U.S. patent application number 12/210918 was filed with the patent office on 2009-06-11 for indeterminate gametophyte 1 (ig1), mutations of ig1, orthologs of ig1, and uses thereof.
This patent application is currently assigned to Carnegie Institution of Washington. Invention is credited to Matthew Evans.
Application Number | 20090151025 12/210918 |
Document ID | / |
Family ID | 36121686 |
Filed Date | 2009-06-11 |
United States Patent
Application |
20090151025 |
Kind Code |
A1 |
Evans; Matthew |
June 11, 2009 |
Indeterminate Gametophyte 1 (ig1), Mutations of ig1, Orthologs of
ig1, and Uses Thereof
Abstract
This invention provides the genes for indeterminate gametophyte1
(ig1), mutants of ig1, homologs of ig1, and orthologs of ig1, as
well as the proteins encoded by these genes. This invention also
provides compositions and methods which utilize these genes and
proteins.
Inventors: |
Evans; Matthew; (Stanford,
CA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Assignee: |
Carnegie Institution of
Washington
Washington
DC
|
Family ID: |
36121686 |
Appl. No.: |
12/210918 |
Filed: |
September 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11030329 |
Jan 7, 2005 |
7439416 |
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12210918 |
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60534967 |
Jan 9, 2004 |
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Current U.S.
Class: |
800/298 ;
435/471; 435/6.16; 530/387.3 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8287 20130101 |
Class at
Publication: |
800/298 ;
530/387.3; 435/6; 435/471 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07K 16/18 20060101 C07K016/18; C12Q 1/68 20060101
C12Q001/68; C12N 15/82 20060101 C12N015/82 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] This invention was partially made with government support
under United States National Science Foundation Grant No.
IBN-0296074, entitled "Genetic Control of Polar Nuclei Number in
Maize." The U.S. government has certain rights in this invention.
Claims
1-8. (canceled)
9. An isolated polypeptide produced by the method of claim 7.
10. An isolated polypeptide or protein selected from the group
consisting of: (a) an isolated polypeptide comprising the amino
acid sequence of IG1, IG1-O, IG1-MUM, or SEQ ID NO: 2; (b) an
isolated polypeptide comprising a fragment of at least 6 amino
acids of IG1, IG1-O, IG1-MUM, or SEQ ID NO: 2; (c) an isolated
polypeptide comprising conservative amino acid substitutions of
IG1, IG1-O, IG1-MUM, or SEQ ID NO: 2; (d) naturally occurring amino
acid sequence variants of SEQ ID NO: 2; and (e) an isolated
polypeptide exhibiting at least about 80%, or at least about 85%,
or at least about 90%, or at least about 95%, or at least about 99%
amino acid sequence identity with IG1, IG1-O, IG1-MUM, or SEQ ID
NO: 2.
11-17. (canceled)
18. A method of identifying and isolating an ortholog of ig1 in a
non-Zea mays plant species, said method comprising using at least
one nucleic acid of claim 1 as a nucleic acid probe.
19. A method of modulating a plant cell comprising an ig1 gene,
said method comprising the step of introducing into said plant cell
an isolated nucleic acid molecule according to claim 1, whereby the
function and/or structure of the ig1 gene is modulated.
20. The method according to claim 19, wherein the isolated nucleic
acid molecule is a knock-out or knock-in construct.
21. A transgenic knock-out plant comprising disruption in the
endogenous ig1 gene, wherein said disruption has been introduced
into its genome by homologous recombination with a DNA targeting
construct such that the targeting construct is stably integrated in
the genome of said plant, wherein the disruption of the ig1 gene
results in a reduction of production of endogenous ig1 RNA
levels.
22-23. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent
application Ser. No. 60/534,967 filed Jan. 9, 2004, which is herein
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates generally to the genetic control of
polar nuclei number in plants. More specifically, this invention
relates to the indeterminate gametophyte1 (ig1) gene cloned from
Zea mays, mutations of the ig gene, and orthologs of the ig1 gene
isolated from other plant species. The invention also relates to
constructs and vectors comprising said genes, recombinant
prokaryotic and eukaryotic cells comprising said genes and the use
of said constructs and vectors to create transgenic plant cells,
plant tissues and whole plants. In addition, this invention also
relates to methods of using the genes to produce male sterility for
plant breeding and for generating androgenetic progeny in maize
(i.e., corn, Zea mays) and other plants species.
BACKGROUND OF THE INVENTION
[0004] All publications and patent applications herein are
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.
[0005] The following description includes information that may be
useful in understanding the present invention. It is not an
admission that any of the information provided herein is prior art
or relevant to the presently claimed inventions, or that any
publication specifically or implicitly referenced is prior art.
[0006] Plants have two phases to their life cycle: the diploid
phase, or sporophyte, that ends in meiosis to produce haploid
cells, and the haploid phase, or gametophyte, in which mitotic
proliferation to produce a haploid plant includes differentiation
of a subset of cells as gametes prepared for fertilization to
reconstitute a diploid organism. Both the egg-producing haploid
plant, the megagametophyte, and the sperm-producing haploid plant,
the microgametophyte, are genetically active, hence gametophyte
phenotype reflects haploid allele type. In contrast, gamete
properties in animals are determined almost entirely by gene
expression in progenitor diploid cells. Flowering plants are
further distinguished from animals by the process of double
fertilization. Two cells of the female gametophyte, the egg and
central cell, are fertilized by two typically genetically identical
sperm cells of the male gametophyte to produce the embryo and
endosperm, respectively, of the seed.
[0007] In angiosperms the polygonum type of megasporogenesis is
most common, occurring in 70% of species, including Arabidopsis and
maize. In these plants the chalazal megaspore, one of the meiotic
products of the megaspore mother cell, undergoes three rounds of
free nuclear division followed by cellularization to give rise to a
seven-celled embryo sac (FIG. 1). The free nuclear divisions are
invariant in number, tightly regulated as indicated by their
synchrony, and accompanied by stereotypical nuclear migrations. The
angiosperm female gametophyte, called the embryo sac, consists of
four cell types: synergids, antipodals, egg, and central cell
(Drews et al., 1998; Grossniklaus and Schneitz, 1998; Yang and
Sundaresan, 2000).
[0008] Despite the limited size of the gametophytes in flowering
plants a very large number of genes are essential for haploid
development. The gametophytes undergo mitosis, cell growth, and
organelle biogenesis. Cells exchange signals for differentiation
and for interaction with the surrounding diploid tissues.
Gametophytes acquire attributes important in self vs. non-self
recognition during pollination, and gametes acquire factors
required for successful fertilization. Many basic cellular
processes are required in gametophytes (e.g. tip growth of cells in
the pollen tube of the microgametophyte; gamete fusion; cell-cell
attraction; mitosis; cytokinesis; intracellular trafficking; cell
death).
[0009] Demonstration that the entire genome, rather than specific
chromosomes or a few chromosomal segments, are important comes from
classical cytogenetic analyses in maize. In the .about.3000 cM
maize genetic map, there are only a few regions in which short
deletions still permit production of viable gametes, i.e. 2 cM
deletions from anther ear1 to bronze2 on chromosome 1 and from
shrunken1 to bronze1 on chromosome 9 (Patterson, E. B. 1978). More
convincingly Patterson and others exploited more than 850
reciprocal translocation stocks, representing 1700 deficiencies, to
establish that all caused pollen abortion and about 90% resulted in
megagametophyte lethality (Coe et al., 1988). Presumably some
nutritional defects lethal to pollen, which is sealed from
metabolic exchange with the surrounding diploid tissues for days,
can be compensated for in megagametophytes, which continue to
absorb nutrients from their diploid mother.
[0010] Gametophyte mutations result in characteristic phenotypes
and modes of transmission. Heterozygotes for female gametophyte
mutations are expected to have reduced fertility, because half of
the embryo sacs inherit the mutant allele. Male gametophyte
mutations do not cause reduced seed set because there is normally
excess pollen. However, for both male and female gametophyte
mutations the mutant allele is found at a reduced frequency in
progeny when the affected gamete is involved in the cross. Because
these mutations act after meiosis they are transmitted poorly or
not at all, as are loci linked to them.
[0011] Mutations that act in the gametophyte generation have been
identified recently in several species by screening for poor
transmission through the gametes and for semisterility of mutant
heterozygotes (Feldmann et al., 1997; Moore et al., 1997; Howden et
al., 1998; Christensen et al., 1998; Christensen et al., 2002;
Shimizu and Okada, 2000). The mutants fall into several categories:
gametophytes that arrest early in development; well developed, but
morphologically aberrant gametophytes and morphologically normal
gametophytes that nevertheless fail to function. All developmental
steps depicted in FIG. 1 are represented by at least one mutant.
The largest class of mutants contains those that arrest development
early. Although some of these mutations may be in genes with
specific roles during embryo sac development, many of them likely
are required for functions in all cell types. One example of this
is PROLIFERA of Arabidopsis (Springer et al., 1995). PRO shows
homology to MCM2-3-5 genes required for DNA replication and cell
cycle control. The mutant phenotype, sequence, and expression
pattern of PRO suggest it is required in all dividing cells.
SUMMARY OF THE INVENTION
[0012] The instant invention involves the maize indeterminate
gametophyte1 (ig1) gene and the protein product of the gene. The
instant invention provides a partial nucleic acid sequence for the
maize indeterminate gametophyte1 (ig1) gene (SEQ ID NO: 1) and the
corresponding protein product of the partial gene (SEQ ID NO: 2).
The instant invention is further directed to mutations of the
indeterminate gametophyte1 (ig1) gene, such as ig1-O and ig1-mum,
and the protein products of those genes. The instant invention is
further directed to orthologs of the indeterminate gametophyte1
(ig1) gene, wherein such orthologs are identified and cloned in
other plant species, such as rice, by using the ig1 gene and
mutations of the ig1 gene isolated from maize.
[0013] One object of the present invention is to provide maize ig1
nucleic acids and the IG1 protein produced thereby. The present
invention also provides ig1 nucleic acids of Zea mays and the IG1
proteins they produce. The invention includes isolated nucleic acid
molecules selected from the group consisting of isolated nucleic
acid molecules that encode an amino acid sequence of IG1, IG1-O,
IG1-MUM and orthologs of IG1. The present invention provides an
isolated nucleic acid molecule that encodes a fragment of at least
6 amino acids of SEQ ID NO: 2, and an isolated nucleic acid
molecule which hybridizes to a nucleic acid molecule comprising SEQ
ID NO: 1. A nucleic acid molecule can include functional
equivalents of natural nucleic acid molecules encoding a peptide,
polypeptide or protein of the present invention. Functional
equivalents of natural nucleic acid molecules can include, but are
not limited to, natural allelic variants and modified nucleic acid
molecules in which nucleotides have been inserted, deleted,
substituted, and/or inverted in such a manner that such
modifications do not substantially interfere with the nucleic acid
molecule's ability to encode a molecule of the present invention.
Said amino acid substitutions may be conservative or
non-conservative.
[0014] Preferred functional equivalents include sequences capable
of hybridizing under stringent conditions (i.e., sequences having
at least about 70% identity), to at least a portion of an IG1
peptide, polypeptide or protein encoding nucleic acid molecule
according to conditions described in Sambrook et al., (1989)
Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory Press. By stringent conditions it is meant that
hybridization is carried in a buffer consisting of 0.1% SDS, 200 mM
NaCl, 6 mM Na.sub.2HPO.sub.4, 2 mM EDTA at pH=6.8. More preferred
functional equivalents include sequences capable of hybridizing
under highly stringent conditions (i.e., sequences having at least
about 90% identity), to at least a portion of an IG1 peptide,
polypeptide or protein encoding nucleic acid molecule. By highly
stringent conditions it is meant that hybridization is carried in a
buffer consisting of 0.1% SDS, 10 mM NaCl, 0.3 mM
Na.sub.2HPO.sub.4, 0.1 mM EDTA at pH=6.8. Nucleic acid molecules of
the invention may encode a protein having at least about 50 or 60%
amino acid sequence identity with the sequence set forth in SEQ ID
NO: 2, preferably at least about 70 or 75%, more preferably at
least about 80%, still more preferably at least about 85%, yet more
preferably at least about 90%, even more preferably at least about
95% and most preferably at least about 98% sequence identity with
the protein sequence set forth in SEQ ID NO: 2.
[0015] The present invention further includes the nucleic acid
molecules operably linked to one or more expression control
elements, including vectors comprising the isolated nucleic acid
molecules. The invention further includes host cells transformed to
contain the nucleic acid molecules of the invention and methods for
producing a peptide, polypeptide or protein comprising the step of
culturing a host cell transformed with a nucleic acid molecule of
the invention under conditions in which the protein is
expressed.
[0016] The invention further provides an isolated polypeptide
selected from the group consisting of an isolated polypeptide
comprising the amino acid sequence of SEQ ID NO: 2, an isolated
polypeptide comprising a fragment of at least 6 amino acids of SEQ
ID NO: 2, an isolated polypeptide comprising conservative amino
acid substitutions of SEQ ID NO: 2 and an isolated polypeptide
comprising naturally occurring amino acid sequence variants of SEQ
ID NO: 2. Polypeptides of the invention also include polypeptides
with an amino acid sequence having at least about 50 or 60% amino
acid sequence identity with the sequence set forth in SEQ ID NO: 2,
preferably at least about 70 or 75%, more preferably at least about
80%, still more preferably at least about 85%, yet more preferably
at least about 90%, even more preferably at least about 95% and
most preferably at least about 98% sequence identity with the
protein sequence set forth in SEQ ID NO: 2.
[0017] This invention provides vectors comprising the nucleic acid
constructs of the present invention as well as host cells,
recombinant plant cells and transgenic plants comprising the
vectors of the present invention. More particularly, this invention
provides such cells and transgenic plants that are hemizygotic,
heterozygotic or homozygotic for the nucleic acid constructs,
wherein such plants can be monoploid, diploid or polyploid. It is
an object of the present invention to provide such cells and
transgenic plants wherein they express a single copy or multiple
copies of one or more of the IG1, mutant IG1 (e.g. IG1-O or
IG1-MUM), or IG1 ortholog (e.g., the rice IG1) protein products of
the present invention. Cells or transgenic plants which express
multiple copies of one of the IG1, mutant IG1, or IG1 ortholog
proteins, or which express more than one of the IG1, mutant IG1, or
IG1 ortholog proteins, may be desirable, for example, to produce
male sterility for plant breeding or to generate androgenetic
progeny in maize and other plant species.
[0018] The invention further provides nucleic acid probes for the
detection of expression of IG1, or mutants, or homologs, or
orthologs thereof, in plants which either have been genetically
altered to express at least one of said proteins or which may
naturally express IG1, or mutants, or homologs, or orthologs
thereof.
[0019] The invention further provides the use of antibodies to IG1,
or mutants, or homologs, or orthologs thereof to probe a biological
sample or a tissue section for expression of IG1, or mutants, or
homologs, or orthologs. Said biological sample or tissue section
may be from a plant which has been genetically altered to express
said peptide, polypeptide or protein or which may naturally express
IG1, or mutants, or homologs, or orthologs. Thus, the present
invention provides methods of identifying and isolating an ortholog
of ig1 in a non-Zea mays plant species, said method comprising
using at least one nucleic acid of the present invention as a
nucleic acid probe.
[0020] The present invention also provides methods of modulating a
plant cell comprising an ig1 gene, said method comprising the step
of introducing into said plant cell an isolated polynucleotide
according to the present invention, whereby the function and or
structure of the ig1 gene is modulated. More specifically, the
present invention provides such methods wherein the isolated
polynucleotide is a knock-out or knock-in construct.
[0021] The present invention also provides transgenic knock-out
plants comprising disruption in the endogenous ig1 gene, wherein
said disruption has been introduced into their genomes by
homologous recombination with a DNA targeting construct such that
the targeting construct is stably integrated in the genome of said
plant, wherein the disruption of the ig1 gene results in a
reduction of production of endogenous ig1 RNA levels.
[0022] The present invention also provides methods for down
regulating ig1 RNA levels in a plant, said method comprising the of
step introducing a vector provided by the present invention into
plant tissue, wherein expression of said vector causes
down-regulation of expression of ig1 RNA in said plant tissue.
[0023] Other objects, advantages and features of the present
invention become apparent to one skilled in the art upon reviewing
the specification and the drawings provided herein. Thus, further
objects and advantages of the present invention will be clear from
the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1. Male and female gametophyte development. Key:
a=antipodal cells; cc=central cell; pn=polar nuclei; e=egg cell;
sy=synergid; end=endosperm; emb=embryo; va=vacuole; vn=vegetative
cell; gc=generative cell; sp=sperm cells; pt=pollen tube.
FG1-FG8=female gametophyte stage 1 to 8 (stages adapted from
Christensen et al., 1997). FM=free microspore; VM=vacuolated
microspore; BC=bicellular pollen; MP=mature pollen; and
GP=germinating pollen.
[0025] FIG. 2. IG1 protein and related proteins of rice and
Arabidopsis. Rice proteins are designated as os lbd ch# (chromosome
number) and BAC number. OSIG1 is the rice ortholog of IG1.
Arabidopsis proteins are designated "at" followed by gene name and
number. From the tree it can be seen that there are two rice genes
that are closely related to ig1 and that ig1 and these two rice
genes are most similar to AS2 in Arabidposis.
[0026] FIG. 3. Map position of ig2. Mapping with SSR's using the
miniature seed phenotype as an indicator of the presence of the
mutation placed ig2 midway between phi115 and bnlg1031. Mapping
based on the reduced fertility phenotype using floury3 as a marker
confirmed this position.
[0027] FIG. 4. Mature ig2 seed phenotype. Mature seeds stained with
Evans' Blue to detect cell death, ig2 (FIG. 4A); wild type (FIG.
4B). ig2 homozygotes undergo more extensive cell death in the
endosperm than wild type, and it appears that it begins earlier.
However, the embryos are still alive although abnormally shaped and
smaller than wild type. They usually have a shoot pole with a few
leaf primordia like wild type seeds.
[0028] FIG. 5. Leaf of ig2 homozygote. The epidermis of the leaf
has outgrowths and irregularly shaped cells. The seedling produced
two abnormal leaves and then stopped growing.
[0029] FIG. 6. Embryo sacs from an ig3 heterozygote.
[0030] FIG. 6A. Mutant embryo sac (arrow) arrested at the
one-nucleate stage of development.
[0031] FIG. 6B. Sibling embryo sac at the mature stage.
[0032] FIG. 7. Comparative mapping between rice and maize around
ig1. Bacterial Artificial Chromosome (BAC) clones are shown by the
three uppermost horizontal line segments (i.e., those labeled
pco148714, pco092373, pco121964) and the three lowermost horizontal
line segments (i.e., those labeled ap003407, ap003431, ap003346).
Physical distance between markers on the rice chromosome is shown
by the horizontal lines labeled "65 kb", "130 kb" and "845 kb".
Vertical lines show the position of the closest sequence match in
rice for maize clones from the ig1 region. Markers written above
the maize BACs have been placed on the BAC clones but not on the
genetic map.
[0033] FIG. 8. Annotated genes between genes that should flank the
rice ortholog of ig1 based on mapping of orthologous markers in
maize.
[0034] FIG. 9.
[0035] FIG. 9A. Polymorphisms in two independent ig1 alleles in the
LOB domain gene. Southern blot of EcoR1 digested DNA probed with
DNA flanking the Mu insertion in the LOB domain protein. (The probe
used is probe 2-4 from FIG. 9B) Probe includes the LOB domain. DNA
from mutant heterozygotes for ig1-O and ig1-mum is compared to that
of their homozygous wild type progenitors. Arrowheads point to
novel bands present in mutants that are absent from their wild type
progenitors.
[0036] FIG. 9B. Structure of ig1 gene. Position of Mu insertion is
noted and inferred to be a Mu8 element based on terminal inverted
repeat sequence. Positions of primers used to amplify gene
fragments are indicated. The LOB domain is indicated by the
shading.
DETAILED DESCRIPTION
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
[0038] It will be appreciated from the above that the tools and
methods of the present invention have application to all plants
that produce gametes. Such plants include, but are not limited to,
forage grasses, turf grasses, ornamental grasses, forage legumes,
ground covers, vegetables, field crops (e.g., soybeans, corn, rice,
cotton, tobacco, sorghum, field peas), trees and ornamental
flowers.
I. Definitions
[0039] As used herein, the term "allele" refers to any of several
alternative forms of a gene.
[0040] As used herein, the term "amino acid" refers to the
aminocarboxylic acids that are components of proteins and peptides.
The amino acid abbreviations are as follows:
TABLE-US-00001 A (Ala) C (Cys) D (Asp) E (Glu) F (Phe) G (Gly) H
(His) I (Iso) K (Lys) L (Leu) M (Met) N (Asn) P (Pro) Q (Gln) R
(Arg) S (Ser) T (Thr) V (Val) W (Trp) Y (Tyr)
[0041] As used herein, the term "androgenesis" refers to male
parthenogenesis, i.e., the development of a haploid embryo from a
male nucleus.
[0042] As used herein, the term "androgenetic" refers to the
haploid individual produced by androgenesis which contains in its
cells the genome of the male gamete only.
[0043] As used herein, the term "crop plant" refers to any plant
grown for any commercial purpose, including, but not limited to the
following purposes: seed production, hay production, ornamental
use, fruit production, berry production, vegetable production, oil
production, protein production, forage production, animal grazing,
golf courses, lawns, flower production, landscaping, erosion
control, green manure, improving soil tilth/health, producing
pharmaceutical products/drugs, producing food or food additives,
smoking products, pulp production and wood production.
[0044] As used herein, the term "cross pollination" or
"cross-breeding" refer to the process by which the pollen of one
flower on one plant is applied (artificially or naturally) to the
ovule (stigma) of a flower on another plant.
[0045] As used herein, the term "cultivar" refers to a variety,
strain or race of plant that has been produced by horticultural or
agronomic techniques and is not normally found in wild
populations.
[0046] As used herein, the terms "dicotyledon" and "dicot" refer to
a flowering plant having an embryo containing two seed halves or
cotyledons. Examples include tobacco; tomato; the legumes,
including peas, alfalfa, clover and soybeans; oaks; maples; roses;
mints; squashes; daisies; walnuts; cacti; violets and
buttercups.
[0047] As used herein, the term "ectopic" refers to something
occurring in an unusual place or in an unusual form or manner. For
example, ectopic expression of a gene refers to having a gene
expressed in a tissue or cell that would not normally express that
gene.
[0048] As used herein, the term "endosperm" refers to a triploid
structure resulting from the development of a fusion between two
polar nuclei of the embryo sac and one of the sperm nucleus from
the pollen found in many plant seeds. The endosperm frequently
stores food materials, which are broken down during
germination.
[0049] As used herein, the term "female" refers to a plant that
produces ovules. Female plants generally produce seeds after
fertilization. A plant designated as a "female plant" may contain
both male and female sexual organs. Alternatively, the "female
plant" may only contain female sexual organs either naturally
(e.g., in dioecious species) or due to emasculation (e.g., by
detasselling).
[0050] As used herein, the term "filial generation" refers to any
of the generations of cells, tissues or organisms following a
particular parental generation. The generation resulting from a
mating of the parents is the first filial generation (designated as
"F1" or "F.sub.1"), while that resulting from crossing of F1
individuals is the second filial generation (designated as "F2" or
"F.sub.2").
[0051] As used herein, the term "gamete" refers to a reproductive
cell whose nucleus (and often cytoplasm) fuses with that of another
gamete of similar origin but of opposite sex to form a zygote,
which has the potential to develop into a new individual. Gametes
are haploid and are differentiated into male and female.
[0052] As used herein, the term "gene" refers to any segment of DNA
associated with a biological function. Thus, genes include, but are
not limited to, coding sequences and/or the regulatory sequences
required for their expression. Genes can also include nonexpressed
DNA segments that, for example, form recognition sequences for
other proteins. Genes can be obtained from a variety of sources,
including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters.
[0053] As used herein, the term "genotype" refers to the genetic
makeup of an individual cell, cell culture, tissue, plant, or group
of plants.
[0054] As used herein, the term "hemizygous" refers to a cell,
tissue or organism in which a gene is present only once in a
genotype, as a gene in a haploid cell or organism, a sex-linked
gene in the heterogametic sex, or a gene in a segment of chromosome
in a diploid cell or organism where its partner segment has been
deleted.
[0055] As used herein, the terms "heterologous polynucleotide" or a
"heterologous nucleic acid" or an "exogenous DNA segment" refer to
a polynucleotide, nucleic acid or DNA segment that originates from
a source foreign to the particular host cell, or, if from the same
source, is modified from its original form. Thus, a heterologous
gene in a host cell includes a gene that is endogenous to the
particular host cell, but has been modified. Thus, the terms refer
to a DNA segment which is foreign or heterologous to the cell, or
homologous to the cell but in a position within the host cell
nucleic acid in which the element is not ordinarily found.
Exogenous DNA segments are expressed to yield exogenous
polypeptides.
[0056] As used herein, the term "heterologous trait" refers to a
phenotype imparted to a transformed host cell or transgenic
organism by an exogenous DNA segment, heterologous polynucleotide
or heterologous nucleic acid.
[0057] As used herein, the term "heterozygote" refers to a diploid
or polyploid individual cell or plant having different alleles
(forms of a given gene) present at least at one locus.
[0058] As used herein, the term "heterozygous" refers to the
presence of different alleles (forms of a given gene) at a
particular gene locus.
[0059] As used herein, the terms "homolo-" or "homologue" refer to
a nucleic acid or peptide sequence which has a common origin and
functions similarly to a nucleic acid or peptide sequence from
another species.
[0060] As used herein, the term "homozygote" refers to an
individual cell or plant having the same alleles at one or more
loci.
[0061] As used herein, the term "homozygous" refers to the presence
of identical alleles at one or more loci in homologous chromosomal
segments.
[0062] As used herein, the term "hybrid" refers to any individual
cell, tissue or plant resulting from a cross between parents that
differ in one or more genes.
[0063] As used herein, the term "inbred" or "inbred line" refers to
a relatively true-breeding strain.
[0064] As used herein, the term "line" is used broadly to include,
but is not limited to, a group of plants vegetatively propagated
from a single parent plant, via tissue culture techniques or a
group of inbred plants which are genetically very similar due to
descent from a common parent(s). A plant is said to "belong" to a
particular line if it (a) is a primary transformant (T0) plant
regenerated from material of that line; (b) has a pedigree
comprised of a T0 plant of that line; or (c) is genetically very
similar due to common ancestry (e.g., via inbreeding or selfing).
In this context, the term "pedigree" denotes the lineage of a
plant, e.g. in terms of the sexual crosses effected such that a
gene or a combination of genes, in heterozygous (hemizygous) or
homozygous condition, imparts a desired trait to the plant.
[0065] As used herein, the term "locus" (plural: "loci") refers to
any site that has been defined genetically. A locus may be a gene,
or part of a gene, or a DNA sequence that has some regulatory role,
and may be occupied by different sequences.
[0066] As used herein, the term "male" refers to a plant that
produces pollen grains. The "male plant" generally refers to the
sex that produces gametes for fertilizing ova. A plant designated
as a "male plant" may contain both male and female sexual organs.
Alternatively, the "male plant" may only contain male sexual organs
either naturally (e.g., in dioecious species) or due to
emasculation (e.g., by removing the ovary).
[0067] As used herein, the term "mass selection" refers to a form
of selection in which individual plants are selected and the next
generation propagated from the aggregate of their seeds.
[0068] As used herein, the term "monocotyledon" or "monocot" refer
to any of a subclass (Monocotyledoneae) of flowering plants having
an embryo containing only one seed leaf and usually having
parallel-veined leaves, flower parts in multiples of three, and no
secondary growth in stems and roots. Examples include lilies;
orchids; rice; corn, grasses, such as tall fescue, goat grass, and
Kentucky bluegrass; grains, such as wheat, oats and barley; irises;
onions and palms.
[0069] As used herein, the terms "mutant" or "mutation" refer to a
gene, cell, or organism with an abnormal genetic constitution that
may result in a variant phenotype. For example, "a mutant ig1 gene"
or "an ig1 gene with a mutation" refer to a gene with an alteration
in the nucleotide sequence of the ig1 gene, such as ig1-O and
ig1-mum.
[0070] As used herein, the terms "nucleic acid" or "polynucleotide"
refer to deoxyribonucleotides or ribonucleotides and polymers
thereof in either single- or double-stranded form. Unless
specifically limited, the terms encompass nucleic acids containing
known analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.
degenerate codon substitutions) and complementary sequences as well
as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (Batzer et
al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J.
Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al.
(1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used
interchangeably with gene, cDNA, and mRNA encoded by a gene. The
term "nucleic acid" also encompasses polynucleotides synthesized in
a laboratory using procedures well known to those skilled in the
art.
[0071] As used herein, a DNA segment is referred to as "operably
linked" when it is placed into a functional relationship with
another DNA segment. For example, DNA for a signal sequence is
operably linked to DNA encoding a polypeptide if it is expressed as
a preprotein that participates in the secretion of the polypeptide;
a promoter or enhancer is operably linked to a coding sequence if
it stimulates the transcription of the sequence. Generally, DNA
sequences that are operably linked are contiguous, and in the case
of a signal sequence both contiguous and in reading phase. However,
enhancers need not be contiguous with the coding sequences whose
transcription they control. Linking is accomplished by ligation at
convenient restriction sites or at adapters or linkers inserted in
lieu thereof.
[0072] As used herein, the term "open pollination" refers to a
plant population that is freely exposed to some gene flow, as
opposed to a closed one in which there is an effective barrier to
gene flow.
[0073] As used herein, the terms "open-pollinated population" or
"open-pollinated variety" refer to plants normally capable of at
least some cross-fertilization, selected to a standard, that may
show variation but that also have one or more genotypic or
phenotypic characteristics by which the population or the variety
can be differentiated from others. A hybrid, which has no barriers
to cross-pollination, is an open-pollinated population or an
open-pollinated variety.
[0074] As used herein, the terms "ortholog" and "orthologue" refer
to a nucleic acid or peptide sequence which functions similarly to
a nucleic acid or peptide sequence from another species. For
example, where one gene from one plant species has a high nucleic
acid sequence similarity and codes for a protein with a similar
function to another gene from another plant species, such genes
would be orthologs.
[0075] As used herein, the term "ovule" refers to the female
gametophyte, whereas the term "pollen" means the male
gametophyte.
[0076] As used herein, the term "ovule-specific promoter" refers
broadly to a nucleic acid sequence that regulates the expression of
nucleic acid sequences selectively in the cells or tissues of a
plant essential to ovule formation and/or function and/or limits
the expression of a nucleic acid sequence to the period of ovule
formation in a plant.
[0077] As used herein, the term "peptide" refers to a class of
compounds of low molecular weight which yield two or more amino
acids on hydrolysis and form the constituent parts of proteins. As
used herein, an "oligopeptide" refers to any molecule that contains
a small number (two to about 20) of amino-acid residues connected
by peptide linkages.
[0078] As used herein, the term "phenotype" refers to the
observable characters of an individual cell, cell culture, plant,
or group of plants which results from the interaction between that
individual's genetic makeup (i.e., genotype) and the
environment.
[0079] As used herein, the term "plant" refers to whole plants,
plant organs (e.g., leaves, stems, roots, etc.), seeds and plant
cells and progeny of it. The class of plants that can be used in
the methods of the invention is generally as broad as the class of
higher plants amenable to transformation techniques, including both
monocotyledonous and dicotyledonous plants.
[0080] As used herein, the term "plant line" is used broadly to
include, but is not limited to, a group of plants vegetatively
propagated from a single parent plant, via tissue culture
techniques or a group of inbred plants which are genetically very
similar due to descent from a common parent(s). A plant is said to
"belong" to a particular line if it (a) is a primary transformant
(T0) plant regenerated from material of that line; (b) has a
pedigree comprised of a T0 plant of that line; or (c) is
genetically very similar due to common ancestry (e.g. via
inbreeding or selfing). In this context, the term "pedigree"
denotes the lineage of a plant, e.g. in terms of the sexual crosses
effected such that a gene or a combination of genes, in
heterozygous (hemizygous) or homozygous condition, imparts a
desired trait to the plant.
[0081] As used herein, the term "plant organ" refers to any part of
a plant. Examples of plant organs include, but are not limited to
the leaf, stem, root, tuber, seed, branch, pubescence, nodule, leaf
axil, flower, pollen, stamen, pistil, petal, peduncle, stalk,
stigma, style, bract, fruit, trunk, carpel, sepal, anther, ovule,
pedicel, needle, cone, rhizome, stolon, shoot, pericarp, endosperm,
placenta, berry, stamen, and leaf sheath.
[0082] As used herein, the terms "plant transcription unit" or
"PTU" refer to a nucleic acid sequence encoding a promoter
sequence, a coding sequence and a 3' termination sequence.
[0083] As used herein, the term "polypeptide" refers to a linear
polymer of amino acids linked via peptide bonds. A polypeptide may
be as short as 2 amino acids to virtually any length.
[0084] As used herein, the term "promoter" refers to a region of
DNA involved in binding RNA polymerase to initiate
transcription.
[0085] As used herein, the terms "protein," "peptide" or
polypeptide" refer to amino acid residues and polymers thereof.
Unless specifically limited, the terms encompass amino acids
containing known analogues of natural amino acid residues that have
similar binding properties as the reference amino acid and are
metabolized in a manner similar to naturally occurring amino acid
residues. Unless otherwise indicated, a particular amino acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g. conservative substitutions) as well as the
sequence explicitly indicated. The term "polypeptide" also
encompasses polypeptides synthesized in a laboratory using
procedures well known to those skilled in the art.
[0086] As used herein, the term "recombinant" refers to a cell,
tissue or organism that has undergone transformation with
recombinant DNA. The original recombinant is designated as "R0" or
"R.sub.0." Selfing the R0 produces a first transformed generation
designated as "R1" or "R.sub.1."
[0087] As used herein, the term "self pollinated" or
"self-pollination" means the pollen of one flower on one plant is
applied (artificially or naturally) to the ovule (stigma) of the
same or a different flower on the same plant.
[0088] As used herein, the term "signal sequence" refers to an
amino acid sequence (the signal peptide) attached to the
polypeptide which binds the polypeptide to the endoplasmic
reticulum and is essential for protein secretion.
[0089] As used herein, the term "synthetic" refers to a set of
progenies derived by intercrossing a specific set of clones or
seed-propagated lines. A synthetic may contain mixtures of seed
resulting from cross-, self-, and sib-fertilization.
[0090] As used herein, the term "transcript" refers to a product of
a transcription process.
[0091] As used herein, the term "transformation" refers to the
transfer of nucleic acid (i.e., a nucleotide polymer) into a cell.
As used herein, the term "genetic transformation" refers to the
transfer and incorporation of DNA, especially recombinant DNA, into
a cell.
[0092] As used herein, the term "transformant" refers to a cell,
tissue or organism that has undergone transformation. The original
transformant is designated as "T0" or "T.sub.0." Selfing the T0
produces a first transformed generation designated as "T1" or
"T.sub.1."
[0093] As used herein, the term "transgene" refers to a nucleic
acid that is inserted into an organism, host cell or vector in a
manner that ensures its function.
[0094] As used herein, the term "transgenic" refers to cells, cell
cultures, organisms, plants, and progeny of plants which have
received a foreign or modified gene by one of the various methods
of transformation, wherein the foreign or modified gene is from the
same or different species than the species of the plant, or
organism, receiving the foreign or modified gene.
[0095] As used herein, the term "transposition event" refers to the
movement of a transposon from a donor site to a target site.
[0096] As used herein, the term "transposon" refers to a genetic
element, including but not limited to segments of DNA or RNA that
can move from one chromosomal site to another.
[0097] As used herein, the term "variety" refers to a subdivision
of a species, consisting of a group of individuals within the
species that are distinct in form or function from other similar
arrays of individuals. As used herein, the terms "untranslated
region" or "UTR" refer to any part of a mRNA molecule not coding
for a protein (e.g., in eukaryotes the poly(A) tail).
[0098] As used herein, the term "vector" refers broadly to any
plasmid or virus encoding an exogenous nucleic acid. The term
should also be construed to include non-plasmid and non-viral
compounds which facilitate transfer of nucleic acid into virions or
cells, such as, for example, polylysine compounds and the like. The
vector may be a viral vector that is suitable as a delivery vehicle
for delivery of the nucleic acid, or mutant thereof, to a cell, or
the vector may be a non-viral vector which is suitable for the same
purpose. Examples of viral and non-viral vectors for delivery of
DNA to cells and tissues are well known in the art and are
described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci.
U.S.A. 94:12744-12746). Examples of viral vectors include, but are
not limited to, a recombinant vaccinia virus, a recombinant
adenovirus, a recombinant retrovirus, a recombinant
adeno-associated virus, a recombinant avian pox virus, and the like
(Cranage et al., 1986, EMBO J. 5:3057-3063; International Patent
Application No. WO94/17810, published Aug. 18, 1994; International
Patent Application No. WO94/23744, published Oct. 27, 1994).
Examples of non-viral vectors include, but are not limited to,
liposomes, polyamine derivatives of DNA, and the like.
II. Intermediate Gametophyte 1 (ig1)
[0099] Intermediate gametophyte (ig1) is an example of a mutant
whose embryo sacs are viable but structurally variable (Lin 1978,
1981). ig1 embryo sacs undergo extra rounds of free nuclear
divisions resulting in extra eggs, extra central cells, extra polar
nuclei within central cells, and other defects (Lin 1978,1981;
Huang and Sheridan, 1996). The exact number and placement of nuclei
at cellularization is variable in ig1. The phenotypes of ig1 embryo
sacs suggest a position-based determination of cellular identity.
The ability of the extra cells and nuclei to function as egg cells
or polar nuclei, for example, may depend on their position in the
embryo sac. Many of these defective embryo sacs give rise to
abnormal seeds, displaying polyembryony, heterofertilization,
haploid embryos, miniature endosperms, and early abortion
(Kermiicle, 1971). However, because ig1 embryo sacs are viable,
homozygous lines can be established. These homozygous plants have
normal vegetative development and reproductive morphology but in
some genetic backgrounds are male sterile, failing to shed pollen
or even exert anthers in most plants (Kermicle, 1994). Many of the
aspects of the ig1 phenotype are consistent with reduced negative
regulation of nuclear divisions in the embryo sac. ig1 is unique
among published female gametophyte mutants, in having extra rounds
of nuclear division prior to cellularization.
[0100] One function of the ig1 gene is to restrict the embryogenic
potential of cells that lack one of the two parental genomes. ig1
mutant embryo sacs produce haploid progeny, of both maternal and
paternal origin, at a higher rate than wild type (Kermicle, 1969).
The increased frequency of androgenesis (production of progeny with
only a paternal genomic contribution) when ig1 is the female parent
has been exploited agronomically in maize. Haploids can be
generated to produce homozygous lines rapidly, and nuclear
genotypes can be combined with different cytoplasms, such as those
conditioning male sterility (Albertsen and Trimnell, 1990; Kindiger
and Hamann, 1993; Kermicle, 1994). This is accomplished while
simultaneously removing the ig1 mutation, because it is only
present in the female parent. This eliminates the need for
recurrent backcrossing and thus dramatically reduces the time
needed to transfer the germplasm of new elite lines into a male
sterile cytoplasm. The ability either to identify or engineer this
phenotype in other agronomically important crops that use
cytoplasmic male sterile lines for hybrid production would improve
the efficiency of breeding in these species.
III. Nucleic Acids
[0101] A. Promoters
[0102] An inducible promoter is a promoter where the rate of RNA
polymerase binding and initiation is modulated by external stimuli.
Such stimuli include light, heat, anaerobic stress, alteration in
nutrient conditions, presence or absence of a metabolite, presence
of a ligand, microbial attack, wounding and the like.
[0103] A viral promoter is a promoter with a DNA sequence
substantially similar to the promoter found at the 5' end of a
viral gene. For example, a typical viral promoter is found at the
5' end of the gene coding for the p2I protein of MMTV described by
Huang et al., Cell 27:245 (1981).
[0104] A synthetic promoter is a promoter that was chemically
synthesized rather than biologically derived. Usually synthetic
promoters incorporate sequence changes that optimize the efficiency
of RNA polymerase initiation.
[0105] A constitutive promoter is a promoter that promotes the
expression of a gene product throughout an organism, such as a
plant. Examples of constitutive promoters include the cauliflower
mosaic virus 35S and 19S promoters (for example, Poszkowski et al.,
EMBO J. 3: 2719 (1989); Odell et al, Nature 313:810 (1985)); and
the maize ubiquitin-1 promoter (for example, U.S. Pat. Nos.
5,510,474; 5,614,399; 6,020,190 and 6,054,574).
[0106] A temporally regulated promoter is a promoter where the rate
of RNA polymerase binding and initiation is modulated at a specific
time during development. Examples of temporally regulated promoters
are given in, for example, Chua et al., Science, 244:174-181
(1989).
[0107] A spatially regulated promoter is a promoter where the rate
of RNA polymerase binding and initiation is modulated in a specific
structure of the organism such as the leaf, stem, seed or root.
Examples of spatially regulated promoters are given in Chua et al.,
Science 244:174-181 (1989). Such tissue-specific or organ-specific
promoters are well known in the art and include but are not limited
to seed-specific promoters, organ-primordia specific promoters,
stem-specific promoters, leaf specific promoters, mesophyl-specific
promoters (such as the light-inducible Rubisco promoters),
root-specific promoters, tuber-specific promoters, vascular tissue
specific promoters, stamen-selective promoters, dehiscence zone
specific promoters and the like. The most preferred promoters for
use in the instant invention will be most active in seed, fruit and
tuber.
[0108] A spatiotemporally regulated promoter is a promoter where
the rate of RNA polymerase binding and initiation is modulated in a
specific structure of the organism at a specific time during
development. An example of a typical spatio-temporally regulated
promoter is the EPSP synthase-35S promoter described by Chua et
al., Science 244:174-181 (1989).
[0109] There are many excellent examples of suitable promoters to
drive pollen-specific expression in plants. Pollen-specific
promoters have been identified in many plant species such as maize,
rice, tomato, tobacco, Arabidopsis, Brassica, and others (Odell, T.
O., et al. (1985) Nature 313:810-812; Marrs, K. A., et al, (1993)
Dev Genet, Vol. 14/1:27-41; Kim, (1992) Transgenic Res, Vol.
1/4:188-94; Carpenter, J. L., et al. (1992) Plant Cell Vol.
4/5:557-71; Albani, D. et al., (1992) Plant J. 213:331-42; Rommens,
C. M., et al. (1992), Mol. Gen. Genet., Vol. 231/3:433-41;
Kloeckener-Gruissem, et al., (1992) Embo J, Vol. 11/1:157-66;
Hamilton, D. A. et al., (1992), Plant Mol Biol, Vol. 18/2:211-18;
Kyozuka, J., et al. (1991), Mol. Gen. Genet., Vol. 22811-2:40-8;
Albani, D. et. al (1991) Plant Mol Biol Vol. 16/4:501-13; Twell, D.
et al. (1991) Genes Dev. 5/3:496-507; Thorsness, M. K. et al.,
(1991) Dev. Biol Vol. 143/1:173-84; McCormick, S. et al. (1991)
Symp Soc Exp Biol Vol. 45:229-44; Guerrero, F. D. et al. (1990) Mol
Gen Genet Vol 224/2:161-8; Twell, D. et al., (1990) Development
Vol. 109/3:705-13; Bichler, J. et al. (1990), Eur J Biochem Vol.
190/2:415-26; van Tunen, et al. (1990), Plant Cell Vol 2/5:393-401;
Siebertz, B. et al, (1989) Plant Cell Vol 1/10:961-8; Sullivan, T.
D. et al., (1989) Dev Genet Vol 10/6:412-24; Chen, J. et al.
(1987), Genetics Vol 116/3:469-77). Several other examples of
pollen-specific promoters can be found in GenBank. Additional
promoters are also provided in U.S. Pat. Nos. 5,086,169; 5,756,324;
5,633,438; 5,412,085; 5,545,546 and 6,172,279.
[0110] There are also several other eukaryotic sex-specific
promoters suitable for use in the instant invention. Examples
include: the mouse spermatocyte-specific Pgk-2 promoter (Ando et
al. (2000) Biochem. Biophys. Res. Comm. 272/1:125-8); the PACAP
testis-specific promoter (Daniel et al. (2000) Endocrinology,
141/3:1218-27); the mouse mSP-10 spermatid-specific promoter (Reddi
et al. (1999) Biology of Reproduction, 61/5:1256-66); the mouse
sperm-specific promoter (Ramara et al. (1998) J. Clin. Invest.
102/2:371-8); the mouse and rat H1t promoters (vanWert et al.
(1996) J. Cell. Biochem. 60/3:348-62); the human PRM1, PRM2 and
TNP2 spermatid-specific promoters (Nelson et al. (1995) DNA
Sequence 5/6:329-37); the Drosophila exu sex-specific promoter
(Crowley et al. (1995) Molec. Gen. Genet. 248/3:370-4); the mouse
testis ACE promoter (Zhou et al. (1995) Dev. Genet. 16/2:201-9);
the rat GHRH spermatogenic-specific promoter (Srivastava et al.
(1995) Endocrinology 136/4:1502-8); the Drosophila testis-specific
promoter (Lankenau et al. (1994) Mol. Cell. Biol. 14/3:1764-75);
the spermatocyte-specific hst70 gene promoter (Widlak et al. (1994)
Acta Biochim. Polonica 41/2:103-5); and the mouse Prm-1
spermatid-specific promoter (Zambrowicz et al. (1993) Proc. Nat'l.
Acad. Sci. USA 90/11:5071-5).
[0111] Expression of seed-specific genes has been studied in great
detail (see reviews, for example, by Goldberg et al., Cell
56:149-160 (1989) and Higgins et al., Ann. Rev. Plant Physiol.
35:191-221 (1984)). Promoter analysis of seed-specific genes is
reviewed in Goldberg et al., Cell 56: 149-160 (1989) and Thomas,
Plant Cell 5: 1401-1410 (1993). Research indicates that no plant
gene is more tightly regulated in terms of spatial expression than
those encoding seed storage proteins.
[0112] Many seed storage protein genes have been cloned from
diverse plant species, and their promoters have been analyzed in
detail (Thomas, Plant Cell 5: 1401-1410 (1993)). There are
currently numerous examples of seed-specific expression of seed
storage protein genes in transgenic plants. See, for example,
b-phaseolin (Sengupta-Gopalan et al., Proc. Natl. Acad, Sci. USA
82:3320-3324 (1985); Hoffman et al., Plant Mol. Biol. 11, 717-729
(1988)); bean lectin (Voelker et al., EMBO J. 6: 3571-3577 (1987));
soybean lectin (Okamuro et al., Proc. Natl. Acad. Sci. USA
83:8240-8244 (1986)); soybean Kunitz trypsin inhibitor (Perez-Grau
et al., Plant Cell 1:095-1109 (1989)); soybean b-conglycinin
(Beachy et al., EMBO J. 4:3047-3053 (1985); pea vicilin (Higgins et
al., Plant Mol. Biol. 11:683-695 (1988)); pea convicilin (Newbigin
et al., Planta 180:461-470 (1990)); pea legumin (Shirsat et al.,
Mol. Gen. Genetics 215:326-331 (1989)); rapeseed napin (Radke et
al., Theor. Appl. Genet. 75:685-694 (1988)); maize 18 kD oleosin
(Lee et al., Proc Natl. Acad. Sci. USA 888:6181-6185 (1991));
barley b-hordein (Marris et al., Plant Mol. Biol. 10:359-366
(1988); wheat glutenin (Colot et al., EMBO J. 6:3559-3564 (1987)).
For additional sources of seed-specific promoters, see, for
example, U.S. Pat. Nos. 5,623,067; 6,100,450; 6,177,613; 6,225,529;
6,342,657 and 6,403,371; Knutzon et al., Proc. Natl. Acad. Sci. USA
89:2624 (1992); Bustos et al., EMBO J. 10:1469 (1991), Lam and
Chua, Science 248:471 (1991); Stayton et al., Aust. J. Plant.
Physiol. 18:507 (1991), each of which is incorporated by reference
in its entirety. Moreover, seed-specific promoter genes operably
linked to heterologous coding sequences in chimeric gene constructs
also maintain their temporal and spatial expression pattern in
transgenic plants. Such examples include use of Arabidopsis
thaliana 2S seed storage protein gene promoter to express
enkephalin peptides in Arabidopsis and B. napus seeds (see, for
example, Vandekerckhove et al., Bio/Technology 7:929-932 (1989));
bean lectin and bean b-phaseolin promoters to express luciferase
(see, for example, Riggs et al., Plant Sci. 63:47-57 (1989)); and
wheat glutenin promoters to express chloramphenicol acetyl
transferase (see, for example, Colot et al., EMBO J. 6:3559-3564
(1987)).
[0113] B. Transgenes and Heterologous Nucleic Acids
[0114] There are numerous examples of genes successfully introduced
into plants using recombinant DNA methodologies including, but not
limited to, those coding for the following traits: seed storage
proteins, including modified 7S legume seed storage proteins (U.S.
Pat. Nos. 5,508,468, 5,592,223 and 5,576,203); herbicide tolerance
or resistance (U.S. Pat. Nos. 5,498,544 and 5,554,798; Powell et
al., Science 232:738-743 (1986); Kaniewski et al., Bio/Tech.
8:750-754 (1990); Day et al., Proc. Natl. Acad. Sci. USA
88:6721-6725 (1991)); phytase (U.S. Pat. No. 5,593,963); resistance
to bacterial, fungal, nematode and insect pests, including
resistance to the lepidoptera insects conferred by the Bt gene
(U.S. Pat. Nos. 5,597,945 and 5,597,946; Hilder et al., Nature
330:160-163; Johnson et al., Proc. Natl. Acad. Sci. USA,
86:9871-9875 (1989); Perlak et al., Bio/tech. 8:939-943 (1990));
lectins (U.S. Pat. No. 5,276,269); and flower color (Meyer et al.,
Nature 330:677-678 (1987); Napoli et al., Plant Cell 2:279-289
(1990); van der Krol et al., Plant Cell 2:291-299 (1990)).
[0115] C. Site-Specific Recombination Systems
[0116] Methods and constructs for targeting of DNA sequences for
insertion into a particular DNA locus, while enabling removal of
randomly inserted DNA sequences that occur as a by-product of
transformation procedures, are described in U.S. Pat. Nos.
5,527,695 and 6,114,600. One manner of removing these random
insertions is to utilize a site-specific recombinase system. In
general, a site-specific recombinase system consists of three
elements: two pairs of DNA sequence (the site-specific
recombination sequences) and a specific enzyme (the site-specific
recombinase). The site-specific recombinase will catalyze a
recombination reaction only between two site-specific recombination
sequences.
[0117] A number of different site-specific recombinase systems can
be used, including but not limited to the Cre/lox system of
bacteriophage P1, the FLP/FRT system of yeast, the Gin recombinase
of phage Mu, the Pin recombinase of E. coli, and the R/RS system of
the pSR1 plasmid. The two preferred site-specific recombinase
systems are the bacteriophage P1 Cre/lox and the yeast FLP/TRT
systems. In these systems a recombinase (Cre or FLP) will interact
specifically with its respective site-specific recombination
sequence (10.times. or FRT respectively) to invert or excise the
intervening sequences. The sequence for each of these two systems
is relatively short (34 bp for 10.times. and 47 bp for FRT).
Currently the FLP/FRT system of yeast is the preferred
site-specific recombinase system since it normally functions in a
eukaryotic organism (yeast), and is well characterized. It is
thought that the eukaryotic origin of the FLP/FRT system allows the
FLP/FRT system to function more efficiently in eukaryotic cells
than the prokaryotic site-specific recombinase systems.
[0118] The FLP/FRT recombinase system has been demonstrated to
function efficiently in plant cells. Experiments on the performance
of the FLP/FRT system in both maize and rice protoplasts indicates
that FRT site structure, and amount of the FLP protein present,
affects excision activity. In general, short incomplete FRT sites
leads to higher accumulation of excision products than the complete
full-length FRT sites. Site-specific recombination systems can
catalyze both intra- and intermolecular reactions in maize
protoplasts, indicating that the system can be used for DNA
excision as well as integration reactions. The recombination
reaction is reversible and this reversibility can compromise the
efficiency of the reaction in each direction. Altering the
structure of the site-specific recombination sequences is one
approach to remedying this situation. The site-specific
recombination sequence can be mutated in a manner that the product
of the recombination reaction is no longer recognized as a
substrate for the reverse reaction, thereby stabilizing the
integration or excision event.
[0119] D. Vectors
[0120] Expression Units to Express Exogenous DNA in a Plant
[0121] As provided above, several embodiments of the present
invention employ expression units (or expression vectors or
systems) to express an exogenously supplied nucleic acid sequence
in a plant. Methods for generating expression units/systems/vectors
for use in plants are well known in the art and can readily be
adapted for use in the instant invention. A skilled artisan can
readily use any appropriate plant/vector/expression system in the
present methods following the outline provided herein.
[0122] The expression control elements used to regulate the
expression of the protein can either be the expression control
element that is normally found associated with the coding sequence
(homologous expression element) or can be a heterologous expression
control element. A variety of homologous and heterologous
expression control elements are known in the art and can readily be
used to make expression units for use in the present invention.
Transcription initiation regions, for example, can include any of
the various opine initiation regions, such as octopine, mannopine,
nopaline and the like that are found in the Ti plasmids of
Agrobacterium tumafacians. Alternatively, plant viral promoters can
also be used, such as the cauliflower mosaic virus 19S and 35S
promoters (CaMV 19S and CaMV 35S promoters, respectively) to
control gene expression in a plant (U.S. Pat. Nos. 5,352,605;
5,530,196 and 5,858,742 for example). Enhancer sequences derived
from the CaMV can also be utilized (U.S. Pat. Nos. 5,164,316;
5,196,525; 5,322,938; 5,530,196; 5,352,605; 5,359,142; and
5,858,742 for example).
[0123] Lastly, plant promoters such as prolifera promoter,
fruit-specific promoters, Ap3 promoter, heat shock promoters,
seed-specific promoters, etc. can also be used.
[0124] Either a gamete-specific promoter, a constitutive promoter
(such as the CaMV or Nos promoter), an organ-specific promoter
(such as the E8 promoter from tomato) or an inducible promoter is
typically ligated to the protein or antisense encoding region using
standard techniques known in the art. The expression unit may be
further optimized by employing supplemental elements such as
transcription terminators and/or enhancer elements.
[0125] Thus, for expression in plants, the expression units will
typically contain, in addition to the protein sequence, a plant
promoter region, a transcription initiation site and a
transcription termination sequence. Unique restriction enzyme sites
at the 5' and 3' ends of the expression unit are typically included
to allow for easy insertion into a preexisting vector.
[0126] In the construction of heterologous promoter/structural gene
or antisense combinations, the promoter is preferably positioned
about the same distance from the heterologous transcription start
site as it is from the transcription start site in its natural
setting. As is known in the art, however, some variation in this
distance can be accommodated without loss of promoter function.
[0127] In addition to a promoter sequence, the expression cassette
can also contain a transcription termination region downstream of
the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes. If the
mRNA encoded by the structural gene is to be efficiently processed,
DNA sequences which direct polyadenylation of the RNA are also
commonly added to the vector construct. Polyadenylation sequences
include, but are not limited to the Agrobacterium octopine synthase
signal (Gielen et al., EMBO J 3:835-846 (1984)) or the nopaline
synthase signal (Depicker et al., Mol. and Appl. Genet. 1:561-573
(1982)).
[0128] The resulting expression unit is ligated into or otherwise
constructed to be included in a vector that is appropriate for
higher plant transformation. The vector will also typically contain
a selectable marker gene by which transformed plant cells can be
identified in culture. Usually, the marker gene will encode
antibiotic resistance. These markers include resistance to G418,
hygromycin, bleomycin, kanamycin, and gentamicin. After
transforming the plant cells, those cells having the vector will be
identified by their ability to grow on a medium containing the
particular antibiotic. Replication sequences, of bacterial or viral
origin, are generally also included to allow the vector to be
cloned in a bacterial or phage host, preferably a broad host range
prokaryotic origin of replication is included. A selectable marker
for bacteria should also be included to allow selection of
bacterial cells bearing the desired construct. Suitable prokaryotic
selectable markers also include resistance to antibiotics such as
ampicillin, kanamycin or tetracycline.
[0129] Other DNA sequences encoding additional functions may also
be present in the vector, as is known in the art. For instance, in
the case of Agrobacterium transformations, T-DNA sequences will
also be included for subsequent transfer to plant chromosomes.
[0130] The sequences of the present invention can also be fused to
various other nucleic acid molecules such as Expressed Sequence
Tags (ESTs), epitopes or fluorescent protein markers.
[0131] ESTs are gene fragments, typically 300 to 400 nucleotides in
length, sequenced from the 3' or 5' end of complementary-DNA (cDNA)
clones. Nearly 30,000 Arabidopsis thaliana ESTs have been produced
by a French and an American consortium (Delseny et al., FEBS Lett.
405(2):129-132 (1997); Arabidopsis thaliana Database,
http://genome.www.stanford.edu/Arabidopsis). For a discussion of
the analysis of gene-expression patterns derived from large EST
databases, see, e.g., M. R. Fannon, TIBTECH 14:294-298 (1996).
[0132] Biologically compatible fluorescent protein probes,
particularly the self-assembling green fluorescent protein (GFP)
from the jellyfish Aequorea victoria, have revolutionized research
in cell, molecular and developmental biology because they allow
visualization of biochemical events in living cells (Murphy et al.,
Curr. Biol. 7(11):870-876 (1997); Grebenok et al., Plant J.
11(3):573-586 (1997); Pang et al., Plant Physiol 112(3) (1996);
Chiu et al., Curr. Biol. 6(3):325-330 (1996); Plautz et al., Gene
173(1):83-87 (1996); Sheen et al., Plant J. 8(5):777-784
(1995)).
[0133] Site-directed mutagenesis has been used to develop a more
soluble version of the codon-modified GFP called soluble-modified
GFP (smGFP). When introduced into Arabidopsis, greater fluorescence
was observed when compared to the codon-modified GFP, implying that
smGFP is `brighter` because more of it is present in a soluble and
functional form (Davis et al., Plant Mol. Biol. 36(4):521-528
(1998)). By fusing genes encoding GFP and beta-glucuronidase (GUS),
researchers were able to create a set of bifunctional reporter
constructs which are optimized for use in transient and stable
expression systems in plants, including Arabidopsis (Quaedvlieg et
al., Plant Mol. Biol. 37(4):715-727 (1998)).
[0134] Berger et al. (Dev. Biol. 194(2):226-234 (1998)) report the
isolation of a GFP marker line for Arabidopsis hypocotyl epidermal
cells. GFP-fusion proteins have been used to localize and
characterize a number of Arabidopsis genes, including
geranylgeranyl pyrophosphate (GGPP) (Zhu et al., Plant Mol. Biol.
35(3):331-341 (1997).
IV. Transformation
[0135] To introduce a desired gene or set of genes by conventional
methods requires a sexual cross between two lines, and then
repeated back-crossing between hybrid offspring and one of the
parents until a plant with the desired characteristics is obtained.
This process, however, is restricted to plants that can sexually
hybridize, and genes in addition to the desired gene will be
transferred.
[0136] Recombinant DNA techniques allow plant researchers to
circumvent these limitations by enabling plant geneticists to
identify and clone specific genes for desirable traits, such as
resistance to an insect pest, and to introduce these genes into
already useful varieties of plants. Once the foreign genes have
been introduced into a plant, that plant can then be used in
conventional plant breeding schemes (e.g., pedigree breeding,
single-seed-descent breeding schemes, reciprocal recurrent
selection) to produce progeny which also contain the gene of
interest.
[0137] Genes can be introduced in a site directed fashion using
homologous recombination. Homologous recombination permits
site-specific modifications in endogenous genes and thus inherited
or acquired mutations may be corrected, an or novel alterations may
be engineered into the genome. Homologous recombination and
site-directed integration in plants are discussed in, for example,
U.S. Pat. Nos. 5,451,513; 5,501,967 and 5,527,695.
[0138] Methods of producing transgenic plants are well known to
those of ordinary skill in the art. Transgenic plants can now be
produced by a variety of different transformation methods
including, but not limited to, electroporation; microinjection;
microprojectile bombardment, also known as particle acceleration or
biolistic bombardment; viral-mediated transformation; and
Agrobacterium-mediated transformation. See, for example, U.S. Pat.
Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318;
5,641,664; 5,736,369 and 5,736369; Watson et al., Recombinant DNA,
Scientific American Books (1992); Hinchee et al, Bio/Tech.
6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988);
Toriyama et al., Bio/Tech. 6:1072-1074 (1988); Fromm et al.,
Bio/Tech. 8:833-839 (1990); Mullins et al. Bio/Tech. 8:833-839
(1990); Hiei et al., Plant Molecular Biology 35:205-218 (1997);
Ishida et al., Nature Biotechnology 14:745-750 (1996); Zhang et
al., Molecular Biotechnology 8:223-231 (1997); Ku et al., Nature
Biotechnology 17:76-80 (1999); and, Raineri et al., Bio/Tech.
8:33-38 (1990)), each of which is expressly incorporated herein by
reference in their entirety.
[0139] Agrobacterium tumefaciens is a naturally occurring bacterium
that is capable of inserting its DNA (genetic information) into
plants, resulting in a type of injury to the plant known as crown
gall. Most species of plants can now be transformed using this
method, including alfalfa. See, for example, Wang et al.,
Australian Journal of Plant Physiology 23(3): 265-270 (1996);
Hoffman et al., Molecular Plant-Microbe Interactions 10(3): 307-315
(1997); and, Trieu et al., Plant Cell Reports 16:6-11 (1996).
[0140] Microprojectile bombardment is also known as particle
acceleration, biolistic bombardment, and the gene gun
(Biolistic.RTM. Gene Gun). The gene gun is used to shoot pellets
that are coated with genes (e.g., for desired traits) into plant
seeds or plant tissues in order to get the plant cells to then
express the new genes. The gene gun uses an actual explosive (0.22
caliber blank) to propel the material. Compressed air or steam may
also be used as the propellant. The Biolistic.RTM. Gene Gun was
invented in 1983-1984 at Cornell University by John Sanford, Edward
Wolf, and Nelson Allen. It and its registered trademark are now
owned by E. I. du Pont de Nemours and Company. Most species of
plants have been transformed using this method, including alfalfa
(U.S. Pat. No. 5,324,646) and clover (Voisey et al., Biocontrol
Science and Technology 4(4): 475-481 (1994); Quesbenberry et al.,
Crop Science 36(4): 1045-1048 (1996); Khan et al., Plant Physiology
105(1): 81-88 (1994); and, Voisey et al., Plant Cell Reports 13(6):
309-314 (1994)).
[0141] Developed by ICI Seeds Inc. (Garst Seed Company) in 1993,
WHISKERS.TM. is an alternative to other methods of inserting DNA
into plant cells (e.g., the Biolistic.RTM. Gene Gun, Agrobacterium
tumefaciens, the "Shotgun" Method, etc.); and it consists of
needle-like crystals ("whiskers") of silicon carbide. The fibers
are placed into a container along with the plant cells, then mixed
at high speed, which causes the crystals to pierce the plant cell
walls with microscopic "holes" passages). Then the new DNA (gene)
is added, which causes the DNA to flow into the plant cells. The
plant cells then incorporate the new gene(s); and thus they have
been genetically engineered.
[0142] The essence of the WHISKERS.TM. technology is the small
needle-like silicon carbide "whisker" (0.6 microns in diameter and
5-80 microns in length) which is used in the following manner. A
container holding a "transformation cocktail" composed of DNA
(e.g., agronomic gene plus a selectable marker gene), embryogenic
corn tissue, and silicon carbide "whiskers" is mixed or shaken in a
robust fashion on either a dental amalgam mixer or a paint shaker.
The subsequent collisions between embryogenic corn cells and the
sharp silicon carbide "whiskers" result in the creation of small
holes in the plant cell wall through which DNA (the agronomic gene)
is presumed to enter the cell. Those cells receiving and
incorporating a new gene are then induced to grow and ultimately
develop into fertile transgenic plants.
[0143] Silicon carbide "whisker" transformation has now produced
stable transformed calli and/or plants in a variety of plants
species such as Zea mays. See, for example, U.S. Pat. Nos.
5,302,523 and 5,464,765, each of which is incorporated herein by
reference in their entirety; Frame et al., The Plant Journal 6:
941-948 (1994); Kaeppler et al., Plant Cell Reports 9:415-418
(1990); Kaeppler et al., Theoretical and Applied Genetics
84:560-566 (1992); Petolino et al., Plant Cell Reports
19(8):781-786 (2000); Thompson et al., Euphytica 85:75-80 (1995);
Wang et al., In Vitro Cellular and Developmental Biology 31:101-104
(1995); Song et al., Plant Cell Reporter 20:948-954 (2002);
Petolino et al., Molecular Methods of Plant Analysis, In Genetic
Transformation of Plants, Vol. 23, pp. 147-158, Springer-Verlag,
Berlin (2003). Other examples include Lolium multiflorum, Lolium
perenne, Festuca arundinacea, Agrostis stolonifera (Dalton et al.,
Plant Science 132:31-43 (1997)), Oryza sativa Nagatani et al.,
Biotechnology Techniques 11:471-473 (1997)), and Triticum aestivum
and Nicotiana tobacum (Kaeppler et al., Theoretical and Applied
Genetics 84:560-566 (1992)). Even Chlamydomonas (see, for example,
Dunahay, T. G., Biotechniques 15:452-460 (1993)) can be transformed
with a "whiskers" approach. As it is currently practiced on higher
plants, the "whisker" system is one of the least complex ways to
transform some plant cells.
[0144] Genes successfully introduced into plants using recombinant
DNA methodologies include, but are not limited to, those coding for
the following traits: seed storage proteins, including modified 7S
legume seed storage proteins (see, for example, U.S. Pat. Nos.
5,508,468, 5,559,223 and 5,576,203); herbicide tolerance or
resistance (see, for example, De Greef et al., Bio/Technology 7:61
(1989); U.S. Pat. No. 4,940,835; U.S. Pat. No. 4,769,061; U.S. Pat.
No. 4,975,374; Marshall et al. (1992) Theor. Appl. Genet. 83, 435;
U.S. Pat. No. 5,489,520; U.S. Pat. No. 5,498,544; U.S. Pat. No.
5,554,798; Powell et al, Science 232:738-743 (1986); Kaniewski et
al., Bio/Tech. 8:750-754 (1990)); Day et al., Proc. Natl. Acad.
Sci. USA 88:6721-6725 (1991)); phytase (see, for example, U.S. Pat.
No. 5,593,963); resistance to bacterial, fungal, nematode and
insect pests, including resistance to the lepidoptera insects
conferred by the Bt gene (see, for example, U.S. Pat. Nos.
5,597,945 and 5,597,946; Johnson et al., Proc. Natl. Acad. Sci.
USA, 86:9871-9875 (1989); Perlak et al., Bio/Tech. 8:939-943
(1990)); lectins (U.S. Pat. No. 5,276,269); flower color (Meyer et
al., Nature 330:677-678 (1987); Napoli et al., Plant Cell 2:279-289
(1990); van der Krol et al., Plant Cell 2:291-299 (1990)); Bt genes
(Voisey et al., supra); neomycin phosphotransferase II
(Quesbenberry et al., supra); the pea lectin gene (Diaz et al.,
Plant Physiology 109(4):1167-1177 (1995); Eijsden et al., Plant
Molecular Biology 29(3):431-439 (1995)); the auxin-responsive
promoter GH3 (Larkin et al., Transgenic Research 5(5):325-335
(1996)); seed albumin gene from sunflowers (Khan et al., Transgenic
Research 5(3):179-185 (1996)); and genes encoding the enzymes
phosphinothricin acetyl transferase, beta-glucuronidase (GUS)
coding for resistance to the Basta.RTM., herbicide, neomycin
phosphotransferase, and an alpha-amylase inhibitor (Khan et al.,
supra), each of which is expressly incorporated herein by reference
in their entirety.
[0145] For certain purposes, different antibiotic or herbicide
selection markers may be preferred. Selection markers used
routinely in transformation include the nptII gene which confers
resistance to kanamycin and related antibiotics (see, for example,
Messing & Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature
304:184-187 (1983)), the bar gene which confers resistance to the
herbicide phosphinothricin (White et al., Nucl Acids Res 18:1062
(1990), Spencer et al., Theor Appl Genet. 79: 625-631 (1990)), and
the dhfr gene, which confers resistance to methotrexate (Bourouis
et al., EMBO J. 2(7): 1099-1104 (1983)).
[0146] Transgenic alfalfa plants have been produced using a number
of different genes isolated from both alfalfa and non-alfalfa
species including, but not limited to, the following: the promoter
of an early nodulin gene fused to the reporter gene gusA (Bauer et
al., The Plant Journal 10(1):91-105 (1996)); the early nodulin gene
(Charon et al., Proc. Natl. Acad. of Sci. USA 94(16):8901-8906
(1997); Bauer et al., Molecular Plant-Microbe Interactions
10(1):39-49 (1997)); NADH-dependent glutamate synthase (Gantt, The
Plant Journal 8(3):345-358 (1995)); promoter-gusA fusions for each
of three lectin genes (Bauchrowitz et al., The Plant Journal
9(1):31-43 (1996); the luciferase enzyme of the marine soft coral
Renilla reniforms fused to the CaMV promoter (Mayerhofer et al.,
The Plant Journal 7(6):1031-1038 (1995)); Mn-superoxide dismutase
cDNA (McKersie et al., Plant Physiology 111(4):1177-1181 (1996));
synthetic cryIC genes encoding a Bacillus thuringiensis
delta-endotoxin (Strizhov et al., Proc. Natl. Acad. Sci. USA
93(26):15012-15017 (1996)); glucanse (Dixon et al., Gene
179(1):61-71 (1996); and leaf senescence gene (U.S. Pat. No.
5,689,042).
[0147] Genetic transformation has also been reported in numerous
forage and turfgrass species (Conger B. V., Genetic Transformation
of Forage Grasses in Molecular and Cellular Technologies for Forage
Improvement, CSSA Special Publication No. 26, Crop Science Society
of America, Inc. E. C. Brummer et al. Eds. 1998, pages 49-58).
These include, but are not limited to, orchardgrass (Dactylis
glomerata L.), tall fescue (Festuca arundinacea Schreb.) red fescue
(Festuca rubra L.), meadow fescue (Festuca pratensis Huds.)
perennial ryegrass (Lolium perenne L.) creeping bentgrass (Agrostis
palustris Huds.) and redtop (Agrostis alba L.).
V. Hemizygosity
[0148] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single gene on one chromosome,
although multiple copies are possible. Such transgenic plants can
be referred to as being hemizygous for the added gene. A more
accurate name for such a plant is an independent sergeant, because
each transformed plant represents a unique T-DNA integration event
(U.S. Pat. No. 6,156,953). A transgene locus is generally
characterized by the presence and/or absence of the transgene. A
heterozygous genotype in which one allele corresponds to the
absence of the transgene is also designated hemizygous (U.S. Pat.
No. 6,008,437).
[0149] Assuming normal hemizygosity, selfing will result in maximum
genotypic segregation in the first selfed recombinant generation,
also known as the R1 or R.sub.1, generation. The R1 generation is
produced by selfing the original recombinant line, also known as
the R0 or R.sub.0 generation. Because each insert acts as a
dominant allele, in the absence of linkage and assuming only one
hemizygous insert is required for tolerance expression, one insert
would segregate 3:1, two inserts, 15:1, three inserts, 63:1, etc.
Therefore, relatively few R1 plants need to be grown to find at
least one resistance phenotype (U.S. Pat. Nos. 5,436,175 and
5,776,760).
[0150] As mentioned above, self-pollination of a hemizygous
transgenic regenerated plant should produce progeny equivalent to
an F2 in which approximately 25% should be homozygous transgenic
plants. Self-pollination and testcrossing of the F2 progeny to
non-transformed control plants can be used to identify homozygous
transgenic plants and to maintain the line. If the progeny
initially obtained for a regenerated plant were from
cross-pollination, then identification of homozygous transgenic
plants will require an additional generation of self-pollination
(U.S. Pat. No. 5,545,545).
VI. Disabling Genes
[0151] It may be desirable to disable certain plant genes to gain
the expression of the transgene and or to obtain the desired
protein produced as a result of the expression of the transgene.
For example, in the instant invention, it may be desirable to
disable certain enzymes that are native to the transgenic plant,
for example one or more specific plant transferases. Methods of
disabling genes are well known to those of ordinary skill in the
art.
[0152] For example, an effective disabling modification is the
introduction of a single nucleotide deletion occurring at the
beginning of a gene that would produce a translational reading
frameshift. Such a frameshift would disable the gene, resulting in
non-expressible gene product and thereby disrupting functional
protein production by that gene. If the unmodified gene encodes a
protease, for example, protease production by the gene could be
disrupted if the regulatory regions or the coding regions of the
protease gene are disrupted.
[0153] In addition to disabling genes by deleting nucleotides,
causing a transitional reading frameshift, disabling modifications
would also be possible by other techniques well known to those of
ordinary skill, including insertions, substitutions, inversions or
transversions of nucleotides within the gene's DNA that would
effectively prevent the formation of the protein encoded by the
DNA.
[0154] It is also within the capabilities of one skilled in the art
to disable genes by the use of less specific methods. Examples of
less specific methods would be the use of chemical mutagens such as
hydroxylamine or nitrosoguanidine or the use of radiation mutagens
such as gamma radiation or ultraviolet radiation to randomly mutate
genes. Such mutated strains could, by chance, contain disabled
genes such that the genes were no longer capable of producing
functional proteins for any one or more of the domains. The
presence of the desired disabled genes could be detected by routine
screening techniques. For further guidance, see, for example, U.S.
Pat. No. 5,759,538.
VII. Down Regulation
[0155] Down-regulation of expression of a target gene may be
achieved using anti-sense technology or "sense regulation"
("co-suppression").
[0156] In using anti-sense genes or partial gene sequences to
down-regulate gene expression, a nucleotide sequence is placed
under the control of a promoter in a "reverse orientation" such
that transcription yields RNA which is complementary to normal mRNA
transcribed from the "sense" strand of the target gene. See, for
example, Smith et al, (1988) Nature 334:724-726; Zhang et al,
(1992) The Plant Cell 4:1575-1588, English et al., (1996) The Plant
Cell 8:179-188. Antisense technology is also reviewed in Bourque,
(1995), Plant Science 105:125-149, and Flavell, (1994) PNAS USA
91:3490-3496. Methods for inhibiting expression in plants using
antisense constructs, including generation of antisense sequences
in situ are well known to those of ordinary skill in the art and
are also described, for example, in U.S. Pat. Nos. 5,107,065;
5,254,800; 5,356,799; 5,728,926; and 6,184,439.
[0157] "Co-suppression" refers to the production of sense RNA
transcripts capable of suppressing the expression of identical or
substantially similar foreign or endogenous genes (U.S. Pat. No.
5,231,020). An alternative is to use a copy of all or part of the
target gene inserted in sense, that is the same, orientation as the
target gene, to achieve reduction in expression of the target gene
by co-suppression. See, for example, van der Krol et al., (1990)
The Plant Cell 2:291-299; Napoli et al., (1990) The Plant Cell
2:279-289; Zhang et al., (1992) The Plant Cell 4:1575-1588, and
U.S. Pat. No. 5,231,020. Further refinements of gene silencing or
co-suppression technology may be found in WO95/34668 (Biosource);
Angell & Baulcombe (1997) The EMBO Journal 16(12):3675-3684;
and Voinnet & Baulcombe (1997) Nature 389, pg 553.
[0158] The complete sequence corresponding to the coding sequence
(in reverse orientation for anti-sense) need not be used. For
example fragments of sufficient length, such as SEQ ID NO: 1, may
be used. It is a routine matter for the person skilled in the art
to screen fragments of various sizes and from various parts of the
coding sequence to optimise the level of anti-sense inhibition. It
may be advantageous to include the initiating methionine ATG codon,
and perhaps one or more nucleotides upstream of the initiating
codon. A further possibility is to target a conserved sequence of a
gene, e.g. a sequence that is characteristic of one or more genes,
such as a regulatory sequence.
[0159] The sequence employed may be about 500 nucleotides or less,
possibly about 400 nucleotides, about 300 nucleotides, about 200
nucleotides, or about 100 nucleotides. It may be possible to use
oligonucleotides of much shorter lengths, 14-23 nucleotides,
although longer fragments, and generally even longer than about 500
nucleotides are preferable where possible, such as longer than
about 600 nucleotides, than about 700 nucleotides, than about 800
nucleotides, than about 1000 nucleotides or more.
[0160] It may be preferable that there is complete sequence
identity in the sequence used for down-regulation of expression of
a target sequence, and the target sequence, though total
complementarity or similarity of sequence is not essential. One or
more nucleotides may differ in the sequence used from the target
gene. Thus, a sequence employed in a down-regulation of gene
expression in accordance with the present invention may be a
wild-type sequence (e.g. gene) selected from those available, or a
mutant, derivative, variant or allele, by way of insertion,
addition, deletion or substitution of one or more nucleotides, of
such a sequence. The sequence need not include an open reading
frame or specify an RNA that would be translatable. It may be
preferred for there to be sufficient homology for the respective
anti-sense and sense RNA molecules to hybridise. There may be down
regulation of gene expression even where there is about 5%, 10%,
15% or 20% or more mismatch between the sequence used and the
target gene.
[0161] Generally, the transcribed nucleic acid may represent a
fragment of an ig1 gene, such as including a nucleotide sequence
provided in SEQ ID NO: 1, or the complement thereof, or may be a
mutant, ortholog, derivative, variant or allele thereof, in similar
terms as discussed above in relation to alterations being made to
an ig1 coding sequence and the homology of the altered sequence.
The homology may be sufficient for the transcribed anti-sense RNA
to hybridise with nucleic acid within cells of the plant, though
irrespective of whether hybridisation takes place the desired
effect is down-regulation of gene expression.
[0162] Other methods that can be used to inhibit expression of an
endogenous gene in a plant may also be used in the present methods.
For example, formation of a triple helix at an essential region of
a duplex gene serves this purpose. The triplex code, permitting
design of the proper single stranded participant is also known in
the art. (See, for example, H. E. Moser et al., Science 238:645-650
(1987) and M. Cooney et al., Science 241:456-459 (1988)). Regions
in the control sequences containing stretches of purine bases are
particularly attractive targets. Triple helix formation along with
photocrosslinking is described, e.g., in D. Praseuth et al., Proc.
Nat'l Acad. Sci. USA 85:1349-1353 (1988).
VIII. Knock-Ins and Knock-Outs
[0163] As used herein, the term "knock-in" refers to a cell, tissue
or organism that has had a gene introduced into its genome, wherein
the gene can be of exogenous or endogenous origin. Generally, if
the introduced gene is endogenous in origin, it will be a modified
gene. An introduced gene that is exogenous in origin can be in its
wild-type form or in a modified form.
[0164] As used herein, a "knock-out" refers to a cell, tissue or
organism in which there is partial or complete suppression of the
expression of an endogenous gene (e.g., based on deletion of at
least a portion of the gene, replacement of at least a portion of
the gene with a second sequence, introduction of stop codons, the
mutation of bases encoding critical amino acids, or the removal of
an intron junction, etc.). The targeted gene can be partially or
completely suppressed by disruption, inactivation or deletion. Said
partial suppression may also be referred to herein as a
"knock-down." Knock-outs can be performed using both in vitro and
in vivo recombination techniques. In order to study gene functions,
usually the cell, tissue or organism is genetically engineered with
specified wild-type alleles replaced with mutated ones. Knock-outs
can be made using homologous recombination between the target gene
and a piece of cloned DNA to insert a piece of "junk" DNA into the
gene desired to be disrupted. If the organism is haploid, then this
technique will result in that organism's only copy of the gene
being knocked out. If it is diploid, then only one of the two
alleles will be knocked out, and it will be necessary to do
conventional breeding to produce a diploid organism that has two
copies of the gene knocked out.
IX. Over-Expression
[0165] "Overexpression" refers to the production of a gene product
in transgenic organisms that exceeds levels of production in normal
or non-transformed organisms. In the present invention,
over-expression of ig1 may be achieved by introduction of the
nucleotide sequence of ig1 in a sense orientation.
[0166] Thus, the present invention provides a method of influencing
a physical, e.g. flowering characteristic of a plant, the method
including causing or allowing expression of the product
(polypeptide or nucleic acid transcript) encoded by heterologous
nucleic acid according to the invention from that nucleic acid
within cells of the plant.
[0167] Methods of over-expressing genes are generally known by
those skilled in the art. For examples of producing cells which
over-express specific genes, see, for example, U.S. Pat. Nos.
5,905,146; 5,849,999; 5,859,311; 5,602,309; 5,952,169 and
5,772,997; Saito et al., "Modulation of Cystein Biosynthesis in
Chloroplasts of Transgenic Tobacco Overexpressing Cysteine Synthase
[O-Acetylserine(thiol)-lyase]", Plant Physiology (1996)106:
887-895.
X. Double Stranded RNA Interference (dsRNAi)
[0168] Reduction or inhibition of a gene can also be accomplished
through the use of a RNA interference (RNAi). As is well known to
those skilled in the art, this is a phenomenon in which the
introduction of double-stranded RNA (dsRNA) into a diverse range of
organisms and cell types causes degradation of the complementary
mRNA. In the cell, long dsRNAs are cleaved into short 21-25
nucleotide small interfering RNAs, or siRNAs, by a ribonuclease
known as Dicer. The siRNAs subsequently assemble with protein
components into an RNA-induced silencing complex (RISC), unwinding
in the process. Activated RISC then binds to complementary
transcript by base pairing interactions between the siRNA antisense
strand and the mRNA. The bound mRNA is cleaved and sequence
specific degradation of mRNA results in gene silencing. See, for
example, U.S. Pat. No. 6,506,559; Fire et al., Nature (1998)
391(19):306-311; Timmons et al., Nature (1998) 395:854; Montgomery
et al., TIG (1998) 14(7):255-258; David R. Engelke, Ed., RNA
Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press
(2003); Gregory J. Hannon, Ed., and RNAi A Guide to Gene Silencing,
Cold Spring Harbor Laboratory Press (2003). For plant-specific
information on dsRNAi see, for example, WVO 99/53050; and WO
99/49029. Therefore, the present invention also includes methods of
silencing genes by using RNAi technology.
XI. Breeding Methods
[0169] Open-Pollinated Populations. The improvement of
open-pollinated populations of such crops as rye, many maizes and
sugar beets, herbage grasses, legumes such as alfalfa and clover,
and tropical tree crops such as cacao, coconuts, oil palm and some
rubber, depends essentially upon changing gene-frequencies towards
fixation of favorable alleles while maintaining a high (but far
from maximal) degree of heterozygosity. Uniformity in such
populations is impossible and trueness-to-type in an
open-pollinated variety is a statistical feature of the population
as a whole, not a characteristic of individual plants. Thus, the
heterogeneity of open-pollinated populations contrasts with the
homogeneity (or virtually so) of inbred lines, clones and
hybrids.
[0170] Population improvement methods fall naturally into two
groups, those based on purely phenotypic selection, normally called
mass selection, and those based on selection with progeny testing.
Interpopulation improvement utilizes the concept of open breeding
populations; allowing genes for flow from one population to
another. Plants in one population (cultivar, strain, ecotype, or
any germplasm source) are crossed either naturally (e.g., by wind)
or by hand or by bees (commonly Apis mellifera L. or Megachile
rotundata F.) with plants from other populations. Selection is
applied to improve one (or sometimes both) population(s) by
isolating plants with desirable traits from both sources.
[0171] There are basically two primary methods of open-pollinated
population improvement. First, there is the situation in which a
population is changed en masse by a chosen selection procedure. The
outcome is an improved population that is indefinitely propagable
by random-mating within itself in isolation. Second, the synthetic
variety attains the same end result as population improvement but
is not itself propagable as such; it has to be reconstructed from
parental lines or clones. These plant breeding procedures for
improving open-pollinated populations are well known to those
skilled in the art and comprehensive reviews of breeding procedures
routinely used for improving cross-pollinated plants are provided
in numerous texts and articles, including: Allard, Principles of
Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds,
Principles of Crop Improvement, Longman Group Limited (1979);
Hallauer and Miranda, Quantitative Genetics in Maize Breeding, Iowa
State University Press (1981); and, Jensen, Plant Breeding
Methodology, John Wiley & Sons, Inc. (1988).
[0172] Mass Selection. In mass selection, desirable individual
plants are chosen, harvested, and the seed composited without
progeny testing to produce the following generation. Since
selection is based on the maternal parent only, and there is no
control over pollination, mass selection amounts to a form of
random mating with selection. As stated above, the purpose of mass
selection is to increase the proportion of superior genotypes in
the population.
[0173] Synthetics. A synthetic variety is produced by crossing
inter se a number of genotypes selected for good combining ability
in all possible hybrid combinations, with subsequent maintenance of
the variety by open pollination. Whether parents are (more or less
inbred) seed-propagated lines, as in some sugar beet and beans
(Vicia) or clones, as in herbage grasses, clovers and alfalfa,
makes no difference in principle. Parents are selected on general
combining ability, sometimes by test crosses or topcrosses, more
generally by polycrosses. Parental seed lines may be deliberately
inbred (e.g. by selfing or sib crossing). However, even if the
parents are not deliberately inbred, selection within lines during
line maintenance will ensure that some inbreeding occurs. Clonal
parents will, of course, remain unchanged and highly
heterozygous.
[0174] Whether a synthetic can go straight from the parental seed
production plot to the farmer or must first undergo one or two
cycles of multiplication depends on seed production and the scale
of demand for seed. In practice, grasses and clovers are generally
multiplied once or twice and are thus considerably removed from the
original synthetic.
[0175] While mass selection is sometimes used, progeny testing is
generally preferred for polycrosses, because of their operational
simplicity and obvious relevance to the objective, namely
exploitation of general combining ability in a synthetic.
[0176] The number of parental lines or clones that enter a
synthetic vary widely. In practice, numbers of parental lines range
from 10 to several hundred, with 100-200 being the average. Broad
based synthetics formed from 100 or more clones would be expected
to be more stable during seed multiplication than narrow based
synthetics.
[0177] Hybrids. A hybrid is an individual plant resulting from a
cross between parents of differing genotypes. Commercial hybrids
are now used extensively in many crops, including corn (maize),
sorghum, sugarbeet, sunflower and broccoli. Hybrids can be formed
in a number of different ways, including by crossing two parents
directly (single cross hybrids), by crossing a single cross hybrid
with another parent (three-way or triple cross hybrids), or by
crossing two different hybrids (four-way or double cross
hybrids).
[0178] Strictly speaking, most individuals in an out breeding
(i.e., open-pollinated) population are hybrids, but the term is
usually reserved for cases in which the parents are individuals
whose genomes are sufficiently distinct for them to be recognized
as different species or subspecies. Hybrids may be fertile or
sterile depending on qualitative and/or quantitative differences in
the genomes of the two parents. Heterosis, or hybrid vigor, is
usually associated with increased heterozygosity that results in
increased vigor of growth, survival, and fertility of hybrids as
compared with the parental lines that were used to form the hybrid.
Maximum heterosis is usually achieved by crossing two genetically
different, highly inbred lines.
[0179] The production of hybrids is a well-developed industry,
involving the isolated production of both the parental lines and
the hybrids which result from crossing those lines. For a detailed
discussion of the hybrid production process, see, e.g., Wright,
Commercial Hybrid Seed Production 8:161-176, In Hybridization of
Crop Plants.
XII. Genetic Probes
[0180] The present invention further provides methods of
recognizing variations in the DNA sequence of Zea mays ig1 as well
as for detecting the gene or its homologs or orthologs in other
plant genera, species, strains, varieties or cultivars. One method
involves the introduction of a nucleic acid molecule (also known as
a probe or nucleic acid probe) having a sequence identical or
complementary to at least a portion of ig1 (SEQ ID NO: 1) of the
invention under sufficient hybridizing conditions as would be
understood by those in the art, such as the moderately stringent or
highly stringent hybridization conditions as described elsewhere
within the instant description. Said probe would share identity
with the DNA sequence of SEQ ID NO: 1 over at least about 10
contiguous nucleic acid residues. Preferably, said identity would
be over at least about 25 or 30 contiguous nucleic acid residues.
More preferably, said identity would be over at least about 40 or
50 contiguous nucleic acid residues. Even more preferably, said
identity would be over at least about 60 or 75 contiguous nucleic
acid residues. Still more preferably, said identity would be over
at least about 100 or 150 contiguous nucleic acid residues. Yet
more preferably, said identity would be over at least about 200 or
250 contiguous nucleic acid residues. Most preferably, said
identity would be over at least about 300 contiguous nucleic acid
residues or would math the entire open reading frame of SEQ ID NO:
1 or its complement.
[0181] Another method of recognizing DNA sequence variation is
direct DNA sequence analysis by multiple methods well known in the
art. Another embodiment involves the detection of DNA sequence
variation in IG1 proteins as represented by different plant genera,
species, strains, varieties or cultivars. Another embodiment
involves using said nucleic acid probes for the detection of ig1
sequences in a sample or tissue section using in situ hybridization
according to any method known to those of skill in the art. The ig1
sequence used for the probe can be from any plant for which the
presence of ig1 has been determined. A particularly good probe for
a monocotyledonous plant would be that coding for the IG 1 of
maize. In one embodiment, the sequence will bind specifically to
one allele of a IG1-encoding gene, or a fragment thereof, and in
another embodiment will bind to multiple alleles. Such detection
methods include the polymerase chain reaction, restriction fragment
length polymorphism (RFLP) analysis and single stranded
conformational analysis.
[0182] Diagnostic probes useful in such assays of the invention
include antibodies to IG1 proteins. The antibodies to IG1 may be
either monoclonal or polyclonal, produced using standard techniques
well known in the art (See Harlow & Lane's Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). They
can be used to detect IG1, or a homolog or ortholog thereof,
protein by binding to the protein and subsequent detection of the
antibody-protein complex by ELISA, Western blot or the like. The
IG1 sequence used to elicit these antibodies can be any of the IG1
variants discussed herein, including IG1-0 and IG1-MUM. Antibodies
are also produced from peptide sequences of IG 1 using standard
techniques in the art (See Protocols in Immunology, John Wiley
& Sons, 1994). Fragments of the monoclonals or the polyclonal
antisera which contain the immunologically significant portion can
also be prepared.
[0183] Assays to detect or measure IG1 polypeptide in a biological
sample with an antibody probe may be based on any available format.
For instance, in immunoassays where IG1 polypeptides are the
analyte, the test sample, typically a biological sample, is
incubated with anti-IG1 antibodies under conditions that allow the
formation of antigen-antibody complexes. Various formats can be
employed, such as "sandwich" assay where antibody bound to a solid
support is incubated with the test sample; washed, incubated with a
second, labeled antibody to the analyte; and the support is washed
again. Analyte is detected by determining if the second antibody is
bound to the support. In a competitive format, which can be either
heterogeneous or homogeneous, a test sample is usually incubated
with an antibody and a labeled competing antigen, either
sequentially or simultaneously. These and other formats are well
known in the art. Alternatively, a test sample may be a tissue
section of a plant which is probed with an antibody to IG1 using
methods well known to those in the art for detection of proteins in
a tissue section with an antibody. Said tissue section may be from
a plant being tested for natural expression of IG1 or a homolog or
ortholog thereof. Alternatively said tissue section may be from a
plant which has been genetically altered by the means of the
present invention or by some other means to express at least one
protein selected from the group consisting of IG1, IG1-0, IG1-MUM,
and homologs or orthologs thereof.
EXAMPLES
Example 1
Characterize Newly Established Mutants with Extra Polar Nuclei
[0184] ig2 has been mapped using Simple Sequence Repeat (SSR)
markers and the floury3(fl3) mutation to chromosome 8 (FIG. 3).
ig*-15791 does not show linkage to markers linked to ig1 or ig2 and
consequently has been named ig3.
[0185] ig1, ig2, and ig3 have all been crossed to several inbred
lines to determine their spectrum of phenotypic expression. ig1
shows good expression in W23, Mol7, W64A, and A158 but is
suppressed in B73. ig2 shows good expression in B73, A158, and W64A
but is suppressed in W23 and Mol7. ig3 shows good expression in B73
and W23 and moderate expression in Mol7 and is suppressed in W64A.
From these results double mutants are being constructed of ig1 and
ig2 in an A158 background. The frequency of miniature kernels
produced by ig1, ig2 double mutants is the same as predicted if ig1
and ig2 interact additively.
[0186] Self-pollinations of ig2 and ig3 heterozygotes suggest
mutant phenotypes of homozygous sporophytes. ig2 homozygotes are
likely embryo lethal with an early aborting endosperm phenotype. In
a W64A inbred background, the ig2 homozygous phenotype is slightly
less severe. The endosperm undergoes extensive cell death and the
embryo is small and abnormally shaped (FIG. 4). Some endosperm
development occurs and seeds germinate to produce abnormal
seedlings (FIG. 5). The epidermis of these plants is highly
abnormal and irregular with swollen cells. ig3 homozygotes on the
other hand are likely viable but have minor seed defects as seen by
the increase in a variety of abnormal seed types in
self-pollinations. Additionally, ig3 homozygotes may be male
sterile in some backgrounds.
[0187] To test for heterofertilization of ig2 and ig3 embryo sacs
mutant heterozgotes in a B73 inbred background have been pollinated
by B73 inbreds heterozygous at the r locus, carrying R-sc
(conferring aleurone and embryo color) and r-g (conferring a
colorless aleurone and embryo). Control crosses onto standard B73
plants have also been made. The frequency of heterofertilization in
crosses onto ig2 and ig3 heterozygotes is not significantly
different from wild type. Early examination of ig3 embryo sacs has
demonstrated that some of the mutant embryo sacs arrest early in
megagametogenesis (FIG. 6).
Example 2
Cloning of Indeterminate Gametophyte1
[0188] Fine mapping of the ig1 gene was performed using the rice
genome as a framework to order maize genes in the region of ig1.
First an overall level of synteny was established between rice and
maize around ig1 by taking the sequence of maize genetic markers
and performing a Blast search against the rice genome sequence to
find the most similar rice gene to each maize marker. This
established rice chromosome 1 as the correct region to search for
ig1. This analysis demonstrated that the majority of the maize
markers fell in the same contig of rice chromosome 1 and the
relative order of the markers was conserved.
[0189] Fine mapping of ig1 against these markers placed ig1 between
umc1311 and umc1973 which have orthologs on rice chromosome 1
approximately 845 kilobases (kb) apart (FIG. 7). By generating
mapping populations with multiple inbreds polymorphisms were found
with other markers that further reduced this interval in the rice
map to 378 kb on three rice Bacterial Artificial Chromosomes
(BACs). At this point the available markers in the region in maize
had been exhausted, so an attempt was made to use the rice genome
sequence as displayed in the Gramene database (ww.gramene.org) to
develop more maize markers. In addition to displaying the annotated
rice genome DNA sequence of other grasses including maize was also
displayed that had high similarity to the annotated rice genes.
[0190] Gene content of the region was inferred from the rice
contig, and the sequences of the maize clones that matched these
genes was used to design primers to develop PCR based mapping
markers. Primers were first designed to amplify within exons, and
amplicons were digested with several different restriction enzymes.
These amplicons did not show any polymorphisms that could be used
on the ig1 mapping population. However, by using the exon-intron
structure of the rice genes as a guide for maize gene structure,
primers were designed around maize introns and four out of eight
were polymorphic. Two of these were useful in the mapping
population. This reduced the interval in the rice genome predicted
to contain the ig1 ortholog to 65 kb. The annotated genes in this
interval are shown in FIG. 8.
[0191] Non-complementation screens of 65,000 individuals produced
seven male sterile selections as potential Mu alleles of ig1. After
backcrossing, one of these new alleles has been proven to be
heritable and to cause characteristic ig1 seed phenotypes. Mutant
heterozygotes produce miniatures, aborted kernels, and twins. A
cosegregating Mu element was found in a small population using a
modified version of the Amplification of Insertion Mutagenized
Sites protocol (AIMS) (Frey et al., 1998). This band was cloned and
sequenced and found to be a Wu insertion in the LOB domain protein,
the closest homoloa of which lies in the rice genomic interval
defined by the comparative mapping experiments. The DNA sequence
flanking this insertion was used as a probe on a Southern blot with
DNA from plants heterozygous for either the ig1-mum allele compared
to its progenitor or the ig1-O allele compared to its progenitor.
Both alleles have a novel band not found in their progenitors (i.e.
both alleles have a new mutation in this gene) demonstrating that
this is the ig1 gene (FIG. 9).
[0192] As additional evidence that the reference allele ig1-O
carries a mutation in this same gene, PCR amplification of this
gene was performed on homozygotes for ig1-O and its progenitor for
several primer combinations (FIG. 9B). Primer R1 in combination
with primer F1 or F2 generated a PCR product in wild type but
failed to do so in ig1-O homozygotes. Primer R2 with either F1 or
F2 generated a PCR product in wild type and mutant. These data
suggest that the mutation in ig1-O lies between Primers R1 and R2
or within primer R1.
[0193] Thus, the ig1 gene was isolated using a combination of
directed tagging and comparative mapping approaches. The partial
nucleotide sequence of ig1 is provided in SEQ ID NO: 1 and the
corresponding partial amino acid sequence of ig1 is provided in SEQ
ID NO: 2. The ig1-O mutation is of unknown nature and occurs within
base pair number 506 to base pair number 614 of SEQ ID NO: 1. The
ig1-mum is a Mutator transposable element insertion between base
pair number 173 and base pair number 174 of SEQ ID NO: 1.
[0194] The ig1 gene encodes a member of the LATERAL ORGANS BOUNDARY
(LOB) protein family (Shuai et al., 2002). This is a plant-specific
gene family with 42 members in Arabidopsis (Shuai et al., 2002;
Iwakawa et al., 2002). ig1 and its rice ortholog are most similar
to ASYMMETRIC LEAVES2 in Arabidopsis (FIG. 2). The AS2 gene has
been shown to repress the expression of the Knotted1-like homeobox
(knox) genes K-NAT1/BPEVIPEDICELLUS1 (BP)), KCVAT2, and KVNAT6 in
leaf primordia (On et al., 2000; Semiarti et al., 2001; Xu et al.,
2003; Lin et al., 2003). These genes are normally expressed in the
shoot apical meristem but not in leaf primordia (Long et al., 1996;
Lincoln et al., 1994). as2 loss-of-function mutations result in
ectopic expression of know genes in leaf primordia. This phenotype
is also caused by loss-of-function mutations in the MYB-domain gene
ASYMMETRIC LEAVES1 (Ori et al., 2000; Semiarti et al., 2001).
[0195] The maize ortholog of AS1 rough sheath2, has a similar role
in repressing knox gene expression in leaf primordia demonstrating
conservation of function in dicots and monocots (Schneeberger et
al., 1998; Timmermans et al., 1999; Tsiantis et al., 1999). AS2 is
a nuclear protein that interacts physically with AS1 protein,
suggesting a role for LOB domain genes in regulating transcription
(in the case of AS2 in combination with AS1) (Iwakawa et al., 2002;
Xu et al., 2003). It is not yet known whether AS1 and AS2 repress
transcription of knox genes directly or indirectly. Interestingly,
regarding the role of ig1 in restricting the embryogenic potential
of cells lacking a maternal or paternal genome, as2 mutants have an
enhanced ability to develop autonomous shoots in vitro (Semiarti et
al., 2001). To date no effect of as2 on the female gametophyte has
been reported. The molecular identity of ig1 suggests that common
mechanisms have been used in regulating the number of
nuclear-cytoplasmic domains in the embryo sac and stem cell
identity in the shoot.
Example 3
Screening for Additional Mutants with Extra Polar Nuclei
[0196] Two new isolates were identified that produced plump
endosperms and triploid embryos when pollinated with pollen from a
tetraploid plant. One of these was recovered by outcrossing as a
pollen parent followed by retesting as a female and shown to have
ig1-like seed phenotypes. This verified polar nuclei number mutant
arose in a Mutator transposable element line.
Example 4
Expression Pattern of ig1
[0197] Developing tassels and ears of wild-type plants are fixed
and sectioned according to the protocol of Jackson et al. (1991).
The 3' portion of the ig1 gene is to be used for a probe for in
situ hybridization because the amino terminus of the protein
carries the conserved LOB domain while the carboxy terminus is
divergent among members of the LOB gene family. Specifically, the
time of expression of ig1 during megagametogenesis is
established.
Example 5
Test for a Role for as2 or One of the Closely Related Genes in the
Arabidopsis Embryo Sac
[0198] AS2 falls into a lade of LOB genes with 4 other members that
are more closely related than the others (FIG. 2 and Iwakawa et
al., 2002). These are LBD36/ASL1, LBD10/ASL2, LBD25/ASL3, and
LOBa/ASL4.
[0199] Testing for expression of the most closely related LOB genes
in the embryo. Knowing which of these genes is expressed in the
embryo sac is useful in determining which may be carrying out ig1
function in Arabidopsis thaliana. Microarray data has been analyzed
on the expression of some of these genes in flowers and other
tissues and they have been compared to the expression of genes
known to be required in the embryo sac: DEMETER (DME), MEDEA (MEA),
and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) (Table 1). None of
these genes is expressed at high levels in any tissue tested.
Unfortunately, the small number of cells contributed to the flower
by the embryo sac makes it difficult or impossible to distinguish
embryo sac gene expression in this manner, as evidenced by the
extremely low values for MEA in the flower despite the fact that it
is required in the embryo sac. Consequently, expression of AS2 and
its four closest relatives are examined in the embryo sac using in
situ hybridization. Probes from the 3' end of the cDNAs for these
genes are used to prevent cross hybridization. These experiments
are used to determine which of these family members are expressed
in the embryo sac. This information helps establish which one(s)
carry out the role of the ig1 gene in the embryo sac of
Arabidopsis.
TABLE-US-00002 TABLE 1 Microarray analysis of expression levels of
some LOB domain genes and gametophyte required genes in Arabidopsis
AS2 LBD10 LBD25 DME MEA FIE flower average 309 337 42 147 16 235
leaf average 191 524 50 244 171 283 root average 84 175 93 133 12
254 seedling average 100 215 183 124 11 184 silique average 238 193
20 60 14 293 stem average 27 307 52 119 34 166
[0200] Examination of mutant phenotypes of knockouts of the genes
that are expressed in the embryo sac. If expression pattern does
not distinguish them, they are examined in order of similarity to
ig1. Since ig1 bears the most similarity to AS2, as2 are examined
first for embryo sac defects using Confocal Laser Scanning
Microscopy according to the method of Christensen et al. (1997).
Specifically, embryo sacs are examined for the presence of extra
rounds of free nuclear divisions and improper placement of nuclei
during the syncytial phase of development. If no defects are
detected in as2, knockouts in the five most closely related genes
are examined in a similar fashion next; these genes are LBD36/ASL1,
LBD10/ASL2, LBD25/ASL3, and LOBa/ASL4. T-DNA insertions are
available for all of these genes except LBD36/ASL1 for which there
are two EMS induced TILLING alleles in conserved amino acid
residues. It is possible that there is genetic redundancy present
in Arabidopsis that is not present in maize (and vice versa). If
none of the single mutants display an ig-like phenotype, then
double mutants are tested starting with as2 and its closest
relative lbd36/asl1 followed by as2 lbd10/asl2 doubles, lbd36
lbd10, and lbd25 loba. If these all have a wild type embryo sac
phenotype, the triple mutant between as2, lbd36/asl1, and
lbd10/asl2 will be constructed and tested next because these three
are grouped together more closely than LBD25/ASL3 and
LOBa/ASL4.
Example 6
Test for a Role of Knox Genes in the ig1 Phenotype
[0201] Test for ectopic expression of knox genes in ig1 mutant
embryo sacs. Because ig1 is most similar to AS2 in Arabidopsis it
raises the possibility that the mutant phenotype is caused by a
similar mechanism, i.e. misexpression of knox genes, but in this
case in the embryo sac rather than in leaves. The lack of a leaf
phenotype in ig1 mutants may reflect genetic redundancy in maize
not present in Arabidopsis. However, it is not yet known if either
mutant allele is a complete loss of function.
[0202] Expression of rs1, kn1, lg3, lg4, and gn1 in ig1 mutant and
wild type ovules is examined by in situ hybridization to determine
if any of these genes are ectopically expressed in mutant embryo
sacs.
[0203] Phenocopying the ig1 mutant by misexpressing knox genes. The
as2 and rs2 leaf phenotypes are mimicked by ectopically expressing
knox genes in leaf primordia in both Arabidopsis and maize (Chuck
et al., 1996; Schneeberger et al., 1995). To test if ig1 are
mimicked by similarly expressing knox genes in the embryo sac,
knat1/bp1 and stm are expressed using promoters that can drive
expression specifically in the embryo sac. Three different
promoters are to be tested, those of DEMETER (DME), MEDEA (MEA),
and FERTILIZATION INDEPENDENT SEED2 (FIS2). The promoter sequences
to be used are: for DME the 2282 base pairs 5' of the start site;
for MEA the 2,070 base pairs upstream of the translational start
site; and for FIS2 the 3,189 base pairs upstream of the
translational start site. These promoters have been shown to drive
expression of B-glucuronidase and Green fluorescent protein in
embryo sacs without expression in other parts of the ovule,
although MEA and FIS2 show expression after fertilization (Luo et
al., 2000; Choi et al., 2002).
[0204] However, expression from these promoters in other parts of
the plant has not been reported. If these constructs cause defects
in the sporophyte that make it difficult to score the embryo sac
phenotype, a different strategy will be taken. The DAME promoter is
used to drive expression of a protein fusion of BP1/KNAT1 and the
steroid binding domain of the glucocorticoid receptor. A similar
protein fusion has been used to ectopically express KNOTTED1 in
leaves with the CaMV35S promoter (Hay et al., 2003). Dexamethasone
is added to floral buds to activate the protein fusion in the
embryo sac cells expressing it. Ovules are then be cleared and
examined for extra rounds of free nuclear divisions. This should
prevent expression of bp1/knat1 in tissues outside of the
flower.
[0205] Test if kn1 loss-of-function suppresses the ig1 phenotype.
If misexpression of kn1 causes the ig1 embryo sac phenotype, then
kn1 loss of function should suppress the ig1 phenotype. Double
mutants between ig1 and kn1 are constructed to test this. kn1
homozygotes have meristem defects that interfere with floral
production. However, since the action of ig1 is during the haploid
generation, the plants to be tested are ig1/+kn1/+double
heterozygotes which will produce the critical ig1 kn1 double mutant
embryo sacs. Seeds with ig1 phenotypes (i.e. miniatures and twins)
are tested for the presence of the kn1 mutation. If kn1 suppresses
ig1, then these seeds should be less likely to carry the mutant
allele than normal seeds.
[0206] If knockouts of any of the LOB domain genes have an ig1
phenotype in Arabidopsis, double (or triple) mutants are
constructed between bp1 and the appropriate lob mutant(s). The
frequency of extra rounds of free nuclear divisions in the embryo
sac are compared between bp1 lob double mutants and lob single
mutants. If the ig1-like phenotype is a consequence of ectopic
expression of bp1, then this phenotype should be suppressed by the
bp1 mutation.
Example 7
Test for Interactions Between ig1 and rs2
[0207] Use of yeast 2 hybrids to look for physical interaction
between IG1 and RS2 protein. To further test if ig1 is the ortholog
of AS2 the IG1 protein is tested for the ability to interact with
RS2 protein (the AS1 ortholog). This is performed in yeast
two-hybrid assays similarly to that done for AS1 and AS2 (Xu et
al., 2003). Briefly, IG1 and RS2 is tested as both bait and prey in
the MATCHMAKER two-hybrid vectors pGADT7 and pGBKT7 (Clonetech,
USA). Interaction is indicated by the ability of yeast to grow on
media lacking tryptophan, leucine, histidine, and adenine only in
the presence of both proteins but not in the presence of only one
or neither of them.
[0208] Examination of ig1:rs2 double mutants for both embryo sac
and leaf phenotypes. A genetic test for interaction between ig1 and
rs2 is performed by constructing double mutants. Although rs2 has
no reported embryo sac phenotype and ig1 has no leaf phenotype,
these mutations may enhance one another. Plants heterozygous for
ig1 and heterozygous for rs2 are scored for severity of ig1 embryo
sac phenotypes (i.e. frequency of miniatures, twins, and
transmission of ig1 and rs2) to test if rs2 can enhance ig1
phenotypes. Double homozygotes for rs2 and ig1 are examined for the
severity of rs2 leaf phenotypes to test if ig1 can enhance rs2
phenotypes.
Example 8
Knock Out the Rice ig1 Ortholog and Test for an Increase in
Maternal and Paternal Haploid Production
[0209] Because of the agronomic utility of the ig1 mutant in maize
breeding the ability to apply this to other species could be
beneficial to their breeding programs. This is tested in rice by
knocking out the rice ig1 ortholog, Otyza sativa ig1 (osig1)). A
T-DNA insertion has been identified in osig1 in the 5' region of
the gene in the Plant Functional Genomics Lab. Dept. of Life
Science, POSTECH in Kyoungbuk, Korea. This line is tested for
reduction in the mRNA levels of the gene. If osig1 RNA levels are
reduced in the mutant, then it is be examined for ig1 like
phenotypes. These plants are tested for paternal haploid production
by pollinating with a different rice strain and testing the progeny
by PCR for loss of maternal alleles of polymorphic Simple Sequence
Repeat loci in seedlings. As plants homozygous for paternal alleles
cannot be generated by self-pollination, rare self-pollination
contaminants will not interfere with their identification.
[0210] If the T-DNA insertion does not reduce osig1 RNA levels,
transgenic rice plants are to be generated carrying a construct
that can reduce osig1 expression levels. The 3' end of osig1 is to
be cloned into an RNTA interference (RNAi) vector in both
orientations around a central linker and transformed into rice
plants. This will cause expression of the mRNA as a double stranded
RNA leading to degradation of osig1 mRNA by post-transcriptional
gene silencing. RNAi has been shown to work in rice as a tool to
reduce expression of the rice APETALA3 ortholog (Xiao et al.,
2003). By using the less conserved 3' end any affect on other lob
genes in rice should be prevented. However, because of the
possibility of redundancy of ig function in rice that may or may
not be present in maize (see FIG. 2), the sequence to clone into
the RNAi vector is chosen to match osig1 and its closest partner,
the os lbd gene on chromosome 5 BAC OSJNBa0084P24. As a first test,
this interfering RNA is expressed under control of the native osig1
promoter using four kilobases of sequence upstream of the
translation start site; this includes all of the sequence upstream
of osig1 up to the next annotated gene. If this causes severe
sporophyte phenotypes that interfere with seed production, then the
native osig1 promoter is used to drive expression of the
glucocorticoid receptor. A glucocorticoid responsive promoter
similar to that of Aoyama and Chua (1997) is then be used to drive
expression of the interfering RNA, the expression of which can be
induced in flowers using dexamethasone.
Example 9
Disabling or Eliminating Expression of ig1
[0211] The isolated ig1 gene and vectors comprising the gene as
provided by the present invention can be used to disable or
eliminate ig1 gene function in the tassel to obtain male sterility
in maize and in other plant species. For example, using the
knock-out/knock-in technology described previously, the normal or
wild type ig1 gene can be replaced with a construct comprising an
ig1 gene operably linked to a tassel-specific promoter that
activates a gene that causes the ig1 gene not to be expressed or
results in the loss of function of the ig1 gene in the pollen
and/or tassel. Examples of pollen-specific and tassel-specific
promoters were discussed previously. Thus, when the introduced
construct is expressed in the tassel, the ig1 gene is disabled or
eliminated, thereby leading to the production of sterile pollen.
Using this procedure would not result in the disablement or
elimination of the ig1 gene in the cells that are not located in
the tassel.
[0212] Alternatively, the tassel-specific promoter could be linked
to another gene that inactivates the ig1 gene in the tassel only
when a certain compound is present. For example, the gene that
inactivates the ig1 gene may only be expressed when the tassels are
sprayed with a specific compound.
Example 10
Rescuing the ig1 Mutant Phenotype in the Tassel
[0213] Rescuing an ig1 mutant phenotype in all tissues except in
the tassel can also produce male sterile maize and male sterility
in other plant species. For example, utilizing the nucleic acid
sequence of the ig1 gene as provided by the present invention, a
mutation could be introduced into the ig1 gene using knock-in
technology or via site-directed mutation, or a mutant ig1 gene
could replace the normal or wild type ig1 gene by using
knock-out/knock-in technology. In this system, a construct could
either be introduced with the knock-in technology or separately
wherein the construct includes a genetic system that will rescue
the ig1 mutant so that it functions in all of the cells except
those in the tassel.
Example 11
Downregulation of ig1 Orthologs
[0214] As discussed previously, the isolated ig1 maize gene of the
present invention can be used as a probe to locate orthologs of the
ig1 gene in other plant species. Then, down-regulation of the ig1
ortholog(s) in plants other than maize (e.g., rice, wheat, sorghum,
soybean) will effectively generate ig1 mutants in these species
leading to the production of androgenic haploid and diploid plants.
This down-regulation can be achieved either by identifying
mutations in the gene(s) or by using transgenic technology.
Transgenic methods of down-regulation were discussed previously,
including but not limited to anti-sense technology and
co-suppression.
Example 12
Ectopically Expressing Genes Repressed by Expression of ig1
[0215] Ectopically expressing genes (e.g., knotted-like homeobox
genes) that would normally be repressed by the ig1 gene in the
female gametophyte of maize or other plants can be used to mimic
loss-of-function mutant phenotypes for the purpose of generating
androgenic haploid and diploid progeny (i.e., progeny that lack a
maternal contribution to their genome).
Example 13
Ectopic Expression of ig1
[0216] Ectopic expression of the ig1 gene can also be used to
create plant cultivars that produce embryos that are clones of the
parent plant by apomixis.
[0217] It must be noted that as used in this specification and the
appended claims, the singular forms "a," "and," and "the" include
plural referents unless the contexts clearly dictates otherwise.
Thus, for example, reference to "a metal" includes mixtures and
large numbers of such metals and heavy metals, reference to "a
transgenic plant" includes large numbers of transgenic plants and
mixtures thereof, and reference to "the method" includes one or
more methods or steps of the type described herein.
[0218] Unless defined otherwise, all technical and scientific terms
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials, similar or equivalent to those described
herein, can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein. All publications cited herein are incorporated herein by
reference for the purpose of disclosing and describing specific
aspects of the invention for which the publication is cited.
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[0259] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention.
[0260] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
Sequence CWU 1
1
21649DNAZea maysmisc_feature(173)..(174)transposon insertion site
1accagcaggt aggtcttcct cacctcgctg ctgtgcgaaa gcgagcacac aacaaaccct
60aacttactat ttgtattcgg atctgatcta ctggatccgc ccctgatttg aggcagtatt
120cctcatattt ggcaaaggag gccgaattcc agccagattc tctattctcc
ccctgatttc 180cagcagcttc caaaactttc taaagaaaca aaagaagagt
ctaacacagc tcgtgatcct 240tccgccaggg cagcagacgg atg gct tcg tcg gtg
ccg gcg cca tcg ggg tcg 293Met Ala Ser Ser Val Pro Ala Pro Ser Gly
Ser1 5 10gtg atc acc gtg gcg tcg tct tct tcc tca gca gcc gcg gcc
gcg gtg 341Val Ile Thr Val Ala Ser Ser Ser Ser Ser Ala Ala Ala Ala
Ala Val 15 20 25tgc ggc acg ggc tcc cca tgc gct gcg tgc aag ttc ctg
cgt cgc aag 389Cys Gly Thr Gly Ser Pro Cys Ala Ala Cys Lys Phe Leu
Arg Arg Lys 30 35 40tgc cag ccg gac tgc gtg ttc gcg ccc tac ttc cca
ccg gac aac ccg 437Cys Gln Pro Asp Cys Val Phe Ala Pro Tyr Phe Pro
Pro Asp Asn Pro 45 50 55cag aag ttc gtg cac gtg cac cgc gtc ttc ggc
gcg agc aac gtg acc 485Gln Lys Phe Val His Val His Arg Val Phe Gly
Ala Ser Asn Val Thr60 65 70 75aag ctg ctg aac gag ctc cac ccc ttc
cag cgc gag gac gcc gcg aac 533Lys Leu Leu Asn Glu Leu His Pro Phe
Gln Arg Glu Asp Ala Ala Asn 80 85 90tcc ctc gcc tac gag gcc gac atg
cgc ctc cgc gac ccc gtc tac ggc 581Ser Leu Ala Tyr Glu Ala Asp Met
Arg Leu Arg Asp Pro Val Tyr Gly 95 100 105tgc gtc ggc gtc atc tcc
atc ctc cag cac aac cta cga cag ctc cag 629Cys Val Gly Val Ile Ser
Ile Leu Gln His Asn Leu Arg Gln Leu Gln 110 115 120cag gac ctc ccc
ccg cgc ca 649Gln Asp Leu Pro Pro Arg 1252129PRTZea mays 2Met Ala
Ser Ser Val Pro Ala Pro Ser Gly Ser Val Ile Thr Val Ala1 5 10 15Ser
Ser Ser Ser Ser Ala Ala Ala Ala Ala Val Cys Gly Thr Gly Ser 20 25
30Pro Cys Ala Ala Cys Lys Phe Leu Arg Arg Lys Cys Gln Pro Asp Cys
35 40 45Val Phe Ala Pro Tyr Phe Pro Pro Asp Asn Pro Gln Lys Phe Val
His 50 55 60Val His Arg Val Phe Gly Ala Ser Asn Val Thr Lys Leu Leu
Asn Glu65 70 75 80Leu His Pro Phe Gln Arg Glu Asp Ala Ala Asn Ser
Leu Ala Tyr Glu 85 90 95Ala Asp Met Arg Leu Arg Asp Pro Val Tyr Gly
Cys Val Gly Val Ile 100 105 110Ser Ile Leu Gln His Asn Leu Arg Gln
Leu Gln Gln Asp Leu Pro Pro 115 120 125Arg
* * * * *
References