U.S. patent application number 14/019695 was filed with the patent office on 2014-03-13 for compositions and methods comprising male fertility sequences.
The applicant listed for this patent is Pioneer Hi Bred International Inc.. Invention is credited to Marc C Albertsen, Howard P. Hershey, Michael W. Lassner, Yongzhong Wu.
Application Number | 20140075597 14/019695 |
Document ID | / |
Family ID | 49209557 |
Filed Date | 2014-03-13 |
United States Patent
Application |
20140075597 |
Kind Code |
A1 |
Albertsen; Marc C ; et
al. |
March 13, 2014 |
COMPOSITIONS AND METHODS COMPRISING MALE FERTILITY SEQUENCES
Abstract
Compositions and methods for modulating male fertility in a
plant are provided. Compositions comprise nucleotide sequences, and
active fragments and variants thereof, which modulate male
fertility. Further provided are expression cassettes comprising the
male fertility polynucleotides, or active fragments or variants
thereof, operably linked to a promoter, wherein expression of the
polynucleotides modulates the male fertility of a plant. Various
methods are provided wherein the level and/or activity of the
sequences that influence male fertility is modulated in a plant or
plant part.
Inventors: |
Albertsen; Marc C; (Grimes,
IA) ; Hershey; Howard P.; (West Chester, PA) ;
Lassner; Michael W.; (Urbandale, IA) ; Wu;
Yongzhong; (Johnston, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pioneer Hi Bred International Inc. |
Johnston |
IA |
US |
|
|
Family ID: |
49209557 |
Appl. No.: |
14/019695 |
Filed: |
September 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61697590 |
Sep 6, 2012 |
|
|
|
Current U.S.
Class: |
800/287 ;
435/320.1; 435/414; 435/415; 435/416; 435/419; 530/375; 800/278;
800/298 |
Current CPC
Class: |
C12N 15/8287 20130101;
C12N 15/8289 20130101; C07K 14/415 20130101 |
Class at
Publication: |
800/287 ;
435/414; 435/415; 435/416; 435/419; 435/320.1; 530/375; 800/278;
800/298 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A polynucleotide comprising an expression cassette comprising a
first polynucleotide operably linked to a first heterologous
promoter that drives expression in a plant, wherein said first
polynucleotide comprises a nucleotide sequence selected from the
group consisting of: (a) a nucleotide sequence comprising SEQ ID
NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17; (b) a nucleotide sequence
encoding an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12,
14, 16 or 18; (c) a nucleotide sequence comprising at least 95%
sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17,
wherein said polynucleotide encodes a polypeptide that modulates
male fertility; and (e) a nucleotide sequence encoding a
polypeptide having at least 95% identity to SEQ ID NO: 2, 4, 6, 8,
10, 12, 14, 16 or 18, wherein said polynucleotide encodes a
polypeptide that modulates male fertility.
2. The polynucleotide of claim 1, wherein said first heterologous
promoter of the expression cassette is a constitutive, inducible,
tissue-preferred, or growth-stage-preferred promoter.
3. The polynucleotide of claim 2, wherein said promoter is a
male-tissue-preferred promoter.
4. The polynucleotide of claim 1, further comprising a second
polynucleotide operably linked to a second promoter that drives
expression in a plant.
5. The polynucleotide of claim 4, wherein said second
polynucleotide encodes a polynucleotide or polypeptide that
interferes with male gamete function, formation or dispersal.
6. The polynucleotide of claim 5, wherein said second
polynucleotide encodes barnase, DAM-methylase, amylase or ADP
ribosylase.
7. The polynucleotide of claim 5, further comprising a third
polynucleotide operably linked to a third promoter, wherein said
third polynucleotide encodes a marker gene product.
8. The polynucleotide of claim 7, wherein said marker gene product
comprises an antibiotic resistance marker gene product or a visual
marker gene product.
9. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18; and (b) an
amino acid sequence comprising at least 95% sequence identity to
SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18, wherein said
polypeptide modulates male fertility,
10. A vector comprising the expression cassette of claim 1.
11. A plant cell comprising the expression cassette of claim 1.
12. The plant cell of claim 11, wherein said plant is maize,
barley, millet, wheat, rice, sorghum, rye, soybean, canola,
alfalfa, sunflower, safflower, sugarcane, tobacco, Arabidopsis or
cotton.
13. A plant comprising the plant cell of claim 11.
14. The plant of claim 13, wherein expression of said first
polynucleotide operably linked to a heterologous promoter modulates
the male fertility of said plant when compared to a control
plant.
15. The plant of claim 14, wherein said plant has increased male
fertility when compared to a control plant.
16. The plant of claim 14, wherein the expression of said
polynucleotide confers male fertility to a male sterile plant.
17. The plant of claim 14, wherein said plant is a female-fertile
plant.
18. The plant of claim 14, wherein the formation of at least one
male tissue of said plant is modulated, following expression of
said first polynucleotide, when compared to a control plant.
19. The plant of claim 18, wherein said male tissue comprises the
stamen, anther, filament or pollen.
20. A seed of the plant of claim 13, wherein said seed comprises
said expression cassette.
21. A method of increasing the activity and/or level of a
polypeptide in a plant, comprising introducing into said plant a
polynucleotide operably linked to a promoter active in said plant,
said polynucleotide comprising a nucleotide sequence selected from
the group consisting of: (a) a nucleotide sequence comprising SEQ
ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17; (b) a nucleotide sequence
encoding an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12,
14, 16 or 18; (c) a nucleotide sequence comprising at least 95%
sequence identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17,
wherein said polynucleotide encodes a polypeptide that modulates
male fertility; and (e) a nucleotide sequence encoding a
polypeptide having at least 95% identity to SEQ ID NO: 2, 4, 6, 8,
10, 12, 14, 16 or 18, wherein said polypeptide modulates male
fertility.
22. The method of claim 21, wherein said polynucleotide comprises
the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 9, 11, 13,
15 or 17.
23. The method of claim 21, wherein expression of said
polynucleotide modulates male fertility in said plant when compared
to a control plant.
24. The method of claim 21, wherein said plant is a female-fertile
plant.
25. The method of claim 21, wherein said promoter is a constitutive
promoter, inducible promoter, tissue-preferred promoter, or
growth-stage-preferred promoter.
26. The method of claim 25, wherein said promoter is a
male-tissue-preferred promoter.
27. The method of claim 21, wherein expression of said
polynucleotide modulates formation of at least one male tissue when
compared to a control plant.
Description
CROSS-REFERENCE
[0001] This utility application claims the benefit of U.S.
Provisional Application Ser. No. 61/697,590, filed Sep. 6, 2012
which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of plant
molecular biology, more particularly to influencing male
fertility.
REFERENCE TO ELECTRONICALLY-SUBMITTED SEQUENCE LISTING
[0003] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with the file name 20130903.sub.--5282USNP_SeqList.txt, last
modified on Sep. 5, 2013 and having a size of 78 KB, and is filed
concurrently with the specification. The sequence listing contained
in this ASCII formatted document is part of the specification and
is herein incorporated by reference in its entirety. This substance
of this sequence listing file is identical to that filed with U.S.
Provisional Application Ser. No. 61/697,590.
BACKGROUND OF THE INVENTION
[0004] Development of hybrid plant breeding has made possible
considerable advances in quality and quantity of crops produced.
Increased yield and combination of desirable characteristics, such
as resistance to disease and insects, heat and drought tolerance,
along with variations in plant composition are all possible because
of hybridization procedures. These procedures frequently rely
heavily on providing for a male parent contributing pollen to a
female parent to produce the resulting hybrid.
[0005] Field crops are bred through techniques that take advantage
of the plant's method of pollination. A plant is self-pollinated if
pollen from one flower is transferred to the same or another flower
of the same plant or a genetically identical plant. A plant is
cross-pollinated if the pollen comes from a flower on a genetically
different plant.
[0006] In certain species, such as Brassica campestris, the plant
is normally self-sterile and can only be cross-pollinated. In
self-pollinating species, such as soybeans and cotton, the male and
female plants are anatomically juxtaposed. During natural
pollination, the male reproductive organs of a given flower
pollinate the female reproductive organs of the same flower.
[0007] Bread wheat (Triticum aestivum) is a hexaploid plant having
three pairs of homologous chromosomes defining genomes A, B and D.
The endosperm of wheat grain comprises 2 haploid complements from a
maternal cell and 1 from a paternal cell. The embryo of wheat grain
comprises one haploid complement from each of the maternal and
paternal cells. Hexaploidy has been considered a significant
obstacle in researching and developing useful variants of wheat. In
fact, very little is known regarding how homologous genes of wheat
interact, how their expression is regulated and how the different
proteins produced by homologous genes function separately or in
concert.
[0008] An essential aspect of much of the work underway with
genetic male sterility systems is the identification of genes
influencing male fertility. Such a gene can be used in a variety of
systems to control male fertility including those described
herein.
BRIEF SUMMARY OF THE INVENTION
[0009] Compositions and methods for modulating male fertility in a
plant are provided. Compositions comprise nucleotide sequences, and
active fragments and variants thereof, which modulate male
fertility. Further provided are expression cassettes comprising one
or more of the male fertility polynucleotides or active fragments
or variants thereof, operably linked to a promoter, wherein
expression of the polynucleotides modulates the male fertility of a
plant. Various methods are provided wherein the level and/or
activity of a polynucleotide that influences male fertility is
modulated in a plant or plant part.
DETAILED DESCRIPTION
[0010] 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 inventions 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.
[0011] 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.
I. Male Fertility Polynucleotides
[0012] Compositions disclosed herein include polynucleotides and
polypeptides that influence male fertility. In particular, isolated
polynucleotides are provided comprising nucleotide sequences
encoding the amino acid sequences set forth in SEQ ID NOS: 2, 4, 6,
8, 10, 12, 14, 16 or 18 or active fragments or variants thereof.
Further provided are polypeptides having an amino acid sequence
encoded by a polynucleotide described herein, for example those set
forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15 or 17 or active
fragments or variants thereof.
[0013] Sexually reproducing plants develop specialized tissues
specific for the production of male and female gametes. Successful
production of male gametes relies on proper formation of the male
reproductive tissues. The stamen, which embodies the male
reproductive organ of plants, contains various cell types,
including for example, the filament, anther, tapetum and pollen. As
used herein, "male tissue" refers to the specialized tissue in a
sexually reproducing plant that is responsible for production of
the male gamete. Male tissues include, but are not limited to, the
stamen, filament, anther, tapetum and pollen.
[0014] The process of mature pollen grain formation begins with
microsporogenesis, wherein meiocytes are formed in the sporogenous
tissue of the anther. Microgametogenesis follows, wherein
microspores divide mitotically and develop into the
microgametophyte, or pollen grains. The condition of "male
fertility" or "male fertile" refers to those plants producing a
mature pollen grain capable of fertilizing a female gamete to
produce a subsequent generation of offspring. The term "influences
male fertility" or "modulates male fertility", as used herein,
refers to any increase or decrease in the ability of a plant to
produce a mature pollen grain when compared to an appropriate
control. A "mature pollen grain" or "mature pollen" refers to any
pollen grain capable of fertilizing a female gamete to produce a
subsequent generation of offspring. Likewise, the term "male
fertility polynucleotide" or "male fertility polypeptide" refers to
a polynucleotide or polypeptide that modulates male fertility. A
male fertility polynucleotide may, for example, encode a
polypeptide that participates in the process of microsporogenesis
or microgametogenesis.
[0015] Male fertility polynucleotides disclosed herein include
homologs and orthologs of polynucleotides shown to influence male
fertility. For example, male fertility polynucleotides, and active
fragments and variants thereof, disclosed herein include homologs
and orthologs of Ms22 (also referred to as Msca1). Mutagenesis
studies of Ms22 resulted in phenotypically male sterile maize
plants with anthers that did not extrude from the tassel and lacked
sporogenous tissue. West and Albertsen, (1985) Maize Newsletter
59:87; Neuffer, et al., (1977) Mutants of maize. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
[0016] Plants deficient in Ms22 expression exhibit physiological
changes early in reproductive-tissue development. Ms22 is believed
to have a role in another development that occurs earlier than that
of Ms45 or Ms26. Certain male sterility genes such as MAC1, EMS1 or
GNE2 (Sorensen, et al., (2002) Plant J. 29:581-594) prevent cell
growth in the quartet stage. Mutations in the SPOROCYTELESS/NOZZLE
gene act early in development, but impact both anther and ovule
formation such that plants are male and female sterile. The
SPOROCYTELESS gene of Arabidopsis is required for initiation of
sporogenesis and encodes a novel nuclear protein (Genes Dev.
(1999)13(16):2108-17). Because Ms22 is critical for the progression
of microsporogenesis, maintenance of male sterility in Ms22 mutants
is very reliable compared to other male sterility mutant
constructs. As disclosed elsewhere herein, Ms22 polynucleotides
from wheat are set forth in SEQ ID NOS: 1, 3 and 5.
[0017] Additional male fertility polynucleotides include the Ms26
polynucleotide and homologs and orthologs thereof. Ms26
polypeptides have been reported to have significant homology to
P450 enzymes found in yeast, plants, and mammals. P450 enzymes have
been widely studied and characteristic protein domains have been
elucidated. The Ms26 protein contains several structural motifs
characteristic of eukaryotic P450's, including the heme-binding
domain FxxGxRxCxG (domain D; SEQ ID NO: 19), domain A A/GGXD/ETT/S
(dioxygen-binding; SEQ ID NO: 20), domain B (steroid-binding) and
domain C. Phylogenetic tree analysis revealed that Ms26 is most
closely related to P450s involved in fatty acid omega-hydroxylation
found in Arabidopsis thaliana and Vicia sativa. See, for example,
US Patent Publication Number. 2012/0005792, herein incorporated by
reference. As disclosed elsewhere herein, Ms26 polynucleotides from
wheat are set forth in SEQ ID NOS: 7, 9 and 11.
[0018] Additional male fertility polynucleotides and active
fragments and variants thereof, disclosed herein may also include
homologs and orthologs of Ms45 polynucleotides. The Ms45
polynucleotide is a male fertility polynucleotide characterized in
maize. Mutations of Ms45 can result in breakdown of
microsporogenesis during vacuolation of the microspores rendering
the mutated plants male sterile. When the cloned maize Ms45
polynucleotide is introduced into such mutated male sterile plants,
the gene can complement the mutation and confer male fertility. As
disclosed elsewhere herein, Ms45 polynucleotides from wheat are set
forth in SEQ ID NOS: 13, 15 and 17.
[0019] Strategies for manipulation of expression of male-fertility
polynucleotides in wheat will require consideration of the ploidy
level of the individual wheat variety. Triticum aestivum is a
hexaploid containing three genomes designated A, B and D (N=21);
each genome comprises seven pairs of nonhomologous chromosomes.
Einkorn wheat varieties are diploids (N=7) and emmer wheat
varieties are tetraploids (N=14).
[0020] Isolated or substantially purified nucleic acid molecules or
protein compositions are disclosed herein. An "isolated" or
"purified" nucleic acid molecule, polynucleotide, or protein, or
biologically active portion thereof, is substantially or
essentially free from components that normally accompany or
interact with the polynucleotide or protein as found in its
naturally occurring environment. Thus, an isolated or purified
polynucleotide or protein is substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized. Optimally, an "isolated"
polynucleotide is free of sequences (optimally protein encoding
sequences) that naturally flank the polynucleotide (i.e., sequences
located at the 5' and 3' ends of the polynucleotide) in the genomic
DNA of the organism from which the polynucleotide is derived. For
example, in various embodiments, the isolated polynucleotide can
contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1
kb of nucleotide sequence that naturally flank the polynucleotide
in genomic DNA of the cell from which the polynucleotide is
derived. A protein that is substantially free of cellular material
includes preparations of protein having less than about 30%, 20%,
10%, 5% or 1% (by dry weight) of contaminating protein. When the
polypeptides disclosed herein or biologically active portion
thereof is recombinantly produced, optimally culture medium
represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight)
of chemical precursors or non-protein-of-interest chemicals.
[0021] A "subject plant" or "subject plant cell" is one in which
genetic alteration, such as transformation, has been affected as to
a gene of interest or is a plant or plant cell which is descended
from a plant or cell so altered and which comprises the alteration.
A "control" or "control plant" or "control plant cell" provides a
reference point for measuring changes in phenotype of the subject
plant or plant cell.
[0022] A control plant or plant cell may comprise, for example: (a)
a wild-type plant or plant cell, i.e., of the same genotype as the
starting material for the genetic alteration which resulted in the
subject plant or cell; (b) a plant or plant cell of the same
genotype as the starting material but which has been transformed
with a null construct (i.e., with a construct which has no known
effect on the trait of interest, such as a construct comprising a
marker gene); (c) a plant or plant cell which is a non-transformed
segregant among progeny of a subject plant or plant cell; (d) a
plant or plant cell genetically identical to the subject plant or
plant cell but which is not exposed to conditions or stimuli that
would induce expression of the gene of interest; or (e) the subject
plant or plant cell itself, under conditions in which the gene of
interest is not expressed.
[0023] A. Fragments and Variants of Male Fertility Sequences
[0024] Fragments and variants of the disclosed polynucleotides and
proteins encoded thereby are also provided. By "fragment" is
intended a portion of the polynucleotide or a portion of the amino
acid sequence and hence protein encoded thereby. Fragments of a
polynucleotide may encode protein fragments that retain the
biological activity of the native protein and hence influence male
fertility. Alternatively, fragments of a polynucleotide that are
useful as hybridization probes generally do not encode fragment
proteins retaining biological activity. Thus, fragments of a
nucleotide sequence may range from at least about 20 nucleotides,
about 50 nucleotides, about 100 nucleotides and up to the
full-length polynucleotide encoding the polypeptides disclosed
herein.
[0025] A fragment of a polynucleotide that encodes a biologically
active portion of a polypeptide that influences male fertility will
encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400,
450, 500, 525 or 537 contiguous amino acids, or up to the total
number of amino acids present in a full-length polypeptide that
influences male fertility (for example, SEQ ID NO: 2, 4, 6, 8, 10,
12, 14, 16 and 18, respectively). Fragments of a polynucleotide
encoding a polypeptide that influences male fertility that are
useful as hybridization probes or PCR primers generally need not
encode a biologically active portion of a polypeptide that
influences male fertility.
[0026] Thus, a fragment of a male fertility polynucleotide as
disclosed herein may encode a biologically active portion of a male
fertility polypeptide or it may be a fragment that can be used as a
hybridization probe or PCR primer using methods disclosed below. A
biologically active portion of a male fertility polypeptide can be
prepared by isolating a portion of one of the male fertility
polynucleotides disclosed herein, expressing the encoded portion of
the male fertility protein (e.g., by recombinant expression in
vitro) and assessing the activity of the encoded portion of the
male fertility polypeptide. Polynucleotides that are fragments of a
male fertility polynucleotide comprise at least 16, 20, 50, 75,
100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,
800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600 or 1629
nucleotides or up to the number of nucleotides present in a
full-length male fertility polynucleotide disclosed herein (i.e.,
SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15 or 17, respectively).
[0027] "Variants" is intended to mean substantially similar
sequences. For polynucleotides, a variant comprises a deletion
and/or addition of one or more nucleotides at one or more internal
sites within the native polynucleotide and/or a substitution of one
or more nucleotides at one or more sites in the native
polynucleotide. As used herein, a "native" or "wild type"
polynucleotide or polypeptide comprises a naturally occurring
nucleotide sequence or amino acid sequence, respectively. For
polynucleotides, conservative variants include those sequences
that, because of the degeneracy of the genetic code, encode the
amino acid sequence of one of the male fertility polypeptides
disclosed herein. Naturally occurring allelic variants such as
these can be identified with the use of well-known molecular
biology techniques, as, for example, with polymerase chain reaction
(PCR) and hybridization techniques as outlined below. Variant
polynucleotides also include synthetically derived polynucleotides,
such as those generated, for example, by using site-directed
mutagenesis but which still encode a male fertility polypeptide.
Generally, variants of a particular polynucleotides disclosed
herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more sequence identity to that particular polynucleotide (e.g., any
one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15 or 17) as determined
by sequence alignment programs and parameters described elsewhere
herein.
[0028] Variants of a particular polynucleotide disclosed herein
(i.e., the reference polynucleotide) can also be evaluated by
comparison of the percent sequence identity between the polypeptide
encoded by a variant polynucleotide and the polypeptide encoded by
the reference polynucleotide. Thus, for example, an isolated
polynucleotide that encodes a polypeptide with a given percent
sequence identity to the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10,
12, 14, 16 or 18 are disclosed. Percent sequence identity between
any two polypeptides can be calculated using sequence alignment
programs and parameters described elsewhere herein. Where any given
pair of polynucleotides disclosed herein is evaluated by comparison
of the percent sequence identity shared by the two polypeptides
they encode, the percent sequence identity between the two encoded
polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more sequence identity.
[0029] "Variant" protein is intended to mean a protein derived from
the native protein by deletion or addition of one or more amino
acids at one or more internal sites in the native protein and/or
substitution of one or more amino acids at one or more sites in the
native protein. Variant proteins disclosed herein are biologically
active, that is they continue to possess the desired biological
activity of the native protein, that is, male fertility activity as
described herein. Such variants may result from, for example,
genetic polymorphism or from human manipulation. Biologically
active variants of a male fertility protein disclosed herein will
have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to the amino acid sequence for the native protein
(e.g., any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18) as
determined by sequence alignment programs and parameters described
elsewhere herein. A biologically active variant of a protein
disclosed herein may differ from that protein by as few as 1-15
amino acid residues, as few as 1-10, such as 6-10, as few as 5, as
few as 4, 3, 2 or even 1 amino acid residue.
[0030] The proteins disclosed herein may be altered in various ways
including amino acid substitutions, deletions, truncations, and
insertions. Methods for such manipulations are generally known in
the art. For example, amino acid sequence variants and fragments of
the male fertility polypeptides can be prepared by mutations in the
DNA. Methods for mutagenesis and polynucleotide alterations are
well known in the art. See, for example, Kunkel, (1985) Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel, et al., (1987) Methods in
Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra,
eds. (1983) Techniques in Molecular Biology (MacMillan Publishing
Company, New York) and the references cited therein. Guidance as to
appropriate amino acid substitutions that do not affect biological
activity of the protein of interest may be found in the model of
Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure
(Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated
by reference. Conservative substitutions, such as exchanging one
amino acid with another having similar properties, may be
optimal.
[0031] Thus, the genes and polynucleotides disclosed herein include
both the naturally occurring sequences as well as DNA sequence
variants which retain function. Likewise, the male fertility
polypeptides and proteins encompass both naturally occurring
polypeptides as well as variations and modified forms thereof. Such
polynucleotide and polypeptide variants will continue to possess
the desired male fertility activity. The mutations that will be
made in the DNA encoding the variant must not place the sequence
out of reading frame and optimally will not create complementary
regions that could produce secondary mRNA structure. See, EP Patent
Application Publication Number 75,444.
[0032] The deletions, insertions and substitutions of the protein
sequences encompassed herein are not expected to produce radical
changes in the characteristics of the protein. However, when it is
difficult to predict the exact effect of the substitution, deletion
or insertion in advance of doing so, one skilled in the art will
appreciate that the effect will be evaluated by routine screening
assays. That is, the activity can be evaluated by assaying for male
fertility activity.
[0033] Increases or decreases in male fertility can be assayed in a
variety of ways. One of ordinary skill in the art can readily
assess activity of the variant or fragment by introducing the
polynucleotide into a plant homozygous for a stable male sterile
allele of the polynucleotide and observing male tissue development
in the plant. For example, to assay for male fertility activity of
Ms22 (i.e., SEQ ID NO: 1, 3 or 5), one of skill in the art can
begin by constructing a plant homozygous for a mutation in the
native Ms22 gene resulting in male sterility. Subsequently, one
could complement the mutation by providing the Ms22 polynucleotide
or active fragment or variant thereof and observing whether the
male tissues of the plant develop normally and are able to produce
mature pollen. Likewise, the same procedure can be carried out to
assay for the male fertility activity of variants or fragments of
Ms26 (i.e., SEQ ID NO: 7, 9 or 11) or Ms45 (i.e., SEQ ID NO: 13, 15
or 17), also disclosed herein.
[0034] Variant functional polynucleotides and proteins also
encompass sequences and proteins derived from a mutagenic and
recombinogenic procedure such as DNA shuffling. With such a
procedure, one or more different male fertility sequences can be
manipulated to create a new male fertility polypeptide possessing
the desired properties. In this manner, libraries of recombinant
polynucleotides are generated from a population of related sequence
polynucleotides comprising sequence regions that have substantial
sequence identity and can be homologously recombined in vitro or in
vivo. For example, using this approach, sequence motifs encoding a
domain of interest may be shuffled between the male fertility
polynucleotides disclosed herein and other known male fertility
polynucleotides to obtain a new gene coding for a protein with an
improved property of interest, such as an increased K.sub.m in the
case of an enzyme. Strategies for such DNA shuffling are known in
the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci.
USA 91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et
al., (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J.
Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad.
Sci. USA 94:4504-4509; Crameri, et al., (1998) Nature 391:288-291
and U.S. Pat. Nos. 5,605,793 and 5,837,458.
II. Sequence Analysis
[0035] As used herein, "sequence identity" or "identity" in the
context of two polynucleotides or polypeptide sequences makes
reference to the residues in the two sequences that 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. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that 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., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0036] 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.
[0037] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using GAP Weight of 50 and Length Weight of 3,
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using GAP Weight of 8 and Length Weight
of 2 and the BLOSUM62 scoring matrix; or any equivalent program
thereof. By "equivalent program" is intended any sequence
comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide or amino acid
residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by GAP Version
10.
[0038] The use of the term "polynucleotide" is not intended to
limit the present disclosure to polynucleotides comprising DNA.
Those of ordinary skill in the art will recognize that
polynucleotides can comprise ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides
and ribonucleotides include both naturally occurring molecules and
synthetic analogues. The polynucleotides disclosed herein also
encompass all forms of sequences including, but not limited to,
single-stranded forms, double-stranded forms, hairpins,
stem-and-loop structures, and the like.
III. Expression Cassettes
[0039] The male fertility polynucleotides disclosed herein can be
provided in expression cassettes for expression in an organism of
interest. The cassette can include 5' and 3'regulatory sequences
operably linked to a male fertility polynucleotide as disclosed
herein. "Operably linked" is intended to mean a functional linkage
between two or more elements. For example, an operable linkage
between a polynucleotide of interest and a regulatory sequence
(e.g., a promoter) is a functional link that allows for expression
of the polynucleotide of interest. Operably linked elements may be
contiguous or non-contiguous. When used to refer to the joining of
two protein coding regions, by operably linked is intended that the
coding regions are in the same reading frame.
[0040] The expression cassettes disclosed herein may include in the
5'-3' direction of transcription, a transcriptional and
translational initiation region (i.e., a promoter), a
polynucleotide of interest and a transcriptional and translational
termination region (i.e., termination region) functional in the
host cell (i.e., the plant). Expression cassettes are also provided
with a plurality of restriction sites and/or recombination sites
for insertion of the male fertility polynucleotide to be under the
transcriptional regulation of the regulatory regions described
elsewhere herein. The regulatory regions (i.e., promoters,
transcriptional regulatory regions, and translational termination
regions) and/or the polynucleotide of interest may be
native/analogous to the host cell or to each other. Alternatively,
the regulatory regions and/or the polynucleotide of interest may be
heterologous to the host cell or to each other. As used herein,
"heterologous" in reference to a polynucleotide or polypeptide
sequence is a sequence 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 polynucleotide is from a species different from the
species from which the polynucleotide was derived, or, if from the
same/analogous species, one or both are substantially modified from
their original form and/or genomic locus, or the promoter is not
the native promoter for the operably linked polynucleotide. As used
herein, a chimeric polynucleotide comprises a coding sequence
operably linked to a transcription initiation region that is
heterologous to the coding sequence.
[0041] In certain embodiments the polynucleotides disclosed herein
can be stacked with any combination of polynucleotide sequences of
interest or expression cassettes as disclosed elsewhere herein. For
example, the male fertility polynucleotides disclosed herein may be
stacked with any other polynucleotides encoding male-gamete
disruptive polynucleotides or polypeptides, cytotoxins, markers or
other male fertility sequences as disclosed elsewhere herein. The
stacked polynucleotides may be operably linked to the same promoter
as the male fertility polynucleotide, or may be operably linked to
a separate promoter polynucleotide.
[0042] As described elsewhere herein, expression cassettes may
comprise a promoter operably linked to a polynucleotide of
interest, along with a corresponding termination region. The
termination region may be native to the transcriptional initiation
region, may be native to the operably linked male fertility
polynucleotide of interest or with the male fertility promoter
sequences, may be native to the plant host, or may be derived from
another source (i.e., foreign or heterologous). Convenient
termination regions are available from the Ti-plasmid of A.
tumefaciens, such as the octopine synthase and nopaline synthase
termination regions. See also, Guerineau, et al., (1991) Mol. Gen.
Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et
al., (1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell
2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Belles, et
al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al.,
(1987) Nucleic Acids Res. 15:9627-9639.
[0043] Where appropriate, the polynucleotides of interest may be
optimized for increased expression in the transformed plant. That
is, the polynucleotides can be synthesized using plant-preferred
codons for improved expression. See, for example, Campbell and
Gowri, (1990) Plant Physiol. 92:1-11 for a discussion of
host-preferred codon usage. Methods are available in the art for
synthesizing plant-preferred genes. See, for example, U.S. Pat.
Nos. 5,380,831 and 5,436,391 and Murray, et al., (1989) Nucleic
Acids Res. 17:477-498, herein incorporated by reference.
[0044] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
[0045] The expression cassettes may additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'
noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad.
Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238),
MDMV leader (Maize Dwarf Mosaic Virus) (Johnson, et al., (1986)
Virology 154:9-20) and human immunoglobulin heavy-chain binding
protein (BiP) (Macejak, et al., (1991) Nature 353:90-94);
untranslated leader from the coat protein mRNA of alfalfa mosaic
virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625);
tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in
Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256);
and maize chlorotic mottle virus leader (MCMV) (Lommel, et al.,
(1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987)
Plant Physiol. 84:965-968. Other methods known to enhance
translation can also be utilized, for example, introns, and the
like.
[0046] In preparing the expression cassette, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate, in the
proper reading frame. Toward this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resubstitutions, e.g., transitions and transversions,
may be involved.
[0047] A. Expression Cassettes Comprising a Male Fertility
Polynucleotide
[0048] In particular embodiments, the expression cassettes
disclosed herein comprise a promoter operably linked to a male
fertility polynucleotide or active fragment or variant thereof, as
disclosed herein. In certain embodiments, a male fertility promoter
or an active fragment or variant thereof, is operably linked to a
male fertility polynucleotide disclosed herein, such as the male
fertility polynucleotide set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11,
13, 15 or 17 or an active fragment or variant thereof.
[0049] In certain embodiments, plant promoters can preferentially
initiate transcription in certain tissues, such as stamen, anther,
filament and pollen or developmental growth stages, such as
sporogenous tissue, microspores and microgametophyte. Such plant
promoters are referred to as "tissue-preferred", "cell
type-preferred" or "growth-stage preferred". Promoters which
initiate transcription only in certain tissue are referred to as
"tissue-specific". Likewise, promoters which initiate transcription
only at certain growth stages are referred to as "growth
stage-specific". A "cell type-specific" promoter drives expression
only in certain cell types in one or more organs, for example,
stamen cells or individual cell types within the stamen such as
anther, filament or pollen cells.
[0050] Male fertility polynucleotides disclosed herein, and active
fragments and variants thereof, can be operably linked to
male-tissue-specific or male-tissue-preferred promoters including,
for example, stamen-specific or stamen-preferred promoters,
anther-specific or anther-preferred promoters, pollen-specific or
pollen-preferred promoters, tapetum-specific promoters or
tapetum-preferred promoters, and the like. Promoters can be
selected based on the desired outcome. For example, the
polynucleotides of interest can be operably linked to constitutive,
tissue-preferred, growth stage-preferred or other promoters for
expression in plants.
[0051] In one embodiment, the promoters may be those which
preferentially express a polynucleotide of interest in the male
tissues of the plant. No particular male fertility tissue-preferred
promoter must be used in the process, and any of the many such
promoters known to one skilled in the art may be employed. One such
promoter is the 5126 promoter, which preferentially directs
expression of the polynucleotide to which it is linked to male
tissue of the plants, as described in U.S. Pat. Nos. 5,837,851 and
5,689,051. Other examples include the maize Ms45 promoter described
at U.S. Pat. No. 6,037,523; SF3 promoter described at U.S. Pat. No.
6,452,069; the BS92-7 promoter described at WO 2002/063021; a SGB6
regulatory element described at U.S. Pat. No. 5,470,359; the TA29
promoter (Koltunow, et al., (1990) Plant Cell 2:1201-1224;
Goldberg, et al., (1993) Plant Cell 5:1217-1229 and U.S. Pat. No.
6,399,856); the type 2 metallothionein-like gene promoter
(Charbonnel-Campaa, et al., Gene (2000) 254:199-208) and the
Brassica Bca9 promoter (Lee, et al., (2003) Plant Cell Rep.
22:268-273).
[0052] In some embodiments, expression cassettes comprise
male-gamete-preferred promoters operably linked to a male fertility
polynucleotide. Male-gamete-preferred promoters include the PG47
promoter (U.S. Pat. No. 5,412,085; U.S. Pat. No. 5,545,546; Plant J
3(2):261-271 (1993)), as well as ZM13 promoter (Hamilton, et al.,
(1998) Plant Mol. Biol. 38:663-669); actin depolymerizing factor
promoters (such as Zmabp1, Zmabp2; see, for example Lopez, et al.,
(1996) Proc. Natl. Acad. Sci. USA 93:7415-7420); the promoter of
the maize pectin methylesterase-like gene, ZmC5 (Wakeley, et al.,
(1998) Plant Mol. Biol. 37:187-192); the profilin gene promoter
Zmpro1 (Kovar, et al., (2000) The Plant Cell 12:583-598); the
sulphated pentapeptide phytosulphokine gene ZmPSK1 (Lorbiecke, et
al., (2005) Journal of Experimental Botany 56(417):1805-1819); the
promoter of the calmodulin binding protein Mpcbp (Reddy, et al.,
(2000) J. Biol. Chem. 275(45):35457-70).
[0053] As disclosed herein, constitutive promoters include, for
example, the core promoter of the Rsyn7 promoter and other
constitutive promoters disclosed in WO 1999/43838 and U.S. Pat. No.
6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature
313:810-812); rice actin (McElroy, et al., (1990) Plant Cell
2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol. Biol.
12:619-632 and Christensen, et al., (1992) Plant Mol. Biol.
18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet.
81:581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS
promoter (U.S. Pat. No. 5,659,026) and the like. Other constitutive
promoters include, for example, U.S. Pat. Nos. 5,608,149;
5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;
5,608,142 and 6,177,611.
[0054] "Seed-preferred" promoters include both those promoters
active during seed development such as promoters of seed storage
proteins as well as those promoters active during seed germination.
See, Thompson, et al., (1989) BioEssays 10:108, herein incorporated
by reference. Such seed-preferred promoters include, but are not
limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa
zein); milps (myo-inositol-1-phosphate synthase) (see, WO
2000/11177 and U.S. Pat. No. 6,225,529; herein incorporated by
reference). Gamma-zein is an endosperm-specific promoter.
Globulin-1 (Glob-1) is a representative embryo-specific promoter.
For dicots, seed-specific promoters include, but are not limited
to, bean .beta.-phaseolin, napin, .beta.-conglycinin, soybean
lectin, cruciferin, and the like. For monocots, seed-specific
promoters include, but are not limited to, maize 15 kDa zein, 22
kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2,
globulin 1, etc. See also, WO 2000/12733, where seed-preferred
promoters from end1 and end2 genes are disclosed, herein
incorporated by reference. Additional embryo specific promoters are
disclosed in Sato, et al., (1996) Proc. Natl. Acad. Sci.
93:8117-8122; Nakase, et al., (1997) Plant J 12:235-46 and
Postma-Haarsma et al. (1999) Plant Mol. Biol. 39:257-71. Additional
endosperm specific promoters are disclosed in Albani, et al.,
(1984) EMBO 3:1405-15; Albani, et al., (1999) Theor. Appl. Gen.
98:1253-62; Albani, et al., (1993) Plant J. 4:343-55; Mena, et al.,
(1998) The Plant Journal 116:53-62 and Wu, et al., (1998) Plant
Cell Physiology 39:885-889.
[0055] Dividing cell or meristematic tissue-preferred promoters
have been disclosed in Ito, et al., (1994) Plant Mol. Biol.
24:863-878; Reyad, et al., (1995) Mo. Gen. Genet. 248:703-711;
Shaul, et al., (1996) Proc. Natl. Acad. Sci. 93:4868-4872; Ito, et
al., (1997) Plant J. 11:983-992 and Trehin, et al., (1997) Plant
Mol. Biol. 35:667-672.
[0056] Stress inducible promoters include salt/water
stress-inducible promoters such as P5CS (Zang, et al., (1997) Plant
Sciences 129:81-89); cold-inducible promoters, such as, cor15a
(Hajela, et al., (1990) Plant Physiol. 93:1246-1252), cor15b
(Wlihelm, et al., (1993) Plant Mol Biol 23:1073-1077), wsc120
(Ouellet, et al., (1998) FEBS Lett. 423:324-328), ci7 (Kirch, et
al., (1997) Plant Mol. Biol. 33:897-909), ci21A (Schneider, et al.,
(1997) Plant Physiol. 113:335-45); drought-inducible promoters,
such as, Trg-31 (Chaudhary, et al., (1996) Plant Mol. Biol.
30:1247-57), rd29 (Kasuga, et al., (1999) Nature Biotechnology
18:287-291); osmotic inducible promoters, such as, Rab17
(Vilardell, et al., (1991) Plant Mol. Biol. 17:985-93) and osmotin
(Raghothama, et al., (1993) Plant Mol Biol 23:1117-28) and, heat
inducible promoters, such as, heat shock proteins (Barros, et al.,
(1992) Plant Mol. 19:665-75; Marrs, et al., (1993) Dev. Genet.
14:27-41) and smHSP (Waters, et al., (1996) J. Experimental Botany
47:325-338). Other stress-inducible promoters include rip2 (U.S.
Pat. No. 5,332,808 and US Patent Application Publication Number
2003/0217393) and rp29a (Yamaguchi-Shinozaki, et al., (1993) Mol.
Gen. Genetics 236:331-340).
[0057] As discussed elsewhere herein, the expression cassettes
comprising male fertility polynucleotides may be stacked with other
polynucleotides of interest. Any polynucleotide of interest may be
stacked with the male fertility polynucleotide, including for
example, male-gamete-disruptive polynucleotides and marker
polynucleotides.
[0058] Male fertility polynucleotides disclosed herein may be
stacked in or with expression cassettes comprising a promoter
operably linked to a polynucleotide which is
male-gamete-disruptive; that is, a polynucleotide which interferes
with the function, formation or dispersal of male gametes. A
male-gamete-disruptive polynucleotide can operate to prevent
function, formation or dispersal of male gametes by any of a
variety of methods. By way of example but not limitation, this can
include use of polynucleotides which encode a gene product such as
DAM-methylase or barnase (see, for example, U.S. Pat. No. 5,792,853
or 5,689,049; PCT/EP89/00495); encode a gene product which
interferes with the accumulation of starch or affects osmotic
balance in pollen (see, for example, U.S. Pat. Nos. 7,875,764;
8,013,218; 7,696,405); inhibit formation of a gene product
important to male gamete function, formation or dispersal (see, for
example, U.S. Pat. Nos. 5,859,341; 6,297,426); encode a gene
product which combines with another gene product to prevent male
gamete formation or function (see, U.S. Pat. Nos. 6,162,964;
6,013,859; 6,281,348; 6,399,856; 6,248,935; 6,750,868; 5,792,853);
are antisense to, or cause co-suppression of, a gene critical to
male gamete function, formation or dispersal (see U.S. Pat. Nos.
6,184,439; 5,728,926; 6,191,343; 5,728,558; 5,741,684); interfere
with expression of a male fertility polynucleotide through use of
hairpin formations (Smith, et al., (2000) Nature 407:319-320; WO
1999/53050 and WO 1998/53083) or the like.
[0059] Male-gamete-disruptive polynucleotides include dominant
negative genes such as methylase genes and growth-inhibiting genes.
See, U.S. Pat. No. 6,399,856 Dominant negative genes include
diphtheria toxin A-chain gene (Czako and An, (1991) Plant Physiol.
95:687-692; Greenfield, et al., (1983) PNAS 80:6853); cell cycle
division mutants such as CDC in maize (Colasanti, et al., (1991)
PNAS 88:3377-3381); the WT gene (Farmer, et al., (1994) Mol. Genet.
3:723-728) and P68 (Chen, et al., (1991) PNAS 88:315-319).
[0060] Further examples of male-gamete-disruptive polynucleotides
include, but are not limited to, pectate lyase gene pelE from
Erwinia chrysanthermi (Kenn, et al., (1986) J. Bacteriol. 168:595);
CytA toxin gene from Bacillus thuringiensis Israeliensis (McLean,
et al., (1987) J. Bacteriol. 169:1017, U.S. Pat. No. 4,918,006);
DNAses, RNAses, proteases, or polynucleotides expressing anti-sense
RNA. A male-gamete-disruptive polynucleotide may encode a protein
involved in inhibiting pollen-stigma interactions, pollen tube
growth, fertilization or a combination thereof.
[0061] Male fertility polynucleotides disclosed herein may be
stacked with expression cassettes disclosed herein comprising a
promoter operably linked to a polynucleotide of interest encoding a
reporter or marker product. Examples of suitable reporter
polynucleotides known in the art can be found in, for example,
Jefferson, et al., (1991) in Plant Molecular Biology Manual, ed.
Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet, et
al., (1987) Mol. Cell. Biol. 7:725-737; Goff, et al., (1990) EMBO
J. 9:2517-2522; Kain, et al., (1995) BioTechniques 19:650-655 and
Chiu, et al., (1996) Current Biology 6:325-330. In certain
embodiments, the polynucleotide of interest encodes a selectable
reporter. These can include polynucleotides that confer antibiotic
resistance or resistance to herbicides. Examples of suitable
selectable marker polynucleotides include, but are not limited to,
genes encoding resistance to chloramphenicol, methotrexate,
hygromycin, streptomycin, spectinomycin, bleomycin, sulfonamide,
bromoxynil, glyphosate and phosphinothricin.
[0062] In some embodiments, the expression cassettes disclosed
herein comprise a polynucleotide of interest encoding scorable or
screenable markers, where presence of the polynucleotide produces a
measurable product. Examples include a 13-glucuronidase, or uidA
gene (GUS), which encodes an enzyme for which various chromogenic
substrates are known (for example, U.S. Pat. Nos. 5,268,463 and
5,599,670); chloramphenicol acetyl transferase, and alkaline
phosphatase. Other screenable markers include the
anthocyanin/flavonoid polynucleotides including, for example, a
R-locus polynucleotide, which encodes a product that regulates the
production of anthocyanin pigments (red color) in plant tissues,
the genes which control biosynthesis of flavonoid pigments, such as
the maize C1 and C2, the B gene, the p1 gene and the bronze locus
genes, among others. Further examples of suitable markers encoded
by polynucleotides of interest include the cyan fluorescent protein
(CYP) gene, the yellow fluorescent protein gene, a lux gene, which
encodes a luciferase, the presence of which may be detected using,
for example, X-ray film, scintillation counting, fluorescent
spectrophotometry, low-light video cameras, photon counting cameras
or multiwell luminometry, a green fluorescent protein (GFP) and
DsRed2 where plant cells transformed with the marker gene are red
in color and thus visually selectable. Additional examples include
a p-lactamase gene encoding an enzyme for which various chromogenic
substrates are known (e.g., PADAC, a chromogenic cephalosporin), a
xylE gene encoding a catechol dioxygenase that can convert
chromogenic catechols, an .alpha.-amylase gene and a tyrosinase
gene encoding an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone, which in turn condenses to form the easily detectable
compound melanin.
[0063] The expression cassette can also comprise a selectable
marker gene for the selection of transformed cells. Selectable
marker genes are utilized for the selection of transformed cells or
tissues. Marker genes include genes encoding antibiotic resistance,
such as those encoding neomycin phosphotransferase II (NEO) and
hygromycin phosphotransferase (HPT), as well as genes conferring
resistance to herbicidal compounds, such as glufosinate ammonium,
bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D).
Additional selectable markers include phenotypic markers such as
.beta.-galactosidase and fluorescent proteins such as green
fluorescent protein (GFP) (Su, et al., (2004) Biotechnol Bioeng
85:610-9 and Fetter, et al., (2004) Plant Cell 16:215-28), cyan
florescent protein (CYP) (Bolte, et al., (2004) J. Cell Science
117:943-54 and Kato, et al., (2002) Plant Physiol 129:913-42) and
yellow florescent protein (PhiYFP.TM. from Evrogen, see, Bolte, et
al., (2004) J. Cell Science 117:943-54). For additional selectable
markers, see generally, Yarranton, (1992) Curr. Opin. Biotech.
3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci.
USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff,
(1992) Mol. Microbiol. 6:2419-2422; Barkley, et al., (1980) in The
Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et
al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722;
Deuschle, et al., (1989) Proc. Natl. Acad. Ad. USA 86:5400-5404;
Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553;
Deuschle, et al., (1990) Science 248:480-483; Gossen, (1993) Ph.D.
Thesis, University of Heidelberg; Reines, et al., (1993) Proc.
Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell.
Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad.
Sci. USA 89:3952-3956; Bairn, et al., (1991) Proc. Natl. Acad. Sci.
USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res.
19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol.
10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother.
35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry
27:1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg;
Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551;
Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919;
Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol.
78 (Springer-Verlag, Berlin); Gill, et al., (1988) Nature
334:721-724. Such disclosures are herein incorporated by reference.
The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the
compositions and methods disclosed herein.
[0064] In some embodiments, the expression cassettes disclosed
herein comprise a first polynucleotide of interest encoding a male
fertility polynucleotide operably linked to a first promoter
polynucleotide stacked with a second polynucleotide of interest
encoding a male-gamete-disruptive gene product operably linked to a
male tissue-preferred promoter polynucleotide. In other
embodiments, the expression cassettes described herein may also be
stacked with a third polynucleotide of interest encoding a marker
polynucleotide operably linked to a third promoter
polynucleotide.
[0065] In specific embodiments, the expression cassettes disclosed
herein comprise a first polynucleotide of interest encoding a wheat
male fertility gene disclosed herein, such as Ms22, Ms26 or Ms45
operably linked to a constitutive promoter, such as the cauliflower
mosaic virus (CaMV) 35S promoter. The expression cassettes may
further comprise a second polynucleotide of interest encoding a
male-gamete-disruptive gene product operably linked to a male
tissue-preferred promoter. In certain embodiments, the expression
cassettes disclosed herein may further comprise a third
polynucleotide of interest encoding a marker gene, such as the
phosphinothricin acetyltransferase (PAT) gene from Streptomyces
viridochomagenes operably linked to a constitutive promoter, such
as the cauliflower mosaic virus (CaMV) 35S promoter.
IV. Plants
[0066] A. Plants Having Altered Levels/Activity of Male Fertility
Polypeptide
[0067] Further provided are plants having altered levels and/or
activities of a male fertility polypeptide and/or altered levels of
male fertility. In some embodiments, the plants disclosed herein
have stably incorporated into their genomes a heterologous male
fertility polynucleotide, or active fragments or variants thereof,
as disclosed herein. Thus, plants, plant cells, plant parts and
seeds are provided which comprise at least one heterologous male
fertility polynucleotide as set forth in any one of SEQ ID NO: 1,
3, 5, 7, 9, 11, 13, 15 or 17 or any active fragments or variants
disclosed herein.
[0068] Plants are further provided comprising the expression
cassettes disclosed herein comprising a male fertility
polynucleotide operably linked to a promoter that is active in the
plant. In some embodiments, expression of the male fertility
polynucleotide modulates male fertility of the plant. In certain
embodiments, expression of the male fertility polynucleotide
increases male fertility of the plant. For example, plants are
provided comprising an expression cassette comprising an Ms22
polynucleotide as set forth in SEQ ID NO: 1, 3 or 5 or an active
fragment or variant thereof, operably linked to a constitutive
promoter, such as the CaMV 35S promoter. Upon expression of the
Ms22 polynucleotide, male fertility of the plant is increased.
[0069] In certain embodiments, expression cassettes comprising a
heterologous male fertility polynucleotide as disclosed herein, or
an active fragment or variant thereof, operably linked to a
promoter active in a plant, are provided to a male sterile plant.
Upon expression of the heterologous male fertility polynucleotide,
the male fertility of the plant is restored. In specific
embodiments, the plants disclosed herein comprise an expression
cassette comprising a heterologous male fertility polynucleotide as
disclosed herein, or an active fragment or variant thereof,
operably linked to a promoter, stacked with one or more expression
cassettes comprising a polynucleotide of interest operably linked
to a promoter active in the plant. For example, the stacked
polynucleotide of interest can comprise a male-gamete-disruptive
polynucleotide and/or a marker polynucleotide.
[0070] Plants disclosed herein may also comprise stacked expression
cassettes described herein comprising at least two polynucleotides
such that the at least two polynucleotides are inherited together
in more than 50% of meioses, i.e., not randomly. Accordingly, when
a plant or plant cell comprising stacked expression cassettes with
two polynucleotides undergoes meiosis, the two polynucleotides
segregate into the same progeny (daughter) cell. In this manner,
stacked polynucleotides will likely be expressed together in any
cell for which they are present. For example, a plant may comprise
an expression cassette comprising a male fertility polynucleotide
stacked with an expression cassette comprising a
male-gamete-disruptive polynucleotide such that the male fertility
polynucleotide and the male-gamete-disruptive polynucleotide are
inherited together. Specifically, a male sterile plant could
comprise an expression cassette comprising a male fertility
polynucleotide disclosed herein operably linked to a constitutive
promoter, stacked with an expression cassette comprising a
male-gamete-disruptive polynucleotide operably linked to a male
tissue-preferred promoter, such that the plant produces mature
pollen grains. However, in such a plant, development of the
daughter pollen cells comprising the male fertility polynucleotide
will be prevented by expression of the male-gamete-disruptive
polynucleotide.
[0071] B. Plants and Methods of Introduction
[0072] As used herein, the term plant includes plant cells, plant
protoplasts, plant cell tissue cultures from which a plant can be
regenerated, plant calli, plant clumps and plant cells that are
intact in plants or parts of plants such as embryos, pollen,
ovules, seeds, leaves, flowers, branches, fruit, kernels, ears,
cobs, husks, stalks, roots, root tips, anthers, grain and the like.
As used herein "grain" is intended the mature seed produced by
commercial growers for purposes other than growing or reproducing
the species. Progeny, variants and mutants of the regenerated
plants are also included within the scope of the disclosure,
provided that these parts comprise the introduced nucleic acid
sequences.
[0073] The methods disclosed herein comprise introducing a
polypeptide or polynucleotide into a plant cell. "Introducing" is
intended to mean presenting to the plant the polynucleotide or
polypeptide in such a manner that the sequence gains access to the
interior of a cell. The methods disclosed herein do not depend on a
particular method for introducing a sequence into the host cell,
only that the polynucleotide or polypeptides gains access to the
interior of at least one cell of the host. Methods for introducing
polynucleotide or polypeptides into host cells (i.e., plants) are
known in the art and include, but are not limited to, stable
transformation methods, transient transformation methods and
virus-mediated methods.
[0074] "Stable transformation" is intended to mean that the
nucleotide construct introduced into a host (i.e., a plant)
integrates into the genome of the plant and is capable of being
inherited by the progeny thereof. "Transient transformation" is
intended to mean that a polynucleotide is introduced into the host
(i.e., a plant) and expressed temporally or a polypeptide is
introduced into a host (i.e., a plant).
[0075] Transformation protocols as well as protocols for
introducing polypeptides or polynucleotide sequences into plants
may vary depending on the type of plant or plant cell, i.e.,
monocot or dicot, targeted for transformation. Suitable methods of
introducing polypeptides and polynucleotides into plant cells
include microinjection (Crossway, et al., (1986) Biotechniques
4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl.
Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation
(Townsend, et al., U.S. Pat. No. 5,563,055; Zhao, et al., U.S. Pat.
No. 5,981,840), 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; Tomes, et al.,
U.S. Pat. No. 5,879,918; Tomes, et al., U.S. Pat. No. 5,886,244;
Bidney, et al., U.S. Pat. No. 5,932,782; Tomes, et al., (1995)
"Direct DNA Transfer into Intact Plant Cells via Microprojectile
Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental
Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);
McCabe, et al., (1988) Biotechnology 6:923-926) and Lec1
transformation (WO 2000/28058). Also see, 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); McCabe, et al.,
(1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen,
(1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et
al., (1998) Theor. Appl. Genet. 96:319-324 (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); Tomes, U.S. Pat. No.
5,240,855; Buising, et al., U.S. Pat. Nos. 5,322,783 and 5,324,646;
Tomes, et al., (1995) "Direct DNA Transfer into Intact Plant Cells
via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ
Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)
(maize); Klein, et al., (1988) Plant Physiol. 91:440-444 (maize);
Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van
Slogteren, et al., (1984) Nature (London) 311:763-764; Bowen, et
al., U.S. Pat. No. 5,736,369 (cereals); 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.
Chapman, et al., (Longman, New York), pp. 197-209 (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); 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 Biotechnology 14:745-750 (maize via
Agrobacterium tumefaciens), all of which are herein incorporated by
reference.
[0076] In specific embodiments, the male fertility polynucleotides
or expression cassettes disclosed herein can be provided to a plant
using a variety of transient transformation methods. Such transient
transformation methods include, but are not limited to, the
introduction of the male fertility polypeptide or variants and
fragments thereof directly into the plant or the introduction of a
male fertility transcript into the plant. Such methods include, for
example, microinjection or particle bombardment. See, for example,
Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et
al., (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl.
Acad. Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of
Cell Science 107:775-784, all of which are herein incorporated by
reference. Alternatively, the male fertility polynucleotide or
expression cassettes disclosed herein can be transiently
transformed into the plant using techniques known in the art. Such
techniques include viral vector system and the precipitation of the
polynucleotide in a manner that precludes subsequent release of the
DNA. Thus, the transcription from the particle-bound DNA can occur,
but the frequency with which it is released to become integrated
into the genome is greatly reduced. Such methods include the use of
particles coated with polyethylimine (PEI; Sigma #P3143).
[0077] In other embodiments, the male fertility polynucleotides or
expression cassettes disclosed herein may be introduced into plants
by contacting plants with a virus or viral nucleic acids.
Generally, such methods involve incorporating a nucleotide
construct of disclosed herein within a viral DNA or RNA molecule.
It is recognized that a male fertility sequence disclosed herein
may be initially synthesized as part of a viral polyprotein, which
later may be processed by proteolysis in vivo or in vitro to
produce the desired recombinant protein. Methods for introducing
polynucleotides into plants and expressing a protein encoded
therein, involving viral DNA or RNA molecules, are known in the
art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,
5,866,785, 5,589,367, 5,316,931 and Porta, et al., (1996) Molecular
Biotechnology 5:209-221, herein incorporated by reference.
[0078] Methods are known in the art for the targeted insertion of a
polynucleotide at a specific location in the plant genome. In one
embodiment, the insertion of the polynucleotide at a desired
genomic location is achieved using a site-specific recombination
system. See, for example, WO 1999/25821, WO 1999/25854, WO
1999/25840, WO 1999/25855 and WO 1999/25853, all of which are
herein incorporated by reference. Briefly, the polynucleotide
disclosed herein can be contained in transfer cassette flanked by
two non-identical recombination sites. The transfer cassette is
introduced into a plant having stably incorporated into its genome
a target site which is flanked by two non-identical recombination
sites that correspond to the sites of the transfer cassette. An
appropriate recombinase is provided and the transfer cassette is
integrated at the target site. The polynucleotide of interest is
thereby integrated at a specific chromosomal position in the plant
genome.
[0079] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants
may then be grown, and pollinated with either the same transformed
strain or different strains, and the resulting progeny having
desired expression of the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that
expression of the desired phenotypic characteristic is stably
maintained and inherited and then seeds harvested to ensure
expression of the desired phenotypic characteristic has been
achieved. In this manner, the present disclosure provides
transformed seed (also referred to as "transgenic seed") having a
male fertility polynucleotide disclosed herein, for example, an
expression cassette disclosed herein, stably incorporated into
their genome. Seed comprising any expression cassette disclosed
herein can be sorted based on size parameters, including but not
limited to, seed length, seed width, seed density or any
combination thereof.
[0080] The male fertility polynucleotides and expression cassettes
disclosed herein may be used for transformation of any plant
species, including, but not limited to, monocots and dicots.
Examples of plant species of interest include, but are not limited
to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.
juncea), alfalfa (Medicago sativa), rice (Oryza sativa), rye
(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare),
millet (e.g., pearl millet (Pennisetum glaucum), proso millet
(Panicum miliaceum), foxtail millet (Setaria italica), finger
millet (Eleusine coracana)), sunflower (Helianthus annuus),
safflower (Carthamus tinctorius), wheat (Triticum aestivum),
soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum
tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium
barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus),
cassaya (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos
nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.),
cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa
spp.), avocado (Persea americana), fig (Ficus casica), guava
(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),
papaya (Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, grasses and conifers.
[0081] In particular embodiments, wheat plants are used in the
methods and compositions disclosed herein. As used herein, the term
"wheat" refers to any species of the genus Triticum, including
progenitors thereof, as well as progeny thereof produced by crosses
with other species. Wheat includes "hexaploid wheat" which has
genome organization of AABBDD, comprised of 42 chromosomes, and
"tetraploid wheat" which has genome organization of AABB, comprised
of 28 chromosomes. Hexaploid wheat includes T. aestivum, T. spelta,
T. mocha, T. compactum, T. sphaerococcum, T. vavilovii and
interspecies cross thereof. Tetraploid wheat includes T. durum
(also referred to as durum wheat or Triticum turgidum ssp. durum),
T. dicoccoides, T. dicoccum, T. polonicum, and interspecies cross
thereof. In addition, the term "wheat" includes possible
progenitors of hexaploid or tetraploid Triticum sp. such as T.
uartu, T. monococcum or T. boeoticum for the A genome, Aegilops
speltoides for the B genome, and T. tauschii (also known as
Aegilops squarrosa or Aegilops tauschii) for the D genome. A wheat
cultivar for use in the present disclosure may belong to, but is
not limited to, any of the above-listed species. Also encompassed
are plants that are produced by conventional techniques using
Triticum sp. as a parent in a sexual cross with a non-Triticum
species, such as rye Secale cereale, including but not limited to
Triticale. In some embodiments, the wheat plant is suitable for
commercial production of grain, such as commercial varieties of
hexaploid wheat or durum wheat, having suitable agronomic
characteristics which are known to those skilled in the art.
[0082] Vegetables include tomatoes (Lycopersicon esculentum),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.) and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima) and chrysanthemum.
[0083] Conifers that may be employed in practicing the present
methods and compositions include, for example, pines such as
loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa
pine (Pinus ponderosa), lodgepole pine (Pinus contorta) and
Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii);
Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca);
redwood (Sequoia sempervirens); true firs such as silver fir (Abies
amabilis) and balsam fir (Abies balsamea) and cedars such as
Western red cedar (Thuja plicata) and Alaska yellow-cedar
(Chamaecyparis nootkatensis). In specific embodiments, plants
disclosed herein are crop plants (for example, corn, alfalfa,
sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum,
wheat, millet, tobacco, etc.). In other embodiments, corn and
soybean plants are optimal and in yet other embodiments corn plants
are optimal.
[0084] Other plants of interest include grain plants that provide
seeds of interest, oil-seed plants, and leguminous plants. Seeds of
interest include grain seeds, such as corn, wheat, barley, rice,
sorghum, rye, etc. Oil-seed plants include cotton, soybean,
safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
Leguminous plants include beans and peas. Beans include guar,
locust bean, fenugreek, soybean, garden beans, cowpea, mungbean,
lima bean, fava bean, lentils, chickpea, etc.
[0085] Typically, an intermediate host cell will be used in the
practice of the methods and compositions disclosed herein to
increase the copy number of the cloning vector. With an increased
copy number, the vector containing the nucleic acid of interest can
be isolated in significant quantities for introduction into the
desired plant cells. In one embodiment, plant promoters that do not
cause expression of the polypeptide in bacteria are employed.
[0086] 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 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.
[0087] The vector is selected to allow introduction 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 disclosed herein are available using Bacillus sp. and
Salmonella (Palva, et al., (1983) Gene 22:229-235); Mosbach, et
al., (1983) Nature 302:543-545).
[0088] In some embodiments, the expression cassette or male
fertility polynucleotides disclosed herein are maintained in a
hemizygous state in a plant. Hemizygosity is a genetic condition
existing when there is only one copy of a gene (or set of genes)
with no allelic counterpart on the sister chromosome. In certain
embodiments, the expression cassettes disclosed herein comprise a
first promoter operably linked to a male fertility polynucleotide
which is stacked with a male-gamete-disruptive polynucleotide
operably linked to a male tissue-preferred promoter and such
expression cassettes are introduced into a male sterile plant in a
hemizygous condition. When the male fertility polynucleotide is
expressed, the plant is able to successfully produce mature pollen
grains because the male fertility polynucleotide restores the plant
to a fertile condition. Given the hemizygous condition of the
expression cassette, only certain daughter cells will inherit the
expression cassette in the process of pollen grain formation. The
daughter cells that inherit the expression cassette containing the
male fertility polynucleotide will not develop into mature pollen
grains due to the male tissue-preferred expression of the stacked
encoded male-gamete-disruptive gene product. Those pollen grains
that do not inherit the expression cassette will continue to
develop into mature pollen grains and be functional, but will not
contain the male fertility polynucleotide of the expression
cassette and therefore will not transmit the male fertility
polynucleotide to progeny through pollen.
V. Modulating the Concentration and/or Activity of Male Fertility
Polypeptides
[0089] A method for modulating the concentration and/or activity of
the male fertility polypeptides disclosed herein in a plant is
provided. The term "influences" or "modulates", as used herein with
reference to the concentration and/or activity of the male
fertility polypeptides, refers to any increase or decrease in the
concentration and/or activity of the male fertility polypeptides
when compared to an appropriate control. In general, concentration
and/or activity of a male fertility polypeptide disclosed herein is
increased or decreased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80% or 90% relative to a native control plant, plant part
or cell. Modulation as disclosed herein may occur during and/or
subsequent to growth of the plant to the desired stage of
development. In specific embodiments, the male fertility
polypeptides disclosed herein are modulated in monocots,
particularly wheat.
[0090] A variety of methods can be employed to assay for modulation
in the concentration and/or activity of a male fertility
polypeptide. For instance, the expression level of the male
fertility polypeptide may be measured directly, for example, by
assaying for the level of the male fertility polypeptide in the
plant (i.e., Western or Northern blot) or indirectly, for example,
by assaying the male fertility activity of the male fertility
polypeptide in the plant. Methods for measuring the male fertility
activity are described elsewhere herein. In specific embodiments,
modulation of male fertility polypeptide concentration and/or
activity comprises the modulation (i.e., an increase or a decrease)
in the level of male fertility polypeptide in the plant. Methods to
measure the level and/or activity of male fertility polypeptides
are known in the art and are discussed elsewhere herein. In still
other embodiments, the level and/or activity of the male fertility
polypeptide is modulated in vegetative tissue, in reproductive
tissue or in both vegetative and reproductive tissue.
[0091] In one embodiment, the activity and/or concentration of the
male fertility polypeptide is increased by introducing the
polypeptide or the corresponding male fertility polynucleotide into
the plant. Subsequently, a plant having the introduced male
fertility sequence is selected using methods known to those of
skill in the art such as, but not limited to, Southern blot
analysis, DNA sequencing, PCR analysis or phenotypic analysis. In
certain embodiments, marker polynucleotides are introduced with the
male fertility polynucleotide to aid in selection of a plant having
or lacking the male fertility polynucleotide disclosed herein. A
plant or plant part altered or modified by the foregoing
embodiments is grown under plant forming conditions for a time
sufficient to modulate the concentration and/or activity of the
male fertility polypeptide in the plant. Plant forming conditions
are well known in the art.
[0092] As discussed elsewhere herein, many methods are known the
art for providing a polypeptide to a plant including, but not
limited to, direct introduction of the polypeptide into the plant,
or introducing into the plant (transiently or stably) a
polynucleotide construct encoding a male fertility polypeptide. It
is also recognized that the methods disclosed herein 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 male fertility polypeptide may be increased by
altering the gene encoding the male fertility 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 male fertility genes, where the mutations increase
expression of the male fertility gene or increase the activity of
the encoded male fertility polypeptide are provided.
[0093] In other embodiments, the concentration and/or activity of a
male fertility polypeptide is increased by introduction into a
plant of an expression cassette comprising a male fertility
polynucleotide (e.g. SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17) or
an active fragment or variant thereof, as disclosed elsewhere
herein. The male fertility polynucleotide may be operably linked to
promoter that is heterologous to the plant or native to the plant.
By increasing the concentration and/or activity of a male fertility
polypeptide in a plant, the male fertility of the plant is likewise
increased. Thus, the male fertility of a plant can be increased by
increasing the concentration and/or activity of a male fertility
polypeptide. For example, male fertility can be restored to a male
sterile plant by increasing the concentration and/or activity of a
male fertility polypeptide.
[0094] It is also recognized that the level and/or activity of the
polypeptide may be modulated by employing a polynucleotide that is
not capable of directing, in a transformed plant, the expression of
a protein or an RNA. For example, the polynucleotides disclosed
herein may be used to design polynucleotide constructs that can be
employed in methods for altering or mutating a genomic nucleotide
sequence in an organism. Such polynucleotide constructs include,
but are not limited to, RNA:DNA vectors, RNA:DNA mutational
vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides,
self-complementary RNA:DNA oligonucleotides and recombinogenic
oligonucleobases. Such nucleotide constructs and methods of use are
known in the art. See, U.S. Pat. Nos. 5,565,350; 5,731,181;
5,756,325; 5,760,012; 5,795,972 and 5,871,984, all of which are
herein incorporated by reference. See also, WO 1998/49350, WO
1999/07865, WO 1999/25821 and Beetham, et al., (1999) Proc. Natl.
Acad. Sci. USA 96:8774-8778, herein incorporated by reference. It
is therefore recognized that methods disclosed herein do not depend
on the incorporation of the entire polynucleotide into the genome,
only that the plant or cell thereof is altered as a result of the
introduction of the polynucleotide into a cell.
[0095] In one embodiment, the genome may be altered following the
introduction of the polynucleotide into a cell. For example, the
polynucleotide, or any part thereof, may incorporate into the
genome of the plant. Alterations to the genome disclosed herein
include, but are not limited to, additions, deletions and
substitutions of nucleotides into the genome. While the methods
disclosed herein do not depend on additions, deletions and
substitutions of any particular number of nucleotides, it is
recognized that such additions, deletions or substitutions
comprises at least one nucleotide.
TABLE-US-00001 TABLE 1 Summary of SEQ ID NOS SEQ ID: Description 1
Wheat (T. urartu, A genome) Ms22 polynucleotide 2 Wheat (T. urartu,
A genome) Ms22 polypeptide 3 Wheat (Ae. Speltoides, B genome) Ms22
polynucleotide 4 Wheat (Ae. Speltoides, B genome) Ms22 polypeptide
5 Wheat (Ae. Tauschii, D genome) Ms22 polynucleotide 6 Wheat (Ae.
Tauschii, D genome) Ms22 polypeptide 7 Wheat (T. urartu, A genome)
Ms26 polynucleotide 8 Wheat (T. urartu, A genome) Ms26 polypeptide
9 Wheat (Ae. Speltoides, B genome) Ms26 polynucleotide 10 Wheat
(Ae. Speltoides, B genome) Ms26 polypeptide 11 Wheat (Ae. Tauschii,
D genome) Ms26 polynucleotide 12 Wheat (Ae. Tauschii, D genome)
Ms26 polypeptide 13 Wheat (T. urartu, A genome) Ms45 polynucleotide
14 Wheat (T. urartu, A genome) Ms45 polypeptide 15 Wheat (Ae.
Speltoides, B genome) Ms45 polynucleotide 16 Wheat (Ae. Speltoides,
B genome) Ms45 polypeptide 17 Wheat (Ae. Tauschii, D genome) Ms45
polynucleotide 18 Wheat (Ae. Tauschii, D genome) Ms45 polypeptide
19 Heme-binding domain of Ms26: FxxGxRxCxG 20 Dioxygen binding
domain A of Ms26 A/GGXD/ETT/S
[0096] The article "a" and "an" are used herein to refer to one or
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "an element" means one or more
element.
[0097] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this disclosure pertains. 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 to be incorporated by
reference.
[0098] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
EXPERIMENTAL
Example 1
Identification of Male Fertility Polynucleotides in Wheat
[0099] Male-fertility polynucleotides disclosed herein may be
identified using bioinformatic approaches. As an example, sequences
putatively representing male-fertility genes in wheat are initially
identified by an in silico search of proprietary databases using
known fertility genes from other species, such as maize. Candidate
ESTs are selected based on protein-level homology to the reference
sequences and consideration of the library from which the candidate
sequence originated, e.g., representing expression in male
reproductive tissue.
[0100] Based on the candidate EST sequences, primers are created
and used to screen a proprietary wheat BAC library. Super-pools
identified are further screened with appropriate primers to
identify specific BAC clones comprising the ESTs. Touchdown PCR may
be performed (GeneAmp.RTM. PCR System 9700, Applied Biosystems),
using the following cycling parameters: 94.degree. C. for 3 min
(one cycle), 94.degree. C. for 1 min, 55.degree. C. for 1 min and
72.degree. C. for 1 min 30s, (35 cycles), 72.degree. C. for 7 min
and termination at 4.degree. C. Pfu Ultra Hotstart.TM. DNA
polymerase (Stratagene) may be preferred for its very low average
error rate (less than 0.5% per 500-bp fragment amplified).
[0101] Wheat insert DNA isolated from the BAC clones is digested
for Southern blot confirmation using a candidate EST clone as a
probe. BAC fragments are subcloned into pBluescript.RTM.
(Stratagene Inc., La Jolla, Calif.). White colonies are grown in LB
medium and transferred onto a membrane using a dot-blot procedure.
After denaturation the membrane is probed with a candidate EST
clone. Positive clones are identified and sequenced.
Example 2
Comparison of Wheat Male Fertility Polynucleotide With Known
Sequences
TABLE-US-00002 [0102] TABLE 2 Global Identity of Wheat, Maize, and
Rice Ms22 Polynucleotides and Polypeptides ##STR00001##
(Polynucleotide result is listed in shaded box; polypeptide result
is listed in unshaded box.)
TABLE-US-00003 TABLE 3 Global Identity of Wheat, Maize, and Rice
Ms26 Polynucleotides and Polypeptides ##STR00002## (Polynucleotide
result is listed in shaded box; polypeptide result is listed in
unshaded box.)
TABLE-US-00004 TABLE 3 Global Identity of Wheat, Maize, and Rice
Ms45 Polynucleotides and Polypeptides ##STR00003## (Polynucleotide
result is listed in shaded box; polypeptide result is listed in
unshaded box.)
Example 3
Wheat Transformation
[0103] Wheat transformation protocols are available to one of skill
in the art. See, for example, He, et al., (2010) J. Exp. Botany
61(6):1567-1581; Wu, et al., (2008) Transgenic Res. 17:425-436;
Nehra, et al., (1994) Plant J. 5(2):285-297; Rasco-Gaunt, et al.,
(2001) J. Exp. Botany 52(357):865-874; Razzaq, et al., (2011)
African J. Biotech. 10(5):740-750.
Sequence CWU 1
1
201414DNATriticum urartumisc_feature(1)..(411)Ms22 1atgttgagga
tgcagcagca ggtggagggc gtggtgggcg gcggcatcgt ggccgaggcg 60gaggaggcgg
cggtgtacga gcgggtggct cgcatggcca gcggcaacgc ggtggtcgtc
120ttcagcgccm gcggctgctg catgtgccac gtcgtcaagc gcctcctgct
tggcctgggg 180gtcggcccca ccgtctacga gttggaccag atgggcggcg
ccgggcgaga gatccaggcg 240gcgctggcgc agctgctgcc ccccggaccc
ggcgccggcc accaccagca gccgccagtg 300cccgtggtgt tcgtcggcgg
gaggctcctg ggcggcgtgg agaaggtcat ggcgtgccac 360atcaacggca
cgctcgtccc gctcctcaag gacgccggcg cgctctggct ctga 4142137PRTTriticum
urartumisc_feature(1)..(137)Xaa = any amino acid 2Met Leu Arg Met
Gln Gln Gln Val Glu Gly Val Val Gly Gly Gly Ile 1 5 10 15 Val Ala
Glu Ala Glu Glu Ala Ala Val Tyr Glu Arg Val Ala Arg Met 20 25 30
Ala Ser Gly Asn Ala Val Val Val Phe Ser Ala Xaa Gly Cys Cys Met 35
40 45 Cys His Val Val Lys Arg Leu Leu Leu Gly Leu Gly Val Gly Pro
Thr 50 55 60 Val Tyr Glu Leu Asp Gln Met Gly Gly Ala Gly Arg Glu
Ile Gln Ala 65 70 75 80 Ala Leu Ala Gln Leu Leu Pro Pro Gly Pro Gly
Ala Gly His His Gln 85 90 95 Gln Pro Pro Val Pro Val Val Phe Val
Gly Gly Arg Leu Leu Gly Gly 100 105 110 Val Glu Lys Val Met Ala Cys
His Ile Asn Gly Thr Leu Val Pro Leu 115 120 125 Leu Lys Asp Ala Gly
Ala Leu Trp Leu 130 135 3414DNAAegilops speltoides 3atgttgagga
tgcagcagca ggtggagggc gtggtgggcg gcggcatcgt ggcggaggcg 60gaggaggcgg
ccgtgtacga gcgggtggct cgcatggcca gcggcaacgc ggtggtcgtc
120ttcagcgcca gcggctgctg catgtgccac gtcgtcaagc gcctcctgct
tggcctggga 180gtcggcccca ccgtgtacga gttggaccag atgggcggcg
ccgggcggga gatccaggcg 240gccctggcgc agctgctgcc ccccggaccc
ggcgccggcc accaccagca gccgccggtg 300cccgtggtgt tcgtyggcgg
gaggctcctg ggcggcgtgg agaaggtgat ggcgtgccac 360atcaacggca
cgctcgtccc gctcctcaag gacgccggcg cgctctggct ctga 4144137PRTAegilops
speltoidesmisc_featureXaa = any amino acid 4Met Leu Arg Met Gln Gln
Gln Val Glu Gly Val Val Gly Gly Gly Ile 1 5 10 15 Val Ala Glu Ala
Glu Glu Ala Ala Val Tyr Glu Arg Val Ala Arg Met 20 25 30 Ala Ser
Gly Asn Ala Val Val Val Phe Ser Ala Ser Gly Cys Cys Met 35 40 45
Cys His Val Val Lys Arg Leu Leu Leu Gly Leu Gly Val Gly Pro Thr 50
55 60 Val Tyr Glu Leu Asp Gln Met Gly Gly Ala Gly Arg Glu Ile Gln
Ala 65 70 75 80 Ala Leu Ala Gln Leu Leu Pro Pro Gly Pro Gly Ala Gly
His His Gln 85 90 95 Gln Pro Pro Val Pro Val Val Phe Xaa Gly Gly
Arg Leu Leu Gly Gly 100 105 110 Val Glu Lys Val Met Ala Cys His Ile
Asn Gly Thr Leu Val Pro Leu 115 120 125 Leu Lys Asp Ala Gly Ala Leu
Trp Leu 130 135 5414DNAAegilopsm tauschii 5atgttgagga tgcagcagca
ggtggagggc gtggtgggcg gcggcatcat ggcggaggcg 60gaggaggcgg cggtgtacga
gcgggtggct cgcatggcca gcggcaacgc ggtggtcgtc 120ttcagcgcca
gcggctgctg catgtgccac gtcgtcaagc gcctcctgct tggcctgggg
180gtcggcccca ccgtctacga gttggaccag atgggcggcg ccgggcgaga
gatccaggcg 240gcgctggcgc agctgctgcc ccccggaccc ggcgccggcc
accaccagca gccgccagtg 300cccgtggtgt tcgtcggcgg gaggctcctg
ggcggcgtgg agaaggtgat ggcgtgccac 360atcaacggca cgctcgtccc
gctcctcaag gacgccggcg cgctctggct ctga 4146137PRTAegilops tauschii
6Met Leu Arg Met Gln Gln Gln Val Glu Gly Val Val Gly Gly Gly Ile 1
5 10 15 Met Ala Glu Ala Glu Glu Ala Ala Val Tyr Glu Arg Val Ala Arg
Met 20 25 30 Ala Ser Gly Asn Ala Val Val Val Phe Ser Ala Ser Gly
Cys Cys Met 35 40 45 Cys His Val Val Lys Arg Leu Leu Leu Gly Leu
Gly Val Gly Pro Thr 50 55 60 Val Tyr Glu Leu Asp Gln Met Gly Gly
Ala Gly Arg Glu Ile Gln Ala 65 70 75 80 Ala Leu Ala Gln Leu Leu Pro
Pro Gly Pro Gly Ala Gly His His Gln 85 90 95 Gln Pro Pro Val Pro
Val Val Phe Val Gly Gly Arg Leu Leu Gly Gly 100 105 110 Val Glu Lys
Val Met Ala Cys His Ile Asn Gly Thr Leu Val Pro Leu 115 120 125 Leu
Lys Asp Ala Gly Ala Leu Trp Leu 130 135 71934DNATriticum urartu
7atggaggaag ctcacggcgg catgccgtcg acgacgacgg cgttcttccc gctggcaggg
60ctccacaagt tcatggccat cttcctcgtg ttcctctcgt ggatcttggt ccactggtgg
120agcctgagga agcagaaggg gccgaggtca tggccggtca tcggcgcgac
gctggagcag 180ctgaggaact actaccggat gcacgactgg ctcgtggagt
acctgtccaa gcaccggacg 240gtcaccgtcg acatgccctt cacctcctac
acctacatcg ccgaccccgt gaacgtcgag 300catgtgctca agaccaattt
caacaattac cccaaggtga aacaatcctc gagatgtcag 360acaaggttca
gtaatcggta ctgacagtgt tacaaatgtc tgaaatctga aattgtatgt
420ctagggggag gtgtacaggt cctacatgga cgtgctgctc ggcgacggca
tattcaacgc 480cgacggcgag ctctggagga agcagaggaa gacggcgagc
ttcgagttcg cttccaagaa 540cctgagagac ttcagcacga tcgtgttcag
ggagtactcg ctgaagctgt ccagcatcct 600gagccaggct tgcaaggccg
gcaaagtcgt ggacatgcag gcaactgaac tcattccctt 660ggtcatctga
acgttgattt cttggacaaa atttcaagat tctgacgcga gcggacgaat
720tcaggagctg tacatgagga tgacgctgga ctcgatctgc aaggtcgggt
tcggggtcga 780gatcggcacg ctgtcgccgg agctgccgga gaacagcttc
gcgcaggcgt tcgacgccgc 840caacatcatc gtgacgctgc ggttcatcga
cccgctgtgg cgcgtgaaga agttcctgca 900cgtcggctcg gaggcgctgc
tggagcagag catcaagctc gtcgacgagt tcacctacag 960cgtcatccgc
cggcgcaagg ccgagatcgt gcaggcccgg gccagcggca agcaggagaa
1020ggtgcgtgcg tgatcatcgt cattcgtcaa gctccggatc gctggtttgt
gtagtaggtg 1080ccattgatca ctgacacgtt aactgggtgc gcagatcaag
cacgacatac tgtcgcggtt 1140catcgagctg ggcgaggccg gcggcgacga
cggcggcagc ctgttcgggg acgacaaggg 1200cctccgcgac gtggtgctca
acttcgtgat cgccgggcgg gacaccacgg ccacgacgct 1260gtcctggttc
acctacatgg ccatgacgca cccggccgtg gccgagaagc tccgccgcga
1320gctggccgcc ttcgaggcgg atcgcgcccg cgaggagggc gtcgctctgg
tcccctgcag 1380cgacggcgag ggcgccgacg aggccttcgc cgcccgcgtg
gcgcagttcg cggggctcct 1440gagctacgac gggctcggga agctggtgta
cctccacgcg tgcgtgacgg agacgctgcg 1500gctgtacccg gcggtgccgc
aggaccccaa gggcatcgcg gaggacgacg tgctcccgga 1560cggcaccaag
gtgcgcgccg gcgggatggt gacgtacgtg ccctactcca tggggcggat
1620ggagtataac tggggccccg acgccgccag cttccggccg gagcggtgga
tcggcgacga 1680cggcgcgttc cgcaacgcgt cgccgttcaa gttcacggcg
ttccaggcgg ggccgcggat 1740ctgcctcggc aaggactcgg cgtacctgca
gatgaagatg gcgctggcca tactgtgcag 1800gttcttcagg ttcgagctcg
tggagggcca ccccgtcaag taccgcatga tgaccatcct 1860ctccatggcg
cacggcctca aggtccgcgt ctccagggcg ccgctcgcct gatcttgatc
1920tgggttccgg cgag 19348548PRTTriticum urartu 8Met Glu Glu Ala His
Gly Gly Met Pro Ser Thr Thr Thr Ala Phe Phe 1 5 10 15 Pro Leu Ala
Gly Leu His Lys Phe Met Ala Ile Phe Leu Val Phe Leu 20 25 30 Ser
Trp Ile Leu Val His Trp Trp Ser Leu Arg Lys Gln Lys Gly Pro 35 40
45 Arg Ser Trp Pro Val Ile Gly Ala Thr Leu Glu Gln Leu Arg Asn Tyr
50 55 60 Tyr Arg Met His Asp Trp Leu Val Glu Tyr Leu Ser Lys His
Arg Thr 65 70 75 80 Val Thr Val Asp Met Pro Phe Thr Ser Tyr Thr Tyr
Ile Ala Asp Pro 85 90 95 Val Asn Val Glu His Val Leu Lys Thr Asn
Phe Asn Asn Tyr Pro Lys 100 105 110 Gly Glu Val Tyr Arg Ser Tyr Met
Asp Val Leu Leu Gly Asp Gly Ile 115 120 125 Phe Asn Ala Asp Gly Glu
Leu Trp Arg Lys Gln Arg Lys Thr Ala Ser 130 135 140 Phe Glu Phe Ala
Ser Lys Asn Leu Arg Asp Phe Ser Thr Ile Val Phe 145 150 155 160 Arg
Glu Tyr Ser Leu Lys Leu Ser Ser Ile Leu Ser Gln Ala Cys Lys 165 170
175 Ala Gly Lys Val Val Asp Met Gln Glu Leu Tyr Met Arg Met Thr Leu
180 185 190 Asp Ser Ile Cys Lys Val Gly Phe Gly Val Glu Ile Gly Thr
Leu Ser 195 200 205 Pro Glu Leu Pro Glu Asn Ser Phe Ala Gln Ala Phe
Asp Ala Ala Asn 210 215 220 Ile Ile Val Thr Leu Arg Phe Ile Asp Pro
Leu Trp Arg Val Lys Lys 225 230 235 240 Phe Leu His Val Gly Ser Glu
Ala Leu Leu Glu Gln Ser Ile Lys Leu 245 250 255 Val Asp Glu Phe Thr
Tyr Ser Val Ile Arg Arg Arg Lys Ala Glu Ile 260 265 270 Val Gln Ala
Arg Ala Ser Gly Lys Gln Glu Lys Ile Lys His Asp Ile 275 280 285 Leu
Ser Arg Phe Ile Glu Leu Gly Glu Ala Gly Gly Asp Asp Gly Gly 290 295
300 Ser Leu Phe Gly Asp Asp Lys Gly Leu Arg Asp Val Val Leu Asn Phe
305 310 315 320 Val Ile Ala Gly Arg Asp Thr Thr Ala Thr Thr Leu Ser
Trp Phe Thr 325 330 335 Tyr Met Ala Met Thr His Pro Ala Val Ala Glu
Lys Leu Arg Arg Glu 340 345 350 Leu Ala Ala Phe Glu Ala Asp Arg Ala
Arg Glu Glu Gly Val Ala Leu 355 360 365 Val Pro Cys Ser Asp Gly Glu
Gly Ala Asp Glu Ala Phe Ala Ala Arg 370 375 380 Val Ala Gln Phe Ala
Gly Leu Leu Ser Tyr Asp Gly Leu Gly Lys Leu 385 390 395 400 Val Tyr
Leu His Ala Cys Val Thr Glu Thr Leu Arg Leu Tyr Pro Ala 405 410 415
Val Pro Gln Asp Pro Lys Gly Ile Ala Glu Asp Asp Val Leu Pro Asp 420
425 430 Gly Thr Lys Val Arg Ala Gly Gly Met Val Thr Tyr Val Pro Tyr
Ser 435 440 445 Met Gly Arg Met Glu Tyr Asn Trp Gly Pro Asp Ala Ala
Ser Phe Arg 450 455 460 Pro Glu Arg Trp Ile Gly Asp Asp Gly Ala Phe
Arg Asn Ala Ser Pro 465 470 475 480 Phe Lys Phe Thr Ala Phe Gln Ala
Gly Pro Arg Ile Cys Leu Gly Lys 485 490 495 Asp Ser Ala Tyr Leu Gln
Met Lys Met Ala Leu Ala Ile Leu Cys Arg 500 505 510 Phe Phe Arg Phe
Glu Leu Val Glu Gly His Pro Val Lys Tyr Arg Met 515 520 525 Met Thr
Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Arg 530 535 540
Ala Pro Leu Ala 545 91929DNAAegilops speltoides 9atggaggaag
ctcaccttgg catgccgtcg acgacggcct tcttcccgct ggcagggctc 60cacaagttca
tggccatctt cctcgtgttc ctctcgtgga tcctggtcca ctggtggagc
120ctgaggaagc agaaggggcc gaggtcatgg ccggtcatcg gcgccacgct
ggagcagctg 180aggaactact accggatgca cgactggctc gtggagtacc
tgtccaagca ccggacggtc 240accgtcgaca tgcccttcac ctcctacacc
tacatcgccg acccggtgaa cgtcgagcat 300gtgctcaaga ccaacttcaa
caattacccc aaggtgaaac aatcctcgag atgtcagtca 360aggttcggta
taatcggtac tgacagtgtt acaaatgtct gaaatctgaa attgtgtgtg
420tagggggagg tgtacaggtc ctacatggac gtgctgctcg gcgacggcat
attcaacgcc 480gacggcgagc tctggaggaa gcagaggaag acggcgagct
tcgagttcgc ttccaagaac 540ctgagagact tcagcacgat cgtgttccgg
gagtactccc tgaagctgtc cagcatcctg 600agccaggctt gcaaggccgg
caaagttgtg gacatgcagg taactgaact ctttcccttg 660gtcatctgaa
cgttgatttc ttggacaaaa tttcaagatt gtgacgcgag cgagccaatt
720caggagctgt acatgaggat gacgctggac tcgatctgca aggtggggtt
cggggtggag 780atcggcacgc tgtcgccgga gctgccggag aacagcttcg
cgcaggcctt cgacgccgcc 840aacatcatcg tgacgctgcg gttcatcgac
ccgctgtggc gcgtgaagaa gttcctgcac 900gtcggctcgg aggcgctgct
ggagcagagc atcaagctcg tcgacgagtt cacctacagc 960gtcatccgcc
ggcgcaaggc cgagatcgtg caggcccggg ccagcggcaa gcaggagaag
1020gtgcgtacgc ggtcatcgtc attcgtcaag ctcccgatcg ctggtttgtg
cagatgccat 1080tgatcactga cacattaact gggcgcgcag atcaagcacg
acatactgtc gcggttcatc 1140gagctgggcg aggccggcgg cgacgacggc
ggcagcctgt tcggggacga caagggcctc 1200cgcgacgtgg tgctcaactt
cgtgatcgcc gggcgggaca ccacggccac gacgctctcc 1260tggttcacct
acatggccat gacgcacccg gacgtggccg agaagctccg ccgcgagctg
1320gccgccttcg agtccgagcg cgcccgcgag gagggcgtcg ctctggtccc
ctgcagcgac 1380ggcgagggct ccgacgaggc cttcgccgcc cgcgtggcgc
agttcgcggg gctcctgagc 1440tacgacgggc tcgggaagct ggtgtacctc
cacgcgtgcg tgacggagac gctgcgcctg 1500tacccggcgg tgccgcagga
tcccaagggc atcgcggagg acgacgtgct cccggacggc 1560accaaggtgc
gcgccggcgg gatggtgacg tacgtgccct actccatggg gcggatggag
1620tacaactggg gccccgacgc cgccagcttc cggccggagc ggtggatcgg
cgacgatggc 1680gccttccgca acgcgtcgcc gttcaagttc acggcgttcc
aggcggggcc gcggatctgc 1740ctgggcaagg actcggcgta cctgcagatg
aagatggcgc tggccatcct gtgcaggttc 1800ttcaggttcg agctcgtgga
gggccacccc gtcaagtacc gcatgatgac catcctctcc 1860atggcgcacg
gcctcaaggt ccgcgtctcc agggcgccgc tcgcctgatc ttgatctggt
1920tccggcgag 192910547PRTAegilops speltoides 10Met Glu Glu Ala His
Leu Gly Met Pro Ser Thr Thr Ala Phe Phe Pro 1 5 10 15 Leu Ala Gly
Leu His Lys Phe Met Ala Ile Phe Leu Val Phe Leu Ser 20 25 30 Trp
Ile Leu Val His Trp Trp Ser Leu Arg Lys Gln Lys Gly Pro Arg 35 40
45 Ser Trp Pro Val Ile Gly Ala Thr Leu Glu Gln Leu Arg Asn Tyr Tyr
50 55 60 Arg Met His Asp Trp Leu Val Glu Tyr Leu Ser Lys His Arg
Thr Val 65 70 75 80 Thr Val Asp Met Pro Phe Thr Ser Tyr Thr Tyr Ile
Ala Asp Pro Val 85 90 95 Asn Val Glu His Val Leu Lys Thr Asn Phe
Asn Asn Tyr Pro Lys Gly 100 105 110 Glu Val Tyr Arg Ser Tyr Met Asp
Val Leu Leu Gly Asp Gly Ile Phe 115 120 125 Asn Ala Asp Gly Glu Leu
Trp Arg Lys Gln Arg Lys Thr Ala Ser Phe 130 135 140 Glu Phe Ala Ser
Lys Asn Leu Arg Asp Phe Ser Thr Ile Val Phe Arg 145 150 155 160 Glu
Tyr Ser Leu Lys Leu Ser Ser Ile Leu Ser Gln Ala Cys Lys Ala 165 170
175 Gly Lys Val Val Asp Met Gln Glu Leu Tyr Met Arg Met Thr Leu Asp
180 185 190 Ser Ile Cys Lys Val Gly Phe Gly Val Glu Ile Gly Thr Leu
Ser Pro 195 200 205 Glu Leu Pro Glu Asn Ser Phe Ala Gln Ala Phe Asp
Ala Ala Asn Ile 210 215 220 Ile Val Thr Leu Arg Phe Ile Asp Pro Leu
Trp Arg Val Lys Lys Phe 225 230 235 240 Leu His Val Gly Ser Glu Ala
Leu Leu Glu Gln Ser Ile Lys Leu Val 245 250 255 Asp Glu Phe Thr Tyr
Ser Val Ile Arg Arg Arg Lys Ala Glu Ile Val 260 265 270 Gln Ala Arg
Ala Ser Gly Lys Gln Glu Lys Ile Lys His Asp Ile Leu 275 280 285 Ser
Arg Phe Ile Glu Leu Gly Glu Ala Gly Gly Asp Asp Gly Gly Ser 290 295
300 Leu Phe Gly Asp Asp Lys Gly Leu Arg Asp Val Val Leu Asn Phe Val
305 310 315 320 Ile Ala Gly Arg Asp Thr Thr Ala Thr Thr Leu Ser Trp
Phe Thr Tyr 325 330 335 Met Ala Met Thr His Pro Asp Val Ala Glu Lys
Leu Arg Arg Glu Leu 340 345 350 Ala Ala Phe Glu Ser Glu Arg Ala Arg
Glu Glu Gly Val Ala Leu Val 355 360 365 Pro Cys Ser Asp Gly Glu Gly
Ser Asp Glu Ala Phe Ala Ala Arg Val 370 375 380 Ala Gln Phe Ala Gly
Leu Leu Ser Tyr Asp Gly Leu Gly Lys Leu Val 385 390 395 400 Tyr Leu
His Ala Cys Val Thr Glu Thr Leu Arg Leu Tyr Pro Ala Val 405 410 415
Pro Gln Asp Pro Lys Gly Ile Ala Glu Asp Asp Val Leu Pro Asp Gly 420
425 430 Thr Lys Val Arg Ala Gly Gly Met Val Thr Tyr Val Pro Tyr Ser
Met 435 440 445 Gly Arg Met Glu Tyr Asn Trp Gly Pro Asp Ala Ala Ser
Phe Arg Pro 450 455 460 Glu Arg Trp Ile Gly Asp Asp Gly Ala Phe Arg
Asn Ala Ser Pro Phe 465 470 475 480 Lys Phe Thr Ala Phe Gln Ala Gly
Pro Arg Ile Cys Leu Gly Lys Asp 485 490 495 Ser Ala Tyr Leu
Gln Met Lys Met Ala Leu Ala Ile Leu Cys Arg Phe 500 505 510 Phe Arg
Phe Glu Leu Val Glu Gly His Pro Val Lys Tyr Arg Met Met 515 520 525
Thr Ile Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Arg Ala 530
535 540 Pro Leu Ala 545 111930DNAAegilops tauschii 11atggaggaag
ctcacggcgg catgccgtcg acgacggcct tcttcccgct ggcagggctc 60cacaagttca
tggccatctt cctcgtgttc ctctcgtgga tcttggtcca ctggtggagc
120ctgaggaagc agaaggggcc gaggtcatgg ccggtcatcg gcgcgacgct
ggagcagctg 180aggaactact accggatgca cgactggctc gtggagtacc
tgtccaagca ccggacggtg 240accgtcgaca tgcccttcac ctcctacacc
tacatcgccg acccggtgaa cgtcgagcat 300gtgctcaaga ccaacttcaa
caattacccc aaggtgaaac aatcctcgag atgtcagtaa 360aggttcagta
taatcggtac tgacagtgtt acaaatgtct gaaatctgaa attgtatgtg
420tagggggagg tgtacaggtc ctacatggac gtgctgctcg gcgacggcat
attcaacgcc 480gacggcgagc tctggaggaa gcagaggaag acggcgagct
tcgagttcgc ttccaagaac 540ttgagagact tcagcacgat cgtgttcagg
gagtactccc tgaagctgtc cagcatactg 600agccaggctt gcaaggccgg
caaagttgtg gacatgcagg taactgaact cattcccttg 660gtcatctgaa
cgttgatttc ttggacaaaa tttcaagatt ctgacgcgag cgagcgaatt
720caggagctgt atatgaggat gacgctggac tcgatctgca aagtggggtt
cggagtcgag 780atcggcacgc tgtcgccgga gctgccggag aacagcttcg
cgcaggcgtt cgacgccgcc 840aacatcatcg tgacgctgcg gttcatcgac
ccgctgtggc gcgtgaagaa gttcctgcac 900gtcggctcgg aggcgctgct
ggagcagagc atcaagctcg tcgacgagtt cacctacagc 960gtcatccgcc
ggcgcaaggc cgagatcgtg caggcccggg ccagcggcaa gcaggagaag
1020gtgcgtgcgt ggtcatcgtc attcgtcaag ctcccggtcg ctggtttgtg
tagatgccat 1080ggatcactga cacactaact gggcgcgcag atcaagcacg
acatactgtc gcggttcatc 1140gagctgggcg aggccggcgg cgacgacggc
ggcagtctgt tcggggacga caagggcctc 1200cgcgacgtgg tgctcaactt
cgtgatcgcc gggcgggaca ccacggccac gacgctgtcc 1260tggttcacct
acatggccat gacgcacccg gacgtggccg agaagctccg ccgcgagctg
1320gccgccttcg aggcggagcg cgcccgcgag gatggcgtcg ctctggtccc
ctgcggcgac 1380ggcgagggct ccgacgaggc cttcgctgcc cgcgtggcgc
agttcgcggg gttcctgagc 1440tacgacggcc tcgggaagct ggtgtacctc
cacgcgtgcg tgacggagac gctgcgcctg 1500tacccggcgg tgccgcagga
ccccaagggc atcgcggagg acgacgtgct cccggacggc 1560accaaggtgc
gcgccggcgg gatggtgacg tacgtgccct actccatggg gcggatggag
1620tacaactggg gccccgacgc cgccagcttc cggccggagc ggtggatcgg
cgacgacggc 1680gccttccgca acgcgtcgcc gttcaagttc acggcgttcc
aggcggggcc gcggatttgc 1740ctcggcaagg actcggcgta cctgcagatg
aagatggcgc tggcaatcct gtgcaggttc 1800ttcaggttcg agctcgtgga
gggccacccc gtcaagtacc gcatgatgac catcctctcc 1860atggcgcacg
gcctcaaggt ccgcgtctcc agggcgccgc tcgcctgatc ttgatctggt
1920tccggcgagg 193012547PRTAegilops tauschii 12Met Glu Glu Ala His
Gly Gly Met Pro Ser Thr Thr Ala Phe Phe Pro 1 5 10 15 Leu Ala Gly
Leu His Lys Phe Met Ala Ile Phe Leu Val Phe Leu Ser 20 25 30 Trp
Ile Leu Val His Trp Trp Ser Leu Arg Lys Gln Lys Gly Pro Arg 35 40
45 Ser Trp Pro Val Ile Gly Ala Thr Leu Glu Gln Leu Arg Asn Tyr Tyr
50 55 60 Arg Met His Asp Trp Leu Val Glu Tyr Leu Ser Lys His Arg
Thr Val 65 70 75 80 Thr Val Asp Met Pro Phe Thr Ser Tyr Thr Tyr Ile
Ala Asp Pro Val 85 90 95 Asn Val Glu His Val Leu Lys Thr Asn Phe
Asn Asn Tyr Pro Lys Gly 100 105 110 Glu Val Tyr Arg Ser Tyr Met Asp
Val Leu Leu Gly Asp Gly Ile Phe 115 120 125 Asn Ala Asp Gly Glu Leu
Trp Arg Lys Gln Arg Lys Thr Ala Ser Phe 130 135 140 Glu Phe Ala Ser
Lys Asn Leu Arg Asp Phe Ser Thr Ile Val Phe Arg 145 150 155 160 Glu
Tyr Ser Leu Lys Leu Ser Ser Ile Leu Ser Gln Ala Cys Lys Ala 165 170
175 Gly Lys Val Val Asp Met Gln Glu Leu Tyr Met Arg Met Thr Leu Asp
180 185 190 Ser Ile Cys Lys Val Gly Phe Gly Val Glu Ile Gly Thr Leu
Ser Pro 195 200 205 Glu Leu Pro Glu Asn Ser Phe Ala Gln Ala Phe Asp
Ala Ala Asn Ile 210 215 220 Ile Val Thr Leu Arg Phe Ile Asp Pro Leu
Trp Arg Val Lys Lys Phe 225 230 235 240 Leu His Val Gly Ser Glu Ala
Leu Leu Glu Gln Ser Ile Lys Leu Val 245 250 255 Asp Glu Phe Thr Tyr
Ser Val Ile Arg Arg Arg Lys Ala Glu Ile Val 260 265 270 Gln Ala Arg
Ala Ser Gly Lys Gln Glu Lys Ile Lys His Asp Ile Leu 275 280 285 Ser
Arg Phe Ile Glu Leu Gly Glu Ala Gly Gly Asp Asp Gly Gly Ser 290 295
300 Leu Phe Gly Asp Asp Lys Gly Leu Arg Asp Val Val Leu Asn Phe Val
305 310 315 320 Ile Ala Gly Arg Asp Thr Thr Ala Thr Thr Leu Ser Trp
Phe Thr Tyr 325 330 335 Met Ala Met Thr His Pro Asp Val Ala Glu Lys
Leu Arg Arg Glu Leu 340 345 350 Ala Ala Phe Glu Ala Glu Arg Ala Arg
Glu Asp Gly Val Ala Leu Val 355 360 365 Pro Cys Gly Asp Gly Glu Gly
Ser Asp Glu Ala Phe Ala Ala Arg Val 370 375 380 Ala Gln Phe Ala Gly
Phe Leu Ser Tyr Asp Gly Leu Gly Lys Leu Val 385 390 395 400 Tyr Leu
His Ala Cys Val Thr Glu Thr Leu Arg Leu Tyr Pro Ala Val 405 410 415
Pro Gln Asp Pro Lys Gly Ile Ala Glu Asp Asp Val Leu Pro Asp Gly 420
425 430 Thr Lys Val Arg Ala Gly Gly Met Val Thr Tyr Val Pro Tyr Ser
Met 435 440 445 Gly Arg Met Glu Tyr Asn Trp Gly Pro Asp Ala Ala Ser
Phe Arg Pro 450 455 460 Glu Arg Trp Ile Gly Asp Asp Gly Ala Phe Arg
Asn Ala Ser Pro Phe 465 470 475 480 Lys Phe Thr Ala Phe Gln Ala Gly
Pro Arg Ile Cys Leu Gly Lys Asp 485 490 495 Ser Ala Tyr Leu Gln Met
Lys Met Ala Leu Ala Ile Leu Cys Arg Phe 500 505 510 Phe Arg Phe Glu
Leu Val Glu Gly His Pro Val Lys Tyr Arg Met Met 515 520 525 Thr Ile
Leu Ser Met Ala His Gly Leu Lys Val Arg Val Ser Arg Ala 530 535 540
Pro Leu Ala 545 131581DNATriticum urartu 13atggaagaga agaagccgcg
gcggcaggga gccgcaggac gcgatggcat cgtgcagtac 60ccgcacctct tcatcgcggc
cctggcgctg gccctggtcc tcatggaccc cttccacctc 120ggcccgctgg
ccgggatcga ctaccggccg gtgaagcacg agctggcgcc gtacagggag
180gtcatgcagc gctggccgag ggacaacggc agccgcctca ggctcggcag
gctcgagttc 240gtcaacgagg tgttcgggcc ggagtccatc gagttcgacc
gccagggccg cgggccctac 300gccgggctcg ccgacggccg cgtcgtgcgg
tggatggggg acaaggccgg gtgggagacg 360ttcgccgtca tgaatcctga
ctggtattgg cttactgcag aaaaaccata gcttacctgt 420gtgtgtgcaa
actaaaatag tttctttcgg aaaaaaaaag gtcggagaaa gtttgtgcta
480acggagtgga gtcgacgacg aagaagcagc acgggaagga gaagtggtgc
ggccggcctc 540tcgggctgag gttccacagg gagaccggcg agctcttcat
cgccgacgcg tactatgggc 600tcatggccgt tggcgaaagc ggcggcgtgg
cgacctccct ggcgagggag gccggcgggg 660acccggtcca cttcgccaac
gacctcgaca tccacatgaa cggctcgata ttcttcaccg 720acacgagcac
gagatacagc agaaagtgag cggagtactg ctgccgatct cctttttctg
780ttcttgagat ttgtgtttga caaatgactg atcatgcagg gaccatttga
acattttgct 840ggaaggagaa ggcacgggga ggctgctgag atatgaccga
gaaaccggtg ccgttcatgt 900cgtgctcaac gggctggtct tcccaaacgg
cgtgcagatc tcacaggacc agcaatttct 960cctcttctcc gagacaacaa
actgcaggtg agataaactc aggttttcag tatgatccgg 1020ctcgagagat
ccaggaactg atgacgcctt tattaatcgg ctcatgcatg cacactagga
1080tcatgaggta ctggctggaa ggtccaagag cgggccaggt ggaggtgttc
gcgaacctgc 1140cggggttccc cgacaacgtg cgcttgaaca gcaaggggca
gttctgggtg gcgatcgact 1200gctgccggac gccgacgcag gaggtgttcg
cgcggtggcc gtggctgcgg accgcctact 1260tcaagatccc ggtgtcgatg
aagacgctgg ggaagatggt gagcatgaag atgtacacgc 1320ttctcgcgct
cctcgacggc gaggggaacg tggtcgaggt actcgaggac cggggcggcg
1380aggtgatgaa gctggtgagc gaggtgaggg aggtggaccg gaggctgtgg
atcgggaccg 1440ttgcgcacaa ccacatcgcc acgatccctt acccgttgga
ctagagtgtg tagtgtctca 1500tttgatttgc tggttttata ttagcaagga
ggtgtatcag tttatggttt gcttgtttat 1560tgggttcgtg tgatgatcgt g
158114413PRTTriticum urartu 14Met Glu Glu Lys Lys Pro Arg Arg Gln
Gly Ala Ala Gly Arg Asp Gly 1 5 10 15 Ile Val Gln Tyr Pro His Leu
Phe Ile Ala Ala Leu Ala Leu Ala Leu 20 25 30 Val Leu Met Asp Pro
Phe His Leu Gly Pro Leu Ala Gly Ile Asp Tyr 35 40 45 Arg Pro Val
Lys His Glu Leu Ala Pro Tyr Arg Glu Val Met Gln Arg 50 55 60 Trp
Pro Arg Asp Asn Gly Ser Arg Leu Arg Leu Gly Arg Leu Glu Phe 65 70
75 80 Val Asn Glu Val Phe Gly Pro Glu Ser Ile Glu Phe Asp Arg Gln
Gly 85 90 95 Arg Gly Pro Tyr Ala Gly Leu Ala Asp Gly Arg Val Val
Arg Trp Met 100 105 110 Gly Asp Lys Ala Gly Trp Glu Thr Phe Ala Val
Met Asn Pro Asp Trp 115 120 125 Ser Glu Lys Val Cys Ala Asn Gly Val
Glu Ser Thr Thr Lys Lys Gln 130 135 140 His Gly Lys Glu Lys Trp Cys
Gly Arg Pro Leu Gly Leu Arg Phe His 145 150 155 160 Arg Glu Thr Gly
Glu Leu Phe Ile Ala Asp Ala Tyr Tyr Gly Leu Met 165 170 175 Ala Val
Gly Glu Ser Gly Gly Val Ala Thr Ser Leu Ala Arg Glu Ala 180 185 190
Gly Gly Asp Pro Val His Phe Ala Asn Asp Leu Asp Ile His Met Asn 195
200 205 Gly Ser Ile Phe Phe Thr Asp Thr Ser Thr Arg Tyr Ser Arg Lys
Asp 210 215 220 His Leu Asn Ile Leu Leu Glu Gly Glu Gly Thr Gly Arg
Leu Leu Arg 225 230 235 240 Tyr Asp Arg Glu Thr Gly Ala Val His Val
Val Leu Asn Gly Leu Val 245 250 255 Phe Pro Asn Gly Val Gln Ile Ser
Gln Asp Gln Gln Phe Leu Leu Phe 260 265 270 Ser Glu Thr Thr Asn Cys
Arg Ile Met Arg Tyr Trp Leu Glu Gly Pro 275 280 285 Arg Ala Gly Gln
Val Glu Val Phe Ala Asn Leu Pro Gly Phe Pro Asp 290 295 300 Asn Val
Arg Leu Asn Ser Lys Gly Gln Phe Trp Val Ala Ile Asp Cys 305 310 315
320 Cys Arg Thr Pro Thr Gln Glu Val Phe Ala Arg Trp Pro Trp Leu Arg
325 330 335 Thr Ala Tyr Phe Lys Ile Pro Val Ser Met Lys Thr Leu Gly
Lys Met 340 345 350 Val Ser Met Lys Met Tyr Thr Leu Leu Ala Leu Leu
Asp Gly Glu Gly 355 360 365 Asn Val Val Glu Val Leu Glu Asp Arg Gly
Gly Glu Val Met Lys Leu 370 375 380 Val Ser Glu Val Arg Glu Val Asp
Arg Arg Leu Trp Ile Gly Thr Val 385 390 395 400 Ala His Asn His Ile
Ala Thr Ile Pro Tyr Pro Leu Asp 405 410 151536DNAAegilops
speltoides 15atggaagaga agaagccgcg gcggcaggga gccgcagtac gcgatggcat
cgtgcagtac 60ccgcacctct tcatcgcggc cctggcgctg gccctggtcg tcatggaccc
cttccacctc 120ggcccgctgg ccgggatcga ctaccggccg gtgaagcacg
agctggcgcc gtacagggag 180gtcatgcagc gctggccgag ggacaacggc
agccggctga gactcggcag gctcgagttc 240gtcaacgagg tgttcgggcc
ggagtccatc gagttcgacc gccagggccg cgggccctac 300gccggcctcg
ccgacggccg cgtcgtgcgg tggatggggg agaaggccgg gtgggagacg
360ttcgccgtca tgaatcctga ctggtattgg cttactgcag ataaatccat
agcttacctg 420tgtgtttgca aactaaaatg gtttcttgga aaaaaaaagg
tcggagaaag tttgtgctaa 480cggagtggag tcaacgacga agaagcagca
cgggaaggag aagtggtgcg gccggcctct 540cgggctgagg ttccacaggg
agaccggcga gctcttcatc gccgacgcgt actatgggct 600catggccgtc
ggcgaaagcg gcggcgtggc gacctccctg gcaagggagg ccggcgggga
660cccggtccac ttcgccaacg accttgacat ccacatgaac ggctcgatat
tcttcaccga 720cacgagcacg agatacagca gaaagtgagc gaactgctgc
cgctgttctc catttttgtt 780aatgagatgt tgtgtttgag tgtctgacac
catgactgat catgcaggga ccatttgaac 840attttgctgg aaggagaagg
cacggggagg ctgctgagat atgaccgaga aaccggtgcc 900gttcatgtcg
tgctcaacgg gctggtcttc ccaaacggcg tgcagatttc acaggaccag
960caatttctcc tcttctccga gacaacaaac tgcaggtgag ataaactcag
attttcagta 1020tgatccggct cgagagatcc aggaactgat gacggctcat
gcacgcacgc taggatcatg 1080aggtactggc tggaaggtcc aagagcgggc
caggtggagg tgttcgcgaa cctgccgggg 1140ttccccgaca acgtgcgcct
gaacagcaag gggcagttct gggtggcgat cgactgctgc 1200cggacgccga
cgcaggaggt gttcgcgcgg tggccgtggc tgcggaccgc ctacttcaag
1260atcccggtgt cgatgaagac gctggggaag atggtgagca tgaagatgta
cacgcttctc 1320gcgctcctcg acggcgaggg gaacgtcgtg gaggtgctcg
aggaccgggg cggcgaggtg 1380atgaagctgg tgagcgaggt gagggaggtg
gaccggaggc tgtggatcgg gaccgttgcg 1440cacaaccaca tcgccacgat
cccttacccg ctggactaga gggagtgtgt agtgtccatt 1500tgctggttta
tattagcaag gaggtgtatc agttta 153616413PRTAegilops speltoides 16Met
Glu Glu Lys Lys Pro Arg Arg Gln Gly Ala Ala Val Arg Asp Gly 1 5 10
15 Ile Val Gln Tyr Pro His Leu Phe Ile Ala Ala Leu Ala Leu Ala Leu
20 25 30 Val Val Met Asp Pro Phe His Leu Gly Pro Leu Ala Gly Ile
Asp Tyr 35 40 45 Arg Pro Val Lys His Glu Leu Ala Pro Tyr Arg Glu
Val Met Gln Arg 50 55 60 Trp Pro Arg Asp Asn Gly Ser Arg Leu Arg
Leu Gly Arg Leu Glu Phe 65 70 75 80 Val Asn Glu Val Phe Gly Pro Glu
Ser Ile Glu Phe Asp Arg Gln Gly 85 90 95 Arg Gly Pro Tyr Ala Gly
Leu Ala Asp Gly Arg Val Val Arg Trp Met 100 105 110 Gly Glu Lys Ala
Gly Trp Glu Thr Phe Ala Val Met Asn Pro Asp Trp 115 120 125 Ser Glu
Lys Val Cys Ala Asn Gly Val Glu Ser Thr Thr Lys Lys Gln 130 135 140
His Gly Lys Glu Lys Trp Cys Gly Arg Pro Leu Gly Leu Arg Phe His 145
150 155 160 Arg Glu Thr Gly Glu Leu Phe Ile Ala Asp Ala Tyr Tyr Gly
Leu Met 165 170 175 Ala Val Gly Glu Ser Gly Gly Val Ala Thr Ser Leu
Ala Arg Glu Ala 180 185 190 Gly Gly Asp Pro Val His Phe Ala Asn Asp
Leu Asp Ile His Met Asn 195 200 205 Gly Ser Ile Phe Phe Thr Asp Thr
Ser Thr Arg Tyr Ser Arg Lys Asp 210 215 220 His Leu Asn Ile Leu Leu
Glu Gly Glu Gly Thr Gly Arg Leu Leu Arg 225 230 235 240 Tyr Asp Arg
Glu Thr Gly Ala Val His Val Val Leu Asn Gly Leu Val 245 250 255 Phe
Pro Asn Gly Val Gln Ile Ser Gln Asp Gln Gln Phe Leu Leu Phe 260 265
270 Ser Glu Thr Thr Asn Cys Arg Ile Met Arg Tyr Trp Leu Glu Gly Pro
275 280 285 Arg Ala Gly Gln Val Glu Val Phe Ala Asn Leu Pro Gly Phe
Pro Asp 290 295 300 Asn Val Arg Leu Asn Ser Lys Gly Gln Phe Trp Val
Ala Ile Asp Cys 305 310 315 320 Cys Arg Thr Pro Thr Gln Glu Val Phe
Ala Arg Trp Pro Trp Leu Arg 325 330 335 Thr Ala Tyr Phe Lys Ile Pro
Val Ser Met Lys Thr Leu Gly Lys Met 340 345 350 Val Ser Met Lys Met
Tyr Thr Leu Leu Ala Leu Leu Asp Gly Glu Gly 355 360 365 Asn Val Val
Glu Val Leu Glu Asp Arg Gly Gly Glu Val Met Lys Leu 370 375 380 Val
Ser Glu Val Arg Glu Val Asp Arg Arg Leu Trp Ile Gly Thr Val 385 390
395 400 Ala His Asn His Ile Ala Thr Ile Pro Tyr Pro Leu Asp 405 410
171573DNAAegilops tauschii 17atggaagaga agaaaccgcg gcggcaggga
gccgcagtac gcgatggcat cgtgcagtac 60ccgcacctct tcatcgcggc cctggcgctg
gccctggtcc tcatggaccc gttccacctc 120ggcccgctgg ccgggatcga
ctaccgaccg gtgaagcacg agctggcgcc gtacagggag 180gtcatgcagc
gctggccgag ggacaacggc agccgcctca ggctcggcag gctcgagttc
240gtcaacgagg tgttcgggcc ggagtccatc gagttcgacc gccagggccg
cgggccttac 300gccgggctcg ccgacggccg cgtcgtgcgg tggatggggg
acaaggccgg gtgggagacg 360ttcgccgtca tgaatcctga ctggtactgg
cttactgcag aaaaacccat agcttacctg
420tgtgtgtgca gactaaaata gtttctttca taaaaaaaaa ggtcggagaa
agtttgtgct 480aacggagtgg agtcgacgac gaagaagcag cacgggaagg
agaagtggtg cggccggcct 540ctcggcctga ggttccacag ggagaccggc
gagctcttca tcgccgacgc gtactatggg 600ctcatggccg tcggcgaaag
gggcggcgtg gcgacctccc tggcgaggga ggccggcggg 660gacccggtcc
acttcgccaa cgaccttgac atccacatga acggctcgat attcttcacc
720gacacgagca cgagatacag cagaaagtga gcggagtact gctgccgatc
tcctttttct 780gttcttgaga tttgtgtttg acaaatgact gatcatgcag
ggaccatttg aacattttgc 840tggaaggaga aggcacgggg aggctgctga
gatatgaccg agaaaccggt gccgttcatg 900tcgtgctcaa cgggctggtc
ttcccaaacg gcgtgcagat atcacaggac cagcaatttc 960tcctcttctc
cgagacaaca aactgcaggt gagataaact caggttttca gtatgatccg
1020gctcgagaga tccaggaact gatgacggct catgcatgca cactaggatc
atgaggtact 1080ggctggaagg tccaagagcg ggccaggtgg aggtgttcgc
gaacctgccg gggttccccg 1140acaatgtgcg cctgaacagc aaggggcagt
tctgggtggc catcgactgc tgccgtacgc 1200cgacgcagga ggtgttcgcg
cggtggccgt ggctgcggac cgcctacttc aagatcccgg 1260tgtcgatgaa
gacgctgggg aagatggtga gcatgaagat gtacacgctt ctcgcgctcc
1320tcgacggcga ggggaacgtc gtggaggtgc tcgaggaccg gggcggcgag
gtgatgaagc 1380tggtgagcga ggtgagggag gtggaccgga ggctgtggat
cgggaccgtt gcgcacaacc 1440acatcgccac gatcccttac ccgctggact
agagggagtg tgtagtgtcc catttgattt 1500gctggtttta tattagcaag
gaggtgtatc agtttatggt ttgcttgttc attgggttcg 1560tgtgatgatc gtg
157318415PRTAegilops tauschii 18Met Glu Glu Lys Lys Pro Arg Arg Gln
Gly Ala Ala Val Arg Asp Gly 1 5 10 15 Ile Val Gln Tyr Pro His Leu
Phe Ile Ala Ala Leu Ala Leu Ala Leu 20 25 30 Val Leu Met Asp Pro
Phe His Leu Gly Pro Leu Ala Gly Ile Asp Tyr 35 40 45 Arg Pro Val
Lys His Glu Leu Ala Pro Tyr Arg Glu Val Met Gln Arg 50 55 60 Trp
Pro Arg Asp Asn Gly Ser Arg Leu Arg Leu Gly Arg Leu Glu Phe 65 70
75 80 Val Asn Glu Val Phe Gly Pro Glu Ser Ile Glu Phe Asp Arg Gln
Gly 85 90 95 Arg Gly Pro Tyr Ala Gly Leu Ala Asp Gly Arg Val Val
Arg Trp Met 100 105 110 Gly Asp Lys Ala Gly Trp Glu Thr Phe Ala Val
Met Asn Pro Asp Trp 115 120 125 Tyr Trp Ser Glu Lys Val Cys Ala Asn
Gly Val Glu Ser Thr Thr Lys 130 135 140 Lys Gln His Gly Lys Glu Lys
Trp Cys Gly Arg Pro Leu Gly Leu Arg 145 150 155 160 Phe His Arg Glu
Thr Gly Glu Leu Phe Ile Ala Asp Ala Tyr Tyr Gly 165 170 175 Leu Met
Ala Val Gly Glu Arg Gly Gly Val Ala Thr Ser Leu Ala Arg 180 185 190
Glu Ala Gly Gly Asp Pro Val His Phe Ala Asn Asp Leu Asp Ile His 195
200 205 Met Asn Gly Ser Ile Phe Phe Thr Asp Thr Ser Thr Arg Tyr Ser
Arg 210 215 220 Lys Asp His Leu Asn Ile Leu Leu Glu Gly Glu Gly Thr
Gly Arg Leu 225 230 235 240 Leu Arg Tyr Asp Arg Glu Thr Gly Ala Val
His Val Val Leu Asn Gly 245 250 255 Leu Val Phe Pro Asn Gly Val Gln
Ile Ser Gln Asp Gln Gln Phe Leu 260 265 270 Leu Phe Ser Glu Thr Thr
Asn Cys Arg Ile Met Arg Tyr Trp Leu Glu 275 280 285 Gly Pro Arg Ala
Gly Gln Val Glu Val Phe Ala Asn Leu Pro Gly Phe 290 295 300 Pro Asp
Asn Val Arg Leu Asn Ser Lys Gly Gln Phe Trp Val Ala Ile 305 310 315
320 Asp Cys Cys Arg Thr Pro Thr Gln Glu Val Phe Ala Arg Trp Pro Trp
325 330 335 Leu Arg Thr Ala Tyr Phe Lys Ile Pro Val Ser Met Lys Thr
Leu Gly 340 345 350 Lys Met Val Ser Met Lys Met Tyr Thr Leu Leu Ala
Leu Leu Asp Gly 355 360 365 Glu Gly Asn Val Val Glu Val Leu Glu Asp
Arg Gly Gly Glu Val Met 370 375 380 Lys Leu Val Ser Glu Val Arg Glu
Val Asp Arg Arg Leu Trp Ile Gly 385 390 395 400 Thr Val Ala His Asn
His Ile Ala Thr Ile Pro Tyr Pro Leu Asp 405 410 415
1910PRTArtificialheme-binding domain of MS26 19Phe Xaa Xaa Gly Xaa
Arg Xaa Cys Xaa Gly 1 5 10 209PRTArtificialdioxygen binding domain
20Ala Gly Gly Xaa Asp Glu Thr Thr Ser 1 5
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