U.S. patent application number 11/255714 was filed with the patent office on 2007-01-11 for nucleic acids that control seed and fruit development in plants.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Robert L. Fischer, Robert B. Goldberg, John Harada, Tomohiro Kiyosue, Linda Margossian, Nir Ohad, Ramin Yadegari.
Application Number | 20070011760 11/255714 |
Document ID | / |
Family ID | 22103917 |
Filed Date | 2007-01-11 |
United States Patent
Application |
20070011760 |
Kind Code |
A1 |
Fischer; Robert L. ; et
al. |
January 11, 2007 |
Nucleic acids that control seed and fruit development in plants
Abstract
The invention provides methods of controlling endosperm
development in plants.
Inventors: |
Fischer; Robert L.; (El
Cerrito, CA) ; Ohad; Nir; (Jerusalem, IL) ;
Kiyosue; Tomohiro; (Okazaka, JP) ; Yadegari;
Ramin; (San Jose, CA) ; Margossian; Linda; (El
Cerrito, CA) ; Harada; John; (Davis, CA) ;
Goldberg; Robert B.; (Topanga, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
22103917 |
Appl. No.: |
11/255714 |
Filed: |
October 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10213512 |
Aug 6, 2002 |
7049488 |
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11255714 |
Oct 20, 2005 |
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09071838 |
May 1, 1998 |
7029917 |
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10213512 |
Aug 6, 2002 |
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Current U.S.
Class: |
800/278 ;
435/419; 435/468; 435/69.1; 530/370; 536/23.6; 800/285 |
Current CPC
Class: |
C12N 15/8287 20130101;
C12N 15/8267 20130101; Y02A 40/146 20180101; C07K 2319/00 20130101;
C07K 14/415 20130101; C12N 15/8261 20130101 |
Class at
Publication: |
800/278 ;
435/069.1; 435/419; 435/468; 800/285; 530/370; 536/023.6 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C07K 14/415 20060101 C07K014/415; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C12N 15/82 20060101
C12N015/82; C12N 5/04 20060101 C12N005/04 |
Claims
1. An isolated nucleic acid molecule comprising a FIE
polynucleotide sequence, which polynucleotide sequence specifically
hybridizes to SEQ ID NO:1 or SEQ ID NO:3 under stringent
conditions.
2. The isolated nucleic acid molecule of claim I, wherein the FIE
polynucleotide is between about at least about 100 nucleotides in
length.
3. The isolated nucleic acid molecule of claim 1, wherein the FIE
polynucleotide is SEQ ID NO:1.
4. The isolated nucleic acid molecule of claim 1, wherein the FIE
polynucleotide is SEQ ID NO:3.
5. The isolated nucleic acid molecule of claim 1, further
comprising a plant promoter operably linked to the FIE
polynucleotide.
6. The isolated nucleic acid molecule of claim 5, wherein the plant
promoter is from a FIE1 gene.
7. The isolated nucleic acid of claim 6, wherein the FIE
polynucleotide is linked to the promoter in an antisense
orientation.
8. An isolated nucleic acid molecule comprising a FIE
polynucleotide sequence, which polynucleotide sequence encodes FIE
polypeptide as shown in SEQ ID NO:2 or SEQ ID NO:4.
9. a transgenic plant comprising an expression cassette containing
a plant promoter operably linked to a heterologous FIE
polynucleotide of claim 1.
10. The transgenic plant of claim 9, wherein the heterologous FIE
polynucleotide encodes a FIE polypeptide.
11. The transgenic plant of claim 10, wherein the FIE polypeptide
is as shown in SEQ ID NO:2 or SEQ ID NO:4.
12. The transgenic plant of claim 9, wherein the heterologous FIE
polynucleotide is linked to the promoter in an antisense
orientation.
13. The transgenic plant of claim 9, wherein the plant promoter is
from a FIE gene.
14. The transgenic plant of claim 13, wherein the FIE gene is as
shown in SEQ ID NO:1 or SEQ ID NO:3.
15. A method of modulating endosperm development in a plant, the
method comprising introducing into the plant an expression cassette
containing a plant promoter operably linked to a heterologous FIE
polynucleotide.
16. The method of claim 15, wherein the heterologous FIE
polynucleotide encodes an FIE polypeptide.
17. The method of claim 16, wherein the FIE polypeptide has an
amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:4.
18. The method of claim 15, wherein the heterologous FIE
polynucleotide is linked to the promoter in an antisense
orientation.
19. The method of claim 15, wherein the heterologous FIE
polynucleotide is SEQ ID NO:1 or SEQ ID NO:3.
20. The method of claim 15, wherein the plant promoter is from a
FIE gene.
21. The method of claim 15, wherein the expression cassette is
introduced into the plant through a sexual cross.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to plant genetic
engineering. In particular, it relates to modulation of expression
of genes controlling endosperm development in plants.
BACKGROUND OF THE INVENTION
[0002] A fundamental problem in biology is to understand how
fertilization initiates reproductive development. In higher plants,
the ovule generates the female gametophyte which is composed of
egg, central, synergid and antipodal cells (Reiser, et al., Plant
Cell, 1291-1301 (1993)). All are haploid except the central cell
which contains two daughter nuclei that fuse prior to
fertilization. One sperm nucleus fertilizes the egg to form the
zygote, whereas another sperm nucleus fuses with the diploid
central cell nucleus to form the triploid endosperm nucleus (van
Went, et al., Embryology of Angiosperms, pp. 273-318 (1984)). The
two fertilization products undergo distinct patterns of
development. In Arabidopsis, the embryo passes through a series of
stages that have been defined morphologically as preglobular,
globular, heart, cotyledon and maturation (Goldberg, R. B., et al.,
Science (1994) 266: 605-614; Mansfield, S. G., et al., Arabidopsis:
An Atlas of Morphology and Development, pp. 367-383 (1994)). The
primary endosperm nucleus undergoes a series of mitotic divisions
to produce nuclei that migrate into the expanding central cell
(Mansfield, S. G., et al., Arab Inf Serv 27: 53-64 (1990); Webb, M.
C., et al., Planta 184: 187-195 (1991)). Cytokinesis sequesters
endosperm cytoplasm and nuclei into discrete cells (Mansfield, S.
G., et al., Arab Inf Serv 27: 65-72 (1990)) that produce storage
proteins, starch, and lipids which support embryo growth (Lopes, M.
A. et al., Plant Cell 5: 1383-1399 (1993)). Fertilization also
activates development of the integument cell layers of the ovule
that become the seed coat, and induces the ovary to grow and form
the fruit, or silique, in Arabidopsis.
[0003] Control of the expression of genes that control egg and
central cell differentiation, or those that activate reproductive
development in response to fertilization is useful in the
production of plants with a range of desired traits. These and
other advantages are provided by the present application.
SUMMARY OF THE INVENTION
[0004] The present invention provides methods of modulating fruit
and seed development and other traits in plants. The methods
involve providing a plant comprising a recombinant expression
cassette containing an FIE nucleic acid linked to a plant
promoter.
[0005] In some embodiments, transcription of the FIE nucleic acid
inhibits expression of an endogenous FIE gene or activity the
encoded protein. This embodiment is particularly useful, for
instance, making embryo-less seed and parthenocarpic fruit.
Alternatively, expression of the FIE nucleic acid may enhance
expression of an endogenous FIE gene or FIE activity
[0006] In the expression cassettes, the plant promoter may be a
constitutive promoter, for example, the CaMV 35S promoter.
Alternatively, the promoter may be a tissue-specific promoter.
Examples of tissue specific expression useful in the invention
include ovule-specific or embryo-specific expression. For instance,
the promoter sequence from the FIE genes disclosed here can be used
to direct expression in relevant plant tissues.
[0007] The invention also provides seed or fruit produced by the
methods described above. The seed or fruit of the invention
comprise a recombinant expression cassette containing an FIE
nucleic acid.
DEFINITIONS
[0008] The phrase "nucleic acid sequence" refers to a single or
double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. It includes chromosomal DNA,
self-replicating plasmids, infectious polymers of DNA or RNA and
DNA or RNA that performs a primarily structural role.
[0009] A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription of an operably linked nucleic
acid. As used herein, a "plant promoter" is a promoter that
functions in plants. Promoters include necessary nucleic acid
sequences near the start site of transcription, such as, in the
case of a polymerase II type promoter, a TATA element. A promoter
also optionally includes distal enhancer or repressor elements,
which can be located as much as several thousand base pairs from
the start site of transcription. A "constitutive" promoter is a
promoter that is active under most environmental and developmental
conditions. An "inducible" promoter is a promoter that is active
under environmental or developmental regulation. The term "operably
linked" refers to a functional linkage between a nucleic acid
expression control sequence (such as a promoter, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence directs
transcription of the nucleic acid corresponding to the second
sequence.
[0010] The term "plant" includes whole plants, plant organs (e.g.,
leaves, stems, flowers, roots, etc.), seeds and plant cells and
progeny of same. The class of plants which can be used in the
method of the invention is generally as broad as the class of
higher plants amenable to transformation techniques, including
angiosperms (monocotyledonous and dicotyledonous plants), as well
as gymnosperms. It includes plants of a variety of ploidy levels,
including polyploid, diploid, haploid and hemizygous.
[0011] A polynucleotide sequence is "heterologous to" an organism
or a second polynucleotide sequence if it originates from a foreign
species, or, if from the same species, is modified from its
original form. For example, a promoter operably linked to a
heterologous coding sequence refers to a coding sequence from a
species different from that from which the promoter was derived,
or, if from the same species, a coding sequence which is different
from any naturally occurring allelic variants.
[0012] A polynucleotide "exogenous to" an individual plant is a
polynucleotide which is introduced into the plant by any means
other than by a sexual cross. Examples of means by which this can
be accomplished are described below, and include
Agrobacterium-mediated transformation, biolistic methods,
electroporation, and the like. Such a plant containing the
exogenous nucleic acid is referred to here as an R.sub.1 generation
transgenic plant. Transgenic plants which arise from sexual cross
or by selfing are descendants of such a plant.
[0013] A "FIE nucleic acid" or "FIE polynucleotide sequence" of the
invention is a subsequence or full length polynucleotide sequence
of a gene which encodes a polypeptide involved in control of
reproductive development and which, when mutated, allows for
aspects of fertilization independent reproductive development. In
some embodiments, the polypeptides of the invention have
substantial sequence identity (as defined below) to a polycomb
group gene of Drosophila. An exemplary nucleic acid of the
invention is the Arabidopsis FIE1 and FIE3 sequences disclosed
below. FIE polynucleotides are defined by their ability to
hybridize under defined conditions to the exemplified nucleic acids
or PCR products derived from them. An FIE polynucleotide is
typically at least about 30-40 nucleotides to about 3000, usually
less than about 5000 nucleotides in length. The nucleic acids
contain coding sequence of from about 100 to about 2000
nucleotides, often from about 500 to about 1700 nucleotides in
length.
[0014] FIE nucleic acids are a new class of plant regulatory genes
that encode polypeptides with sequence identity to members of the
polycomb group genes first identified in Drosophila. Polycomb group
gene products and their homologues in other species are responsible
for repression of homeotic genes. The proteins are a heterogenous
group that interact with each other to form large complexes that
bind DNA and thereby control gene expression. For a review of the
current understanding of polycomb complex genes see, Pirrotta Cur.
Op. Genet. Dev. 7:249-258 (1997). Nine groups of polycomb genes
have been identified. FIE1 (SEQ ID NO: 1) is related to the group
of polycomb genes encoding protein comprising a SET domain (see,
e.g., Jenuwein et al. Cell. Mol. Life Sci. 54:80-93 (1998). FIE3
(SEQ ID NO:3) is related to the group encoding proteins comprising
WD40 repeats (see, Gutjahr et al. EMBO J. 14:4296-4306 (1995).
[0015] In the case of both expression of transgenes and inhibition
of endogenous genes (e.g., by antisense, or sense suppression) one
of skill will recognize that the inserted polynucleotide sequence
need not be identical, but may be only "substantially identical" to
a sequence of the gene from which it was derived. As explained
below, these substantially identical variants are specifically
covered by the term FIE nucleic acid.
[0016] In the case where the inserted polynucleotide sequence is
transcribed and translated to produce a functional polypeptide, one
of skill will recognize that because of codon degeneracy a number
of polynucleotide sequences will encode the same polypeptide. These
variants are specifically covered by the terms "FIE nucleic acid".
In addition, the term specifically includes those sequences
substantially identical (determined as described below) with an FIE
polynucleotide sequence disclosed here and that encode polypeptides
that are either mutants of wild type FIE polypeptides or retain the
function of the FIE polypeptide (e.g., resulting from conservative
substitutions of amino acids in the FIE polypeptide). In addition,
variants can be those that encode dominant negative mutants as
described below.
[0017] Two nucleic acid sequences or polypeptides are said to be
"identical" if the sequence of nucleotides or amino acid residues,
respectively, in the two sequences is the same when aligned for
maximum correspondence as described below. The terms "identical" or
percent "identity," in the context of two or more nucleic acids or
polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same, when compared
and aligned for maximum correspondence over a comparison window, as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. When percentage of
sequence identity is used in reference to proteins or peptides, it
is recognized that residue positions that are not identical often
differ by conservative amino acid substitutions, where amino acids
residues are substituted for other amino acid residues with similar
chemical properties (e.g., charge or hydrophobicity) and therefore
do not change the functional properties of the molecule. Where
sequences differ in conservative substitutions, the percent
sequence identity may be adjusted upwards to correct for the
conservative nature of the substitution. 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 according to, e.g., the algorithm of Meyers &
Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as
implemented in the program PC/GENE (Intelligenetics, Mountain View,
Calif., USA).
[0018] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides, refers to sequences or subsequences
that have at least 60%, preferably 80%, most preferably 90-95%
nucleotide or amino acid residue identity when aligned for maximum
correspondence over a comparison window as measured using one of
the following sequence comparison algorithms or by manual alignment
and visual inspection. This definition also refers to the
complement of a test sequence, which has substantial sequence or
subsequence complementarity when the test sequence has substantial
identity to a reference sequence.
[0019] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0020] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection.
[0021] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method
used is similar to the method described by Higgins & Sharp,
CABIOS 5:151-153 (1989). The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. For example, a reference sequence can be
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps.
[0022] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul et al., J. Mol.
Biol. 215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul et al, supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Extension of the word
hits in each direction are halted when: the cumulative alignment
score falls off by the quantity X from its maximum achieved value;
the cumulative score goes to zero or below, due to the accumulation
of one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T,
and X determine the sensitivity and speed of the alignment. The
BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0023] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0024] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine) can be modified to yield a
functionally identical molecule. Accordingly, each silent variation
of a nucleic acid which encodes a polypeptide is implicit in each
described sequence.
[0025] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art.
[0026] The following six groups each contain amino acids that are
conservative substitutions for one another: [0027] 1) Alanine (A),
Serine (S), Threonine (T); [0028] 2) Aspartic acid (D), Glutamic
acid (E); [0029] 3) Asparagine (N), Glutamine (Q); [0030] 4)
Arginine (R), Lysine (K); [0031] 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); and [0032] 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W). (see, e.g., Creighton, Proteins
(1984)).
[0033] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules or their complements hybridize
to each other under stringent conditions, as described below.
[0034] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0035] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, highly
stringent conditions are selected to be about 5-10.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequence
at a defined ionic strength pH. Low stringency conditions are
generally selected to be about 15-30.degree. C. below the T.sub.m.
The T.sub.m is the temperature (under defined ionic strength, pH,
and nucleic concentration) at which 50% of the probes complementary
to the target hybridize to the target sequence at equilibrium (as
the target sequences are present in excess, at T.sub.m, 50% of the
probes are occupied at equilibrium). Stringent conditions will be
those in which the salt concentration is less than about 1.0 M
sodium ion, typically about 0.01 to 1.0 M sodium ion concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least
about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides)
and at least about 60.degree. C. for long probes (e.g., greater
than 50 nucleotides). Stringent conditions may also be achieved
with the addition of destabilizing agents such as formamide. For
selective or specific hybridization, a positive signal is at least
two times background, preferably 10 time background
hybridization.
[0036] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cased, the nucleic acids typically hybridize under moderately
stringent hybridization conditions.
[0037] In the present invention, genomic DNA or cDNA comprising FIE
nucleic acids of the invention can be identified in standard
Southern blots under stringent conditions using the nucleic acid
sequences disclosed here. For the purposes of this disclosure,
suitable stringent conditions for such hybridizations are those
which include a hybridization in a buffer of 40% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and at least one wash in
0.2.times.SSC at a temperature of at least about 50.degree. C.,
usually about 55.degree. C. to about 60.degree. C., for 20 minutes,
or equivalent conditions. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency.
[0038] A further indication that two polynucleotides are
substantially identical is if the reference sequence, amplified by
a pair of oligonucleotide primers, can then be used as a probe
under stringent hybridization conditions to isolate the test
sequence from a cDNA or genomic library, or to identify the test
sequence in, e.g., a northern or Southern blot.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIGS. 1A and 1B show the genetic map used to clone the FIE3
gene.
[0040] FIG. 2 shows the analysis of the sequence in the DNA shown
in FIG. 1 using the GENSCANW program.
[0041] FIG. 3 shows the position of primers used to PCR amplify
sequences from the FIE3 gene region.
[0042] FIG. 4 shows the genetic map used to clone the FIE1
gene.
[0043] FIG. 5 shows the results of complementation tests
establishing that a single gene (FIE1) was present on the
complementing cosmid (6-22) that was not fully encoded on either of
the non-complementing cosmids (2-9 and 2-8).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0044] This invention provides molecular strategies for controlling
seed and fruit development.
[0045] Reproduction in higher plants is unique because it is
initiated by two fertilization events in the haploid female
gametophyte. One sperm nucleus fertilizes the egg to form the
embryo. A second sperm nucleus fertilizes the central cell to form
the endosperm, a unique tissue that supports the growth of the
embryo. Fertilization also activates maternal tissue
differentiation, the ovule integuments form the seed coat and the
ovary forms the fruit.
[0046] The present invention is based, at least in part, on the
discovery of a set of female-gametophytic mutations, termed fie
(fertilization-independent endosperm), and the subsequent cloning
of the genes involved. Three mutants are disclosed here fie1, fie2,
and fie3, which have been mapped to chromosomes 1, 2, and 3 of
Arabidopsis, respectively. The fie mutations affect the central
cell, allowing for replication of the central cell nucleus and
endosperm development without fertilization. FIE/fie seed coat and
fruit undergo fertilization-independent differentiation, showing
that the fie female gametophyte is the source of signals that
activates sporophytic fruit and seed coat development. Generally,
the mutant fie alleles are not transmitted by the female
gametophyte. Inheritance of a mutant fie allele (e.g., fie3) by the
female gametophyte usually results in embryo abortion, even when
the pollen bears the wild-type FIE allele. In the case of fie 1 and
fie2, however, transmission of the trait occurs in about 1% of the
progeny from the female gametophyte. In contrast, the fie 1, fie2,
and fie3 mutant alleles are passed through the male gametophyte
(i.e., pollen) in normal fashion.
[0047] The isolated sequences prepared as described herein, can be
used in a number of techniques, for example, to suppress or enhance
endogenous FIE gene expression. Modulation of FIE gene expression
or FIE activity in plants is particularly useful, for example, in
producing embryo-less seed, parthenocarpic fruit, or as part of a
system to generate apomictic seed.
Isolation of FIE Nucleic Acids
[0048] Generally, the nomenclature and the laboratory procedures in
recombinant DNA technology described below are those well known and
commonly employed in the art. Standard techniques are used for
cloning, DNA and RNA isolation, amplification and purification.
Generally enzymatic reactions involving DNA ligase, DNA polymerase,
restriction endonucleases and the like are performed according to
the manufacturer's specifications. These techniques and various
other techniques are generally performed according to Sambrook et
al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., (1989).
[0049] The isolation of FIE nucleic acids may be accomplished by a
number of techniques. For instance, oligonucleotide probes based on
the sequences disclosed here can be used to identify the desired
gene in a cDNA or genomic DNA library. To construct genomic
libraries, large segments of genomic DNA are generated by random
fragmentation, e.g. using restriction endonucleases, and are
ligated with vector DNA to form concatemers that can be packaged
into the appropriate vector. To prepare a cDNA library, mRNA is
isolated from the desired organ, such as ovules, and a cDNA library
which contains the FIE gene transcript is prepared from the mRNA.
Alternatively, cDNA may be prepared from mRNA extracted from other
tissues in which FIE genes or homologs are expressed.
[0050] The cDNA or genomic library can then be screened using a
probe based upon the sequence of a cloned FIE gene disclosed here.
Probes may be used to hybridize with genomic DNA or cDNA sequences
to isolate homologous genes in the same or different plant species.
Alternatively, antibodies raised against an FIE polypeptide can be
used to screen an mRNA expression library.
[0051] Alternatively, the nucleic acids of interest can be
amplified from nucleic acid samples using amplification techniques.
For instance, polymerase chain reaction (PCR) technology can be
used to amplify the sequences of the FIE genes directly from
genomic DNA, from cDNA, from genomic libraries or cDNA libraries.
PCR and other in vitro amplification methods may also be useful,
for example, to clone nucleic acid sequences that code for proteins
to be expressed, to make nucleic acids to use as probes for
detecting the presence of the desired mRNA in samples, for nucleic
acid sequencing, or for other purposes. For a general overview of
PCR see PCR Protocols: A Guide to Methods and Applications. (Innis,
M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press,
San Diego (1990).
[0052] Appropriate primers and probes for identifying FIE sequences
from plant tissues are generated from comparisons of the sequences
provided here with other polycomb group genes. For instance, FIE1
can be compared to the other polycomb genes containing the SET
domain, such as the Arabidopsis curly leaf gene (Goodrich et al.
Nature 386:44-51 (1997)) or the Drosophila enhancer of zeste (E(z))
gene. FIE3 can be compared to genes containing WD40 repeats, such
as the extra sex combs (esc) gene from Drosophila. Using these
techniques, one of skill can identify conserved regions in the
nucleic acids disclosed here to prepare the appropriate primer and
probe sequences. Primers that specifically hybridize to conserved
regions in FIE1 or FIE3 genes can be used to amplify sequences from
widely divergent plant species.
[0053] Standard nucleic acid hybridization techniques using the
conditions disclosed above can then be used to identify full length
cDNA or genomic clones.
Control of FIE Activity or Gene Expression
[0054] Since FIE genes are involved in controlling seed, in
particular endosperm, development, inhibition of endogenous Fie
activity or gene expression is useful in a number of contexts. For
instance, inhibition of expression is useful in the development of
parthenocarpic fruit (i.e., fruit formed in the absence of
fertilization).
[0055] In addition, inhibition of FIE activity can be used for
production of fruit with small and/or degraded seed (referred to
here as "seedless fruit") after fertilization. In many plants,
particularly dicots, the endosperm is not persistent and eventually
is degraded. Thus, in plants of the invention in which Fie activity
is inhibited, embryo-less seed do not persist and seedless fruit
are produced.
[0056] Alternatively, plants of the invention can be used to
prevent pre-harvest sprouting in seeds, especially those derived
from cereals. In these plants, the endosperm persists and is the
major component of the mature seed. Premature growth of embryos in
stored grain causes release of degradative enzymes which digest
starch and other components of the endosperm. Plants of the present
invention are useful in addressing this problem because the seeds
lack an embryo and thus will not germinate.
[0057] In yet another use, nucleic acids of the invention can be
used in the development of apomictic plant lines (i.e., plants in
which asexual reproductive processes occur in the ovule, see,
Koltunow, A. Plant Cell 5: 1425-1437 (1993) for a discussion of
apomixis). Apomixis provides a novel means to select and fix
complex heterozygous genotypes that cannot be easily maintained by
traditional breeding. Thus, for instance, new hybrid lines with
desired traits (e.g., hybrid vigor) can be obtained and readily
maintained.
[0058] One of skill will recognize that a number of methods can be
used to modulate FIE activity or gene expression. FIE activity can
be modulated in the plant cell at the gene, transcriptional,
posttranscriptional, translational, or posttranslational, levels.
Techniques for modulating FIE activity at each of these levels are
generally well known to one of skill and are discussed briefly
below.
[0059] Methods for introducing genetic mutations into plant genes
are well known. For instance, seeds or other plant material can be
treated with a mutagenic chemical substance, according to standard
techniques. Such chemical substances include, but are not limited
to, the following: diethyl sulfate, ethylene imine, ethyl
methanesulfonate and N-nitroso-N-ethylurea. Alternatively, ionizing
radiation from sources such as, for example, X-rays or gamma rays
can be used.
[0060] Alternatively, homologous recombination can be used to
induce targeted gene disruptions by specifically deleting or
altering the FIE gene in vivo (see, generally, Grewal and Klar,
Genetics 146: 1221-1238 (1997) and Xu et al., Genes Dev. 10:
2411-2422 (1996)). Homologous recombination has been demonstrated
in plants (Puchta et al., Experientia 50: 277-284 (1994), Swoboda
et al., EMBO J. 13: 484-489 (1994); Offringa et al., Proc. Natl.
Acad. Sci. USA 90: 7346-7350 (1993); and Kempin et al. Nature
389:802-803 (1997)).
[0061] In applying homologous recombination technology to the genes
of the invention, mutations in selected portions of an FIE gene
sequences (including 5' upstream, 3' downstream, and intragenic
regions) such as those disclosed here are made in vitro and then
introduced into the desired plant using standard techniques. Since
the efficiency of homologous recombination is known to be dependent
on the vectors used, use of dicistronic gene targeting vectors as
described by Mountford et al. Proc. Natl. Acad. Sci. USA 91:
4303-4307 (1994); and Vaulont et al. Transgenic Res. 4: 247-255
(1995) are conveniently used to increase the efficiency of
selecting for altered FIE gene expression in transgenic plants. The
mutated gene will interact with the target wild-type gene in such a
way that homologous recombination and targeted replacement of the
wild-type gene will occur in transgenic plant cells, resulting in
suppression of FIE activity.
[0062] Alternatively, oligonucleotides composed of a contiguous
stretch of RNA and DNA residues in a duplex conformation with
double hairpin caps on the ends can be used. The RNA/DNA sequence
is designed to align with the sequence of the target FIE gene and
to contain the desired nucleotide change. Introduction of the
chimeric oligonucleotide on an extrachromosomal T-DNA plasmid
results in efficient and specific FIE gene conversion directed by
chimeric molecules in a small number of transformed plant cells.
This method is described in Cole-Strauss et al. Science
273:1386-1389 (1996) and Yoon et al. Proc. Natl. Acad. Sci. USA 93:
2071-2076 (1996).
[0063] Gene expression can be inactivated using recombinant DNA
techniques by transforming plant cells with constructs comprising
transposons or T-DNA sequences. FIE mutants prepared by these
methods are identified according to standard techniques. For
instance, mutants can be detected by PCR or by detecting the
presence or absence of FIE mRNA, e.g., by Northern blots. Mutants
can also be selected by assaying for development of endosperm in
the absence of fertilization.
[0064] The isolated nucleic acid sequences prepared as described
herein, can also be used in a number of techniques to control
endogenous FIE gene expression at various levels. Subsequences from
the sequences disclosed here can be used to control, transcription,
RNA accumulation, translation, and the like.
[0065] A number of methods can be used to inhibit gene expression
in plants. For instance, antisense technology can be conveniently
used. To accomplish this, a nucleic acid segment from the desired
gene is cloned and operably linked to a promoter such that the
antisense strand of RNA will be transcribed. The construct is then
transformed into plants and the antisense strand of RNA is
produced. In plant cells, it has been suggested that antisense
suppression can act at all levels of gene regulation including
suppression of RNA translation (see, Bourque Plant Sci. (Limerick)
105: 125-149 (1995); Pantopoulos In Progress in Nucleic Acid
Research and Molecular Biology, Vol. 48. Cohn, W. E. and K. Moldave
(Ed.). Academic Press, Inc.: San Diego, Calif., USA; London,
England, UK. p. 181-238; Heiser et al. Plant Sci. (Shannon) 127:
61-69 (1997)) and by preventing the accumulation of mRNA which
encodes the protein of interest, (see, Baulcombe Plant Mol. Bio.
32:79-88 (1996); Prins and Goldbach Arch. Virol. 141: 2259-2276
(1996); Metzlaff et al. Cell 88: 845-854 (1997), Sheehy et al.,
Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988), and Hiatt et al.,
U.S. Pat. No. 4,801,340).
[0066] The nucleic acid segment to be introduced generally will be
substantially identical to at least a portion of the endogenous FIE
gene or genes to be repressed. The sequence, however, need not be
perfectly identical to inhibit expression. The vectors of the
present invention can be designed such that the inhibitory effect
applies to other genes within a family of genes exhibiting homology
or substantial homology to the target gene.
[0067] For antisense suppression, the introduced sequence also need
not be full length relative to either the primary transcription
product or fully processed mRNA. Generally, higher homology can be
used to compensate for the use of a shorter sequence. Furthermore,
the introduced sequence need not have the same intron or exon
pattern, and homology of non-coding segments may be equally
effective. Normally, a sequence of between about 30 or 40
nucleotides and about full length nucleotides should be used,
though a sequence of at least about 100 nucleotides is preferred, a
sequence of at least about 200 nucleotides is more preferred, and a
sequence of about 500 to about 1700 nucleotides is especially
preferred.
[0068] A number of gene regions can be targeted to suppress FIE
gene expression. The targets can include, for instance, the coding
regions, introns, sequences from exon/intron junctions, 5' or 3'
untranslated regions, and the like. In some embodiments, the
constructs can be designed to eliminate the ability of regulatory
proteins to bind to FIE gene sequences that are required for its
cell- and/or tissue-specific expression. Such transcriptional
regulatory sequences can be located either 5'-, 3'-, or within the
coding region of the gene and can be either promote (positive
regulatory element) or repress (negative regulatory element) gene
transcription. These sequences can be identified using standard
deletion analysis, well known to those of skill in the art. Once
the sequences are identified, an antisense construct targeting
these sequences is introduced into plants to control gene
transcription in particular tissue, for instance, in developing
ovules and/or seed.
[0069] Oligonucleotide-based triple-helix formation can be used to
disrupt FIE gene expression. Triplex DNA can inhibit DNA
transcription and replication, generate site-specific mutations,
cleave DNA, and induce homologous recombination (see, e.g., Havre
and Glazer J. Virology 67:7324-7331 (1993); Scanlon et al. FASEB J.
9:1288-1296 (1995); Giovannangeli et al. Biochemistry
35:10539-10548 (1996); Chan and Glazer J. Mol. Medicine (Berlin)
75: 267-282 (1997)). Triple helix DNAs can be used to target the
same sequences identified for antisense regulation.
[0070] Catalytic RNA molecules or ribozymes can also be used to
inhibit expression of FIE genes. It is possible to design ribozymes
that specifically pair with virtually any target RNA and cleave the
phosphodiester backbone at a specific location, thereby
functionally inactivating the target RNA. In carrying out this
cleavage, the ribozyme is not itself altered, and is thus capable
of recycling and cleaving other molecules, making it a true enzyme.
The inclusion of ribozyme sequences within antisense RNAs confers
RNA-cleaving activity upon them, thereby increasing the activity of
the constructs. Thus, ribozymes can be used to target the same
sequences identified for antisense regulation.
[0071] A number of classes of ribozymes have been identified. One
class of ribozymes is derived from a number of small circular RNAs
which are capable of self-cleavage and replication in plants. The
RNAs replicate either alone (viroid RNAs) or with a helper virus
(satellite RNAs). Examples include RNAs from avocado sunblotch
viroid and the satellite RNAs from tobacco ringspot virus, lucerne
transient streak virus, velvet tobacco mottle virus, solanum
nodiflorum mottle virus and subterranean clover mottle virus. The
design and use of target RNA-specific ribozymes is described in
Zhao and Pick Nature 365:448-451 (1993); Eastham and Ahlering J.
Urology 156:1186-1188 (1996); Sokol and Murray Transgenic Res.
5:363-371 (1996); Sun et al. Mol. Biotechnology 7:241-251 (1997);
and Haseloff et al. Nature, 334:585-591 (1988).
[0072] Another method of suppression is sense cosuppression.
Introduction of nucleic acid configured in the sense orientation
has been recently shown to be an effective means by which to block
the transcription of target genes. For an example of the use of
this method to modulate expression of endogenous genes (see, Assaad
et al. Plant Mol. Bio. 22: 1067-1085 (1993); Flavell Proc. Natl.
Acad. Sci. USA 91: 3490-3496 (1994); Stam et al. Annals Bot. 79:
3-12 (1997); Napoli et al., The Plant Cell 2:279-289 (1990); and
U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184).
[0073] The suppressive effect may occur where the introduced
sequence contains no coding sequence per se, but only intron or
untranslated sequences homologous to sequences present in the
primary transcript of the endogenous sequence. The introduced
sequence generally will be substantially identical to the
endogenous sequence intended to be repressed. This minimal identity
will typically be greater than about 65%, but a higher identity
might exert a more effective repression of expression of the
endogenous sequences. Substantially greater identity of more than
about 80% is preferred, though about 95% to absolute identity would
be most preferred. As with antisense regulation, the effect should
apply to any other proteins within a similar family of genes
exhibiting homology or substantial homology.
[0074] For sense suppression, the introduced sequence, needing less
than absolute identity, also need not be full length, relative to
either the primary. transcription product or fully processed mRNA.
This may be preferred to avoid concurrent production of some plants
which are overexpressers. A higher identity in a shorter than full
length sequence compensates for a longer, less identical sequence.
Furthermore, the introduced sequence need not have the same intron
or exon pattern, and identity of non-coding segments will be
equally effective. Normally, a sequence of the size ranges noted
above for antisense regulation is used. In addition, the same gene
regions noted for antisense regulation can be targetted using
cosuppression technologies.
[0075] Alternatively, FIE activity may be modulated by eliminating
the proteins that are required for FIE cell-specific gene
expression. Thus, expression of regulatory proteins and/or the
sequences that control FIE gene expression can be modulated using
the methods described here.
[0076] Another method is use of engineered tRNA suppression of FIE
mRNA translation. This method involves the use of suppressor tRNAs
to transactivate target genes containing premature stop codons
(see, Betzner et al. Plant J. 11:587-595 (1997); and Choisne et al.
Plant J. 11: 597-604 (1997). A plant line containing a
constitutively expressed FIE gene that contains an amber stop codon
is first created. Multiple lines of plants, each containing tRNA
suppressor gene constructs under the direction of cell-type
specific promoters are also generated. The tRNA gene construct is
then crossed into the FIE line to activate FIE activity in a
targeted manner. These tRNA suppressor lines could also be used to
target the expression of any type of gene to the same cell or
tissue types.
[0077] As noted above, FIE proteins as products of polycomb group
genes are believed to form large complexes in vivo. Thus,
production of dominant-negative forms of FIE polypeptides that are
defective in their abilities to bind to other polycomb group
proteins is a convenient means to inhibit endogenous FIE activity.
This approach involves transformation of plants with constructs
encoding mutant FIE polypeptides that form defective complexes with
endogenous polycomb group proteins and thereby prevent the complex
from forming properly. The mutant polypeptide may vary from the
naturally occurring sequence at the primary structure level by
amino acid substitutions, additions, deletions, and the like. These
modifications can be used in a number of combinations to produce
the final modified protein chain. Use of dominant negative mutants
to inactivate target genes is described in Mizukami et al. Plant
Cell 8:831-845 (1996).
[0078] Another strategy to affect the ability of an FIE protein to
interact with itself or with other proteins involves the use of
antibodies specific to FIE. In this method cell-specific expression
of FIE-specific Abs is used inactivate functional domains through
antibody:antigen recognition (see, Hupp et al. Cell 83:237-245
(1995)).
Use of Nucleic Acids of the Invention to Enhance FIE Gene
Expression
[0079] Isolated sequences prepared as described herein can also be
used to introduce expression of a particular FIE nucleic acid to
enhance or increase endogenous gene expression. For instance,
polycomb genes are known to control cell cycling. Enhanced
expression can therefore be used to control plant morphology by
controlling whether or not cell division takes place in desired
tissues or cells. Enhanced expression can also be used, for
instance, to increase vegetative growth by preventing the plant
from setting seed. Where overexpression of a gene is desired, the
desired gene from a different species may be used to decrease
potential sense suppression effects.
[0080] One of skill will recognize that the polypeptides encoded by
the genes of the invention, like other proteins, have different
domains which perform different functions. Thus, the gene sequences
need not be full length, so long as the desired functional domain
of the protein is expressed.
[0081] Modified protein chains can also be readily designed
utilizing various recombinant DNA techniques well known to those
skilled in the art and described in detail, below. For example, the
chains can vary from the naturally occurring sequence at the
primary structure level by amino acid substitutions, additions,
deletions, and the like. These modifications can be used in a
number of combinations to produce the final modified protein
chain.
Preparation of Recombinant Vectors
[0082] To use isolated sequences in the above techniques,
recombinant DNA vectors suitable for transformation of plant cells
are prepared. Techniques for transforming a wide variety of higher
plant species are well known and described in the technical and
scientific literature. See, for example, Weising et al. Ann. Rev.
Genet. 22:421-477 (1988). A DNA sequence coding for the desired
polypeptide, for example a cDNA sequence encoding a full length
protein, will preferably be combined with transcriptional and
translational initiation regulatory sequences which will direct the
transcription of the sequence from the gene in the intended tissues
of the transformed plant.
[0083] For example, for overexpression, a plant promoter fragment
may be employed which will direct expression of the gene in all
tissues of a regenerated plant. Such promoters are referred to
herein as "constitutive" promoters and are active under most
environmental conditions and states of development or cell
differentiation. Examples of constitutive promoters include the
cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium
tumafaciens, and other transcription initiation regions from
various plant genes known to those of skill. Such genes include for
example, ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol.
33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147,
Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene
encoding stearoyl-acyl carrier protein desaturase from Brassica
napus (Genbank No. X74782, Solocombe et al. Plant Physiol.
104:1167-1176 (1994)), GPc1 from maize (GenBank No. X15596,
Martinez et al. J. Mol. Biol 208:551-565 (1989)), and Gpc2 from
maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol.
33:97-112 (1997)).
[0084] Alternatively, the plant promoter may direct expression of
the FIE nucleic acid in a specific tissue or may be otherwise under
more precise environmental or developmental control. Examples of
environmental conditions that may effect transcription by inducible
promoters include anaerobic conditions, elevated temperature, or
the presence of light. Such promoters are referred to here as
"inducible" or "tissue-specific" promoters. One of skill will
recognize that a tissue-specific promoter may drive expression of
operably linked sequences in tissues other than the target tissue.
Thus, as used herein a tissue-specific promoter is one that drives
expression preferentially in the target tissue, but may also lead
to some expression in other tissues as well.
[0085] Examples of promoters under developmental control include
promoters that initiate transcription only (or primarily only) in
certain tissues, such as fruit, seeds, or flowers. Promoters that
direct expression of nucleic acids in ovules, flowers or seeds are
particularly useful in the present invention. As used herein a
seed-specific promoter is one which directs expression in seed
tissues, such promoters may be, for example, ovule-specific (which
includes promoters which direct expression in maternal tissues
or-the female gametophyte, such as egg cells or the central cell),
embryo-specific, endosperm-specific, integument-specific, seed
coat-specific, or some combination thereof. Examples include a
promoter from the ovule-specific BEL1 gene described in Reiser et
al. Cell 83:735-742 (1995) (GenBank No. U39944). Other suitable
seed specific promoters are derived from the following genes: MAC1
from maize (Sheridan et al. Genetics 142:1009-1020 (1996), Cat3
from maize (GenBank No. L05934, Abler et al. Plant Mol. Biol.
22:10131-1038 (1993), the gene encoding oleosin 18 kD from maize
(GenBank No. J05212, Lee et al. Plant Mol. Biol. 26:1981-1987
(1994)), vivparous-1 from Arabidopsis (Genbank No. U93215), the
gene encoding oleosin from Arabidopsis (Genbank No. Z17657), Atmyc1
from Arabidopsis (Urao et al. Plant Mol. Biol. 32:571-576 (1996),
the 2 s seed storage protein gene family from Arabidopsis
(Conceicao et al. Plant 5:493-505 (1994)) the gene encoding oleosin
20 kD from Brassica napus (GenBank No. M63985), napA from Brassica
napus (GenBank No. J02798, Josefsson et al. JBL 26:12196-1301
(1987), the napin gene family from Brassica napus (Sjodahl et al.
Planta 197:264-271 (1995), the gene encoding the 2S storage protein
from Brassica napus (Dasgupta et al. Gene 133:301-302 (1993)), the
genes encoding oleosin A (Genbank No. U09118) and oleosin B
(Genbank No. U09119) from soybean and the gene encoding low
molecular weight sulphur rich protein from soybean (Choi et al. Mol
Gen, Genet. 246:266-268 (1995)).
[0086] In addition, the promoter sequences from the FIE genes
disclosed here can be used to drive expression of the FIE
polynucleotides of the invention or heterologous sequences. The
sequences of the promoters are identified below.
[0087] If proper polypeptide expression is desired, a
polyadenylation region at the 3'-end of the coding region should be
included. The polyadenylation region can be derived from the
natural gene, from a variety of other plant genes, or from
T-DNA.
[0088] The vector comprising the sequences (e.g., promoters or
coding regions) from genes of the invention will typically comprise
a marker gene which confers a selectable phenotype on plant cells.
For example, the marker may encode biocide resistance, particularly
antibiotic resistance, such as resistance to kanamycin, G418,
bleomycin, hygromycin, or herbicide resistance, such as resistance
to chlorosulfuron or Basta.
Production of Transgenic Plants
[0089] DNA constructs of the invention may be introduced into the
genome of the desired plant host by a variety of conventional
techniques. For example, the DNA construct may be introduced
directly into the genomic DNA of the plant cell using techniques
such as electroporation and microinjection of plant cell
protoplasts, or the DNA constructs can be introduced directly to
plant tissue using ballistic methods, such as DNA particle
bombardment.
[0090] Microinjection techniques are known in the art and well
described in the scientific and patent literature. The introduction
of DNA constructs using polyethylene glycol precipitation is
described in Paszkowski et al. Embo J. 3:2717-2722 (1984).
Electroporation techniques are described in Fromm et al. Proc.
Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation
techniques are described in Klein et al. Nature 327:70-73
(1987).
[0091] Alternatively, the DNA constructs may be combined with
suitable T-DNA flanking regions and introduced into a conventional
Agrobacterium tumefaciens host vector. The virulence functions of
the Agrobacterium tumefaciens host will direct the insertion of the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria. Agrobacterium tumefaciens-mediated
transformation techniques, including disarming and use of binary
vectors, are well described in the scientific literature. See, for
example Horsch et al. Science 233:496-498 (1984), and Fraley et al.
Proc. Natl. Acad. Sci. USA 80:4803 (1983).
[0092] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant which possesses the transformed genotype and thus the
desired phenotype such as increased seed mass. Such regeneration
techniques rely on manipulation of certain phytohormones in a
tissue culture growth medium, typically relying on a biocide and/or
herbicide marker which has been introduced together with the
desired nucleotide sequences. Plant regeneration from cultured
protoplasts is described in Evans et al., Protoplasts Isolation and
Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan
Publishing Company, New York, 1983; and Binding, Regeneration of
Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985.
Regeneration can also be obtained from plant callus, explants,
organs, or parts thereof. Such regeneration techniques are
described generally in Klee et al. Ann. Rev. of Plant Phys.
38:467486 (1987).
[0093] The nucleic acids of the invention can be used to confer
desired traits on essentially any plant. Thus, the invention has
use over a broad range of plants, including species from the genera
Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus,
Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita,
Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus,
Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus,
Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea,
Oryza, Panieum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum,
Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis,
Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis,
Vigna, and Zea.
[0094] One of skill will recognize that after the expression
cassette is stably incorporated in transgenic plants and confirmed
to be operable, it can be introduced into other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed.
[0095] Seed obtained from plants of the present invention can be
analyzed according to well known procedures to identify plants with
the desired trait. If antisense or other techniques are used to
control Fie gene expression, Northern blot analysis can be used to
screen for desired plants. In addition, the presence of
fertilization independent reproductive development can be detected.
Plants can be screened, for instance, for the ability to form
embryo-less seed, form seed that abort after fertilization, or set
fruit in the absence of fertilization. These procedures will
depend, part on the particular plant species being used, but will
be carried out according to methods well known to those of
skill.
[0096] The following Examples are offered by way of illustration,
not limitation.
EXAMPLE 1
[0097] The following example describes methods used to identify the
fie mutants. The methods described here are generally as described
in Ohad et al., Proc. Natl. Acad. Sci. USA 93:5319-5324 (1996).
Materials and Methods
Growth and Phenotype of Plants.
[0098] Plants were grown under low humidity conditions (less than
50%) in glass houses under 16 hr light/8 hr dark photoperiods
generated by supplemental lighting. Plants were grown at high
humidity (greater than 80%) in a lighted incubator (Percival,
Boone, Iowa).
[0099] To test for fertilization-independent development, flower
buds from plants that had not yet begun to shed pollen (stage 12;
(Smyth, D. R., et al., Plant Cell 2:755-767 (1990))) were opened,
immature anthers were removed, and the flower bud was covered with
a plastic bag. Seven days later, the silique was measured,
dissected, and the number of seed-like structures and degenerating
ovules were counted. To determine the frequency of seed abortion
following fertilization, siliques were harvested 10 days after
self-pollination, dissected, and wild-type and aborted seeds were
counted.
Genetic Mapping.
[0100] Heterozygous FIE/fie (Landsberg erecta ecotype) plants were
crossed as males with female plants (Columbia ecotype). Because the
mutant fie allele is only transmitted through the male gametophyte,
FIE/fie progeny were crossed as males a second time to female
gl1/gl1 (Columbia ecotype) plants. Approximately fifty-five progeny
were scored for the segregation of the wild-type FIE and mutant fie
alleles and for alleles of molecular markers as described
previously (Bell, C., et al., Genomics 19:137-144 (1994)). This
analysis indicated that fie3 is located at approximately position
30 on chromosome three, fie2 is located at approximately position
65 on chromosome two, and fie1 is located at approximately position
2 on chromosome one. Genetic recombination frequencies and map
distances were calculated according to Koornneef and Stam
(Koornneef, M., et al., Methods in Arabidopsis Research, pp. 83-99
(1992)) and Kosambi (Kosambi, Ann. Eugen., 12: 172-175 (1944)).
Light Microscopy.
[0101] Nomarski photographs of whole-mount embryos and endosperm
were obtained by fixing longitudinally slit siliques in an
ethanol:acetic acid (9:1) solution overnight, followed by two
washes in 90% and 70% ethanol, respectively. Siliques were cleared
with a chloral hydrate:glycerol:water solution (8:1:2, w:v:v)
(Berleth, T., et al., Devel 118: 575-587 (1993)). Whole mount
preparations were fixed and stained with hematoxylin (Beeckman, T.,
et al., Plant Mol Biol Rep 12: 37-42 (1994)). Embryo and endosperm
were photographed with a Zeiss Axioskop microscope (Carl Zeiss,
Inc., Oberkochen, Germany) using Nomarski optics that permits
visualization of optical sections within the seed.
GUS Histochemical Assays.
[0102] GUS activity was detected histochemically as described
previously by (Beeckman, T., et al., Plant Mol Biol Rep 12: 37-42
(1994)).
Image Processing.
[0103] Photographs were scanned using a Microtek scanner. Pictures
were processed for publication using Adobe Photoshop 3.0 and
printed on a Tektronix Phaser 400 color printer.
Results
Isolation of Mutant Lines.
[0104] To begin to understand mechanisms that initiate reproductive
development, we generated mutant Arabidopsis plants that undergo
several reproductive processes in the absence of fertilization.
Arabidopsis plants homozygous for the conditional male sterile pop1
mutation (Preuss, D., et al., Genes and Devel 7: 974-985 (1993))
were used as the parental strain (Landsberg erecta ecotype).
Fertility in pop1 plants is sensitive to humidity because pop1
pollen do not hydrate properly due to a defect in wax biosynthesis.
When grown at permissive condition, high relative humidity
(>80%), pop1 plants were male fertile and produced long siliques
with many viable seeds. By contrast, when grown at non-permissive
condition, low relative humidity (<50%), pop1 plants were male
sterile and produced short siliques with no seeds. Thus, silique
elongation is a marker for reproductive events. To isolate
mutations, homozygous pop1 seeds were mutagenized with
ethylmethansulfonate (EMS) and approximately 50,000 M1 plants were
screened for silique elongation at non-permissive conditions. Rare
M1 plants were identified that displayed heterozygous sectors with
elongated siliques. These plants were transferred to permissive
conditions to insure the production of viable M2 seed. Plants from
M2 and M3 families grown at non-permissive conditions were
rechecked for non-sectored silique elongation. To eliminate any
effects of the pop1 mutation, or other EMS-induced lesions on the
mutant phenotype, mutant plants were backrossed twice, as males, to
wild-type plants. After removing the pop1 mutation,
fertilization-independent phenotypes were confirmed after manual
removal of anthers from immature flowers before pollen was shed. A
total of twelve lines were identified that displayed elongated
siliques in the absence of fertilization.
Fertilization-Independent Endosperm, Seed Coat and Silique
Development.
[0105] In a representative line chosen for further study,
heterozygous plants produced by back crosses to wild-type plants
generated elongated siliques after anther removal with numerous
seed-like structures. These results indicated that heterozygous
mutant plants were capable of silique elongation and seed-like
structure development in the absence of fertilization. We compared
the development of the mutant seed-like structures to that of
wild-type seeds. After fertilization, the endosperm nucleus
replicated and daughter nuclei migrated into the expanding central
cell. Ultimately, a syncytium of endosperm nuclei was produced.
Nuclear divisions of the endosperm preceded the zygotic divisions
that formed the globular stage embryo. Embryo, endosperm or seed
coat development did not occur in wild-type plants in the absence
of fertilization. Development of the ovule and female gametophyte
in heterozygous mutant plants was normal. Just prior to flower
opening, female gametophytes in these plants contained a single,
prominent central cell nucleus. Subsequently, in the absence of
fertilization, central cells with two large nuclei were detected.
Further divisions resulted in the production of additional nuclei
that migrated into the expanded central cell. Later in development,
a nuclear syncytium was formed with abundant endosperm nuclei.
These results indicated that the central cell in mutant female
gametophytes initiated endosperm development in the absence of
fertilization. We have named this mutation fie for
fertilization-independent endosperm. By contrast, replication of
other nuclei in fie female gametophytes (egg, synergid, or
antipodal) was not detected. Thus, the fie mutation specifically
affects replication of the central cell nucleus.
[0106] We analyzed the frequency of multinucleate central cell
formation in fie female gametophytes by comparing the percentage of
multinucleate central cells at three, five, and six days after
emasculation of heterozygous FIE/fie and control wild-type flowers.
At each time point, only 3% to 5% of wild-type central cells had
more than one nucleus. Because none had more than two nuclei, most
likely, these represented central cells with haploid nuclei that
had not fused during female gametophyte development. By contrast,
the percentage of central cells in female gametophytes from FIE/fie
siliques with two or more nuclei increased from 21% to 47% over the
same time period. These results indicated that the fie mutation
caused a significant increase in formation of multinucleate central
cells in the absence of fertilization. The fact that close to 50%
of the female gametophytes in heterozygous plants had multinucleate
central cells suggested that fie is a gametophytic mutation because
a 1:1 segregation of wild-type and mutant fie alleles occurs during
meiosis.
[0107] We compared the fertilization-independent development of the
maternal seed coat in FIE/fie seed-like structures to that of
fertilized wild-type seeds. The seed coat in wild-type Arabidopsis
is generated by the integuments of the ovule and surrounds the
developing embryo and endosperm. Similarly, FIE/fie ovule
integuments formed a seed coat that surrounded the developing
mutant endosperm. These results indicated that the fie mutation
activated both endosperm development and maternal sporophytic seed
coat and silique differentiation that support reproduction. No
other effects on sporophytic growth and development were detected
in FIE/fie plants. The fie3 Mutant Allele Is Not Transmitted by the
Female Gametophyte to the Next Generation.
[0108] To understand the mode of inheritance of the fie mutation,
we analyzed the progeny of reciprocal crosses. FIE3/fie3 females,
crossed to wild-type males, produced siliques with approximately
equal numbers of viable seeds with normal green embryos and
nonviable white seeds with embryos aborted at the heart stage
(344:375, 1:1, c2=1.3, P>0.2). Viable seeds from this cross were
germinated and all 120 F1 progeny generated were wild-type. That
is, none of the F1 progeny had significant levels of F2 aborted
seeds in their siliques after self-pollination. Nor did the F1
progeny demonstrate fertilization-independent development. This
indicated that presence of the fie mutant allele in the female
gametophyte, even when the male provided a wild-type allele,
resulted in embryo abortion. Thus, the fie mutation is not
transmitted by the female gametophyte to the next generation. To
study transmission of fie through the male gametophyte, we
pollinated female wild-type plants with pollen from male FIE3/fie3
plants. Siliques from these crosses contained no aborted F1 seed.
F1 plants were examined and a 1:1 segregation of wild-type and
FIE3/fie3 genotype was observed (62:58, c2=0.13, P>0.5). This
indicated that wild-type and mutant fie3 alleles were transmitted
by the male gametophyte with equal efficiency. That is, fie does
not affect male gametophyte, or pollen grain, function. Results
from reciprocal crosses were verified by analyzing the progeny from
self-pollinated FIE3/fie3 plants. Self-pollinated siliques
displayed 1:1 segregation of normal and aborted seeds (282:286,
c2=0.03, P>0.8). Viable seed from self-pollinated siliques were
germinated and a 1:1 (71:64, c2=0.36, P>0.5) segregation of
wild-type and FIE3/fie3 progeny was observed. These results
confirmed that inheritance of a fie mutant allele by the female
gametophyte resulted in embryo abortion, and that inheritance of a
fie mutant allele by the male gametophyte did not affect pollen
function. Thus, the wild-type FIE3 allele probably carries out a
function unique to the female gametophyte and does not appear to be
needed for male fertility.
[0109] In contrast, fie1 and fie2 mutant alleles were transmitted
at low frequencies (about 1% of normal) through the female
gametophyte. In this way, fie1 homozygous mutants and fie2
homozygous mutants were obtained that appeared to display normal
vegetative growth and development.
Discussion
[0110] In wild-type plants, fertilization initiates embryogenesis
and endosperm formation, and activates maternal seed coat and
silique development. The results presented here indicate that
specific aspects of plant reproductive development can occur in
FIE/fie plants in the absence of fertilization. These include
silique elongation, seed coat formation, and endosperm development.
Morphological analysis shows that early aspects of
fertilization-independent fie endosperm development closely
resemble fertilized wild-type endosperm development. First, the fie
central cell nucleus is stimulated to undergo replication. Second,
nuclei that are produced migrate from the micropylar end of the
central cell and take up new positions in the central cell. Third,
the developing fie central cell expands to form an endosperm
cavity. Thus, the requirement for fertilization to initiate these
early events in endosperm formation has been eliminated by the fie
mutation. This suggests that FIE plays a role in a signal
transduction pathway that links fertilization with the onset of
central cell nuclear replication and early endosperm
development.
Mechanisms for Regulation of Endosperm Development by FIE.
[0111] One can envision two possible mechanisms for how FIE
regulates replication of the central cell nucleus in response to
fertilization. The protein encoded by the FIE gene may be involved
in a positive regulatory interaction. In this model, FIE is
required for the central cell to initiate endosperm development.
Normally, fertilization is needed for the presence of active FIE
protein. The fie mutation results in the presence of active protein
in the absence of fertilization. Alternatively, FIE may by involved
in a negative regulatory interaction. In this model, the function
of FIE protein is to prevent the central cell from initiating
endosperm development, and fertilization results in the
inactivation of FIE protein. The fie mutation results in the
production of inactive protein, so that fertilization is no longer
required to initiate endosperm development. However,
complementation experiments using transgenic plants indicate that
FIE1 and FIE3 alleles are dominant over their respective mutant
alleles. This indicates that the wild-type allele is involved in a
negative regulatory interaction. Recently, it has been shown that
cyclin-dependent kinase complexes, related to those that function
in mammals, control the induction of DNA synthesis and mitosis in
maize endosperm (Grafi, G. et al., Science 269: 1262-1264 (1995)).
Because fie stimulates replication of the central cell, fie may,
either directly or indirectly, impinge upon cell cycle control of
the central cell nucleus, allowing replication to take place in the
absence of fertilization.
Communication Between the Fie Female Gametophyte and the
Sporophytic Ovule and Carpels.
[0112] The analysis of FIE/fie mutant plants has provided clues
about interactions between endosperm and maternal sporophytic
tissues. FIE/fie ovule integuments surrounding a mutant fie female
gametophyte initiate seed coat development, whereas FIE/fie
integuments in contact with a quiescent wild-type female
gametophyte do not develop. This suggests that the FIE/fie ovule
integuments initiate seed coat differentiation in response to a
signal produced by the fie female gametophyte. We propose that the
source of the signal is the mutant fie central cell that has
initiated endosperm development, although we cannot rule out the
participation of other cells in the fie female gametophyte. In
wild-type plants, most likely, fertilization of the central cell
produces an endosperm that activates seed coat development. This is
consistent with experiments showing that the maize endosperm
interacts with nearby maternal cells (Miller, M. E., et al., Plant
Cell 4:297-305 (1992)). FIE/fie plants also display
fertilization-independent elongation of the ovary to form the
silique. We propose that a signal is produced by the developing
seed-like structures to initiate silique elongation. This is in
agreement with experiments suggesting that seeds are the source of
hormones, auxins and gibberellins, that activate fruit development
(Lee, T. D. Plant Reproductive Ecology, pp. 179-202 (1988)). Taken
together, these results suggest that the fertilized female
gametophyte activates maternal developmental programs.
Relationship Between Fie and Apomixis.
[0113] Certain plant species display aspects of
fertilization-independent reproductive development, including
apomictic generation of embryo and endosperm, and development of
the maternal seed coat and fruit (reviewed in (Koltunow, a. Plant
Cell 5: 1425-1437 (1993)). The fie mutation reveals that
Arabidopsis, a sexually reproducing plant, has the genetic
potential for aspects of fertilization-independent reproductive
development. It is not known whether the mechanism of
fertilization-independent endosperm development conferred by the
fie mutation is the same as autonomous endosperm formation observed
in certain apomictic plant species. However, the fact that the fie
phenotype is caused by a single genetic locus substantiates the
view that the number of genetic differences between sexually and
asexually reproducing plants is small (Koltunow, a. M., et al.,
Plant Physiol 108:1345-1352 (1995)).
EXAMPLE 2
[0114] This example describes cloning of two Fie genes, Fie1 and
Fie3.
Cloning The FIE3 Gene.
[0115] a. Mapping the position of the fie3 gene genetically. The
fie3 mutation was initially mapped to position 30 on chromosome 3,
between AXR2 (auxin resistant dwarf) and EMB29 (embryo lethal).
Next, two sets of F2 plants with recombination breakpoints in the
fie3 gene region were obtained. One set was between emb29 and fie3
and the other set was between axr2 and fie3. As shown in FIG. 1A,
these recombinants were used to map the fie3 gene relative to
molecular markers (NDR, CH18, CH18S, BO20, AG20, KN1 and E13F12)
that were obtained from overlapping YAC (yUP13F12), BAC (T1B4 and
T4N1) and cosmid clones (FIG. 1A). YAC and BAC clones were obtained
from the Arabidopsis Stock Center (Ohio State University, USA).
Cosmid subclones were generated in my laboratory. As shown in FIG.
1A and 1B, this genetic analysis indicates that the fie3 gene
resides within the 25 Kb region between the BO20 and AG20
markers.
[0116] b. Mapping the position of the fie3 gene by complementation
experiments. To more precisely localize the fie3 gene, we analyzed
a series of overlapping cosmid clones (BO20, GM15, AG20 and EI12)
that span the fie3 gene region. Each cosmid clone was tested for
its ability to complement the fie3 mutation in transgenic plants.
Only cosmid GM 15complemented the fie3 mutation (FIG. 1A). These
results indicate that an essential portion of the fie3 gene is in
the 10 Kb region that is unique to cosmid GM 15. As shown in FIG.
1B, we have cloned DNA that spans this essential portion of the
fie3 gene and have determined its DNA sequence. As shown in FIG. 2,
analysis of the sequence using the GENSCANW program revealed a gene
with an open reading frame. The predicted cDNA sequence and
predicted amino acid sequence are shown in SEQ ID NO:3 and SEQ ID
NO:4, respectively. Comparing the predicted amino acid sequence to
those in public data bases revealed significant homology to the
WD40 family of Polycomb Group genes, and in particular, the "extra
sex combs" gene in Drosophila. FIG. 3 shows the position of primers
used to PCR amplify this region. SEQ ID NO:5 provides the genomic
DNA sequence of the WD40/Polycomb gene, plus approximately 3.8 Kb
of 5'-flanking sequences and 0.3 Kb of 3'-flanking sequences, plus
the sequence of primers used to PCR amplify this region. The
transcription start site in SEQ ID NO:5 is at position 3,872. Thus,
the promoter sequence for FIE3 is located between position 1 and
3,872. The 5'-flanking and 3'-flanking regions contain regulatory
DNA sequences that control the expression of this gene.
Cloning the FIE1 Gene.
[0117] a. Mapping the position of the FIE1 gene genetically. The
fie1 mutation was initially mapped to position 3 on chromosome 1,
between AXR3 (auxin resistant dwarf) and EMB60 (embryo lethal).
Next, two sets of F2 plants with recombination breakpoints in the
FIE1 gene region were obtained. One set was between emb60 and fie3
and the other set was between axr3 and fie3. These recombinants
were used to map the fie3 gene relative to molecular markers (FIG.
4) that were obtained from an overlapping series of YAC and BAC
clones from the Arabidopsis Stock Center (Ohio State University,
USA).
[0118] b. Mapping the position of the FIE1 gene by complementation
experiments. To more precisely localize the FIE1 gene, a series of
overlapping cosmid clones (2-9, 6-22, 2-8) that span the FIE1 gene
region were analyzed (FIG. 4). Each cosmid clone was tested for its
ability to complement the fie1 mutation in transgenic plants. Only
cosmid 6-22 complemented the fie1 mutation. The cosmids were
analyzed for genes with open reading frames. FIG. 5 shows that a
single gene was present on the complementing cosmid (6-22) that was
not fully encoded on either of the non-complementing cosmids (2-9
and 2-8). By RTPCR and 5'-race, the cDNA sequence of this gene and
predicted amino acid of its protein were obtained (SEQ ID NO: 1 and
SEQ ID NO:2, respectively). Comparison of the predicted amino acid
sequence to those in public data bases revealed significant
homology to the SET family of Polycomb Group Genes (e.g., Enhancer
of Zeste in Drosophila and Curly Leaf in Arabiopsis). We compared
the wild-type and fie1 mutant sequence in 6-22. The only difference
is a single base pair change that creates a premature translation
stop codon in the 5 '-end of the set/polycomb group gene. The base
pair change is at position 823 (C->T) on the cDNA sequence shown
in SEQ ID NO: 1.
[0119] SEQ ID NO:6 shows the genomic sequence of the FIE1
SET/polycomb gene, plus approximately 2 Kb of 5'-flanking sequences
and approximately 0.7 Kb of 3'-flanking sequences. The translation
start site is located at position 2036 of SEQ ID NO:6. Thus, the
promoter sequence is located between position 1 and position
2036.
[0120] The above examples are provided to illustrate the invention
but not to limit its scope. Other variants of the invention will be
readily apparent to one of ordinary skill in the art and are
encompassed by the appended claims. All publications, patents, and
patent applications cited herein are hereby incorporated by
reference.
Sequence CWU 1
1
* * * * *
References