacccttccg acatctccaa
ctggctcgaa accatgcttt ctaattttga ccctctcccc 300tccgaagagc
cggaaaagga ctccgcctcg tcggactacg atcttaaggc tattccgggg
360aaagcaattt atggagctag cgacgcgcta ccaaacccta agcgcgtgaa
agccgacgag 420tcaaggcgcg cggtggtggt cgttgactcg caggagaacg
ggatccgcct cgtgcacagc 480ctcatggcgt gcgcggaggc cgtggagaac
aacaacctcg ccgtggcgga ggcgctggtg 540aagcagatcg gcttcctcgc
tgtgtcgcag gttggagcta tgaggaaagt cgcaatctac 600ttcgccgaag
cgctcgcgag gcgaatctac agagtcttcc ctctgcaaca ctctctctcc
660gattctcttc agattcactt ctacgaaacc tgtccatacc tcaagttcgc
acacttcacc 720gcgaaccagg ttatcctcga agcgttccaa ggaaagaacc
gcgttcacgt gattgatttc 780ggtatcaacc aggggatgca gtggccggcg
ctgatgcaag ccctagcggt tcgcaccggc 840ggtcctccgg ttttccgact
caccggaatc gggccgccgg cggcggacaa ctccgaccac 900ctccaggagg
tagggtggaa gctcgcgcag ctggcggagg agatcaacgt gcagttcgag
960taccgtggct tcgtcgcgaa cagcctcgcc gatctcgacg cctccatgct
cgatctccgg 1020gaaggcgaag ccgtcgctgt gaactctgtc ttcgagtttc
acaagctcct cgcccgcccc 1080ggcgcggtgg agaaagtact ctccgtcgta
cggcagattc ggccggagat tgtcaccgtc 1140gtcgagcaag aagcgaacca
caacagactg agttttgtcg accggttcac ggagtcactg 1200cactattatt
caaccctatt cgactcgctg gagggttcgc ctgtgaaccc taacgataag
1260gccatgtcgg aggtttactt agggaagcaa atctgcaacg tggtggcgtg
cgagggaatg 1320gaccgcgtgg aaaggcacga gacgctgaac cagtggcgga
accggttcgt ttcgaccgga 1380ttttcttcgg ttcacttggg ttcgaacgcg
tacaagcagg ccagcatgtt gctcgcgctt 1440tttgcgggtg gggatgggta
tagggtggaa gagaacaatg gttgtctcat gttgggatgg 1500cacactaggc
ccttgattgc cacctccgcg tggcaactcg ctgcaactcg ctga 1554134215DNAZea
mays 13gatccctgtg gagaaatttt tacgtcgcgg ggatggtatg gggagttatt
cccctgtagg 60aaatgggtga cgcctaagag ggagggtgaa gtaggacttc taaaactttc
actaaactag 120gccacaaata attccctaga gcaaaaccta tgcaaatagt
caaactagaa tgtgcaaacc 180aagttttgtc taagtgttgc tatctctacc
gcaatggcta agtttcaatc tacactatat 240aagtatgaat acaagaatga
aacttaaata cttaatataa atgcggaaac ttaaagagca 300aggtagagat
gcaaattctc gtggatgacg cctgcatttt tatcgaggta tccggaacca
360cgcaaggtcc cgactaatcc tcattggtgc ccctacgcaa agggaagccc
acgcgagggc 420caagcacctc ggtcgagtaa ctctatagag agccgtgggc
cttctccacg cgcaagtggt 480gctctgcttt cagctcctct cagaccctcc
ccgctgtctc cactatcgag cttccggctg 540aaaatgccat gggcctcgtt
ccctccggta cacggtggcg gccgtgacac aaatgcggtt 600atcacggtct
cgcaagactc tcacccccac ttggtacaat ttcaatggct cgcacaagag
660ccgaggggtt gatggtttat ctaatctcac tcaactaact
aggattcatc taaagcaagc 720gctagagcgg tctaactaac ctaagcactt
cacaaagcac ctacgctaat caccgagtga 780ttctatttag cacttgggtg
caagagcact tgagaatgtc tactatatgc cttgctatgt 840ctcttgggct
cccaaacttg gaaatggccg gttggtggtg tatttatagc ccccaacaca
900aaactagccg ttggaggaag ctgctgcttt ttgtggtgca ccggacagtc
cggtggggtc 960accagacagt ccgacgcccc tgtccggtgc ccctgtccga
tgcgcctagt tgttgggtct 1020gtcagcgtag gtgaccgttg gcgcgcaggc
tttttgcacc ggacagtccg gtggtcttcc 1080ctcgacagtg ccacctggag
ctagccgtta gggctactgt tcctggtgca ccggacagta 1140gtccggtgct
cttgtctgga cagtccgact gtggcaacac ttcttctttt cttggacttt
1200acttgatctt catgatgtct tcttttgagg tgttgctttc ctaagtgcct
tggtccaagt 1260aacttatcat cctgtgaact acaaacacaa atagtagcaa
acacattagt ccacaggtta 1320tgttgatcat caaataccaa aatctattaa
gccaaatggc ccagggtcca ttttccttac 1380atccccgacg aagaattctc
cgttgccatc cctatctgtg tacgcactac tggaatccgg 1440gtctttgctg
agtaccgcac tcggcaaagt cctactctcg gtaacgatgc cttttgccga
1500gagcaggact ctcggcacag gaatacactc ggcgaagggc gggtctcggc
aaaggccgtt 1560agccaccgtc caaagctgac ggtcgttacc tatgccgagt
ggtggaaaga tattgtgaag 1620gcctaaggcc gatttcgtcc taagcagggc
ccaaaggaag gaagtacttc agtggatcaa 1680gatgttgatg ttccctgatg
ggtatgcagc taacctgagt aggtggggtg aacttatcta 1740ctctgtgagt
cttagggatg aagagtcatg acttccacat atggattgaa cagattcttc
1800tctgtgcatg gacaatctgg ggcggcatcc aacaaccctc atggatcgcc
cggccaatcg 1860ccgcaccagt ccatccgccc acctcgatga gacttatgtt
cttagtgttg agacttcaga 1920acttattgat aatgctgtat tggatactta
tgtttgtgtt cgatacttat gtgagaactt 1980gagacttatg agacttatgt
tcttgatact tatgtttgtg ttgagaactt ggatatttat 2040gtttgtgttg
gatacttatg tctgtgatga tatatgtgat gtatatatgt gatgtatatg
2100tgacatatgt gatgtatatg tggtatcttt tgtttgtttg gatggaatag
agaaagcaaa 2160taaaaatgtg tatactggtc actttgtcga gtgtaacact
cggcaaaaag gtgctttgcc 2220gagtgttagg gccatagcac tcggtagaga
accaatactt aggcaccggt aaagcttttt 2280tgccgagtgt tgtggccctg
gcactcagct ttgccgagtg cctcacagag cactcgacaa 2340agaacctgac
aaatggaccc gctggtaaat cctttaccga gtgcaggtca gtagacactc
2400ggcaaaggta acttctttgc cgagtgccgc ttagaacatt tgacaaaggg
tcatctccgt 2460tacccggtgt cgtgacggcc gcttttcttt gccgagtgcc
tgatagaaag tactcggcaa 2520agaagtcgtt gccaatgtat tgttcgctga
ggtctctttg tcaagtatta cactcggcaa 2580agactgtgcc gagtgttttt
cagactttgc cgagtggttt aagcactcag caaagcgctc 2640gatttcggta
gtgacggttg tttggcaata gtaaaatcca gccctctccc gtggggaaaa
2700aactggtagg atctggctcg tggctaagat tctctttctt ccctttgtaa
aaaaagagaa 2760gaaaaaaaaa acgactgtca cggtgccttg tctggtaatg
atcgcgcggt cggctctgtc 2820ctaacccgta agatggacgg gagctgatga
tagcgtgacc tccaaataaa caacaagggc 2880gtgttccccg tggtcgaata
ttttaagggc cactgattag gtgcggttga atacatcaac 2940ttcacgaaca
tcatctgatc tgatctgatt tggtctgata tgatctgggt agtcatttct
3000gcaatgagca tctatcaggt gaaccaatta atattgatga cattatgagt
tcgaagatat 3060actctaaagt gttatctaaa tacagaagac attcgttcgt
tctttgccta taactctaaa 3120aggcttgtaa caccctcatt catcctctat
atacgaagac tctctcctat catttttatc 3180gatttatttt ttttatattt
tagacaatgg aattaaatag aactaaaata tatataagaa 3240tctgaggacc
cgagatggta atggggactc gatcctcgat tctccacgga gaattcctct
3300aggatatagg taatttgtcc ccacgaggat tgaaacgggg taatttggtc
cccatgtgcc 3360cgtcccgcga acttctcttg atctaaatta gtctatttcc
atgttaaaac tatactaaaa 3420atttaataca cagtctatta taaaatagca
aactaaattc taaagttgat gcatcttgta 3480attttaaatc tggtttgttc
aagttatatt catttgatat aataaatttg aatttgactc 3540ttaatatcgt
attttttcct aacggggacg gattctccac ggggataaat tccatgatac
3600agatgggatg aaagaaaaat ctcccgtatg aacttttgca ggaatgggga
tgggccagag 3660aaattttctc cctgcgggga cgggggagcc atatcctcgg
tggagaattt cccattatca 3720tccttatttg tggtacatat atatgcataa
tctttttttt ttgactgaca tgtgggaaag 3780tatcccatct caatagtaga
aaatcttggg aacggtagga tcgaacacaa agatcagcta 3840gcttgtaatc
accgagccat atagctagag ggtaatagat catgaatcaa atgttttttt
3900cataaattat taaggctcta aattattttt aatttaaaaa taaataaaaa
tatagttcga 3960ttcttacatt ttatagtgta aaactttaaa gtctattatt
acccctactt attgagttat 4020ggttcagttc ttgtcgacgg agagtaatga
gatatagaat aaggtaccct atagaataaa 4080gaatctttct ctgaaaagtc
tgacgtacgt aaataagata taataaaaaa aatacaaaga 4140gaagcgctgg
actggagatg ctcctatatg cggcaatgcc tgtgcttata aatagccacc
4200tcggtcggca aggac 421514538DNAZea mays 14gtccgccttg tttctcctct
gtctcttgat ctgactaatc ttggtttatg attcgttgag 60taattttggg gaaagcttcg
tccacagttt ttttttcgat gaacagtgcc gcagtggcgc 120tgatcttgta
tgctatcctg caatcgtggt gaacttattt cttttatatc cttcactccc
180atgaaaaggc tagtaatctt tctcgatgta acatcgtcca gcactgctat
taccgtgtgg 240tccatccgac agtctggctg aacacatcat acgatattga
gcaaagatcg atctatcttc 300cctgttcttt aatgaaagac gtcattttca
tcagtatgat ctaagaatgt tgcaacttgc 360aaggaggcgt ttctttcttt
gaatttaact aactcgttga gtggccctgt ttctcggacg 420taaggccttt
gctgctccac acatgtccat tcgaatttta ccgtgtttag caagggcgaa
480aagtttgcat cttgatgatt tagcttgact atgcgattgc tttcctggac ccgtgcag
538151377DNAZea mays 15aattcgccct tgtttaaact taatatttgt ttaaactttt
tactaaattc atgtaataat 60taatgtatgc gttatatata tatgtctagg tttataatta
ttcatatgaa tatgaacata 120aaaatctagg gctaaaacga ctactatttt
gaaaacggaa ggagtagtaa gttatttaag 180cggaggggaa ccatgatggg
ctagtgattt aatttacata tatatattgg tgttctgggc 240tcttacatga
gaagatctag ttaactgttg ttactgaaca gcgaagacaa atatataatt
300taagctcccc aactgctagt gattctgtta agaggtaatg tttaaagtaa
atttacaaga 360gcccgtctag ctcagtcggt agagcgcaag gctcttaacc
ttgtggtcgt gggttcgagc 420cccacggtgg gcgcacaatt ttttgttttt
tgacattttt tgtttgctta gttgcagacg 480gtttttcccc tgctaggaga
tttccgagag aaaaaaaagg cactacaggt taaccaaaac 540caccaacctt
tggagcgtcg aggcgacggg gcatttgcgt agttgaagct tacaaagttg
600catatgagat gagtgccgga catgaagcgg ataacgtttt aaactggcaa
caatatctag 660ctgtttcaaa ttcaggcgtg ggaagctacg cctacgcgcc
ctggacggcg tgtaaagagc 720cagcatcggc atcattgtca aacgatcgac
aaggccaaga aattccaaat atattattaa 780taaaaaagaa ggcaccaaat
tagtttttgt tttttagtat gtgtggcgga ggaaattttg 840agaacgaacg
tatccaaaga aggcacaaga cgatatagat tgacgcggct agaaagttgc
900agcaagacag tgggtacggt cttatatatc ctaataaata aaaaataaaa
ctatagtgtg 960tcaaatgtca acaagaggag gaggcagcca aattagcaga
gggagacaag tagagcacgc 1020cttattagct tgcttattta tcgtggtggt
gtacttgtta attactggca cgcattatca 1080acaacgcagt tctggatgtg
aatctagaca aacatttgtc taggttccgc acgtatagtt 1140ttttttcttt
ttttttgggg ggggggggga acggaagctg taataaacgg tactaggaac
1200gaaagcaacc gccgcgcgca tgtttttgca atagattacg gtgaccttga
tgcaccaccg 1260cgtgctataa aaaccagtgt ccccgagtct actcatcaac
caatccataa ctcgaaacct 1320tttcttgtgc tctgttctgt ctgtgtgttt
ccaaagcaag cgaaagaggt cgagggg 1377161078PRTZea mays 16Met Asp Ala
Gly Ser Val Thr Gly Gly Leu Ala Ala Gly Ser His Met1 5 10 15 Arg
Asp Glu Leu His Val Met Arg Ala Arg Glu Glu Pro Asn Ala Lys 20 25
30 Val Arg Ser Ala Asp Val Lys Thr Cys Arg Val Cys Ala Asp Glu Val
35 40 45 Gly Thr Arg Glu Asp Gly Gln Pro Phe Val Ala Cys Ala Glu
Cys Gly 50 55 60 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg
Ser Glu Gly Thr65 70 75 80 Gln Cys Cys Pro Gln Cys Asn Thr Arg Tyr
Lys Arg Gln Lys Gly Cys 85 90 95 Pro Arg Val Glu Gly Asp Glu Glu
Glu Gly Pro Glu Met Asp Asp Phe 100 105 110 Glu Asp Glu Phe Pro Ala
Lys Ser Pro Lys Lys Pro His Glu Pro Val 115 120 125 Ala Phe Asp Val
Tyr Ser Glu Asn Gly Glu His Pro Ala Gln Lys Trp 130 135 140 Arg Thr
Gly Gly Gln Thr Leu Ser Ser Phe Thr Gly Ser Val Ala Gly145 150 155
160 Lys Asp Leu Glu Ala Glu Arg Glu Met Glu Gly Ser Met Glu Trp Lys
165 170 175 Asp Arg Ile Asp Lys Trp Lys Thr Lys Gln Glu Lys Arg Gly
Lys Leu 180 185 190 Asn His Asp Asp Ser Asp Asp Asp Asp Asp Lys Asn
Glu Asp Glu Tyr 195 200 205 Met Leu Leu Ala Glu Ala Arg Gln Pro Leu
Trp Arg Lys Val Pro Ile 210 215 220 Pro Ser Ser Met Ile Asn Pro Tyr
Arg Ile Val Ile Val Leu Arg Leu225 230 235 240 Val Val Leu Cys Phe
Phe Leu Lys Phe Arg Ile Thr Thr Pro Ala Thr 245 250 255 Asp Ala Val
Pro Leu Trp Leu Ala Ser Val Ile Cys Glu Leu Trp Phe 260 265 270 Ala
Phe Ser Trp Ile Leu Asp Gln Leu Pro Lys Trp Ala Pro Val Thr 275 280
285 Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg Tyr Asp Arg Glu Gly
290 295 300 Glu Ala Cys Arg Leu Ser Pro Ile Asp Phe Phe Val Ser Thr
Val Asp305 310 315 320 Pro Leu Lys Glu Pro Pro Ile Ile Thr Ala Asn
Thr Val Leu Ser Ile 325 330 335 Leu Ala Val Asp Tyr Pro Val Asp Arg
Val Ser Cys Tyr Val Ser Asp 340 345 350 Asp Gly Ala Ser Met Leu Leu
Phe Asp Ala Leu Ser Glu Thr Ala Glu 355 360 365 Phe Ala Arg Arg Trp
Val Pro Phe Cys Lys Lys Phe Ala Val Glu Pro 370 375 380 Arg Ala Pro
Glu Phe Tyr Phe Ser Gln Lys Ile Asp Tyr Leu Lys Asp385 390 395 400
Lys Val Gln Pro Thr Phe Val Lys Glu Arg Arg Ala Met Lys Arg Glu 405
410 415 Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys Ala
Gln 420 425 430 Lys Lys Pro Glu Glu Gly Trp Val Met Gln Asp Gly Thr
Pro Trp Pro 435 440 445 Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile
Gln Val Tyr Leu Gly 450 455 460 Asn Gln Gly Ala Leu Asp Val Glu Gly
His Glu Leu Pro Arg Leu Val465 470 475 480 Tyr Val Ser Arg Glu Lys
Arg Pro Gly Tyr Asn His His Lys Lys Ala 485 490 495 Gly Ala Met Asn
Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn Ala 500 505 510 Pro Phe
Ile Leu Asn Leu Asp Cys Asp His Tyr Val Asn Asn Ser Lys 515 520 525
Ala Val Arg Glu Ala Met Cys Phe Leu Met Asp Pro Gln Leu Gly Lys 530
535 540 Lys Leu Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp
Arg545 550 555 560 His Asp Arg Tyr Ala Asn Arg Asn Val Val Phe Phe
Asp Ile Asn Met 565 570 575 Lys Gly Leu Asp Gly Ile Gln Gly Pro Val
Tyr Val Gly Thr Gly Cys 580 585 590 Val Phe Asn Arg Gln Ala Leu Tyr
Gly Tyr Asp Pro Pro Arg Pro Glu 595 600 605 Lys Arg Pro Lys Met Thr
Cys Asp Cys Trp Pro Ser Trp Cys Cys Cys 610 615 620 Cys Cys Cys Phe
Gly Gly Gly Lys Arg Gly Lys Ala Arg Lys Asp Lys625 630 635 640 Lys
Gly Asp Gly Gly Glu Glu Pro Arg Arg Gly Leu Leu Gly Phe Tyr 645 650
655 Arg Lys Arg Ser Lys Lys Asp Lys Leu Gly Gly Gly Ser Val Ala Gly
660 665 670 Ser Lys Lys Gly Gly Gly Leu Tyr Lys Lys His Gln Arg Ala
Phe Glu 675 680 685 Leu Glu Glu Ile Glu Glu Gly Leu Glu Gly Tyr Asp
Glu Leu Glu Arg 690 695 700 Ser Ser Leu Met Ser Gln Lys Ser Phe Glu
Lys Arg Phe Gly Gln Ser705 710 715 720 Pro Val Phe Ile Ala Ser Thr
Leu Val Glu Asp Gly Gly Leu Pro Gln 725 730 735 Gly Ala Ala Ala Asp
Pro Ala Ala Leu Ile Lys Glu Ala Ile His Val 740 745 750 Ile Ser Cys
Gly Tyr Glu Glu Lys Thr Glu Trp Gly Lys Glu Ile Gly 755 760 765 Trp
Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met 770 775
780 His Cys Arg Gly Trp Lys Ser Val Tyr Cys Thr Pro Thr Arg Pro
Ala785 790 795 800 Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg
Leu His Gln Val 805 810 815 Leu Arg Trp Ala Leu Gly Ser Val Glu Ile
Phe Met Ser Arg His Cys 820 825 830 Pro Leu Arg Tyr Ala Tyr Gly Gly
Arg Leu Lys Trp Leu Glu Arg Phe 835 840 845 Ala Tyr Thr Asn Thr Ile
Val Tyr Pro Phe Thr Ser Ile Pro Leu Leu 850 855 860 Ala Tyr Cys Thr
Ile Pro Ala Val Cys Leu Leu Thr Gly Lys Phe Ile865 870 875 880 Ile
Pro Thr Leu Asn Asn Leu Ala Ser Ile Trp Phe Ile Ala Leu Phe 885 890
895 Leu Ser Ile Ile Ala Thr Ser Val Leu Glu Leu Arg Trp Ser Gly Val
900 905 910 Ser Ile Glu Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile
Gly Gly 915 920 925 Val Ser Ala His Leu Phe Ala Val Phe Gln Gly Phe
Leu Lys Val Leu 930 935 940 Gly Gly Val Asp Thr Ser Phe Thr Val Thr
Ser Lys Ala Ala Gly Asp945 950 955 960 Glu Ala Asp Ala Phe Gly Asp
Leu Tyr Leu Phe Lys Trp Thr Thr Leu 965 970 975 Leu Val Pro Pro Thr
Thr Leu Ile Ile Ile Asn Met Val Gly Ile Val 980 985 990 Ala Gly Val
Ser Asp Ala Val Asn Asn Gly Tyr Gly Ser Trp Gly Pro 995 1000 1005
Leu Phe Gly Lys Leu Phe Phe Ser Phe Trp Val Ile Val His Leu Tyr
1010 1015 1020 Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr
Pro Thr Ile1025 1030 1035 1040Val Val Leu Trp Ser Ile Leu Leu Ala
Ser Ile Phe Ser Leu Val Trp 1045 1050 1055 Val Arg Ile Asp Pro Phe
Ile Pro Lys Ala Lys Gly Pro Ile Leu Lys 1060 1065 1070 Pro Cys Gly
Val Glu Cys 1075 171077PRTZea mays 17Met Glu Gly Asp Ala Asp Gly
Val Lys Ser Gly Arg Arg Gly Gly Gly1 5 10 15 Gln Val Cys Gln Ile
Cys Gly Asp Gly Val Gly Thr Thr Ala Glu Gly 20 25 30 Asp Val Phe
Ala Ala Cys Asp Val Cys Gly Phe Pro Val Cys Arg Pro 35 40 45 Cys
Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln Ala Cys Pro Gln Cys 50 55
60 Lys Thr Lys Tyr Lys Arg His Lys Gly Ser Pro Ala Ile Arg Gly
Glu65 70 75 80 Glu Gly Asp Asp Thr Asp Ala Asp Ser Asp Phe Asn Tyr
Leu Ala Ser 85 90 95 Gly Asn Glu Asp Gln Lys Gln Lys Ile Ala Asp
Arg Met Arg Ser Trp 100 105 110 Arg Met Asn Val Gly Gly Ser Gly Asp
Val Gly Arg Pro Lys Tyr Asp 115 120 125 Ser Gly Glu Ile Gly Leu Thr
Lys Tyr Asp Ser Gly Glu Ile Pro Arg 130 135 140 Gly Tyr Ile Pro Ser
Val Thr Asn Ser Gln Ile Ser Gly Glu Ile Pro145 150 155 160 Gly Ala
Ser Pro Asp His His Met Met Ser Pro Thr Gly Asn Ile Gly 165 170 175
Lys Arg Ala Pro Phe Pro Tyr Val Asn His Ser Pro Asn Pro Ser Arg 180
185 190 Glu Phe Ser Gly Ser Ile Gly Asn Val Ala Trp Lys Glu Arg Val
Asp 195 200 205 Gly Trp Lys Met Lys Gln Asp Lys Gly Thr Ile Pro Met
Thr Asn Gly 210 215 220 Thr Ser Ile Ala Pro Ser Glu Gly Arg Gly Val
Gly Asp Ile Asp Ala225 230 235 240 Ser Thr Asp Tyr Asn Met Glu Asp
Ala Leu Leu Asn Asp Glu Thr Arg 245 250 255 Gln Pro Leu Ser Arg Lys
Val Pro Leu Pro Ser Ser Arg Ile Asn Pro 260 265 270 Tyr Arg Met Val
Ile Val Leu Arg Leu Ile Val Leu Ser Ile Phe Leu 275 280 285 His Tyr
Arg Ile Thr Asn Pro Val Arg Asn Ala Tyr Pro Leu Trp Leu 290 295 300
Leu Ser Val Ile Cys Glu Ile Trp Phe Ala Leu Ser Trp Ile Leu Asp305
310 315 320 Gln Phe Pro Lys Trp Phe Pro Ile Asn Arg Glu Thr Tyr Leu
Asp Arg 325 330 335 Leu Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser
Gln Leu Ala Ala 340 345 350 Val Asp Ile Phe Val Ser Thr Val Asp Pro
Met Lys Glu Pro Pro Leu 355 360 365 Val Thr Ala Asn Thr Val Leu Ser
Ile Leu Ala Val Asp Tyr Pro Val 370 375 380
Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr385
390 395 400 Phe Asp Ala Leu Ala Glu Thr Ser Glu Phe Ala Arg Lys Trp
Val Pro 405 410 415 Phe Val Lys Lys Tyr Asn Ile Glu Pro Arg Ala Pro
Glu Trp Tyr Phe 420 425 430 Ser Gln Lys Ile Asp Tyr Leu Lys Asp Lys
Val His Pro Ser Phe Val 435 440 445 Lys Asp Arg Arg Ala Met Lys Arg
Glu Tyr Glu Glu Phe Lys Val Arg 450 455 460 Val Asn Gly Leu Val Ala
Lys Ala Gln Lys Val Pro Glu Glu Gly Trp465 470 475 480 Ile Met Gln
Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His 485 490 495 Pro
Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr 500 505
510 Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg
515 520 525 Pro Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala
Leu Val 530 535 540 Arg Val Ser Ala Val Leu Thr Asn Gly Gln Tyr Met
Leu Asn Leu Asp545 550 555 560 Cys Asp His Tyr Ile Asn Asn Ser Lys
Ala Leu Arg Glu Ala Met Cys 565 570 575 Phe Leu Met Asp Pro Asn Leu
Gly Arg Ser Val Cys Tyr Val Gln Phe 580 585 590 Pro Gln Arg Phe Asp
Gly Ile Asp Arg Asn Asp Arg Tyr Ala Asn Arg 595 600 605 Asn Thr Val
Phe Phe Asp Ile Asn Leu Arg Gly Leu Asp Gly Ile Gln 610 615 620 Gly
Pro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Thr Ala Leu625 630
635 640 Tyr Gly Tyr Glu Pro Pro Ile Lys Gln Lys Lys Gly Gly Phe Leu
Ser 645 650 655 Ser Leu Cys Gly Gly Arg Lys Lys Ala Ser Lys Ser Lys
Lys Gly Ser 660 665 670 Asp Lys Lys Lys Ser Gln Lys His Val Asp Ser
Ser Val Pro Val Phe 675 680 685 Asn Leu Glu Asp Ile Glu Glu Gly Val
Glu Gly Ala Gly Phe Asp Asp 690 695 700 Glu Lys Ser Leu Leu Met Ser
Gln Met Ser Leu Glu Lys Arg Phe Gly705 710 715 720 Gln Ser Ala Ala
Phe Val Ala Ser Thr Leu Met Glu Tyr Gly Gly Val 725 730 735 Pro Gln
Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile His Val 740 745 750
Ile Ser Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Thr Glu Ile Gly 755
760 765 Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys
Met 770 775 780 His Ala Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Lys
Arg Pro Ala785 790 795 800 Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser
Asp Arg Leu Asn Gln Val 805 810 815 Leu Arg Trp Ala Leu Gly Ser Val
Glu Ile Leu Phe Ser Arg His Cys 820 825 830 Pro Leu Trp Tyr Gly Tyr
Gly Gly Arg Leu Lys Phe Leu Glu Arg Phe 835 840 845 Ala Tyr Ile Asn
Thr Thr Ile Tyr Pro Leu Thr Ser Ile Pro Leu Leu 850 855 860 Ile Tyr
Cys Ile Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile865 870 875
880 Ile Pro Glu Ile Ser Asn Phe Ala Ser Ile Trp Phe Ile Ser Leu Phe
885 890 895 Ile Ser Ile Phe Ala Thr Gly Ile Leu Glu Met Arg Trp Ser
Gly Val 900 905 910 Gly Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe Trp
Val Ile Gly Gly 915 920 925 Ile Ser Ala His Leu Phe Ala Val Phe Gln
Gly Leu Leu Lys Val Leu 930 935 940 Ala Gly Ile Asp Thr Asn Phe Thr
Val Thr Ser Lys Ala Ser Asp Glu945 950 955 960 Asp Gly Asp Phe Ala
Glu Leu Tyr Met Phe Lys Trp Thr Thr Leu Leu 965 970 975 Ile Pro Pro
Thr Thr Ile Leu Ile Ile Asn Leu Val Gly Val Val Ala 980 985 990 Gly
Ile Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu 995
1000 1005 Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu
Tyr Pro 1010 1015 1020 Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg
Thr Pro Thr Ile Val1025 1030 1035 1040Val Val Trp Ala Ile Leu Leu
Ala Ser Ile Phe Ser Leu Leu Trp Val 1045 1050 1055 Arg Ile Asp Pro
Phe Thr Thr Arg Val Thr Gly Pro Asp Thr Gln Thr 1060 1065 1070 Cys
Gly Ile Asn Cys 1075 18525PRTZea mays 18Met Leu Ser Glu Leu Asn Ala
Pro Pro Ala Pro Leu Pro Pro Ala Thr1 5 10 15 Pro Ala Pro Arg Leu
Ala Ser Thr Ser Ser Thr Val Thr Ser Gly Ala 20 25 30 Ala Ala Gly
Ala Gly Tyr Phe Asp Leu Pro Pro Ala Val Asp Ser Ser 35 40 45 Ser
Ser Thr Tyr Ala Leu Lys Pro Ile Pro Ser Pro Val Ala Ala Pro 50 55
60 Ser Ala Asp Pro Ser Thr Asp Ser Ala Arg Glu Pro Lys Arg Met
Arg65 70 75 80 Thr Gly Gly Gly Ser Thr Ser Ser Ser Ser Ser Ser Ser
Ser Ser Met 85 90 95 Asp Gly Gly Arg Thr Arg Ser Ser Val Val Glu
Ala Ala Pro Pro Ala 100 105 110 Thr Gln Ala Ser Ala Ala Ala Asn Gly
Pro Ala Val Pro Val Val Val 115 120 125 Val Asp Thr Gln Glu Ala Gly
Ile Arg Leu Val His Ala Leu Leu Ala 130 135 140 Cys Ala Glu Ala Val
Gln Gln Glu Asn Phe Ser Ala Ala Glu Ala Leu145 150 155 160 Val Lys
Gln Ile Pro Met Leu Ala Ser Ser Gln Gly Gly Ala Met Arg 165 170 175
Lys Val Ala Ala Tyr Phe Gly Glu Ala Leu Ala Arg Arg Val Tyr Arg 180
185 190 Phe Arg Pro Pro Pro Asp Ser Ser Leu Leu Asp Ala Ala Phe Ala
Asp 195 200 205 Leu Leu His Ala His Phe Tyr Glu Ser Cys Pro Tyr Leu
Lys Phe Ala 210 215 220 His Phe Thr Ala Asn Gln Ala Ile Leu Glu Ala
Phe Ala Gly Cys Arg225 230 235 240 Arg Val His Val Val Asp Phe Gly
Ile Lys Gln Gly Met Gln Trp Pro 245 250 255 Ala Leu Leu Gln Ala Leu
Ala Leu Arg Pro Gly Gly Pro Pro Ser Phe 260 265 270 Arg Leu Thr Gly
Val Gly Pro Pro Gln Pro Asp Glu Thr Asp Ala Leu 275 280 285 Gln Gln
Val Gly Trp Lys Leu Ala Gln Phe Ala His Thr Ile Arg Val 290 295 300
Asp Phe Gln Tyr Arg Gly Leu Val Ala Ala Thr Leu Ala Asp Leu Glu305
310 315 320 Pro Phe Met Leu Gln Pro Glu Gly Asp Asp Thr Asp Asp Glu
Pro Glu 325 330 335 Val Ile Ala Val Asn Ser Val Phe Glu Leu His Arg
Leu Leu Ala Gln 340 345 350 Pro Gly Ala Leu Glu Lys Val Leu Gly Thr
Val Arg Ala Val Arg Pro 355 360 365 Arg Ile Val Thr Val Val Glu Gln
Glu Ala Asn His Asn Ser Gly Thr 370 375 380 Phe Leu Asp Arg Phe Thr
Glu Ser Leu His Tyr Tyr Ser Thr Met Phe385 390 395 400 Asp Ser Leu
Glu Gly Ala Gly Ala Gly Ser Gly Gln Ser Thr Asp Ala 405 410 415 Ser
Pro Ala Ala Ala Gly Gly Thr Asp Gln Val Met Ser Glu Val Tyr 420 425
430 Leu Gly Arg Gln Ile Cys Asn Val Val Ala Cys Glu Gly Ala Glu Arg
435 440 445 Thr Glu Arg His Glu Thr Leu Gly Gln Trp Arg Ser Arg Leu
Gly Gly 450 455 460 Ser Gly Phe Ala Pro Val His Leu Gly Ser Asn Ala
Tyr Lys Gln Ala465 470 475 480 Ser Thr Leu Leu Ala Leu Phe Ala Gly
Gly Asp Gly Tyr Arg Val Glu 485 490 495 Glu Lys Asp Gly Cys Leu Thr
Leu Gly Trp His Thr Arg Pro Leu Ile 500 505 510 Ala Thr Ser Ala Trp
Arg Val Ala Ala Ala Ala Ala Pro 515 520 525 1990PRTZea mays 19Met
Leu Val His Ala Leu Leu Ala Cys Ala Glu Ala Val Gln Gln Glu1 5 10
15 Asn Phe Ser Ala Ala Glu Ala Leu Val Lys Gln Ile Pro Met Leu Ala
20 25 30 Ser Ser Gln Gly Gly Ala Met Arg Lys Val Ala Ala Tyr Phe
Gly Glu 35 40 45 Ala Leu Ala Arg Arg Val Tyr Arg Phe Arg Pro Pro
Pro Asp Ser Ser 50 55 60 Leu Leu Asp Ala Ala Phe Ala Asp Leu Leu
His Ala His Phe Tyr Glu65 70 75 80 Ser Cys Pro Tyr Leu Lys Phe Ala
His Phe 85 90 20245PRTZea mays 20Met Gly Arg Gly Lys Val Gln Leu
Lys Arg Ile Glu Asn Lys Ile Asn1 5 10 15 Arg Gln Val Thr Phe Ser
Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20 25 30 His Glu Ile Ser
Val Leu Cys Asp Ala Glu Val Ala Leu Ile Ile Phe 35 40 45 Ser Thr
Lys Gly Lys Leu Tyr Glu Tyr Ser Thr Asp Ser Cys Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu65
70 75 80 Ile Ser Ala Glu Tyr Glu Thr Gln Gly Asn Trp Cys His Glu
Tyr Arg 85 90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys
Gln Lys His Leu 100 105 110 Met Gly Glu Asp Leu Glu Thr Leu Asn Leu
Lys Glu Leu Gln Gln Leu 115 120 125 Glu Gln Gln Leu Glu Ser Ser Leu
Lys His Ile Arg Thr Arg Lys Ser 130 135 140 Gln Leu Met Val Glu Ser
Ile Ser Ala Leu Gln Arg Lys Glu Lys Ser145 150 155 160 Leu Gln Glu
Glu Asn Lys Val Leu Gln Lys Glu Leu Ala Glu Lys Gln 165 170 175 Lys
Asp Gln Arg Gln Gln Val Gln Arg Asp Gln Thr Gln Gln Gln Thr 180 185
190 Ser Ser Ser Ser Thr Ser Phe Met Leu Arg Glu Ala Ala Pro Thr Thr
195 200 205 Asn Val Ser Ile Phe Pro Val Ala Ala Gly Gly Arg Val Val
Glu Gly 210 215 220 Ala Ala Ala Gln Pro Gln Ala Arg Val Gly Leu Pro
Pro Trp Met Leu225 230 235 240 Ser His Leu Ser Cys 245
2186PRTGlycine max 21Gly Ile Arg Leu Val His Ser Leu Met Ala Cys
Ala Glu Ala Val Glu1 5 10 15 Asn Asn Asn Leu Ala Val Ala Glu Ala
Leu Val Lys Gln Ile Gly Phe 20 25 30 Leu Ala Val Ser Gln Val Gly
Ala Met Arg Lys Val Ala Ile Tyr Phe 35 40 45 Ala Glu Ala Leu Ala
Arg Arg Ile Tyr Arg Val Phe Pro Leu Gln His 50 55 60 Ser Leu Ser
Asp Ser Leu Gln Ile His Phe Tyr Glu Thr Cys Pro Tyr65 70 75 80 Leu
Lys Phe Ala His Phe 85 22517PRTGlycine max 22Met Lys Arg Glu Arg
Glu Gln Leu Gly Ser Ile Ala Gly Thr Ser Ser1 5 10 15 Cys Gly Tyr
Ser Ser Gly Lys Ser Asn Leu Trp Glu Glu Glu Gly Gly 20 25 30 Met
Asp Glu Leu Leu Ala Val Val Gly Tyr Lys Val Arg Ser Ser Asp 35 40
45 Met Ala Glu Val Ala Gln Lys Leu Glu Arg Leu Glu Glu Ala Met Gly
50 55 60 Asn Val Gln Asp Asp Leu Pro Glu Ile Ser Asn Asp Val Val
His Tyr65 70 75 80 Asn Pro Ser Asp Ile Ser Asn Trp Leu Glu Thr Met
Leu Ser Asn Phe 85 90 95 Asp Pro Leu Pro Ser Glu Glu Pro Glu Lys
Asp Ser Ala Ser Ser Asp 100 105 110 Tyr Asp Leu Lys Ala Ile Pro Gly
Lys Ala Ile Tyr Gly Ala Ser Asp 115 120 125 Ala Leu Pro Asn Pro Lys
Arg Val Lys Ala Asp Glu Ser Arg Arg Ala 130 135 140 Val Val Val Val
Asp Ser Gln Glu Asn Gly Ile Arg Leu Val His Ser145 150 155 160 Leu
Met Ala Cys Ala Glu Ala Val Glu Asn Asn Asn Leu Ala Val Ala 165 170
175 Glu Ala Leu Val Lys Gln Ile Gly Phe Leu Ala Val Ser Gln Val Gly
180 185 190 Ala Met Arg Lys Val Ala Ile Tyr Phe Ala Glu Ala Leu Ala
Arg Arg 195 200 205 Ile Tyr Arg Val Phe Pro Leu Gln His Ser Leu Ser
Asp Ser Leu Gln 210 215 220 Ile His Phe Tyr Glu Thr Cys Pro Tyr Leu
Lys Phe Ala His Phe Thr225 230 235 240 Ala Asn Gln Val Ile Leu Glu
Ala Phe Gln Gly Lys Asn Arg Val His 245 250 255 Val Ile Asp Phe Gly
Ile Asn Gln Gly Met Gln Trp Pro Ala Leu Met 260 265 270 Gln Ala Leu
Ala Val Arg Thr Gly Gly Pro Pro Val Phe Arg Leu Thr 275 280 285 Gly
Ile Gly Pro Pro Ala Ala Asp Asn Ser Asp His Leu Gln Glu Val 290 295
300 Gly Trp Lys Leu Ala Gln Leu Ala Glu Glu Ile Asn Val Gln Phe
Glu305 310 315 320 Tyr Arg Gly Phe Val Ala Asn Ser Leu Ala Asp Leu
Asp Ala Ser Met 325 330 335 Leu Asp Leu Arg Glu Gly Glu Ala Val Ala
Val Asn Ser Val Phe Glu 340 345 350 Phe His Lys Leu Leu Ala Arg Pro
Gly Ala Val Glu Lys Val Leu Ser 355 360 365 Val Val Arg Gln Ile Arg
Pro Glu Ile Val Thr Val Val Glu Gln Glu 370 375 380 Ala Asn His Asn
Arg Leu Ser Phe Val Asp Arg Phe Thr Glu Ser Leu385 390 395 400 His
Tyr Tyr Ser Thr Leu Phe Asp Ser Leu Glu Gly Ser Pro Val Asn 405 410
415 Pro Asn Asp Lys Ala Met Ser Glu Val Tyr Leu Gly Lys Gln Ile Cys
420 425 430 Asn Val Val Ala Cys Glu Gly Met Asp Arg Val Glu Arg His
Glu Thr 435 440 445 Leu Asn Gln Trp Arg Asn Arg Phe Val Ser Thr Gly
Phe Ser Ser Val 450 455 460 His Leu Gly Ser Asn Ala Tyr Lys Gln Ala
Ser Met Leu Leu Ala Leu465 470 475 480 Phe Ala Gly Gly Asp Gly Tyr
Arg Val Glu Glu Asn Asn Gly Cys Leu 485 490 495 Met Leu Gly Trp His
Thr Arg Pro Leu Ile Ala Thr Ser Ala Trp Gln 500 505 510 Leu Ala Ala
Thr Arg 515 23523PRTGlycine max 23Met Lys Arg Glu Arg Gln Gln Leu
Gly Ser Asn Ala Gly Thr Ser Ser1 5 10 15 Cys Gly Tyr Ser Ser Gly
Lys Ser Asn Leu Trp Glu Glu Glu Gly Gly 20 25 30 Met Asp Glu Leu
Leu Ala Val Val Gly Tyr Lys Val Arg Ser Ser Asp 35 40 45 Met Ala
Glu Val Ala Gln Lys Leu Glu Arg Leu Glu Glu Ala Met Gly 50 55 60
Asn Val Gln Asp Asp Leu Thr Asp Leu Ser Asn Asp Ala Val His Tyr65
70 75 80 Asn Pro Ser Asp Ile Ser Asn Trp Leu Gln Thr Met Leu Ser
Asn Phe 85 90 95 Asp Pro Leu Pro Ser Glu Glu Pro Glu Lys Asp Ser
Ala Ser Ser Asp 100 105 110 Tyr Asp Leu Lys Ala Ile Pro Gly Lys Ala
Ile Tyr Gly Gly Gly Ser 115 120 125 Asp Ala Leu Pro Asn Pro Lys Arg
Val Arg Thr Asp Glu Ser Thr Arg 130 135
140 Ala Val Val Val Val Asp Leu Gln Glu Asn Gly Ile Arg Leu Val
His145 150 155 160 Ser Leu Met Ala Cys Ala Glu Ala Val Glu Asn Asn
Asn Leu Ala Val 165 170 175 Ala Glu Ala Leu Val Lys Gln Ile Gly Phe
Leu Ala Leu Ser Gln Val 180 185 190 Gly Ala Met Arg Lys Val Ala Thr
Tyr Phe Ala Glu Ala Leu Ala Arg 195 200 205 Arg Ile Tyr Arg Val Phe
Pro Gln Gln His Ser Leu Ser Asp Ser Leu 210 215 220 Gln Ile His Phe
Tyr Glu Thr Cys Pro Tyr Leu Lys Phe Ala His Phe225 230 235 240 Thr
Ala Asn Gln Ala Ile Leu Glu Ala Phe Gln Gly Lys Asn Arg Val 245 250
255 His Val Ile Asp Phe Gly Ile Asn Gln Gly Met Gln Trp Pro Ala Leu
260 265 270 Met Gln Ala Leu Ala Leu Arg Asn Asp Gly Pro Pro Val Phe
Arg Leu 275 280 285 Thr Gly Ile Gly Pro Pro Ala Ala Asp Asn Ser Asp
His Leu Gln Glu 290 295 300 Val Gly Trp Lys Leu Ala Gln Leu Ala Glu
Arg Ile His Val Gln Phe305 310 315 320 Glu Tyr Arg Gly Phe Val Ala
Asn Ser Leu Ala Asp Leu Asp Ala Ser 325 330 335 Met Leu Asp Leu Arg
Glu Asp Glu Ser Val Ala Val Asn Ser Val Phe 340 345 350 Glu Phe His
Lys Leu Leu Ala Arg Pro Gly Ala Val Glu Lys Val Leu 355 360 365 Ser
Val Val Arg Gln Ile Arg Pro Glu Ile Leu Thr Val Val Glu Gln 370 375
380 Glu Ala Asn His Asn Gly Leu Ser Phe Val Asp Arg Phe Thr Glu
Ser385 390 395 400 Leu His Tyr Tyr Ser Thr Leu Phe Asp Ser Leu Glu
Gly Ser Pro Val 405 410 415 Asn Pro Asn Asp Lys Ala Met Ser Glu Val
Tyr Leu Gly Lys Gln Ile 420 425 430 Cys Asn Val Val Ala Cys Glu Gly
Met Asp Arg Val Glu Arg His Glu 435 440 445 Thr Leu Asn Gln Trp Arg
Asn Arg Phe Gly Ser Thr Gly Phe Ser Pro 450 455 460 Val His Leu Gly
Ser Asn Ala Tyr Lys Gln Ala Ser Met Leu Leu Ser465 470 475 480 Leu
Phe Gly Gly Gly Asp Gly Tyr Arg Val Glu Glu Asn Asn Gly Cys 485 490
495 Leu Met Leu Gly Trp His Thr Arg Pro Leu Ile Ala Thr Ser Val Trp
500 505 510 Gln Leu Ala Thr Lys Ser Val Val Ala Ala His 515 520
2491PRTGlycine max 24Asn Gly Ile Arg Leu Val His Ser Leu Met Ala
Cys Ala Glu Ala Val1 5 10 15 Glu Asn Asn Asn Leu Ala Val Ala Glu
Ala Leu Val Lys Gln Ile Gly 20 25 30 Phe Leu Ala Leu Ser Gln Val
Gly Ala Met Arg Lys Val Ala Thr Tyr 35 40 45 Phe Ala Glu Ala Leu
Ala Arg Arg Ile Tyr Arg Val Phe Pro Gln Gln 50 55 60 His Ser Leu
Ser Asp Ser Leu Gln Ile His Phe Tyr Glu Thr Cys Pro65 70 75 80 Tyr
Leu Lys Phe Ala His Phe Thr Ala Asn Gln 85 90 25517PRTGlycine max
25Met Lys Arg Glu Arg Glu Gln Leu Gly Ser Ile Ala Gly Thr Ser Ser1
5 10 15 Cys Gly Tyr Ser Ser Gly Lys Ser Asn Leu Trp Glu Glu Glu Gly
Gly 20 25 30 Met Asp Glu Leu Leu Ala Val Val Gly Tyr Lys Val Arg
Ser Ser Asp 35 40 45 Met Ala Glu Val Ala Gln Lys Leu Glu Arg Leu
Glu Glu Ala Met Gly 50 55 60 Asn Val Gln Asp Asp Leu Pro Glu Ile
Ser Asn Asp Val Val His Tyr65 70 75 80 Asn Pro Ser Asp Ile Ser Asn
Trp Leu Glu Thr Met Leu Ser Asn Phe 85 90 95 Asp Pro Leu Pro Ser
Glu Glu Pro Glu Lys Asp Ser Ala Ser Ser Asp 100 105 110 Tyr Asp Leu
Lys Ala Ile Pro Gly Lys Ala Ile Tyr Gly Ala Ser Asp 115 120 125 Ala
Leu Pro Asn Pro Lys Arg Val Lys Ala Asp Glu Ser Arg Arg Ala 130 135
140 Val Val Val Val Asp Ser Gln Glu Asn Gly Ile Arg Leu Val His
Ser145 150 155 160 Leu Met Ala Cys Ala Glu Ala Val Glu Asn Asn Asn
Leu Ala Val Ala 165 170 175 Glu Ala Leu Val Lys Gln Ile Gly Phe Leu
Ala Val Ser Gln Val Gly 180 185 190 Ala Met Arg Lys Val Ala Ile Tyr
Phe Ala Glu Ala Leu Ala Arg Arg 195 200 205 Ile Tyr Arg Val Phe Pro
Leu Gln His Ser Leu Ser Asp Ser Leu Gln 210 215 220 Ile His Phe Tyr
Glu Thr Cys Pro Tyr Leu Lys Phe Ala His Phe Thr225 230 235 240 Ala
Asn Gln Val Ile Leu Glu Ala Phe Gln Gly Lys Asn Arg Val His 245 250
255 Val Ile Asp Phe Gly Ile Asn Gln Gly Met Gln Trp Pro Ala Leu Met
260 265 270 Gln Ala Leu Ala Val Arg Thr Gly Gly Pro Pro Val Phe Arg
Leu Thr 275 280 285 Gly Ile Gly Pro Pro Ala Ala Asp Asn Ser Asp His
Leu Gln Glu Val 290 295 300 Gly Trp Lys Leu Ala Gln Leu Ala Glu Glu
Ile Asn Val Gln Phe Glu305 310 315 320 Tyr Arg Gly Phe Val Ala Asn
Ser Leu Ala Asp Leu Asp Ala Ser Met 325 330 335 Leu Asp Leu Arg Glu
Gly Glu Ala Val Ala Val Asn Ser Val Phe Glu 340 345 350 Phe His Lys
Leu Leu Ala Arg Pro Gly Ala Val Glu Lys Val Leu Ser 355 360 365 Val
Val Arg Gln Ile Arg Pro Glu Ile Val Thr Val Val Glu Gln Glu 370 375
380 Ala Asn His Asn Arg Leu Ser Phe Val Asp Arg Phe Thr Glu Ser
Leu385 390 395 400 His Tyr Tyr Ser Thr Leu Phe Asp Ser Leu Glu Gly
Ser Pro Val Asn 405 410 415 Pro Asn Asp Lys Ala Met Ser Glu Val Tyr
Leu Gly Lys Gln Ile Cys 420 425 430 Asn Val Val Ala Cys Glu Gly Met
Asp Arg Val Glu Arg His Glu Thr 435 440 445 Leu Asn Gln Trp Arg Asn
Arg Phe Val Ser Thr Gly Phe Ser Ser Val 450 455 460 His Leu Gly Ser
Asn Ala Tyr Lys Gln Ala Ser Met Leu Leu Ala Leu465 470 475 480 Phe
Ala Gly Gly Asp Gly Tyr Arg Val Glu Glu Asn Asn Gly Cys Leu 485 490
495 Met Leu Gly Trp His Thr Arg Pro Leu Ile Ala Thr Ser Ala Trp Gln
500 505 510 Leu Ala Ala Thr Arg 515 2691PRTGlycine max 26Asn Gly
Ile Arg Leu Val His Ser Leu Met Ala Cys Ala Glu Ala Val1 5 10 15
Glu Asn Asn Asn Leu Ala Val Ala Glu Ala Leu Val Lys Gln Ile Gly 20
25 30 Phe Leu Ala Val Ser Gln Val Gly Ala Met Arg Lys Val Ala Ile
Tyr 35 40 45 Phe Ala Glu Ala Leu Ala Arg Arg Ile Tyr Arg Val Phe
Pro Leu Gln 50 55 60 His Ser Leu Ser Asp Ser Leu Gln Ile His Phe
Tyr Glu Thr Cys Pro65 70 75 80 Tyr Leu Lys Phe Ala His Phe Thr Ala
Asn Gln 85 90 27595PRTGlycine max 27Met Lys Arg Asp His Lys Asp Ser
Cys Gly Gly Gly Gly Ala Ala Gly1 5 10 15 Gly Thr Val Lys Gly Glu
Cys Ser Ser Met Gln Ser Asn Gly Lys Ala 20 25 30 Lys Met Trp Glu
Glu Glu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 35 40 45 Gln Gln
Gln Gln Gly Met Asp Glu Leu Leu Ala Ala Leu Gly Tyr Lys 50 55 60
Val Arg Ala Ser Asp Met Ala Asp Val Ala Gln Lys Leu Glu Gln Leu65
70 75 80 Glu Met Val Met Gly Cys Ala Gln Glu Asp Gly Ile Ser His
Leu Ala 85 90 95 Ser Asp Thr Val His Tyr Asp Pro Thr Asp Leu Tyr
Ser Trp Val Gln 100 105 110 Ser Met Leu Thr Glu Leu Asn Pro Glu Pro
Asn Asn Asn Leu Asp Pro 115 120 125 Ser Ser Phe Leu Ile Asp Asn Asn
Asn Asn Ile Ile Asn Ser Thr Ala 130 135 140 Pro Val Phe Asn Asp Asp
Ser Glu Tyr Asp Leu Arg Ala Ile Pro Gly145 150 155 160 Ile Ala Ala
Tyr Pro Pro Pro Leu Pro Gln Asp Asn His Leu Asp Glu 165 170 175 Ile
Glu Thr Ala Asn Asn Ile Asn Lys Arg Leu Lys Pro Ser Pro Ala 180 185
190 Glu Ser Ala Asp Ser Ala Ala Ser Glu Pro Thr Arg His Val Val Leu
195 200 205 Val Asp His Gln Glu Ala Gly Val Arg Leu Val His Thr Leu
Leu Ala 210 215 220 Cys Ala Glu Ala Val Gln Gln Glu Asn Leu Lys Leu
Ala Asp Ala Leu225 230 235 240 Val Lys His Val Gly Ile Leu Ala Ala
Ser Gln Ala Gly Ala Met Arg 245 250 255 Lys Val Ala Ser Tyr Phe Ala
Gln Ala Leu Ala Arg Arg Ile Tyr Gly 260 265 270 Ile Phe Pro Glu Glu
Thr Leu Asp Ser Ser Phe Ser Asp Val Leu His 275 280 285 Met His Phe
Tyr Glu Ser Cys Pro Tyr Leu Lys Phe Ala His Phe Thr 290 295 300 Ala
Asn Gln Ala Ile Leu Glu Ala Phe Ala Thr Ala Gly Lys Val His305 310
315 320 Val Ile Asp Phe Gly Leu Lys Gln Gly Met Gln Trp Pro Ala Leu
Met 325 330 335 Gln Ala Leu Ala Leu Arg Pro Gly Gly Pro Pro Thr Phe
Arg Leu Thr 340 345 350 Gly Ile Gly Pro Pro Gln Pro Asp Asn Thr Asp
Ala Leu Gln Gln Val 355 360 365 Gly Leu Lys Leu Ala Gln Leu Ala Gln
Ile Ile Gly Val Gln Phe Glu 370 375 380 Phe Arg Gly Phe Val Cys Asn
Ser Leu Ala Asp Leu Asp Pro Asn Met385 390 395 400 Leu Glu Ile Arg
Pro Gly Glu Ala Val Ala Val Asn Ser Val Phe Glu 405 410 415 Leu His
Arg Met Leu Ala Arg Ser Gly Ser Val Asp Lys Val Leu Asp 420 425 430
Thr Val Lys Lys Ile Asn Pro Gln Ile Val Thr Ile Val Glu Gln Glu 435
440 445 Ala Asn His Asn Gly Pro Gly Phe Leu Asp Arg Phe Thr Glu Ala
Leu 450 455 460 His Tyr Tyr Ser Ser Leu Phe Asp Ser Leu Glu Gly Ser
Ser Ser Ser465 470 475 480 Ser Thr Gly Leu Gly Ser Pro Ser Gln Asp
Leu Leu Met Ser Glu Leu 485 490 495 Tyr Leu Gly Arg Gln Ile Cys Asn
Val Val Ala Tyr Glu Gly Pro Asp 500 505 510 Arg Val Glu Arg His Glu
Thr Leu Thr Gln Trp Arg Gly Arg Leu Asp 515 520 525 Ser Ala Gly Phe
Asp Pro Val His Leu Gly Ser Asn Ala Phe Lys Gln 530 535 540 Ala Ser
Met Leu Leu Ala Leu Phe Ala Gly Gly Asp Gly Tyr Arg Val545 550 555
560 Glu Glu Asn Asn Gly Cys Leu Met Leu Gly Trp His Thr Arg Pro Leu
565 570 575 Ile Ala Thr Ser Ala Trp Lys Leu Pro Ser Ser Ser Glu Ser
Ser Gly 580 585 590 Leu Thr Gln 595 2894PRTGlycine max 28Ala Gly
Val Arg Leu Val His Thr Leu Leu Ala Cys Ala Glu Ala Val1 5 10 15
Gln Gln Glu Asn Leu Lys Leu Ala Asp Ala Leu Val Lys His Val Gly 20
25 30 Ile Leu Ala Ala Ser Gln Ala Gly Ala Met Arg Lys Val Ala Ser
Tyr 35 40 45 Phe Ala Gln Ala Leu Ala Arg Arg Ile Tyr Gly Ile Phe
Pro Glu Glu 50 55 60 Thr Leu Asp Ser Ser Phe Ser Asp Val Leu His
Met His Phe Tyr Glu65 70 75 80 Ser Cys Pro Tyr Leu Lys Phe Ala His
Phe Thr Ala Asn Gln 85 90 29584PRTGlycine max 29Met Lys Arg Asp His
Arg Asp Ser Cys Gly Gly Gly Gly Gly Gly Ser1 5 10 15 Val Lys Gly
Glu Cys Ser Ser Met Pro Ser Asn Gly Lys Ala Asn Met 20 25 30 Trp
Glu Glu Gln Gln Gln Gln Gln Gln Gly Met Asp Glu Leu Leu Ala 35 40
45 Ala Leu Gly Tyr Lys Val Arg Ala Ser Asp Met Ala Asp Val Ala Gln
50 55 60 Lys Leu Glu Gln Leu Glu Met Val Met Gly Cys Ala Gln Glu
Glu Gly65 70 75 80 Ile Ser His Leu Ala Ser Asp Thr Val His Tyr Asp
Pro Thr Asp Leu 85 90 95 Tyr Ser Trp Val Gln Thr Met Leu Thr Glu
Leu Asn Pro Glu Pro Asn 100 105 110 Asn Asn Asn Asn Ser Leu Leu Gly
Pro Ser Ser Leu Leu Ile Asp Asn 115 120 125 Asn Thr Ala Pro Val Phe
Asn Asp Asp Ser Glu Tyr Asp Leu Arg Ala 130 135 140 Ile Pro Gly Ile
Ala Ala Tyr Pro Pro Pro Pro Pro Gln Asp Asn Asn145 150 155 160 Asn
Asn Asn Asn Asn Leu Asp Glu Ile Glu Thr Ala Asn Asn Ile Asn 165 170
175 Lys Arg Leu Lys Pro Ser Pro Val Glu Ser Ala Asp Ser Ala Ser Glu
180 185 190 Pro Thr Arg Thr Val Leu Leu Val Asp His Gln Glu Ala Gly
Val Arg 195 200 205 Leu Val His Thr Leu Leu Ala Cys Ala Glu Ala Val
Gln Gln Glu Asn 210 215 220 Leu Lys Leu Ala Asp Ala Leu Val Lys His
Val Gly Ile Leu Ala Ala225 230 235 240 Ser Gln Ala Gly Ala Met Arg
Lys Val Ala Ser Tyr Phe Ala Gln Ala 245 250 255 Leu Ala Arg Arg Ile
Tyr Gly Ile Phe Pro Glu Glu Thr Leu Asp Ser 260 265 270 Ser Phe Ser
Asp Val Leu His Met His Phe Tyr Glu Ser Cys Pro Tyr 275 280 285 Leu
Lys Phe Ala His Phe Thr Ala Asn Gln Ala Ile Leu Glu Ala Phe 290 295
300 Ala Thr Ala Gly Arg Val His Val Ile Asp Phe Gly Leu Arg Gln
Gly305 310 315 320 Met Gln Trp Pro Ala Leu Met Gln Ala Leu Ala Leu
Arg Pro Gly Gly 325 330 335 Pro Pro Thr Phe Arg Leu Thr Gly Ile Gly
Pro Pro Gln Pro Asp Asn 340 345 350 Thr Asp Ala Leu Gln Gln Val Gly
Trp Lys Leu Ala Gln Leu Ala Gln 355 360 365 Asn Ile Gly Val Gln Phe
Glu Phe Arg Gly Phe Val Cys Asn Ser Leu 370 375 380 Ala Asp Leu Asp
Pro Lys Met Leu Glu Ile Arg Pro Gly Glu Ala Val385 390 395 400 Ala
Val Asn Ser Val Phe Glu Leu His Arg Met Leu Ala Arg Pro Gly 405 410
415 Ser Val Asp Lys Val Leu Asp Thr Val Lys Lys Ile Lys Pro Lys Ile
420 425 430 Val Thr Ile Val Glu Gln Glu Ala Asn His Asn Gly Pro Gly
Phe Leu 435 440 445 Asp Arg Phe Thr Glu Ala Leu His Tyr Tyr Ser Ser
Leu Phe Asp Ser 450 455 460 Leu Glu Gly Ser Ser Ser Ser Thr Gly Leu
Gly Ser Pro Asn Gln Asp465 470 475 480 Leu Leu Met Ser Glu Leu Tyr
Leu Gly Arg Gln Ile Cys Asn Val Val 485 490 495 Ala Asn Glu Gly Ala
Asp Arg Val Glu Arg His Glu Thr Leu Ser Gln 500 505 510 Trp Arg Gly
Arg Leu Asp Ser Ala Gly Phe Asp Pro Val His Leu Gly 515 520 525
Ser
Asn Ala Phe Lys Gln Ala Ser Met Leu Leu Ala Leu Phe Ala Gly 530 535
540 Gly Asp Gly Tyr Arg Val Glu Glu Asn Asn Gly Cys Leu Met Leu
Gly545 550 555 560 Trp His Thr Arg Pro Leu Ile Ala Thr Ser Ala Trp
Lys Leu Pro Ser 565 570 575 Pro Asn Asp Leu His Cys Lys Leu 580
3094PRTGlycine max 30Ala Gly Val Arg Leu Val His Thr Leu Leu Ala
Cys Ala Glu Ala Val1 5 10 15 Gln Gln Glu Asn Leu Lys Leu Ala Asp
Ala Leu Val Lys His Val Gly 20 25 30 Ile Leu Ala Ala Ser Gln Ala
Gly Ala Met Arg Lys Val Ala Ser Tyr 35 40 45 Phe Ala Gln Ala Leu
Ala Arg Arg Ile Tyr Gly Ile Phe Pro Glu Glu 50 55 60 Thr Leu Asp
Ser Ser Phe Ser Asp Val Leu His Met His Phe Tyr Glu65 70 75 80 Ser
Cys Pro Tyr Leu Lys Phe Ala His Phe Thr Ala Asn Gln 85 90
31533PRTArabidopsis thaliana 31Met Lys Arg Asp His His His His His
His Gln Asp Lys Lys Thr Met1 5 10 15 Met Met Asn Glu Glu Asp Asp
Gly Asn Gly Met Asp Glu Leu Leu Ala 20 25 30 Val Leu Gly Tyr Lys
Val Arg Ser Ser Glu Met Ala Asp Val Ala Gln 35 40 45 Lys Leu Glu
Gln Leu Glu Val Met Met Ser Asn Val Gln Glu Asp Asp 50 55 60 Leu
Ser Gln Leu Ala Thr Glu Thr Val His Tyr Asn Pro Ala Glu Leu65 70 75
80 Tyr Thr Trp Leu Asp Ser Met Leu Thr Asp Leu Asn Pro Pro Ser Ser
85 90 95 Asn Ala Glu Tyr Asp Leu Lys Ala Ile Pro Gly Asp Ala Ile
Leu Asn 100 105 110 Gln Phe Ala Ile Asp Ser Ala Ser Ser Ser Asn Gln
Gly Gly Gly Gly 115 120 125 Asp Thr Tyr Thr Thr Asn Lys Arg Leu Lys
Cys Ser Asn Gly Val Val 130 135 140 Glu Thr Thr Thr Ala Thr Ala Glu
Ser Thr Arg His Val Val Leu Val145 150 155 160 Asp Ser Gln Glu Asn
Gly Val Arg Leu Val His Ala Leu Leu Ala Cys 165 170 175 Ala Glu Ala
Val Gln Lys Glu Asn Leu Thr Val Ala Glu Ala Leu Val 180 185 190 Lys
Gln Ile Gly Phe Leu Ala Val Ser Gln Ile Gly Ala Met Arg Lys 195 200
205 Val Ala Thr Tyr Phe Ala Glu Ala Leu Ala Arg Arg Ile Tyr Arg Leu
210 215 220 Ser Pro Ser Gln Ser Pro Ile Asp His Ser Leu Ser Asp Thr
Leu Gln225 230 235 240 Met His Phe Tyr Glu Thr Cys Pro Tyr Leu Lys
Phe Ala His Phe Thr 245 250 255 Ala Asn Gln Ala Ile Leu Glu Ala Phe
Gln Gly Lys Lys Arg Val His 260 265 270 Val Ile Asp Phe Ser Met Ser
Gln Gly Leu Gln Trp Pro Ala Leu Met 275 280 285 Gln Ala Leu Ala Leu
Arg Pro Gly Gly Pro Pro Val Phe Arg Leu Thr 290 295 300 Gly Ile Gly
Pro Pro Ala Pro Asp Asn Phe Asp Tyr Leu His Glu Val305 310 315 320
Gly Cys Lys Leu Ala His Leu Ala Glu Ala Ile His Val Glu Phe Glu 325
330 335 Tyr Arg Gly Phe Val Ala Asn Thr Leu Ala Asp Leu Asp Ala Ser
Met 340 345 350 Leu Glu Leu Arg Pro Ser Glu Ile Glu Ser Val Ala Val
Asn Ser Val 355 360 365 Phe Glu Leu His Lys Leu Leu Gly Arg Pro Gly
Ala Ile Asp Lys Val 370 375 380 Leu Gly Val Val Asn Gln Ile Lys Pro
Glu Ile Phe Thr Val Val Glu385 390 395 400 Gln Glu Ser Asn His Asn
Ser Pro Ile Phe Leu Asp Arg Phe Thr Glu 405 410 415 Ser Leu His Tyr
Tyr Ser Thr Leu Phe Asp Ser Leu Glu Gly Val Pro 420 425 430 Ser Gly
Gln Asp Lys Val Met Ser Glu Val Tyr Leu Gly Lys Gln Ile 435 440 445
Cys Asn Val Val Ala Cys Asp Gly Pro Asp Arg Val Glu Arg His Glu 450
455 460 Thr Leu Ser Gln Trp Arg Asn Arg Phe Gly Ser Ala Gly Phe Ala
Ala465 470 475 480 Ala His Ile Gly Ser Asn Ala Phe Lys Gln Ala Ser
Met Leu Leu Ala 485 490 495 Leu Phe Asn Gly Gly Glu Gly Tyr Arg Val
Glu Glu Ser Asp Gly Cys 500 505 510 Leu Met Leu Gly Trp His Thr Arg
Pro Leu Ile Ala Thr Ser Ala Trp 515 520 525 Lys Leu Ser Thr Asn 530
3295PRTArabidopsis thaliana 32Asn Gly Val Arg Leu Val His Ala Leu
Leu Ala Cys Ala Glu Ala Val1 5 10 15 Gln Lys Glu Asn Leu Thr Val
Ala Glu Ala Leu Val Lys Gln Ile Gly 20 25 30 Phe Leu Ala Val Ser
Gln Ile Gly Ala Met Arg Lys Val Ala Thr Tyr 35 40 45 Phe Ala Glu
Ala Leu Ala Arg Arg Ile Tyr Arg Leu Ser Pro Ser Gln 50 55 60 Ser
Pro Ile Asp His Ser Leu Ser Asp Thr Leu Gln Met His Phe Tyr65 70 75
80 Glu Thr Cys Pro Tyr Leu Lys Phe Ala His Phe Thr Ala Asn Gln 85
90 95 33587PRTArabidopsis thaliana 33Met Lys Arg Asp His His Gln
Phe Gln Gly Arg Leu Ser Asn His Gly1 5 10 15 Thr Ser Ser Ser Ser
Ser Ser Ile Ser Lys Asp Lys Met Met Met Val 20 25 30 Lys Lys Glu
Glu Asp Gly Gly Gly Asn Met Asp Asp Glu Leu Leu Ala 35 40 45 Val
Leu Gly Tyr Lys Val Arg Ser Ser Glu Met Ala Glu Val Ala Leu 50 55
60 Lys Leu Glu Gln Leu Glu Thr Met Met Ser Asn Val Gln Glu Asp
Gly65 70 75 80 Leu Ser His Leu Ala Thr Asp Thr Val His Tyr Asn Pro
Ser Glu Leu 85 90 95 Tyr Ser Trp Leu Asp Asn Met Leu Ser Glu Leu
Asn Pro Pro Pro Leu 100 105 110 Pro Ala Ser Ser Asn Gly Leu Asp Pro
Val Leu Pro Ser Pro Glu Ile 115 120 125 Cys Gly Phe Pro Ala Ser Asp
Tyr Asp Leu Lys Val Ile Pro Gly Asn 130 135 140 Ala Ile Tyr Gln Phe
Pro Ala Ile Asp Ser Ser Ser Ser Ser Asn Asn145 150 155 160 Gln Asn
Lys Arg Leu Lys Ser Cys Ser Ser Pro Asp Ser Met Val Thr 165 170 175
Ser Thr Ser Thr Gly Thr Gln Ile Gly Gly Val Ile Gly Thr Thr Val 180
185 190 Thr Thr Thr Thr Thr Thr Thr Thr Ala Ala Gly Glu Ser Thr Arg
Ser 195 200 205 Val Ile Leu Val Asp Ser Gln Glu Asn Gly Val Arg Leu
Val His Ala 210 215 220 Leu Met Ala Cys Ala Glu Ala Ile Gln Gln Asn
Asn Leu Thr Leu Ala225 230 235 240 Glu Ala Leu Val Lys Gln Ile Gly
Cys Leu Ala Val Ser Gln Ala Gly 245 250 255 Ala Met Arg Lys Val Ala
Thr Tyr Phe Ala Glu Ala Leu Ala Arg Arg 260 265 270 Ile Tyr Arg Leu
Ser Pro Pro Gln Asn Gln Ile Asp His Cys Leu Ser 275 280 285 Asp Thr
Leu Gln Met His Phe Tyr Glu Thr Cys Pro Tyr Leu Lys Phe 290 295 300
Ala His Phe Thr Ala Asn Gln Ala Ile Leu Glu Ala Phe Glu Gly Lys305
310 315 320 Lys Arg Val His Val Ile Asp Phe Ser Met Asn Gln Gly Leu
Gln Trp 325 330 335 Pro Ala Leu Met Gln Ala Leu Ala Leu Arg Glu Gly
Gly Pro Pro Thr 340 345 350 Phe Arg Leu Thr Gly Ile Gly Pro Pro Ala
Pro Asp Asn Ser Asp His 355 360 365 Leu His Glu Val Gly Cys Lys Leu
Ala Gln Leu Ala Glu Ala Ile His 370 375 380 Val Glu Phe Glu Tyr Arg
Gly Phe Val Ala Asn Ser Leu Ala Asp Leu385 390 395 400 Asp Ala Ser
Met Leu Glu Leu Arg Pro Ser Asp Thr Glu Ala Val Ala 405 410 415 Val
Asn Ser Val Phe Glu Leu His Lys Leu Leu Gly Arg Pro Gly Gly 420 425
430 Ile Glu Lys Val Leu Gly Val Val Lys Gln Ile Lys Pro Val Ile Phe
435 440 445 Thr Val Val Glu Gln Glu Ser Asn His Asn Gly Pro Val Phe
Leu Asp 450 455 460 Arg Phe Thr Glu Ser Leu His Tyr Tyr Ser Thr Leu
Phe Asp Ser Leu465 470 475 480 Glu Gly Val Pro Asn Ser Gln Asp Lys
Val Met Ser Glu Val Tyr Leu 485 490 495 Gly Lys Gln Ile Cys Asn Leu
Val Ala Cys Glu Gly Pro Asp Arg Val 500 505 510 Glu Arg His Glu Thr
Leu Ser Gln Trp Gly Asn Arg Phe Gly Ser Ser 515 520 525 Gly Leu Ala
Pro Ala His Leu Gly Ser Asn Ala Phe Lys Gln Ala Ser 530 535 540 Met
Leu Leu Ser Val Phe Asn Ser Gly Gln Gly Tyr Arg Val Glu Glu545 550
555 560 Ser Asn Gly Cys Leu Met Leu Gly Trp His Thr Arg Pro Leu Ile
Thr 565 570 575 Thr Ser Ala Trp Lys Leu Ser Thr Ala Ala Tyr 580 585
3495PRTArabidopsis thaliana 34Asn Gly Val Arg Leu Val His Ala Leu
Met Ala Cys Ala Glu Ala Ile1 5 10 15 Gln Gln Asn Asn Leu Thr Leu
Ala Glu Ala Leu Val Lys Gln Ile Gly 20 25 30 Cys Leu Ala Val Ser
Gln Ala Gly Ala Met Arg Lys Val Ala Thr Tyr 35 40 45 Phe Ala Glu
Ala Leu Ala Arg Arg Ile Tyr Arg Leu Ser Pro Pro Gln 50 55 60 Asn
Gln Ile Asp His Cys Leu Ser Asp Thr Leu Gln Met His Phe Tyr65 70 75
80 Glu Thr Cys Pro Tyr Leu Lys Phe Ala His Phe Thr Ala Asn Gln 85
90 95 35511PRTArabidopsis thaliana 35Met Lys Arg Glu His Asn His
Arg Glu Ser Ser Ala Gly Glu Gly Gly1 5 10 15 Ser Ser Ser Met Thr
Thr Val Ile Lys Glu Glu Ala Ala Gly Val Asp 20 25 30 Glu Leu Leu
Val Val Leu Gly Tyr Lys Val Arg Ser Ser Asp Met Ala 35 40 45 Asp
Val Ala His Lys Leu Glu Gln Leu Glu Met Val Leu Gly Asp Gly 50 55
60 Ile Ser Asn Leu Ser Asp Glu Thr Val His Tyr Asn Pro Ser Asp
Leu65 70 75 80 Ser Gly Trp Val Glu Ser Met Leu Ser Asp Leu Asp Pro
Thr Arg Ile 85 90 95 Gln Glu Lys Pro Asp Ser Glu Tyr Asp Leu Arg
Ala Ile Pro Gly Ser 100 105 110 Ala Val Tyr Pro Arg Asp Glu His Val
Thr Arg Arg Ser Lys Arg Thr 115 120 125 Arg Ile Glu Ser Glu Leu Ser
Ser Thr Arg Ser Val Val Val Leu Asp 130 135 140 Ser Gln Glu Thr Gly
Val Arg Leu Val His Ala Leu Leu Ala Cys Ala145 150 155 160 Glu Ala
Val Gln Gln Asn Asn Leu Lys Leu Ala Asp Ala Leu Val Lys 165 170 175
His Val Gly Leu Leu Ala Ser Ser Gln Ala Gly Ala Met Arg Lys Val 180
185 190 Ala Thr Tyr Phe Ala Glu Gly Leu Ala Arg Arg Ile Tyr Arg Ile
Tyr 195 200 205 Pro Arg Asp Asp Val Ala Leu Ser Ser Phe Ser Asp Thr
Leu Gln Ile 210 215 220 His Phe Tyr Glu Ser Cys Pro Tyr Leu Lys Phe
Ala His Phe Thr Ala225 230 235 240 Asn Gln Ala Ile Leu Glu Val Phe
Ala Thr Ala Glu Lys Val His Val 245 250 255 Ile Asp Leu Gly Leu Asn
His Gly Leu Gln Trp Pro Ala Leu Ile Gln 260 265 270 Ala Leu Ala Leu
Arg Pro Asn Gly Pro Pro Asp Phe Arg Leu Thr Gly 275 280 285 Ile Gly
Tyr Ser Leu Thr Asp Ile Gln Glu Val Gly Trp Lys Leu Gly 290 295 300
Gln Leu Ala Ser Thr Ile Gly Val Asn Phe Glu Phe Lys Ser Ile Ala305
310 315 320 Leu Asn Asn Leu Ser Asp Leu Lys Pro Glu Met Leu Asp Ile
Arg Pro 325 330 335 Gly Leu Glu Ser Val Ala Val Asn Ser Val Phe Glu
Leu His Arg Leu 340 345 350 Leu Ala His Pro Gly Ser Ile Asp Lys Phe
Leu Ser Thr Ile Lys Ser 355 360 365 Ile Arg Pro Asp Ile Met Thr Val
Val Glu Gln Glu Ala Asn His Asn 370 375 380 Gly Thr Val Phe Leu Asp
Arg Phe Thr Glu Ser Leu His Tyr Tyr Ser385 390 395 400 Ser Leu Phe
Asp Ser Leu Glu Gly Pro Pro Ser Gln Asp Arg Val Met 405 410 415 Ser
Glu Leu Phe Leu Gly Arg Gln Ile Leu Asn Leu Val Ala Cys Glu 420 425
430 Gly Glu Asp Arg Val Glu Arg His Glu Thr Leu Asn Gln Trp Arg Asn
435 440 445 Arg Phe Gly Leu Gly Gly Phe Lys Pro Val Ser Ile Gly Ser
Asn Ala 450 455 460 Tyr Lys Gln Ala Ser Met Leu Leu Ala Leu Tyr Ala
Gly Ala Asp Gly465 470 475 480 Tyr Asn Val Glu Glu Asn Glu Gly Cys
Leu Leu Leu Gly Trp Gln Thr 485 490 495 Arg Pro Leu Ile Ala Thr Ser
Ala Trp Arg Ile Asn Arg Val Glu 500 505 510 3695PRTArabidopsis
thaliana 36Thr Gly Val Arg Leu Val His Ala Leu Leu Ala Cys Ala Glu
Ala Val1 5 10 15 Gln Gln Asn Asn Leu Lys Leu Ala Asp Ala Leu Val
Lys His Val Gly 20 25 30 Leu Leu Ala Ser Ser Gln Ala Gly Ala Met
Arg Lys Val Ala Thr Tyr 35 40 45 Phe Ala Glu Gly Leu Ala Arg Arg
Ile Tyr Arg Ile Tyr Pro Arg Asp 50 55 60 Asp Val Ala Leu Ser Ser
Phe Ser Asp Thr Leu Gln Ile His Phe Tyr65 70 75 80 Glu Ser Cys Pro
Tyr Leu Lys Phe Ala His Phe Thr Ala Asn Gln 85 90 95
37547PRTArabidopsis thaliana 37Met Lys Arg Gly Tyr Gly Glu Thr Trp
Asp Pro Pro Pro Lys Pro Leu1 5 10 15 Pro Ala Ser Arg Ser Gly Glu
Gly Pro Ser Met Ala Asp Lys Lys Lys 20 25 30 Ala Asp Asp Asp Asn
Asn Asn Ser Asn Met Asp Asp Glu Leu Leu Ala 35 40 45 Val Leu Gly
Tyr Lys Val Arg Ser Ser Glu Met Ala Glu Val Ala Gln 50 55 60 Lys
Leu Glu Gln Leu Glu Met Val Leu Ser Asn Asp Asp Val Gly Ser65 70 75
80 Thr Val Leu Asn Asp Ser Val His Tyr Asn Pro Ser Asp Leu Ser Asn
85 90 95 Trp Val Glu Ser Met Leu Ser Glu Leu Asn Asn Pro Ala Ser
Ser Asp 100 105 110 Leu Asp Thr Thr Arg Ser Cys Val Asp Arg Ser Glu
Tyr Asp Leu Arg 115 120 125 Ala Ile Pro Gly Leu Ser Ala Phe Pro Lys
Glu Glu Glu Val Phe Asp 130 135 140 Glu Glu Ala Ser Ser Lys Arg Ile
Arg Leu Gly Ser Trp Cys Glu Ser145 150 155 160 Ser Asp Glu Ser Thr
Arg Ser Val Val Leu Val Asp Ser Gln Glu Thr 165 170 175 Gly Val Arg
Leu Val His Ala Leu Val Ala Cys Ala Glu Ala Ile His 180 185 190 Gln
Glu Asn Leu Asn Leu Ala Asp Ala Leu Val Lys Arg Val Gly Thr 195 200
205 Leu Ala Gly Ser Gln Ala Gly Ala Met Gly Lys Val Ala Thr
Tyr Phe 210 215 220 Ala Gln Ala Leu Ala Arg Arg Ile Tyr Arg Asp Tyr
Thr Ala Glu Thr225 230 235 240 Asp Val Cys Ala Ala Val Asn Pro Ser
Phe Glu Glu Val Leu Glu Met 245 250 255 His Phe Tyr Glu Ser Cys Pro
Tyr Leu Lys Phe Ala His Phe Thr Ala 260 265 270 Asn Gln Ala Ile Leu
Glu Ala Val Thr Thr Ala Arg Arg Val His Val 275 280 285 Ile Asp Leu
Gly Leu Asn Gln Gly Met Gln Trp Pro Ala Leu Met Gln 290 295 300 Ala
Leu Ala Leu Arg Pro Gly Gly Pro Pro Ser Phe Arg Leu Thr Gly305 310
315 320 Ile Gly Pro Pro Gln Thr Glu Asn Ser Asp Ser Leu Gln Gln Leu
Gly 325 330 335 Trp Lys Leu Ala Gln Phe Ala Gln Asn Met Gly Val Glu
Phe Glu Phe 340 345 350 Lys Gly Leu Ala Ala Glu Ser Leu Ser Asp Leu
Glu Pro Glu Met Phe 355 360 365 Glu Thr Arg Pro Glu Ser Glu Thr Leu
Val Val Asn Ser Val Phe Glu 370 375 380 Leu His Arg Leu Leu Ala Arg
Ser Gly Ser Ile Glu Lys Leu Leu Asn385 390 395 400 Thr Val Lys Ala
Ile Lys Pro Ser Ile Val Thr Val Val Glu Gln Glu 405 410 415 Ala Asn
His Asn Gly Ile Val Phe Leu Asp Arg Phe Asn Glu Ala Leu 420 425 430
His Tyr Tyr Ser Ser Leu Phe Asp Ser Leu Glu Asp Ser Tyr Ser Leu 435
440 445 Pro Ser Gln Asp Arg Val Met Ser Glu Val Tyr Leu Gly Arg Gln
Ile 450 455 460 Leu Asn Val Val Ala Ala Glu Gly Ser Asp Arg Val Glu
Arg His Glu465 470 475 480 Thr Ala Ala Gln Trp Arg Ile Arg Met Lys
Ser Ala Gly Phe Asp Pro 485 490 495 Ile His Leu Gly Ser Ser Ala Phe
Lys Gln Ala Ser Met Leu Leu Ser 500 505 510 Leu Tyr Ala Thr Gly Asp
Gly Tyr Arg Val Glu Glu Asn Asp Gly Cys 515 520 525 Leu Met Ile Gly
Trp Gln Thr Arg Pro Leu Ile Thr Thr Ser Ala Trp 530 535 540 Lys Leu
Ala545 3899PRTArabidopsis thaliana 38Thr Gly Val Arg Leu Val His
Ala Leu Val Ala Cys Ala Glu Ala Ile1 5 10 15 His Gln Glu Asn Leu
Asn Leu Ala Asp Ala Leu Val Lys Arg Val Gly 20 25 30 Thr Leu Ala
Gly Ser Gln Ala Gly Ala Met Gly Lys Val Ala Thr Tyr 35 40 45 Phe
Ala Gln Ala Leu Ala Arg Arg Ile Tyr Arg Asp Tyr Thr Ala Glu 50 55
60 Thr Asp Val Cys Ala Ala Val Asn Pro Ser Phe Glu Glu Val Leu
Glu65 70 75 80 Met His Phe Tyr Glu Ser Cys Pro Tyr Leu Lys Phe Ala
His Phe Thr 85 90 95 Ala Asn Gln39523PRTArabidopsis thaliana 39Met
Lys Arg Ser His Gln Glu Thr Ser Val Glu Glu Glu Ala Pro Ser1 5 10
15 Met Val Glu Lys Leu Glu Asn Gly Cys Gly Gly Gly Gly Asp Asp Asn
20 25 30 Met Asp Glu Phe Leu Ala Val Leu Gly Tyr Lys Val Arg Ser
Ser Asp 35 40 45 Met Ala Asp Val Ala Gln Lys Leu Glu Gln Leu Glu
Met Val Leu Ser 50 55 60 Asn Asp Ile Ala Ser Ser Ser Asn Ala Phe
Asn Asp Thr Val His Tyr65 70 75 80 Asn Pro Ser Asp Leu Ser Gly Trp
Ala Gln Ser Met Leu Ser Asp Leu 85 90 95 Asn Tyr Tyr Pro Asp Leu
Asp Pro Asn Arg Ile Cys Asp Leu Arg Pro 100 105 110 Ile Thr Asp Asp
Asp Glu Cys Cys Ser Ser Asn Ser Asn Ser Asn Lys 115 120 125 Arg Ile
Arg Leu Gly Pro Trp Cys Asp Ser Val Thr Ser Glu Ser Thr 130 135 140
Arg Ser Val Val Leu Ile Glu Glu Thr Gly Val Arg Leu Val Gln Ala145
150 155 160 Leu Val Ala Cys Ala Glu Ala Val Gln Leu Glu Asn Leu Ser
Leu Ala 165 170 175 Asp Ala Leu Val Lys Arg Val Gly Leu Leu Ala Ala
Ser Gln Ala Gly 180 185 190 Ala Met Gly Lys Val Ala Thr Tyr Phe Ala
Glu Ala Leu Ala Arg Arg 195 200 205 Ile Tyr Arg Ile His Pro Ser Ala
Ala Ala Ile Asp Pro Ser Phe Glu 210 215 220 Glu Ile Leu Gln Met Asn
Phe Tyr Asp Ser Cys Pro Tyr Leu Lys Phe225 230 235 240 Ala His Phe
Thr Ala Asn Gln Ala Ile Leu Glu Ala Val Thr Thr Ser 245 250 255 Arg
Val Val His Val Ile Asp Leu Gly Leu Asn Gln Gly Met Gln Trp 260 265
270 Pro Ala Leu Met Gln Ala Leu Ala Leu Arg Pro Gly Gly Pro Pro Ser
275 280 285 Phe Arg Leu Thr Gly Val Gly Asn Pro Ser Asn Arg Glu Gly
Ile Gln 290 295 300 Glu Leu Gly Trp Lys Leu Ala Gln Leu Ala Gln Ala
Ile Gly Val Glu305 310 315 320 Phe Lys Phe Asn Gly Leu Thr Thr Glu
Arg Leu Ser Asp Leu Glu Pro 325 330 335 Asp Met Phe Glu Thr Arg Thr
Glu Ser Glu Thr Leu Val Val Asn Ser 340 345 350 Val Phe Glu Leu His
Pro Val Leu Ser Gln Pro Gly Ser Ile Glu Lys 355 360 365 Leu Leu Ala
Thr Val Lys Ala Val Lys Pro Gly Leu Val Thr Val Val 370 375 380 Glu
Gln Glu Ala Asn His Asn Gly Asp Val Phe Leu Asp Arg Phe Asn385 390
395 400 Glu Ala Leu His Tyr Tyr Ser Ser Leu Phe Asp Ser Leu Glu Asp
Gly 405 410 415 Val Val Ile Pro Ser Gln Asp Arg Val Met Ser Glu Val
Tyr Leu Gly 420 425 430 Arg Gln Ile Leu Asn Leu Val Ala Thr Glu Gly
Ser Asp Arg Ile Glu 435 440 445 Arg His Glu Thr Leu Ala Gln Trp Arg
Lys Arg Met Gly Ser Ala Gly 450 455 460 Phe Asp Pro Val Asn Leu Gly
Ser Asp Ala Phe Lys Gln Ala Ser Leu465 470 475 480 Leu Leu Ala Leu
Ser Gly Gly Gly Asp Gly Tyr Arg Val Glu Glu Asn 485 490 495 Asp Gly
Ser Leu Met Leu Ala Trp Gln Thr Lys Pro Leu Ile Ala Ala 500 505 510
Ser Ala Trp Lys Leu Ala Ala Glu Leu Arg Arg 515 520
4095PRTArabidopsis thaliana 40Thr Gly Val Arg Leu Val Gln Ala Leu
Val Ala Cys Ala Glu Ala Val1 5 10 15 Gln Leu Glu Asn Leu Ser Leu
Ala Asp Ala Leu Val Lys Arg Val Gly 20 25 30 Leu Leu Ala Ala Ser
Gln Ala Gly Ala Met Gly Lys Val Ala Thr Tyr 35 40 45 Phe Ala Glu
Ala Leu Ala Arg Arg Ile Tyr Arg Ile His Pro Ser Ala 50 55 60 Ala
Ala Ile Asp Pro Ser Phe Glu Glu Ile Leu Gln Met Asn Phe Tyr65 70 75
80 Asp Ser Cys Pro Tyr Leu Lys Phe Ala His Phe Thr Ala Asn Gln 85
90 95 4198PRTArtificial SequenceConsensus dimerization domain
sequence 41Gly Val Arg Leu Val His Ala Leu Leu Ala Cys Ala Glu Ala
Val Gln1 5 10 15 Gln Glu Asn Leu Xaa Leu Ala Asp Ala Leu Val Lys
Gln Ile Gly Ile 20 25 30 Leu Ala Ala Ser Gln Ala Gly Ala Met Arg
Lys Val Ala Thr Tyr Phe 35 40 45 Ala Glu Ala Leu Ala Arg Arg Ile
Tyr Arg Ile Phe Pro Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Leu
Asp Xaa Ser Phe Ser Asp Val Leu Gln Met65 70 75 80 His Phe Tyr Glu
Ser Cys Pro Tyr Leu Lys Phe Ala His Phe Thr Ala 85 90 95 Asn
Gln4219DNAArtificial Sequenceprimer 42gtctgcacca tcgtcaacc
194321DNAArtificial Sequenceprimer 43gaagtccagc tgccagaaac c 21
* * * * *