U.S. patent application number 11/787116 was filed with the patent office on 2009-12-17 for methods for altering organ mass, controlling fertility and enhancing asexual reproduction in plants.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Robert L. Fischer, Yukiko Mizukami.
Application Number | 20090313723 11/787116 |
Document ID | / |
Family ID | 22853051 |
Filed Date | 2009-12-17 |
United States Patent
Application |
20090313723 |
Kind Code |
A1 |
Fischer; Robert L. ; et
al. |
December 17, 2009 |
Methods for altering organ mass, controlling fertility and
enhancing asexual reproduction in plants
Abstract
The invention provides methods of altering organ mass,
controlling fertility and enhancing asexual reproduction in plants
through the modulation of plant growth and cell proliferation. The
methods involve producing transgenic plants comprising a
recombinant expression cassette containing an ANT nucleic acid
linked to a plant promoter.
Inventors: |
Fischer; Robert L.; (El
Cerrito, CA) ; Mizukami; Yukiko; (Kensington,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
22853051 |
Appl. No.: |
11/787116 |
Filed: |
April 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10623477 |
Jul 18, 2003 |
7220895 |
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11787116 |
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09479855 |
Jan 7, 2000 |
6639128 |
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10623477 |
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09227421 |
Jan 8, 1999 |
6559357 |
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09479855 |
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Current U.S.
Class: |
800/287 ;
435/320.1; 800/278; 800/290; 800/298 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/829 20130101; C12N 15/8289 20130101; C12N 15/827 20130101;
C12N 15/8222 20130101; C12N 15/8229 20130101; C12N 15/8261
20130101; Y02A 40/146 20180101 |
Class at
Publication: |
800/287 ;
800/278; 800/290; 800/298; 435/320.1 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 5/00 20060101 A01H005/00 |
Claims
1-20. (canceled)
21. A method of increasing cell proliferation in a plant, the
method comprising introducing into the plant an expression cassette
comprising a heterologous plant promoter operably linked to a
heterologous AINTEGUMENTA (ANT) nucleic acid encoding an ANT
polypeptide, wherein the polypeptide is encoded by a polynucleotide
identical to a polynucleotide that is amplified using an ANT
nucleic acid as a template by PCR using a first primer comprising
SEQ ID NO:6 or SEQ ID NO:7 and a second primer comprising SEQ ID
NO:8; and selecting a plant with increased cell number.
22. The method of claim 21, wherein the increased cell
proliferation results in increased plant size, mass or fertility;
and the method comprises the step of selecting plants with
increased size, mass or fertility.
23. The method of claim 21, wherein the heterologous plant promoter
is heterologous to the plant.
24. The method of claim 21, wherein the heterologous plant promoter
is heterologous to the ANT nucleic acid.
25. The method of claim 21, wherein the heterologous plant promoter
is constitutive.
26. The method of claim 21, wherein the heterologous plant promoter
is inducible.
27. The method of claim 21, wherein the heterologous plant promoter
is a tissue-specific promoter.
28. The method of claim 27, wherein the tissue-specific promoter is
a root-specific promoter.
29. The method of claim 27, wherein the tissue-specific promoter is
a vegetative organ-specific promoter.
30. The method of claim 27, wherein the tissue-specific promoter is
a floral organ-specific promoter.
31. The method of claim 27, wherein the tissue-specific promoter is
a seed-specific promoter.
32. The method of claim 27, wherein the tissue-specific promoter is
an ovule-specific promoter.
33. The method of claim 21, wherein the heterologous ANT nucleic
acid encodes an ANT polypeptide having at least 80% sequence
identity to SEQ ID NO:2 or SEQ ID NO:5.
34. The method of claim 21, wherein the template is selected from
Arabidopsis genomic DNA and Arabidopsis cDNA.
35. A plant comprising a heterologous tissue-specific plant
promoter operably linked to a heterologous AINTEGUMENTA (ANT)
nucleic acid encoding an ANT polypeptide, wherein the polypeptide
is encoded by a polynucleotide identical to a polynucleotide that
is amplified using an ANT nucleic acid as a template by PCR using a
first primer comprising SEQ ID NO:6 or SEQ ID NO:7 and a second
primer comprising SEQ ID NO:8.
36. The plant of claim 35, wherein the heterologous plant promoter
is heterologous to the plant.
37. The plant of claim 35, wherein the heterologous plant promoter
is heterologous to the ANT nucleic acid.
38. The plant of claim 35, wherein the heterologous plant promoter
is constitutive.
39. The plant of claim 35, wherein the heterologous plant promoter
is inducible.
40. The plant of claim 35, wherein the tissue-specific promoter is
a root-specific promoter.
41. The plant of claim 35, wherein the tissue-specific promoter is
a vegetative organ-specific promoter.
42. The plant of claim 35, wherein the tissue-specific promoter is
a floral organ-specific promoter.
43. The plant of claim 35, wherein the tissue-specific promoter is
a seed-specific promoter.
44. The plant of claim 35, wherein the tissue-specific promoter is
an ovule-specific promoter.
45. The plant of claim 35, wherein the heterologous ANT nucleic
acid encodes an ANT polypeptide having at least 80% sequence
identity to SEQ ID NO:2 or SEQ ID NO:5.
46. A recombinant expression cassette comprising a heterologous
tissue-specific plant promoter operably linked to a heterologous
AINTEGUMENTA (ANT) nucleic acid encoding an ANT polypeptide,
wherein the polypeptide is encoded by a polynucleotide identical to
a polynucleotide that is amplified using an ANT nucleic acid as a
template by PCR using a first primer comprising SEQ ID NO:6 or SEQ
ID NO:7 and a second primer comprising SEQ ID NO:8, wherein the
recombinant expression cassette promotes the tissue-specific
expression of the ANT polypeptide in a plant.
47. The recombinant expression cassette of claim 46, wherein the
heterologous plant promoter is heterologous to the plant.
48. The recombinant expression cassette of claim 46, wherein the
heterologous plant promoter is heterologous to the ANT nucleic
acid.
49. The recombinant expression cassette of claim 46, wherein the
heterologous plant promoter is constitutive.
50. The recombinant expression cassette of claim 46, wherein the
heterologous plant promoter is inducible.
51. The recombinant expression cassette of claim 46, wherein the
tissue-specific promoter is a root-specific promoter.
52. The recombinant expression cassette of claim 46, wherein the
tissue-specific promoter is a vegetative organ-specific
promoter.
53. The recombinant expression cassette of claim 46, wherein the
tissue-specific promoter is a floral organ-specific promoter.
54. The recombinant expression cassette of claim 46, wherein the
tissue-specific promoter is a seed-specific promoter.
55. The recombinant expression cassette of claim 46, wherein the
tissue-specific promoter is an ovule-specific promoter.
56. The recombinant expression cassette of claim 46, wherein the
heterologous ANT nucleic acid encodes an ANT polypeptide having at
least 80% sequence identity to SEQ ID NO:2 or SEQ ID NO:5.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/623,477, filed Jul. 18, 2003, which is a
continuation of U.S. patent application Ser. No. 09/479,855, filed
Jan. 7, 2000, (now U.S. Pat. No. 6,639,128), which is a
continuation-in-part of U.S. patent application Ser. No.
09/227,421, filed Jan. 8, 1999 (now U.S. Pat. No. 6,559,357), all
of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention is directed to plant genetic
engineering. In particular, it relates to methods of altering organ
mass, controlling fertility and enhancing asexual reproduction in
plants through the modulation of plant growth and cell
proliferation.
BACKGROUND OF THE INVENTION
[0003] Control of organ mass/size and fertility in plants is a
significant goal in commercial agriculture. Plant shoot vegetative
organs and/or structures (e.g. leaves, stems and tubers), roots,
flowers and floral organs (e.g. bracts, sepals, petals, stamens,
carpels, anthers), ovules (including egg and central cells), seed
(including zygote, embryo, endosperm, and seed coat), fruit (the
mature ovary) and seedlings are the harvested product of numerous
agronomically-important crop plants. Therefore the ability to
manipulate the size/mass of these organs/structures through genetic
control would be an important agricultural tool. Similarly,
induction of sterility in plants is useful in limiting plant
pollination and reproduction until it is economically desirable.
For example, male sterile plants are often desirable in crops where
hybrid vigor increases yield.
[0004] The intrinsic plant organ size is determined genetically,
although it can be altered greatly by environment signals (e.g.
growth conditions). In general, larger organs consist of larger
numbers of cells. Since neither cell migration nor cell death plays
a major role during plant development, the number of cells in plant
organs depends on cell proliferation. Precise regulation of cell
proliferation is also necessary for proper development of
reproductive organs that make plants fertile. While some basic
research has identified genes involved in plant organ development
and fertility, little is known about genetic control of cell
proliferation or its link to organogenesis including organ
size/mass control and fertility in plants. Therefore an important
goal is to understand the connection between genes that control
organogenesis and genes that control cell proliferation. A great
deal of basic research has shown that the components (e.g., cyclin
dependent kinases, cyclins and their inhibitors) and mechanisms
(e.g., regulation by phosphorylations, ubiquitin-mediated
proteolysis) that control the cell cycle in yeast and animals are
conserved in higher plants (Burssens, et al. Plant Physiol Biochem.
36:9-19 (1998)).
[0005] In Arabidopsis, the developing flower includes the ovule.
Wild-type ovule development in Arabidopsis has been extensively
analyzed (Robinson-Beers et al., Plant Cell 4:1237-1249 (1992);
Modrusan, et al. Plant Cell. 6:333-349 (1994) and Schneitz et al.,
Plant J. 7:731-749 (1995)). A variety of mutations that affect
ovule development have been identified (Klucher et al., Plant Cell
8:137-153 (1996); Elliott et al., Plant Cell. 8:155-168 (1996);
Baker, et al. Genetics. 145:1109-1124 (1997); Robinson-Beers, et
al., Plant Cell. 4:1237-1249 (1992); Modrusan et al. Plant Cell.
6:333-349 (1994); Ray, A., et al. Proc Natl Acad Sci USA.
91:5761-5765 (1994); Lang, et al., Genetics 137:1101-1110 (1994);
Leon-Kloosterziel Plant Cell. 6:385-392 (1994); Gaiser et al.,
Plant Cell 7:333-345 (1995)), and some of them have been found that
specifically affect patterns of cell division (Schneitz, et al.
Development. 124:1367-1376 (1997)). Of those, several genes have
been cloned; AINTEGUMENTA (ANT) (Klucher et al. Plant Cell.
8:137-153 (1996); Elliott et al., Plant Cell. 8:155-168 (1996)),
AGAMOUS, (Yanofsky et al., Nature. 346:35-39 (1990); Bowman et al.,
Plant Cell. 3:749-758 (1991)), SUPERMAN (Sakai et al., Nature.
378:199-203 (1995)). Because these genes are expressed and function
not only in developing ovules but also in various developing
organs, analysis of these mutations and genes has provided general
information about the control of cell proliferation during plant
development.
[0006] Another trait important to the manipulation of crop species
is the ability to reproduce or propagate plants through asexual
means, particularly vegetative propagation of sterile or hybrid
plants, and regeneration of plants from transformed cells. Asexual
reproduction includes regeneration of plants from cells or tissue,
propagation of plants through cutting by inducing adventitious
shoots and roots, and apomixis by forming somatic embryos. Asexual
reproduction has the advantage that genetic clones of plants with
desirable traits can be readily produced. Not all plants, however,
can produce adventitious shoots or roots, or regenerate whole
plants from cells or tissue.
[0007] In spite of the recent progress in defining the genetic
control of plant cell proliferation, little progress has been
reported in the identification and analysis of genes effecting
agronomically important traits such as organ mass/size, fertility,
asexual reproduction, and the like through regulating cell
proliferation. Characterization of such genes would allow for the
genetic engineering of plants with a variety of desirable traits.
The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
[0008] The present invention provides methods for modulating cell
proliferation and thus cell number in plants by modulating ANT
activity in plants. Typically, the methods comprise modulating the
expression of ANT in plants and selecting for plants with altered
size/mass, fertility, or both. In some preferred embodiments, the
ANT activity is increased and plants with increased cell
proliferation and thus increased cell number are selected. One
method for modulating ANT expression is by introducing into a plant
an expression cassette containing a heterologous ANT nucleic acid
operably linked to a promoter. Examples of possible ANT nucleic
acids that can be used include nucleic acids at least 50% identical
to SEQ ID NO: 1 and SEQ ID NO:4. Other examples include nucleic
acids that encode the polypeptides at least 60% identical to either
SEQ ID NO:2 or SEQ ID NO:5.
[0009] The present invention also provides methods for modulating
cell proliferation and thus the production of adventitious organs
in plants. Typically, the methods comprise increasing the activity
or expression of ANT in plants and selecting for plants with
adventitious shoots, organs or structures such as embryos. One
method for modulating ANT expression is by introducing into a plant
an expression cassette containing a heterologous ANT nucleic acid
operably linked to a promoter. Examples of possible ANT nucleic
acids that can be used include nucleic acids at least 50% identical
to SEQ ID NO:1 and SEQ ID NO:4. Other examples include nucleic
acids that encode the polypeptides at least 60% identical to either
SEQ ID NO:2 or SEQ ID NO:5.
[0010] The present invention also provides methods of reproducing a
plant through asexual means. Typically, the methods comprise
increasing the activity or expression of ANT in plants and
selecting a plant reproduced from the plant cell or tissue. One
method for modulating ANT expression is by introducing into a plant
an expression cassette containing a heterologous ANT nucleic acid
operably linked to a promoter. Examples of possible ANT nucleic
acids that can be used include nucleic acids at least 50% identical
to SEQ ID NO:1 and SEQ ID NO:4. Other examples include nucleic
acids that encode the polypeptides at least 60% identical to either
SEQ ID NO:2 or SEQ ID NO:5. In various embodiments, the plant
arises from an adventitious shoot, a somatic embryo, or a
cutting.
[0011] In another embodiment of the invention, a heterologous gene
is expressed in meristematic tissue of a plant by introducing into
a plant an expression cassette containing an ANT promoter operably
linked to a heterologous polynucleotide. In a preferred embodiment
of this invention, the ANT promoter is shown in SEQ ID NO:3.
[0012] The invention also provides isolated nucleic acid molecules
comprising an ANT nucleic acid that specifically hybridizes to SEQ
ID NO: 4, which is isolated from Brassica napus.
[0013] A variety of plant promoters can be used in the methods of
the invention. The promoter can be constitutive, inducible or
specific for an organ, tissue, or cell. In some embodiments a
promoter from an ANT gene, e.g. SEQ ID NO: 3, is used. Expression
of the ANT nucleic acids of the invention can be directed to any
desired organ, tissue, or cell in the plant. In some preferred
embodiments of the invention, the promoter directs expression of
the ANT nucleic acid in shoot vegetative organs/structures, such as
leaf, stem and tuber. In other preferred embodiments, the promoter
directs expression of the ANT nucleic acid in roots. In other
preferred embodiments, the promoter directs expression of the ANT
nucleic acid in flowers or floral organs/structures, such as
bracts, sepals, petals, stamens, carpels, anthers and ovules. In
different embodiments, the promoter directs expression of the ANT
nucleic acid in seeds (e.g. embryo, endosperm, and seed coat) or
fruit.
DEFINITIONS
[0014] The phrase "nucleic acid sequence" refers to a single or
double-stranded polymer of deoxyribonucleotide or ribonucleotide
bases read from the 5' to the 3' end. It includes chromosomal DNA,
self-replicating plasmids, infectious polymers of DNA or RNA and
DNA or RNA that performs a primarily structural role.
[0015] The term "promoter" refers to regions or sequence located
upstream and/or downstream from the start of transcription and
which are involved in recognition and binding of RNA polymerase and
other proteins to initiate transcription. A "plant promoter" is a
promoter capable of initiating transcription in plant cells.
[0016] The term "plant" includes whole plants, shoot vegetative
organs and/or structures (e.g. leaves, stems and tubers), roots,
flowers and floral organs (e.g. bracts, sepals, petals, stamens,
carpels, anthers), ovules (including egg and central cells), seed
(including zygote, embryo, endosperm, and seed coat), fruit (e.g.,
the mature ovary), seedlings, plant tissue (e.g. vascular tissue,
ground tissue, and the like), cells (e.g. guard cells, egg cells,
trichomes and the like), and progeny of same. The class of plants
that can be used in the method of the invention is generally as
broad as the class of higher and lower plants amenable to
transformation techniques, including angiosperms (monocotyledonous
and dicotyledonous plants), gymnosperms, ferns, and multicellular
algae. It includes plants of a variety of ploidy levels, including
aneuploid, polyploid, diploid, haploid and hemizygous.
[0017] "Increased or enhanced ANT activity or expression of the ANT
gene" refers to an augmented change in ANT activity. Examples of
such increased activity or expression include the following. ANT
activity or expression of the ANT gene is increased above the level
of that in wild-type, non-transgenic control plants (i.e. the
quantity of ANT activity or expression of the ANT gene is
increased). ANT activity or expression of the ANT gene is in an
organ, tissue or cell where it is not normally detected in
wild-type, non-transgenic control plants (i.e. spatial distribution
of ANT activity or expression of the ANT gene is increased). ANT
activity or expression is increased when ANT activity or expression
of the ANT gene is present in an organ, tissue or cell for a longer
period than in a wild-type, non-transgenic controls (i.e. duration
of ANT activity or expression of the ANT gene is increased).
[0018] As used herein, the term "asexual reproduction" refers to
the formation of shoots, roots or a whole plant from a plant cell
without fertilization. If the formation of the whole plant proceeds
through a somatic embryo, the asexual reproduction can be referred
to as apomixis.
[0019] The term "adventitious organ" and "adventitious shoot" refer
to an organ (e.g. stem, leaf, or root) and a shoot arising in a
place other than its usual site, respectively. For example, a root
developing on a stem, or a shoot bud arising on a stem in a place
other than the axil of a leaf. Adventitious organs or shoots may
also arise in callus tissue in vitro. Such adventitious organs or
shoots can then used to regenerate a whole plant using methods well
known to those of skill in the art.
[0020] A polynucleotide sequence is "heterologous to" an organism
or a second polynucleotide sequence if it originates from a foreign
species, or, if from the same species, is modified from its
original form. For example, a promoter operably linked to a
heterologous coding sequence refers to a coding sequence from a
species different from that from which the promoter was derived,
or, if from the same species, a coding sequence which is not
naturally associated with the promoter (e.g. a genetically
engineered coding sequence or an allele from a different ecotype or
variety).
[0021] A polynucleotide "exogenous to" an individual plant is a
polynucleotide which is introduced into the plant by any means
other than by a sexual cross. Examples of means by which this can
be accomplished are described below, and include
Agrobacterium-mediated transformation, biolistic methods,
electroporation, and the like. Such a plant containing the
exogenous nucleic acid is referred to here as a T.sub.1 (e.g. in
Arabidopsis by vacuum infiltration) or R.sub.0 (for plants
regenerated from transformed cells in vitro) generation transgenic
plant. Transgenic plants that arise from sexual cross or by selfing
are descendants of such a plant.
[0022] An "ANT nucleic acid" or "ANT polynucleotide sequence" of
the invention is a subsequence or full length polynucleotide
sequence (SEQ ID NO:1) which, encodes a polypeptide (SEQ ID NO:2)
and its complement, as described, for instance, by Klucher et al.,
Plant Cell 8:137-153 (1996) and Elliott et al., Plant Cell.
8:155-168 (1996) (see, also, GenBank Accession Nos. U40256 and
U41339). SEQ ID NO:4, which encodes SEQ ID NO:5, represents another
"ANT nucleic acid" from Brassica. ANT gene products of the
invention are characterized by the presence of an AP2 domain, first
identified in AP2, this motif is characterized by a region of
approximately 60-70 amino acid residues with a highly conserved
core region with the capacity to form an amphipathic .alpha.-helix
and/or to bind DNA (Jofuku et al., Plant Cell 6:1211-1225 (1994);
Ohme-Takagi and Shinshi, Plant Cell 7: 173-182 (1995). The full
length ANT protein contains two AP2 domains (amino acids 281 to 357
and from 383 to 451 of SEQ ID NO:2) and a linker region (amino
acids 358 to 382), and the homology to other AP2 domain proteins is
restricted to this region. An ANT polynucleotide of the invention
typically comprises a coding sequence at least about 30-40
nucleotides to about 2500 nucleotides in length, usually less than
about 3000 nucleotides in length. Usually the ANT nucleic acids of
the invention are from about 100 to about 5000 nucleotides, often
from about 500 to about 3000 nucleotides in length.
[0023] In the case of both expression of transgenes and inhibition
of endogenous genes (e.g., by antisense, or co-suppression) one of
skill will recognize that the inserted polynucleotide sequence need
not be identical, but may be only "substantially identical" to a
sequence of the gene from which it was derived. As explained below,
these substantially identical variants are specifically covered by
the term ANT nucleic acid.
[0024] In the case where the inserted polynucleotide sequence is
transcribed and translated to produce a functional polypeptide, one
of skill will recognize that because of codon degeneracy a number
of polynucleotide sequences will encode the same polypeptide. These
variants are specifically covered by the terms "ANT nucleic acid",
"ANT polynucleotide" and their equivalents. In addition, the terms
specifically include those full length sequences substantially
identical (determined as described below) with an ANT
polynucleotide sequence and that encode proteins that retain the
function of the ANT polypeptide (e.g., resulting from conservative
substitutions of amino acids in the ANT polypeptide).
[0025] The term "altered fertility" includes any transient or
permanent alteration of fecundity including inducing sterility as
well as altered initiation of floral development (e.g. flowering
time). Sterility can be caused, inter alia, by disruption of pollen
development, dehiscence (i.e. male sterility), by disruption of
ovule development (i.e. female sterility), or by disruption of
pollination/fertilization processes caused by abnormal development
of male/female organs (e.g. stigmatic papillae, transmitting tissue
of septum). Flowering time is the developmental time or stage when
a plant initiates and produces floral tissue.
[0026] Two nucleic acid sequences or polypeptides are said to be
"identical" if the sequence of nucleotides or amino acid residues,
respectively, in the two sequences is the same when aligned for
maximum correspondence as described below. The terms "identical" or
percent "identity," in the context of two or more nucleic acids or
polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same, when compared
and aligned for maximum correspondence over a comparison window, as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. When percentage of
sequence identity is used in reference to proteins or peptides, it
is recognized that residue positions that are not identical often
differ by conservative amino acid substitutions, where amino acids
residues are substituted for other amino acid residues with similar
chemical properties (e.g., charge or hydrophobicity) and therefore
do not change the functional properties of the molecule. Where
sequences differ in conservative substitutions, the percent
sequence identity may be adjusted upwards to correct for the
conservative nature of the substitution. Means for making this
adjustment are well known to those of skill in the art. Typically
this involves scoring a conservative substitution as a partial
rather than a full mismatch, thereby increasing the percentage
sequence identity. Thus, for example, where an identical amino acid
is given a score of 1 and a non-conservative substitution is given
a score of zero, a conservative substitution is given a score
between zero and 1. The scoring of conservative substitutions is
calculated according to, e.g., the algorithm of Meyers &
Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as
implemented in the program PC/GENE (Intelligenetics, Mountain View,
Calif., USA).
[0027] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides, refers to a sequence or subsequence
that has at least 25% sequence identity with a reference sequence.
Alternatively, percent identity can be any integer from 25% to
100%. More preferred embodiments include at least: 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
compared to a reference sequence using the programs described
herein; preferably BLAST using standard parameters, as described
below. This definition also refers to the complement of a test
sequence, when the test sequence has substantial identity to a
reference sequence.
[0028] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0029] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection.
[0030] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The method
used is similar to the method described by Higgins & Sharp,
CABIOS 5:151-153 (1989). The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. For example, a reference sequence can be
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps.
[0031] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul et al., J. Mol.
Biol. 215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul et al, supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Extension of the word
hits in each direction are halted when: the cumulative alignment
score falls off by the quantity X from its maximum achieved value;
the cumulative score goes to zero or below, due to the accumulation
of one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T,
and X determine the sensitivity and speed of the alignment. The
BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62
scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0032] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.01, more preferably
less than about 10.sup.-5, and most preferably less than about
10.sup.-20.
[0033] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine) can be modified to yield a
functionally identical molecule. Accordingly, each silent variation
of a nucleic acid which encodes a polypeptide is implicit in each
described sequence.
[0034] As to amino acid sequences, one of skill will recognize that
individual substitutions, in a nucleic acid, peptide, polypeptide,
or protein sequence which alters a single amino acid or a small
percentage of amino acids in the encoded sequence is a
"conservatively modified variant" where the alteration results in
the substitution of an amino acid with a chemically similar amino
acid. Conservative substitution tables providing functionally
similar amino acids are well known in the art.
[0035] The following six groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
[0036] 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0037] (see, e.g., Creighton, Proteins (1984)).
[0038] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules or their complements hybridize
to each other under stringent conditions, as described below.
[0039] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0040] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, highly
stringent conditions are selected to be about 5-10.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequence
at a defined ionic strength pH. Low stringency conditions are
generally selected to be about 15-30.degree. C. below the T.sub.m.
The T.sub.m is the temperature (under defined ionic strength, pH,
and nucleic concentration) at which 50% of the probes complementary
to the target hybridize to the target sequence at equilibrium (as
the target sequences are present in excess, at T.sub.m, 50% of the
probes are occupied at equilibrium). Stringent conditions will be
those in which the salt concentration is less than about 1.0 M
sodium ion, typically about 0.01 to 1.0 M sodium ion concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least
about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides)
and at least about 60.degree. C. for long probes (e.g., greater
than 50 nucleotides). Stringent conditions may also be achieved
with the addition of destabilizing agents such as formamide. For
selective or specific hybridization, a positive signal is at least
two times background, preferably 10 time background
hybridization.
[0041] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions.
[0042] In the present invention, genomic DNA or cDNA comprising ANT
nucleic acids of the invention can be identified in standard
Southern blots under stringent conditions using the nucleic acid
sequences disclosed here. For the purposes of this disclosure,
suitable stringent conditions for such hybridizations are those
which include a hybridization in a buffer of 40% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and at least one wash in
0.2.times.SSC at a temperature of at least about 50.degree. C.,
usually about 55.degree. C. to about 60.degree. C., for 20 minutes,
or equivalent conditions. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency.
[0043] A further indication that two polynucleotides are
substantially identical is if the reference sequence, amplified by
a pair of oligonucleotide primers, can then be used as a probe
under stringent hybridization conditions to isolate the test
sequence from a cDNA or genomic library, or to identify the test
sequence in, e.g., an RNA gel or DNA gel blot hybridization
analysis.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0044] This invention relates to control of cell proliferation and
thus cell number in plants by modulating ANT activity in plants.
For example, the invention provides molecular strategies for
manipulating plant biomass through controlling the number of cells
and size/mass of plant shoot vegetative organs and/or structures
(e.g. leaves, stems and tubers), roots, flowers and floral organs
(e.g. bracts, sepals, petals, stamens, carpels, anthers), ovules
(including egg and central cells), seed (including zygote, embryo,
endosperm, and seed coat), fruit (the mature ovary) and seedlings
using ANT gene constructs. Thus, by regulating ANT expression
transgenic plants with increased or decreased biomass can be
produced. In addition, modulating expression of the gene in male or
female reproductive organs can lead to sterility through alteration
in the pattern of cell proliferation. Thus, male or female sterile
transgenic plants can be produced by enhancing or inhibiting ANT
gene expression in the appropriate tissues. In yet other
embodiments, formation of adventitious organs, shoots, or
structures such as somatic embryos can be controlled using this
method of the invention. Thus, the efficiency of asexual
reproduction of plants, in particular reproduction of sterile or
hybrid plants with desired traits and regeneration of transgenic
plants from transformed tissue, can be improved.
[0045] Because the ANT gene product most likely functions as a
transcription factor (Vergani et al., FEBS Letters. 400:243-246
(1997)), one of skill will recognize that desired phenotypes
associated with altered ANT activity can be obtained by modulating
the expression or activity of ANT-regulated genes. Any of the
methods described for increasing or decreasing ANT expression or
activity can be used for this purpose.
Increasing ANT Activity or ANT Gene Expression
[0046] Any of a number of means well known in the art can be used
to increase ANT activity in plants. Enhanced expression is useful,
for example, to induce or enhance asexual reproduction, or increase
organ size/mass in desired plant organs. Any organ can be targeted,
such as Plant shoot vegetative organs and/or structures (e.g.
leaves, stems and tubers), roots, flowers and floral organs (e.g.
bracts, sepals, petals, stamens, carpels, anthers), ovules
(including egg and central cells), seed (including zygote, embryo,
endosperm, and seed coat), fruit and seedlings. The beneficial
effects of altering ANT activity need not be the direct result of
increased cell proliferation. For instance, increased leaf
size/mass will lead to an increase in photosynthesis, which will in
turn lead to increased yield. Similarly increased mass/size of
roots will lead to increased nutrient uptake and increased yield.
Increased stem or pedicel thickness can be used to decreases losses
due to breakage, e.g. in cereal crops and fruits.
[0047] Increased ANT activity or ANT expression can also be used to
produce male or female sterile plants. Male or female sterility is
important for agriculture and horticulture, particularly in the
production of hybrid varieties that have commercially advantageous
superior traits. Male or female sterility allows breeders to make
hybrid varieties easily by preventing self-pollen contamination in
parental plants. Thus, for instance targeting ANT expression in
developing anthers will cause male sterility, but not disrupt
female organs thus rendering plants female fertile. Prevention of
dehiscence is also desirable for commercial cut flowers. For
instance, pollination leads to floral senescence, also pollen
grains can be allergenic and in some plants (e.g., lilies) can
cause stains.
[0048] Expression of the ANT gene in transgenic plants can also
cause female sterility. Plants constitutively expressing the ANT
gene in ovules produce large mature ovules that are sterile,
Therefore, introducing female sterility via controlling ANT
function can delay senescence of plants and improve vegetative
yield and quality of crop and horticulturally important plants.
Alternatively, female sterility can result from decreased ANT
expression using methods described below for methods of inhibiting
ANT activity or gene expression.
Increasing ANT Gene Expression
[0049] Isolated sequences prepared as described herein can be used
to introduce expression of a particular ANT nucleic acid to
increase endogenous gene expression using methods well known to
those of skill in the art. Preparation of suitable constructs and
means for introducing them into plants are described below.
[0050] One of skill will recognize that the polypeptides encoded by
the genes of the invention, like other proteins, have different
domains that perform different functions. Thus, the gene sequences
need not be full length, so long as the desired functional domain
of the protein is expressed. The distinguishing features of ANT
polypeptides, including the AP2 domain, nuclear localization
signal, and transcription activation domains, are discussed in
Elliot et al. or Klucher et al. above.
[0051] Modified protein chains can also be readily designed
utilizing various recombinant DNA techniques well known to those
skilled in the art and described in detail, below. For example, the
chains can vary from the naturally occurring sequence at the
primary structure level by amino acid substitutions, additions,
deletions, and the like. These modifications can be used in a
number of combinations to produce the final modified protein
chain.
Modification of Endogenous ANT Genes
[0052] Methods for introducing genetic mutations into plant genes
and selecting plants with desired traits are well known. For
instance, seeds or other plant material can be treated with a
mutagenic chemical substance, according to standard techniques.
Such chemical substances include, but are not limited to, the
following: diethyl sulfate, ethylene imine, ethyl methanesulfonate
and N-nitroso-N-ethylurea. Alternatively, ionizing radiation from
sources such as, X-rays or gamma rays can be used.
[0053] Alternatively, homologous recombination can be used to
induce targeted gene modifications by specifically targeting the
ANT gene in vivo (see, generally, Grewal and Klar, Genetics 146:
1221-1238 (1997) and Xu et al., Genes Dev. 10: 2411-2422 (1996)).
Homologous recombination has been demonstrated in plants (Puchta et
al., Experientia 50: 277-284 (1994), Swoboda et al., EMBO J. 13:
484-489 (1994); Offring a et al., Proc. Natl. Acad. Sci. USA 90:
7346-7350 (1993); and Kempin et al. Nature 389:802-803 (1997)).
[0054] In applying homologous recombination technology to the genes
of the invention, mutations in selected portions of an ANT gene
sequences (including 5' upstream, 3' downstream, and intragenic
regions) such as those disclosed here are made in vitro and then
introduced into the desired plant using standard techniques. Since
the efficiency of homologous recombination is known to be dependent
on the vectors used, use of dicistronic gene targeting vectors as
described by Mountford et al., Proc. Natl. Acad. Sci. USA 91:
4303-4307 (1994); and Vaulont et al., Transgenic Res. 4: 247-255
(1995) are conveniently used to increase the efficiency of
selecting for altered ANT gene expression in transgenic plants. The
mutated gene will interact with the target wild-type gene in such a
way that homologous recombination and targeted replacement of the
wild-type gene will occur in transgenic plant cells, resulting in
suppression of ANT activity.
[0055] Alternatively, oligonucleotides composed of a contiguous
stretch of RNA and DNA residues in a duplex conformation with
double hairpin caps on the ends can be used. The RNA/DNA sequence
is designed to align with the sequence of the target ANT gene and
to contain the desired nucleotide change. Introduction of the
chimeric oligonucleotide on an extrachromosomal T-DNA plasmid
results in efficient and specific ANT gene conversion directed by
chimeric molecules in a small number of transformed plant cells.
This method is described in Cole-Strauss et al. Science
273:1386-1389 (1996) and Yoon et al. Proc. Natl. Acad. Sci. USA 93:
2071-2076 (1996).
Other Means for Increasing ANT Activity
[0056] One method to increase ANT expression is to use "activation
mutagenesis" (see, e.g. Hiyashi et al. Science 258:1350-1353
(1992)). In this method an endogenous ANT gene can be modified to
be expressed constitutively, ectopically, or excessively by
insertion of T-DNA sequences that contain strong/constitutive
promoters upstream of the endogenous ANT gene. As explained below,
preparation of transgenic plants overexpressing ANT can also be
used to increase ANT expression. Activation mutagenesis of the
endogenous ANT gene will give the same effect as overexpression of
the transgenic ANT nucleic acid in transgenic plants.
Alternatively, an endogenous gene encoding an enhancer of ANT
activity or expression of the endogenous ANT gene can be modified
to be expressed by insertion of T-DNA sequences in a similar manner
and ANT activity can be increased.
[0057] Another strategy to increase ANT expression can be the use
of dominant hyperactive mutants of ANT by expressing modified ANT
transgenes. For example expression of modified ANT with a defective
domain that is important for interaction with a negative regulator
of ANT activity can be used to generate dominant hyperactive ANT
proteins. Alternatively, expression of truncated ANT proteins which
have only a domain that interacts with a negative regulator can
titrate the negative regulator and thereby increase endogenous ANT
activity. Use of dominant mutants to hyperactivate target genes is
described in Mizukami et al. Plant Cell 8:831-845 (1996).
Inhibition of ANT Activity or Gene Expression
[0058] As explained above, ANT activity is important in controlling
a number of plant processes through the regulation of cell
proliferation. Inhibition of ANT gene expression activity can be
used, for instance, to decrease plant organ size/mass or to induce
female sterility in plants. In particular, targeted expression of
ANT nucleic acids that inhibit endogenous gene expression (e.g.,
antisense or co-suppression) can be used to inhibit ovule
development at early stages and thus induce female sterility. The
life span of the transgenic plants can therefore be extended
because fertilization (seed formation) can activate and accelerate
senescence processes of plants or organs.
[0059] Inhibition of ANT gene function can also be used to truncate
vegetative growth, resulting in early flowering. Methods that
control flowering time are extremely valuable in agriculture to
optimize harvesting time as desired. Therefore, by regulating the
function of the ANT genes in plants, it is possible to control time
of flowering. For instance, acceleration of fertile plant growth
can be obtained by expressing ANT antisense RNA during vegetative
development to achieve early flowering. Expression of the ANT
transgene can then be shut off during reproductive development to
get fertile plants.
Inhibition of ANT Gene Expression
[0060] The nucleic acid sequences disclosed here can be used to
design nucleic acids useful in a number of methods to inhibit ANT
or related gene expression in plants. For instance, antisense
technology can be conveniently used. To accomplish this, a nucleic
acid segment from the desired gene is cloned and operably linked to
a promoter such that the antisense strand of RNA will be
transcribed. The construct is then transformed into plants and the
antisense strand of RNA is produced. In plant cells, it has been
suggested that antisense suppression can act at all levels of gene
regulation including suppression of RNA translation (see, Bourque
Plant Sci. (Limerick) 105: 125-149 (1995); Pantopoulos In Progress
in Nucleic Acid Research and Molecular Biology, Vol. 48. Cohn, W.
E. and K. Moldave (Ed.). Academic Press, Inc.: San Diego, Calif.,
USA; London, England, UK. p. 181-238; Heiser et al. Plant Sci.
(Shannon) 127: 61-69 (1997)) and by preventing the accumulation of
mRNA which encodes the protein of interest, (see, Baulcombe Plant
Mol. Bio. 32:79-88 (1996); Prins and Goldbach Arch. Virol. 141:
2259-2276 (1996); Metzlaffet al. Cell 88: 845-854 (1997), Sheehy et
al., Proc. Nat. Acad. Sci. USA, 85:8805-8809 (1988), and Hiatt et
al., U.S. Pat. No. 4,801,340).
[0061] The nucleic acid segment to be introduced generally will be
substantially identical to at least a portion of the endogenous ANT
gene or genes to be repressed. The sequence, however, need not be
perfectly identical to inhibit expression. The vectors of the
present invention can be designed such that the inhibitory effect
applies to other genes within a family of genes exhibiting identity
or substantial identity to the target gene.
[0062] For antisense suppression, the introduced sequence also need
not be full length relative to either the primary transcription
product or fully processed mRNA. Generally, higher identity can be
used to compensate for the use of a shorter sequence. Furthermore,
the introduced sequence need not have the same intron or exon
pattern, and identity of non-coding segments may be equally
effective. Normally, a sequence of between about 30 or 40
nucleotides and about full length nucleotides should be used,
though a sequence of at least about 100 nucleotides is preferred, a
sequence of at least about 200 nucleotides is more preferred, and a
sequence of about 500 to about 3500 nucleotides is especially
preferred.
[0063] A number of gene regions can be targeted to suppress ANT
gene expression. The targets can include, for instance, the coding
regions, introns, sequences from exon/intron junctions, 5' or 3'
untranslated regions, and the like.
[0064] Another well known method of suppression is sense
co-suppression. Introduction of nucleic acid configured in the
sense orientation has been recently shown to be an effective means
by which to block the transcription of target genes. For an example
of the use of this method to modulate expression of endogenous
genes (see, Assaad et al. Plant Mol. Bio. 22: 1067-1085 (1993);
Flavell Proc. Natl. Acad. Sci. USA 91: 3490-3496 (1994); Stam et
al. Annals Bot. 79: 3-12 (1997); Napoli et al., The Plant Cell
2:279-289 (1990); and U.S. Pat. Nos. 5,034,323, 5,231,020, and
5,283,184).
[0065] The suppressive effect may occur where the introduced
sequence contains no coding sequence per se, but only intron or
untranslated sequences homologous to sequences present in the
primary transcript of the endogenous sequence. The introduced
sequence generally will be substantially identical to the
endogenous sequence intended to be repressed. This minimal identity
will typically be greater than about 65%, but a higher identity
might exert a more effective repression of expression of the
endogenous sequences. Substantially greater identity of more than
about 80% is preferred, though about 95% to absolute identity would
be most preferred. As with antisense regulation, the effect should
apply to any other proteins within a similar family of genes
exhibiting identity or substantial identity.
[0066] For co-suppression, the introduced sequence, needing less
than absolute identity, also need not be full length, relative to
either the primary transcription product or fully processed mRNA.
This may be preferred to avoid concurrent production of some plants
which are overexpressers. A higher identity in a shorter than full
length sequence compensates for a longer, less identical sequence.
Furthermore, the introduced sequence need not have the same intron
or exon pattern, and identity of non-coding segments will be
equally effective. Normally, a sequence of the size ranges noted
above for antisense regulation is used. In addition, the same gene
regions noted for antisense regulation can be targeted using
co-suppression technologies.
[0067] Oligonucleotide-based triple-helix formation can also be
used to disrupt ANT gene expression. Triplex DNA can inhibit DNA
transcription and replication, generate site-specific mutations,
cleave DNA, and induce homologous recombination (see, e.g., Havre
and Glazer J. Virology 67:7324-7331 (1993); Scanlon et al. FASEB J.
9:1288-1296 (1995); Giovannangeli et al. Biochemistry
35:10539-10548 (1996); Chan and Glazer J. Mol. Medicine. (Berlin)
75: 267-282 (1997)). Triple helix DNAs can be used to target the
same sequences identified for antisense regulation.
[0068] Catalytic RNA molecules or ribozymes can also be used to
inhibit expression of ANT genes. It is possible to design ribozymes
that specifically pair with virtually any target RNA and cleave the
phosphodiester backbone at a specific location, thereby
functionally inactivating the target RNA. In carrying out this
cleavage, the ribozyme is not itself altered, and is thus capable
of recycling and cleaving other molecules, making it a true enzyme.
The inclusion of ribozyme sequences within antisense RNAs confers
RNA-cleaving activity upon them, thereby increasing the activity of
the constructs. Thus, ribozymes can be used to target the same
sequences identified for antisense regulation.
[0069] A number of classes of ribozymes have been identified. One
class of ribozymes is derived from a number of small circular RNAs
which are capable of self-cleavage and replication in plants. The
RNAs replicate either alone (viroid RNAs) or with a helper virus
(satellite RNAs). Examples include RNAs from avocado sunblotch
viroid and the satellite RNAs from tobacco ringspot virus, lucerne
transient streak virus, velvet tobacco mottle virus, solanum
nodiflorum mottle virus and subterranean clover mottle virus. The
design and use of target RNA-specific ribozymes is described in
Zhao and Pick, Nature 365:448-451 (1993); Eastham and Ahlering, J.
Urology 156:1186-1188 (1996); Sokol and Murray, Transgenic Res.
5:363-371 (1996); Sun et al., Mol. Biotechnology. 7:241-251 (1997);
and Haseloff et al., Nature, 334:585-591 (1988).
Modification of Endogenous ANT Genes
[0070] Methods for introducing genetic mutations described above
can also be used to select for plants with decreased ANT
expression.
Other Means for Inhibiting ANT Activity
[0071] ANT activity may be modulated by eliminating the proteins
that are required for ANT cell-specific gene expression. Thus,
expression of regulatory proteins and/or the sequences that control
ANT gene expression can be modulated using the methods described
here.
[0072] Another strategy is to inhibit the ability of an ANT protein
to interact with itself or with other proteins. This can be
achieved, for instance, using antibodies specific to ANT. In this
method cell-specific expression of ANT-specific antibodies is used
to inactivate functional domains through antibody:antigen
recognition (see, Hupp et al. Cell 83:237-245 (1995)). Interference
of activity of an ANT interacting protein(s) can be applied in a
similar fashion. Alternatively, dominant negative mutants of ANT
can be prepared by expressing a transgene that encodes a truncated
ANT protein. Use of dominant negative mutants to inactivate target
genes in transgenic plants is described in Mizukami et al. Plant
Cell 8:831-845 (1996).
Isolation of ANT Nucleic Acids
[0073] Generally, the nomenclature and the laboratory procedures in
recombinant DNA technology described below are those well known and
commonly employed in the art. Standard techniques are used for
cloning, DNA and RNA isolation, amplification and purification.
Generally enzymatic reactions involving DNA ligase, DNA polymerase,
restriction endonucleases and the like are performed according to
the manufacturer's specifications. These techniques and various
other techniques are generally performed according to Sambrook et
al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols
in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc.
(1994-1998).
[0074] The isolation of ANT nucleic acids may be accomplished by a
number of techniques. For instance, oligonucleotide probes based on
the sequences disclosed here can be used to identify the desired
gene in a cDNA or genomic DNA library. To construct genomic
libraries, large segments of genomic DNA are generated by random
fragmentation, e.g. using restriction endonucleases, and are
ligated with vector DNA to form concatemers that can be packaged
into the appropriate vector. To prepare a cDNA library, mRNA is
isolated from the desired organ, such as flowers, and a cDNA
library which contains the ANT gene transcript is prepared from the
mRNA. Alternatively, cDNA may be prepared from mRNA extracted from
other tissues in which ANT genes or homologs are expressed.
[0075] The cDNA or genomic library can then be screened using a
probe based upon the sequence of a cloned ANT gene disclosed here.
Probes may be used to hybridize with genomic DNA or cDNA sequences
to isolate homologous genes in the same or different plant species.
Alternatively, antibodies raised against an ANT polypeptide can be
used to screen an mRNA expression library.
[0076] Alternatively, the nucleic acids of interest can be
amplified from nucleic acid samples using amplification techniques.
For instance, polymerase chain reaction (PCR) technology can be
used to amplify the sequences of the ANT genes directly from
genomic DNA, from cDNA, from genomic libraries or cDNA libraries.
PCR and other in vitro amplification methods may also be useful,
for example, to clone nucleic acid sequences that code for proteins
to be expressed, to make nucleic acids to use as probes for
detecting the presence of the desired mRNA in samples, for nucleic
acid sequencing, or for other purposes. For a general overview of
PCR see PCR Protocols: A Guide to Methods and Applications. (Innis,
M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press,
San Diego (1990). Appropriate primers and probes for identifying
ANT sequences from plant tissues are generated from comparisons of
the sequences provided here (e.g. SEQ ID NO: 4) and those provided
in Klucher et al. and Elliot et al., supra.
[0077] Polynucleotides may also be synthesized by well-known
techniques as described in the technical literature. See, e.g.,
Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418
(1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double
stranded DNA fragments may then be obtained either by synthesizing
the complementary strand and annealing the strands together under
appropriate conditions, or by adding the complementary strand using
DNA polymerase with an appropriate primer sequence. Because at the
very 5' and 3' ends the Arabidopsis ANT nucleotide sequence is very
similar to the Brassica ANT nucleotide sequence but not to other
Arabidopsis AP2-domain containing genes, the primers with
nucleotide sequences shown in SEQ ID NO:6, SEQ ID NO:7, or SEQ ID
NO:8 can be used to screen/isolate ANT orthologs in different
species by RT-PCR.
Preparation of Recombinant Vectors
[0078] To use isolated sequences in the above techniques,
recombinant DNA vectors suitable for transformation of plant cells
are prepared. Techniques for transforming a wide variety of higher
plant species are well known and described in the technical and
scientific literature. See, for example, Weising et al. Ann. Rev.
Genet. 22:421-477 (1988). A DNA sequence coding for the desired
polypeptide, for example a cDNA sequence encoding a full length
protein, will preferably be combined with transcriptional and
translational initiation regulatory sequences which will direct the
transcription of the sequence from the gene in the intended tissues
of the transformed plant.
[0079] For example, for overexpression, a plant promoter fragment
may be employed which will direct expression of the gene in all
tissues of a regenerated plant. Such promoters are referred to
herein as "constitutive" promoters and are active under most
environmental conditions and states of development or cell
differentiation. Examples of constitutive promoters include the
cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium
tumafaciens, and other transcription initiation regions from
various plant genes known to those of skill. Such genes include for
example, ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol.
33:125-139 (1996)), Cat3 from Arabidopsis (GenBank No. U43147,
Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)), the gene
encoding stearoyl-acyl carrier protein desaturase from Brassica
napus (Genbank No. X74782, Solocombe et al. Plant Physiol.
104:1167-1176 (1994)), GPc1 from maize (GenBank No. X15596,
Martinez et al. J. Mol. Biol. 208:551-565 (1989)), and Gpc2 from
maize (GenBank No. U45855, Manjunath et al., Plant Mol. Biol.
33:97-112 (1997)).
[0080] Alternatively, the plant promoter may direct expression of
the ANT nucleic acid in a specific tissue, organ or cell type (i.e.
tissue-specific promoters) or may be otherwise under more precise
environmental or developmental control (i.e. inducible promoters).
Examples of environmental conditions that may effect transcription
by inducible promoters include anaerobic conditions, elevated
temperature, the presence of light, or sprayed with
chemicals/hormones. One of skill will recognize that a
tissue-specific promoter may drive expression of operably linked
sequences in tissues other than the target tissue. Thus, as used
herein a tissue-specific promoter is one that drives expression
preferentially in the target tissue or cell type, but may also lead
to some expression in other tissues as well.
[0081] A number of tissue-specific promoters can also be used in
the invention. For instance, promoters that direct expression of
nucleic acids in flowers, ovules, or anthers (particularly the
tapetum) are useful in methods in which sterility is desired. An
example of a promoter that directs expression in the ovule is the
promoter from the BEL1 gene described in Reiser et al. Cell
83:735-742 (1995) (GenBank No. U39944). Examples of
tapetal-specific promoters include TA29 from tobacco (Mariani et
al., Nature, 347:737-41, (1990)), and A6 and A9 from Brassica (Paul
et al., Plant Mol. Biol., 19:611-22, (1992), Hird et al. Plant
Journal 4:1023-1033 (1993)). Anther-specific promoters could also
be used such as ones isolated by Twell et al. (Mol. Gen. Genet.,
217:240-45, (1991)).
[0082] To introduce male sterility, the 2nd and 3rd floral organ
(petal and stamens)--specific AP3 promoter (Day, et al.,
Development 121:2887, 1995), for example, can be used. The carpel
specific AGL1 (Flanagan and Ma, Plant J. 10:343, 1993) or AGL5
(Savidge, et al., Plant Cell 7:721, 1995) promoter can be applied
for inducing female sterility only. Sterile plants, yet with
increased perianth organs, can be obtained by constitutively
expressing the ANT gene through AG promoter (Sieburth and
Meyerowitz, Plant Cell 9:355, 1997) that is active only in
reproductive organ primordia and developing male and female
organs.
[0083] Using the AP1 promoter (Gustafson-Brown, et al., Cell
76:131, 1994) that is expressed in floral primordia at early stages
of flower development and in developing perianth organs, fertile
flowers with enlarged perianth organs can be produced. For the
increase of aerial vegetative organ biomass, photosynthetic
organ-specific promoters, such as the RBCS promoter (Khoudi, et
al., Gene 197:343, 1997), can be used. Root biomass can be
increased by the constitutive ANT expression under the control of
the root-specific ANR1 promoter (Zhang & Forde, Science,
279:407, 1998). To increase seed size/mass (an agronomically import
trait), seed-specific promoters, such as the LEC promoter (Lotan,
et al., Cell 93:1195 (1998)), the late-embroygenesis-abundant
promoter (West et al. Plant Cell 6:173 (1994)), beta-conglycininin
alpha-subunit promoter (West et al.), the lectin promoter (Goldberg
et al. Science 266:605 (1994)), or the Kunitz trypsin inhibitor 3
promoter (Goldberg et al.) can be used. Any strong, constitutive
promoters, such as the CaMV 35S promoter, can be used for the
increase of total plant biomass.
[0084] If proper polypeptide expression is desired, a
polyadenylation region at the 3'-end of the coding region should be
included. The polyadenylation region can be derived from the
natural gene, from a variety of other plant genes, or from
T-DNA.
[0085] The vector comprising the sequences (e.g., promoters or
coding regions) from genes of the invention will typically comprise
a marker gene that confers a selectable phenotype on plant cells.
For example, the marker may encode biocide resistance, particularly
antibiotic resistance, such as resistance to kanamycin, G418,
bleomycin, hygromycin, or herbicide resistance, such as resistance
to chlorosulfuron or Basta.
[0086] The present invention also provides promoter sequences from
the ANT gene (SEQ ID NO:3), which can be used to direct expression
of the ANT coding sequence or heterologous sequences in desired
tissues. ANT is expressed in meristematic cells throughout the
plant. ANT promoter sequences of the invention are therefore useful
in targeting expression to meristematic cells in lateral roots,
leaf primordia, developing leaves, floral primordial floral organ
primordia, developing floral organs, ovule primordia, developing
ovules, developing embryos, and vascular systems. Genes whose
expression can be targeted to these cells in immature organs
include disease resistance genes, such as the Arabidopsis NPR1 gene
(Cao, et al., Cell 88:57, 1997) and the nematode resistance locus
Gro1 and the Phytophthora infestans resistance locus R7 of potato
(Leister, et al., Nature Genetics 14:421, 1996), for increasing
resistance to pathogens and insects in young, sensitive organs.
[0087] Because the ANT promoter is expressed in developing embryos
at late stages, some genes encoding regulators or key enzymes for
biosynthesis of storage oils, proteins, or starches, such as BiP
(Hatano, et al., Plant and Cell Physiology 38:344, 1997), can be
expressed by the control of the ANT promoter.
Production of Transgenic Plants
[0088] DNA constructs of the invention may be introduced into the
genome of the desired plant host by a variety of conventional
techniques. For example, the DNA construct may be introduced
directly into the genomic DNA of the plant cell using techniques
such as electroporation and microinjection of plant cell
protoplasts, or the DNA constructs can be introduced directly to
plant tissue using ballistic methods, such as DNA particle
bombardment.
[0089] Microinjection techniques are known in the art and well
described in the scientific and patent literature. The introduction
of DNA constructs using polyethylene glycol precipitation is
described in Paszkowski et al. Embo J 3:2717-2722 (1984).
Electroporation techniques are described in Fromm et al. Proc.
Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation
techniques are described in Klein et al. Nature 327:70-73
(1987).
[0090] Alternatively, the DNA constructs may be combined with
suitable T-DNA flanking regions and introduced into a conventional
Agrobacterium tumefaciens host vector. The virulence functions of
the Agrobacterium tumefaciens host will direct the insertion of the
construct and adjacent marker into the plant cell DNA when the cell
is infected by the bacteria. Agrobacterium tumefaciens-mediated
transformation techniques, including disarming and use of binary
vectors, are well described in the scientific literature. See, for
example Horsch et al. Science 233:496-498 (1984), and Fraley et al.
Proc. Natl. Acad. Sci. USA 80:4803 (1983) and Gene Transfer to
Plants, Potrykus, ed. (Springer-Verlag, Berlin 1995).
[0091] Transformed plant cells which are derived by any of the
above transformation techniques can be cultured to regenerate a
whole plant which possesses the transformed genotype and thus the
desired phenotype such as increased seed mass. Such regeneration
techniques rely on manipulation of certain phytohormones in a
tissue culture growth medium, typically relying on a biocide and/or
herbicide marker that has been introduced together with the desired
nucleotide sequences. Plant regeneration from cultured protoplasts
is described in Evans et al., Protoplasts Isolation and Culture,
Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing
Company, New York, 1983; and Binding, Regeneration of Plants, Plant
Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration
can also be obtained from plant callus, explants, organs, or parts
thereof. Such regeneration techniques are described generally in
Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987).
[0092] The nucleic acids of the invention can be used to confer
desired traits on essentially any plant. Thus, the invention has
use over a broad range of plants, including species from the genera
Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica,
Chlamydomonas, Chlorella, Citrus, Citrullus, Capsicum, Carthamus,
Cocos, Coffea, Cucumis, Cucurbita, Cyrtomium, Daucus, Elaeis,
Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum,
Hyoscyamus, Lactuca, Laminaria, Linum, Lolium, Lupinus,
Lycopersicon, Macrocystis, Malus, Manihot, Majorana, Medicago,
Nereocystis, Nicotiana, Olea, Oryza, Osmunda, Panieum, Pannesetum,
Persea, Phaseolus, Pistachia, Pisum, Pyrus, Polypodium, Prunus,
Pteridium, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,
Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and
Zea.
[0093] One of skill will recognize that after the expression
cassette is stably incorporated in transgenic plants and confirmed
to be operable, it can be introduced into other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed.
[0094] Using known procedures one of skill can screen for plants of
the invention by detecting the increase or decrease of ANT mRNA or
protein in transgenic plants. Means for detecting and quantitating
mRNAs or proteins are well known in the art. The plants of the
invention can also be identified by detecting the desired
phenotype. For instance, increased biomass of organs or plants can
be detected according to well-known techniques. Male or female
sterility can be identified by testing for viable pollen and/or the
ability to set seed.
The following Examples are offered by way of illustration, not
limitation.
Example 1
[0095] This example shows that increased ANT expression increases
cell number and the size/mass of roots, leaves, floral organs,
ovules and seeds in Arabidopsis.
[0096] An ANT cDNA with a BamHI site right before the initiation
codon of the ANT coding nucleotide sequence was created by PCR
using synthetic oligonucleotide primers. This ANT nucleic acid
(from C at 268 to T at 2148 (1881 nucleotides) from SEQ ID NO: 1)
was ligated at the BglII site of the plasmid vector pMON530
(Rogers, et al., Meth. Enzymol. 153:253, 1987) under the
constitutive 35S promoter, and the recombinant plasmid DNA which
has an insert of the ANT cDNA in the sense direction with respect
to the CaMV 35S promoter (35S::ANT) were selected. Agrobacterium
cells were transformed with the recombinant plasmid DNA, and used
for Agrobacterium-mediated plant transformation by vacuum
infiltration with Arabidopsis plants (Col-0 ecotype). T.sub.1 seeds
were collected from transformed plants about three weeks after
vacuum infiltration, and planted on MS plates with kanamycin for
screening T.sub.1 transgenic seedlings.
[0097] T.sub.1 seeds include oversized seeds, which were
distinguished because they did not pass through a mesh of defined
size. The majority of these seeds were kanamycin resistant,
carrying the 35S::ANT transgene. This phenotype was not observed in
vector only controls.
[0098] Multiple T.sub.1 seedlings were larger than vector only
control transgenic seedlings. As they develop, T.sub.1 plants
produced a highly branched root system having a larger mass than
vector only controls. In addition, the plants had enlarged leaves,
floral organs, and ovules as compared to the vector only controls.
For example, the average flower and leaf biomass of T.sub.1 lines
was approximately three times and 2.5 times that of the vector only
control, respectively. DIC microscopy and scanning electron
microscopy revealed that this enlarged organ phenotype of T.sub.1
plants was due to the increased cell number in the organs. In
addition, T.sub.1 plants were sterile. Preliminary examination
suggests that anthers fail to shed pollen (which are
morphologically normal) and the ovules were unusually large with an
increased number of nucellar cells that compress/displace the
female gametophyte.
[0099] Because sterility made it difficult to generate and
propagate homozygous transgenic lines, we used a chemical induction
system as described by Aoyama, and Chua Plant J. 11:605-612 (1997)
and McNellis et al. Plant J. 14:247-257 (1998) to regulate ectopic
ANT transcription. This system utilizes a chimeric transcription
factor gene (35S::GVG), consisting of the 35S promoter, the
DNA-binding domain of the yeast transcription factor GAL4, a
transactivating domain, and the receptor domain of the
glucocorticoid receptor (GR). The ANT gene was inserted downstream
from a promoter (UAS::AN7) containing the binding site for the GVG
transcription factor. The 35S::GVG/UAS::ANT construct was
introduced into wild-type Arabidopsis and fertile transgenic lines
were obtained generally as described above.
[0100] Transgenic T.sub.2 plants were germinated on MS agar plates
and transferred to plates either with or without the chemical
inducer, dexamethasone (DEX), a synthetic glucocorticoid hormone
that binds and activates the GVG transcription factor. Multiple
transgenic lines were obtained that displayed an enlarged leaf
phenotype after treatment with DEX. The increase in organ size/mass
is due to an increased number of cells. DEX had no effect on
control transgenic plants with only the 35S::GVG/UAS vector. Taken
together, these results suggest that ectopic ANT expression
increases organ size/mass by increasing cell number.
Example 2
[0101] This example shows that essentially the same phenotypic
changes observed in Arabidopsis were observed in tobacco.
[0102] For generating tobacco transgenic plants expressing ANT cDNA
under the control of the constitutive 35S promoter, the above
recombinant plasmid DNA was used for Agrobacterium-mediated tobacco
callus transformation. Tobacco calli were induced from sterilized
tobacco leaf (SR1 variety) placed on callus-inducing plates, then
co-cultivated with Agrobacterium cells carrying the above
recombinant DNA for three days. After washing bacterial cells out,
leaf calli were placed on shoot-inducing agar plate containing
kanamycin and carbenicillin to generate transformed shoots. These
R.sub.0 shoots were transferred on root inducing agar plates, then
transplanted on soil after regeneration of roots. [deleted a
sentence]
[0103] The R.sub.0 plants in which the ANT gene was constitutively
expressed under the control of the CaMV 35S promoter produced wider
leaves (about 1.5 times that of vector only control transgenic
plants), relatively larger flowers (about 1.7 time greater mass
than vector only control transgenic plants), and sterility as
observed with Arabidopsis. The sterility is largely caused by the
failure of dehiscence of anthers as seen in the Arabidopsis
transgenic anthers. Some R.sub.0 plants produced functional pollen
grains in their closed anthers, and produced seeds (R.sub.1 seeds)
upon self-pollination by hand using pollen grains dissected from
the anther. These R.sub.1 seeds had mass about 1.5 times that of
seed from vector only control plants.
Example 3
[0104] This example describes plant organ size/mass reduction and
altered flowering by co-suppressing endogenous gene activity by the
ANT transgene in Arabidopsis and tobacco.
[0105] Arabidopsis T.sub.1 lines described above included lines
exhibiting reduced organ size/mass and organ cell numbers. These
plants were completely or partially female sterile, as are
loss-of-function ant mutants. In these lines, expression of ANT
mRNA was highly reduced, suggesting that co-suppression of the
endogenous ANT gene, as well as that of the ANT cDNA, took place in
the lines. From partially sterile T.sub.1 lines, transgenic T.sub.2
plants were obtained that segregated for the same co-suppressed
phenotype as in T.sub.1 parental plants. Reduction of organ
size/mass was also observed in co-suppressed R.sub.0 tobacco
plants.
[0106] Multiple co-suppressed lines also exhibited early flowering.
Plants of these lines displayed reduced numbers of rosette leaves
and fewer days before bolting. Because early-flowering phenotype
was not observed in loss-of-function ant mutants, co-suppression by
the ANT transgene could also influence other unknown ANT related
genes that regulate flowering time by itself or together with ANT.
Similar results were also observed in co-suppressed transgenic
tobacco plants.
Example 4
[0107] This example shows that loss of ANT function reduces mature
organ size by decreasing cell numbers.
[0108] Because ANT mRNA accumulated in leaf (Elliott, R. C., et al.
(1996) Plant Cell 8:155-168), we examined the effect of a
loss-of-function ant mutation on vegetative shoot development.
While there was no difference in the timing of leaf primordia
initiation or the number of leaf primordia between ant-1 and
control wild-type plants (not shown), the width and length of
mature ant-1 leaves were both reduced in comparison with those of
corresponding wild-type leaves. Because ant mutant floral organs
were found to be reduced in size (Klucher, K. M., et al. (1996)
Plant Cell 8:137-153; Elliott, R. C., et al. (1996) Plant Cell
8:155-168), these observations demonstrate that loss of ANT
function reduces organ size throughout shoot development.
[0109] A change in organ size can reflect an alteration in the size
or number of cells, or both. To understand why ant-1 organs are
smaller, we examined the size and number of cells in mature ant-1
organs and compared them with those in wild-type controls. The
distal portion of the petal epidermis was observed initially
because it has cells that are diploid and uniform in size and
shape. We found that ant-1 organs had fewer cells per unit area and
per organ than wild type, however ant-1 cells were much larger than
normal. Essentially the same phenotype was observed in the
epidermis and subdermal cell-layers of all ant-1 floral organs and
leaves. Thus, systemic reduction in size of ant-1 organs is
associated with a decrease in cell number, but not a decrease in
cell size.
[0110] Because ant mutants reduce the number of floral organs, it
has been suggested that ANT might be involved in organ primordium
patterning as well as organ growth. To evaluate this possibility,
we observed the pattern of sepal primordia in developing wild type
and ant-1 floral buds under SEM. By the end of floral stage 4
(Smyth, D. R., et al. (1990) Plant Cell 2:755-767), all four sepal
primordia were initiated at the periphery of developing wild-type
floral buds. In ant-1 floral buds at the comparable stage, the
organ primordia initiated were arranged normally in ant-1 floral
buds, although the number of floral organ was reduced (not shown).
Thus, ANT appears to have little role in controlling the position
of floral organ primordium in developing floral buds.
Example 5
[0111] This example shows the isolation of an ANT ortholog from
Brassica napus (Canola).
[0112] The nucleic acid sequence and the encoded protein of the
Brassica cDNA are shown in SEQ ID NO: 4 and SEQ ID NO:5
respectively.
[0113] To prepare this nucleic acid, total RNA was isolated from
young shoot apices of Brassica napus (Canola) seedlings using
TRIZOL as described by Colasanti et al. (Cell. 93:593-603 (1998)).
cDNA was made by reverse transcription, and amplified by PCR using
the high fidelity thermo-tolerant DNA polymerase PFU and
oligonucleotide primers. The primers had the initiation codon and
the anti-parallel nucleotide sequence downstream of the stop codon
of the Arabidopsis ANT nucleotide sequence, respectively. The PCR
products were subcloned into an E. coli vector and screened by PCR
using different sets of oligonucleotide primers having internal ANT
nucleotide sequence. Nucleotide sequence of the inserted Brassica
DNA of selected recombinant plasmid clones was determined and
compared to the Arabidopsis ANT nucleotide sequence for
confirmation. The Brassica ANT (BANT) gene shares 85.5% identity to
the Arabidopsis ANT gene in their coding region at the nucleotide
level and the BANT polypeptide sequence is 83.7% identical to the
ANT polypeptide sequence, respectively.
Example 6
[0114] This example shows use of the ANT5'-upstream nucleotide
sequence (promoter) for expressing heterologous genes in
meristematic cells
[0115] A HindIII-BglII fragment which includes the correctly
oriented ANT promoter was inserted into the pBI101 plasmid vector
DNA (CLONTECH) at the HindIII and BamHI sites which are located
right before the initiation codon of the GUS (beta-glucuronidase)
gene. The same fragment was also inserted into the plasmid pBIN
m-gfp5-ER (Haseloff, et al., Proc. Natl. Acad. Sci. U.S.A.
94:2122-2127, (1997) at the HindIII-BamHI sites located immediately
before the initiation codon of the GFP (green fluorescence protein)
gene. Arabidopsis wild-type plants were transformed by these
recombinant plasmids using the Agrobacterium-mediated vacuum
infiltration method. Multiple T.sub.1 lines, and their following
generations, exhibited GUS activity or GFP expression in
meristematic cells throughout plant development as expected,
proving that the ANT promoter is useful for expressing a
heterologous gene in meristematic cells.
Example 7
[0116] This example shows activation of the cyclin D3 (CYCD3) gene
expression by increasing ANT gene expression in Arabidopsis
plants.
[0117] Cell proliferation is directly controlled by the activity of
cell cycle regulatory genes, such as cyclins and cdks (Nasmyth,
Trends Genet., 12:405-412, (1996); Morgan, Nature, 374:131-134,
1995; and Burssens, et al., Plant Physiol. Biochem., 36:9-19,
(1998)). Because organs from T.sub.1 transgenic lines in which ANT
gene expression is controlled by the CaMV 35S promoter had
increased number of cells, and thus increased cell proliferation
activity, expression of cyclin genes in young and mature organs of
T.sub.1 plants was measured by quantitative RT-PCR analysis. In
young developing organs, where cell proliferation was observed in
both 35S::ANT and control plants, the difference of expression
levels of cyclin genes between them were not significant. However,
in mature organs, while mRNA accumulation of CYCD3, which encodes a
key regulator for G1/S entry in the Arabidopsis cell cycle (Soni,
et al., Plant Cell. 7:85-103 (1995); Fuerst, et al., Plant Physiol.
112:1023-1033 (1996), is no longer detected in control, it was
detected in 35S::ANT lines. These results agree with observations
that no growth differences were detected at early stages of organ
development between 35S::ANT lines and control lines; however, when
organs of control plants were mature and ceased cell proliferation,
cells in the same aged organs of 35S::ANT plants continued to
proliferate and give rise to enlarged organs as the result.
[0118] This result demonstrates that the increased constitutive ANT
activity directly and/or indirectly controls the cell cycle
machinery via regulating expression of a cell cycle regulator
gene(s) and continuously activating cell proliferation in
developing organs. This also indicates that certain genes involved
in cell cycle machinery are targets of the ANT transcription factor
gene (Klucher et al. and Elliot et al.). Taken together, these
results suggest that modulation of expression of these ANT-target
genes could regulate organ size/mass and fertility in plants.
Example 8
[0119] This example shows that ectopic expression of BANT, an ANT
ortholog from Brassica napus (Canola), increases organ mass/size in
Arabidopsis.
[0120] The Brassica ANT (BANT) cDNA, which has the nucleic acid
sequence shown in SEQ ID NO:4, was inserted into the plasmid vector
pMON530 (Rogers, et al., Method. Ezymol. 153:253, 1987) under the
constitutive 35S promoter in the sense direction. The recombinant
plasmid DNA was used for Agrobacterium transformation, and the
Agrobacterium cells transformed with the 35S::BANT plasmid DNA was
used for Agrobacterium mediated plant transformation by vacuum
infiltration with Arabidopsis plants (Col-0 ecotype). T.sub.1 seeds
were collected about three weeks after vacuum infiltration, and
planted on MS agar plates with kanamycin for screening T.sub.1
transgenic seedlings.
[0121] T.sub.1 plants ectopically expressing the 35S::BANT
transgene exhibited multiple organ hyperplasia, as seen in 35S::ANT
transgenic plants described above (Example 1). That is, leaves and
floral organs were, at most, three times larger than control
organs. These transgenic plants were essentially male sterile, and
are often female sterile as well. Some plants, however, produced
seeds upon fertilization with wild-type pollen grains by
hand-pollination, and the T.sub.2 seeds exhibited increased
mass/size. The kanamycin-resistant T.sub.2 seedlings developed into
plants displaying the same phenotype as the T.sub.1 plants,
suggesting that the effect of ANT ectopic expression is
heritable.
Example 9
[0122] This example shows increased ANT expression induces asexual
reproduction and formation of adventitious shoots, organs, and
embryos in Arabidopsis plants.
[0123] Fully matured stems or organs, such as leaves, were
dissected from T.sub.1 plants ectopically expressing ANT and placed
in water or on MS agar plates without any phytohormones. After
about two-week incubation, adventitious root formation was observed
at the cut surface of stems or leaves. Occasionally, adventitious
roots were also produced from the leaf surface. This adventitious
root formation was never observed control stems or leaves treated
in the same way.
[0124] Excised inflorescence (flowering) stems from fully matured
T.sub.1 plants ectopically expressing ANT were placed on MS agar
plates without phytohormones for 10 days. Adventitious root
formation was observed in the cut surface of stems, while
adventitious shoot formation was observed in the senesced floral
buds. These shoots eventually produced roots as well, developing
into complete plants that exhibited the same transgenic trait
(enlarged organ size/mass) as the original plants. The control
inflorescence stems did not show any activity of asexual
reproduction under the same conditions.
[0125] Similar asexual reproduction was observed in embryos excised
from developing 35S::ANT transgenic seeds. The late torpedo-stage
to nearly mature embryos were excised from developing green seeds,
and grew on phytohormone-free MS agar plates containing 50 .mu.g/ml
kanamycin. Although these embryos developed into seedlings, some
cells reproduced secondary embryos or adventitious shoots, which
also developed into complete plants. The control embryos did not
propagate asexually under the same conditions.
CONCLUSION
[0126] In higher plants intrinsic organ size is determined
genetically, although it can be influenced greatly by environmental
factors. The size of organs reflects the number and size of cells.
The total cell number of an organ is determined by the
proliferation of undifferentiated meristematic cells that are
competent to divide. During shoot development, lateral organs are
initiated as primordia from apical and lateral meristems. While
most cells in organ primordia are meristematic and proliferate,
cells lose meristematic competence and withdraw from the cell cycle
as organs develop. Thus, the maintenance of meristematic competence
of cells is a key mechanism that mediates organ growth and cell
proliferation by defining total cell numbers, and thereby the size
of plant organs. However, the molecular nature of meristematic
competence and the developmental regulators that control
meristematic competence are not well understood.
[0127] The Arabidopsis ANT gene encodes a transcription factor of
the AP2-domain family that has been found only in plant systems.
ANT mRNA accumulates in primordia of all lateral shoot organs and
diminishes as organs develop. This suggests that ANT may have a
general function in organ growth. Consistent with ANT expression in
leaf primordia and undifferentiated growing leaves, it was found
that all mature leaves of the loss-of-function ant-1 mutant were
reduced in size in comparison with corresponding wild-type leaves.
Because ant-1 floral organs were also smaller than normal, ANT is
most likely required for organ growth throughout post-embryonic
shoot development. Organ size can be influenced by cell size, cell
number, or both. It was found that ant-1 organs had fewer cells per
unit area and per organ than wild type, however ant-1 cells were
much larger than normal. This demonstrates that the systemic
reduction in size of ant-1 organs is the result of a decrease in
cell number, but not a decrease in cell size. Therefore, ANT
function is necessary to attain the intrinsic cell number of plant
organs.
[0128] The experiments described here demonstrate that ectopic ANT
expression is sufficient to increase organ size and mass by
enhancing organ growth that is coordinated with organ morphogenesis
in Arabidopsis plants. Differentiated cells in fully mature
35S::ANT petals were the same size as those in wild-type petals.
Similarly, no obvious difference in cell size was detected in the
epidermis between control and 35S::ANT organs other than petals.
Thus, an increase of cell numbers, and not cell size, is primarily
responsible for the enlarged 35S::ANT organs. Similar loss- and
gain-of-function effects on organ size was observed when plants
were grown plants grown under short-day, continuous-light
conditions, and in poor or rich media. Thus, ANT function seems to
be independent of the perception of external growth signals. In
contrast to the striking effects on final organ size, ectopic ANT
expression did not perceptibly alter the size or structure of
apical and lateral meristems, nor did it change the size or number
of organ primordia. Although loss of ANT function reduced the
number of floral organs, the organ primordia initiated were
arranged and sized normally in ant-1 floral buds. Therefore, ANT
does not determine organ primordium size, and most likely does not
influence organ primordium number by controlling the organization
of the apical and lateral meristems.
[0129] How does ANT control cell numbers during organogenesis? In
general, plant organ growth involves neither cell migration nor
cell death; thus, organ cell number essentially depends on
proliferation of the meristematic cells in developing organ.
Because ANT is expressed in meristematic cells of the developing
organs, it might modulate cell proliferation during organogenesis
and thereby determine the total cell number in mature organs. To
test this idea, the extent of cell proliferation in control and
ant-1 organs was tested by measuring cell numbers and cell size of
both developing and fully mature petals. During mid-floral stage 9,
the adaxial epidermal cells of wild-type petals were not
differentiated and divided frequently, whereas ant-1 petals had
fewer undifferentiated cells than normal per unit area and per
organ. This reduction in cell numbers became more pronounced in
fully differentiated ant-1 petals at stage 15. Thus, there are
fewer cell divisions than normal in ant-1 petals throughout
organogenesis, particularly during later developmental stages prior
to maturation. Cell growth occurred without cell division in ant-1
petals, resulting in extremely large cells.
[0130] These results suggest that ANT is required for the normal
extent of cell proliferation, but not primarily for cell growth. To
understand how ANT regulates the extent of cell proliferation, we
studied how ectopic ANT expression affects organ size, cell size,
and cell numbers during petal development. In contrast to the early
effect on cell numbers in ant-1 petals, cell numbers and cell size
in 35S::ANT petals at stage 9 were normal. This demonstrates that
ectopic ANT expression does not increase cell growth or the
frequency of cell proliferation in developing petals during early
stages, and suggests that increased ANT activity does not alter the
intrinsic cell cycle time. By stage 15, however, the total cell
number of fully mature 35S::ANT petals reached approximately 2.5
times that of controls, indicating that additional cell divisions
occurred in 35S::ANT petals prior to organ maturation, yet only
after stage 9. Extra cell divisions must be coordinated with cell
growth, since cell size in mature 35S::ANT petals is normal.
Therefore, it is likely that ectopic ANT expression allows petal
cells to proliferate for a longer period than normal without
altering the intrinsic cell cycle time. Similar results were
obtained when comparing growth of rosette leaves of 35S::ANT and
control seedlings. At 16 days after germination (16 DAG), both
35S::ANT and control seedlings had the same number of rosette
leaves, and all leaves of 35S::ANT seedlings were the same size as
corresponding control leaves. However, 35S::ANT leaves continued to
grow beyond the period in which corresponding control leaves ceased
to grow, eventually giving rise to larger leaves than normal. This
observation supports the hypothesis that prolonged cell
proliferation coordinated with cell growth causes hyperplasia in
35S::ANT plants. Taken together, these observations suggests that
ANT regulates the period of cell proliferation by maintaining
meristematic competence of cells during organogenesis. The results
presented here also suggest that ANT does not influence CycD3
expression in tissue where most cells are meristematic. Similar
results were obtained in comparing mRNA levels of CycB1b (Cyc1bAt),
a mitotic cyclin gene. Hence, ANT maintains the meristematic
competence of cells, and consequently sustains expression of cell
cycle regulators.
[0131] Another striking finding that connects ANT function with the
maintenance of meristematic competence is neoplasia found in the
Arabidopsis 35S::ANT organs. That is, clusters of undifferentiated
cells (i.e., calli) were generated from wounds or senesced-surfaces
of 35S::ANT plants, or detached-ends of fully differentiated
35S::ANT organs without external phytohormone treatment. These
calli often differentiated into organs, such as roots, leaves, or
shoots. This neoplasia was observed consistently in 35S::ANT
organs, but never was seen in control organs treated in the same
way. It is well established that differentiated plant tissue can
induce calli after phytohormone treatment. Ectopic ANT expression
in differentiated cells that are normally quiescent preserves
meristematic competence and decreases their dependence on
phytohormones for reentry into the cell cycle.
[0132] The findings presented here demonstrate ANT is an intrinsic
organ size regulator that influences organ growth and the period of
cell proliferation during organogenesis. In a proposed model of ANT
action in plant organ size regulation, developmental growth signals
activate growth regulators, which positively regulate ANT during
organogenesis. ANT functions to maintain meristematic competence of
cells, thereby modulating the expression of cell cycle and cell
growth regulators. As a result, ANT sustains cell proliferation
that is coupled to cell growth in developing organs. Ectopically
expressed ANT, therefore, results in the abnormal retention of
meristematic competence of cells and causes hyperplasia and
neoplasia, while the absence of ANT causes precocious termination
of cell proliferation and organ growth. In plant and animal
systems, growth signaling pathways and the cell cycle machinery
appear to share many common factors. Nevertheless, given the
immobile attributes of plant life and plant cells, which are
surrounded by rigid cell walls, some aspects of plant growth and
cell proliferation are likely to be regulated and coordinated in a
different way from those of animals. Thus, it may not be surprising
that ANT is a plant specific regulator, and identification of
upstream regulators and downstream targets of ANT may reveal how
plants uniquely coordinate cell proliferation with pattern
formation to control organ size. It has been suggested that the
genetic basis for plant interspecies diversity of phenotype might
be minor changes in the structure or expression of orthologous
regulatory genes. Hence, differences in structure and expression
pattern of ANT and its orthologs, at least in part, may be a
mechanism that is responsible for the interspecies diversity of
organ size in higher plants. Finally, increasing organ mass by
ectopic ANT expression might be a new method for improving the
yield of agriculturally important plants.
[0133] The above examples are provided to illustrate the invention
but not to limit its scope. Other variants of the invention will be
readily apparent to one of ordinary skill in the art and are
encompassed by the appended claims. All publications, patents, and
patent applications cited herein are hereby incorporated by
reference for all purposes.
Sequence CWU 1
1
812148DNAArabidopsis thalianaAINTEGUMENTA (ANT) cDNA 1agatcccaac
ggattcaaac agcaaatttg tgctttgctc ttctctctta ttataatatc 60ctctcaaaaa
ccctctccta tatcctccta aagcccccct tccttgtttc tctaccgcaa
120caaagaaaaa acaaaagttt gagaaaaatg gtgtgttcgt tgtgtaacca
atgattgggt 180tttagcttac tacttcgaga gattataaga aagaaagagt
gaagatacat tatagaaaga 240agagaagcag aaaccaaaaa aagaaacc atg aag tct
ttt tgt gat aat gat 292 Met Lys Ser Phe Cys Asp Asn Asp 1 5gat aat
aat cat agc aac acg act aat ttg tta ggg ttc tca ttg tct 340Asp Asn
Asn His Ser Asn Thr Thr Asn Leu Leu Gly Phe Ser Leu Ser 10 15 20tca
aat atg atg aaa atg gga ggt aga gga ggt aga gaa gct att tac 388Ser
Asn Met Met Lys Met Gly Gly Arg Gly Gly Arg Glu Ala Ile Tyr25 30 35
40tca tct tca act tct tca gct gca act tct tct tct tct gtt cca cct
436Ser Ser Ser Thr Ser Ser Ala Ala Thr Ser Ser Ser Ser Val Pro Pro
45 50 55caa ctt gtt gtt ggt gac aac act agc aac ttt ggt gtt tgc tat
gga 484Gln Leu Val Val Gly Asp Asn Thr Ser Asn Phe Gly Val Cys Tyr
Gly 60 65 70tct aac cca aat gga gga atc tat tct cac atg tct gtg atg
cca ctc 532Ser Asn Pro Asn Gly Gly Ile Tyr Ser His Met Ser Val Met
Pro Leu 75 80 85aga tct gat ggt tct ctt tgc tta atg gaa gct ctc aac
aga tct tct 580Arg Ser Asp Gly Ser Leu Cys Leu Met Glu Ala Leu Asn
Arg Ser Ser 90 95 100cac tcg aat cac cat caa gat tca tct cca aag
gtg gag gat ttc ttt 628His Ser Asn His His Gln Asp Ser Ser Pro Lys
Val Glu Asp Phe Phe105 110 115 120ggg acc cat cac aac aac aca agt
cac aaa gaa gcc atg gat ctt agc 676Gly Thr His His Asn Asn Thr Ser
His Lys Glu Ala Met Asp Leu Ser 125 130 135tta gat agt tta ttc tac
aac acc act cat gag ccc aac acg act aca 724Leu Asp Ser Leu Phe Tyr
Asn Thr Thr His Glu Pro Asn Thr Thr Thr 140 145 150aac ttt caa gag
ttc ttt agc ttc cct caa acc aga aac cat gag gaa 772Asn Phe Gln Glu
Phe Phe Ser Phe Pro Gln Thr Arg Asn His Glu Glu 155 160 165gaa act
aga aat tac ggg aat gac cct agt ttg aca cat gga ggg tct 820Glu Thr
Arg Asn Tyr Gly Asn Asp Pro Ser Leu Thr His Gly Gly Ser 170 175
180ttt aat gta ggg gta tat ggg gaa ttt caa cag tca ctg agc tta tcc
868Phe Asn Val Gly Val Tyr Gly Glu Phe Gln Gln Ser Leu Ser Leu
Ser185 190 195 200atg agc cct ggg tca caa tct agc tgc atc act ggc
tct cac cac cac 916Met Ser Pro Gly Ser Gln Ser Ser Cys Ile Thr Gly
Ser His His His 205 210 215caa caa aac caa aac caa aac cac caa agc
caa aac cac cag cag atc 964Gln Gln Asn Gln Asn Gln Asn His Gln Ser
Gln Asn His Gln Gln Ile 220 225 230tct gaa gct ctt gtg gag aca agc
gtt ggg ttt gag acg acg aca atg 1012Ser Glu Ala Leu Val Glu Thr Ser
Val Gly Phe Glu Thr Thr Thr Met 235 240 245gcg gct gcg aag aag aag
agg gga caa gag gat gtt gta gtt gtt ggt 1060Ala Ala Ala Lys Lys Lys
Arg Gly Gln Glu Asp Val Val Val Val Gly 250 255 260cag aaa cag att
gtt cat aga aaa tct atc gat act ttt gga caa cga 1108Gln Lys Gln Ile
Val His Arg Lys Ser Ile Asp Thr Phe Gly Gln Arg265 270 275 280act
tct caa tac cga ggc gtt aca aga cat aga tgg act ggt aga tat 1156Thr
Ser Gln Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Tyr 285 290
295gaa gct cat cta tgg gac aat agt ttc aag aag gaa ggt cac agt aga
1204Glu Ala His Leu Trp Asp Asn Ser Phe Lys Lys Glu Gly His Ser Arg
300 305 310aaa gga aga caa gtt tat ctg gga ggt tat gat atg gag gag
aaa gct 1252Lys Gly Arg Gln Val Tyr Leu Gly Gly Tyr Asp Met Glu Glu
Lys Ala 315 320 325gct cga gca tat gat ctt gct gca ctc aag tac tgg
ggt ccc tct act 1300Ala Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp
Gly Pro Ser Thr 330 335 340cac acc aat ttc tct gcg gag aat tat cag
aaa gag att gaa gac atg 1348His Thr Asn Phe Ser Ala Glu Asn Tyr Gln
Lys Glu Ile Glu Asp Met345 350 355 360aag aac atg act aga caa gaa
tat gtt gca cat ttg aga agg aag agc 1396Lys Asn Met Thr Arg Gln Glu
Tyr Val Ala His Leu Arg Arg Lys Ser 365 370 375agt ggt ttc tct agg
ggt gct tcc atc tat aga gga gtc aca aga cat 1444Ser Gly Phe Ser Arg
Gly Ala Ser Ile Tyr Arg Gly Val Thr Arg His 380 385 390cac cag cat
gga agg tgg caa gca cgg att ggt aga gtc gct gga aac 1492His Gln His
Gly Arg Trp Gln Ala Arg Ile Gly Arg Val Ala Gly Asn 395 400 405aaa
gat ctc tac ctt gga act ttt gga acc caa gaa gaa gct gca gaa 1540Lys
Asp Leu Tyr Leu Gly Thr Phe Gly Thr Gln Glu Glu Ala Ala Glu 410 415
420gct tac gat gta gca gca att aag ttc cgt ggc aca aat gct gtg act
1588Ala Tyr Asp Val Ala Ala Ile Lys Phe Arg Gly Thr Asn Ala Val
Thr425 430 435 440aac ttt gat atc acg agg tac gat gtt gat cgt atc
atg tct agt aac 1636Asn Phe Asp Ile Thr Arg Tyr Asp Val Asp Arg Ile
Met Ser Ser Asn 445 450 455aca ctc ttg tct gga gag tta gcg cga agg
aac aac aac agc att gtc 1684Thr Leu Leu Ser Gly Glu Leu Ala Arg Arg
Asn Asn Asn Ser Ile Val 460 465 470gtc agg aat act gaa gac caa acc
gct cta aat gct gtt gtg gaa ggt 1732Val Arg Asn Thr Glu Asp Gln Thr
Ala Leu Asn Ala Val Val Glu Gly 475 480 485ggt tcc aac aaa gaa gtc
agt act ccc gag aga ctc ttg agt ttt ccg 1780Gly Ser Asn Lys Glu Val
Ser Thr Pro Glu Arg Leu Leu Ser Phe Pro 490 495 500gcg att ttc gcg
ttg cct caa gtt aat caa aag atg ttc gga tca aat 1828Ala Ile Phe Ala
Leu Pro Gln Val Asn Gln Lys Met Phe Gly Ser Asn505 510 515 520atg
ggc gga aat atg agt cct tgg aca tca aac cct aat gct gag ctt 1876Met
Gly Gly Asn Met Ser Pro Trp Thr Ser Asn Pro Asn Ala Glu Leu 525 530
535aag acc gtc gct ctt act ttg cct cag atg ccg gtt ttc gct gct tgg
1924Lys Thr Val Ala Leu Thr Leu Pro Gln Met Pro Val Phe Ala Ala Trp
540 545 550gct gat tct tga tcaacttcaa tgactaactc tggttttctt
ggtttagttg 1976Ala Asp Ser555ctaagtgttt tggtttatct ccggttttat
ccggtttgaa ctacaattcg gtttagtttc 2036gtcggtataa atagtatttg
cttaggagcg gtatatgttt cttttgagta gtattcatgt 2096gaaacagaat
gaatctctct ataacatatt attttaatga atctcctttg ct
21482555PRTArabidopsis thalianaAINTEGUMENTA (ANT) 2Met Lys Ser Phe
Cys Asp Asn Asp Asp Asn Asn His Ser Asn Thr Thr1 5 10 15Asn Leu Leu
Gly Phe Ser Leu Ser Ser Asn Met Met Lys Met Gly Gly 20 25 30Arg Gly
Gly Arg Glu Ala Ile Tyr Ser Ser Ser Thr Ser Ser Ala Ala 35 40 45Thr
Ser Ser Ser Ser Val Pro Pro Gln Leu Val Val Gly Asp Asn Thr 50 55
60Ser Asn Phe Gly Val Cys Tyr Gly Ser Asn Pro Asn Gly Gly Ile Tyr65
70 75 80Ser His Met Ser Val Met Pro Leu Arg Ser Asp Gly Ser Leu Cys
Leu 85 90 95Met Glu Ala Leu Asn Arg Ser Ser His Ser Asn His His Gln
Asp Ser 100 105 110Ser Pro Lys Val Glu Asp Phe Phe Gly Thr His His
Asn Asn Thr Ser 115 120 125His Lys Glu Ala Met Asp Leu Ser Leu Asp
Ser Leu Phe Tyr Asn Thr 130 135 140Thr His Glu Pro Asn Thr Thr Thr
Asn Phe Gln Glu Phe Phe Ser Phe145 150 155 160Pro Gln Thr Arg Asn
His Glu Glu Glu Thr Arg Asn Tyr Gly Asn Asp 165 170 175Pro Ser Leu
Thr His Gly Gly Ser Phe Asn Val Gly Val Tyr Gly Glu 180 185 190Phe
Gln Gln Ser Leu Ser Leu Ser Met Ser Pro Gly Ser Gln Ser Ser 195 200
205Cys Ile Thr Gly Ser His His His Gln Gln Asn Gln Asn Gln Asn His
210 215 220Gln Ser Gln Asn His Gln Gln Ile Ser Glu Ala Leu Val Glu
Thr Ser225 230 235 240Val Gly Phe Glu Thr Thr Thr Met Ala Ala Ala
Lys Lys Lys Arg Gly 245 250 255Gln Glu Asp Val Val Val Val Gly Gln
Lys Gln Ile Val His Arg Lys 260 265 270Ser Ile Asp Thr Phe Gly Gln
Arg Thr Ser Gln Tyr Arg Gly Val Thr 275 280 285Arg His Arg Trp Thr
Gly Arg Tyr Glu Ala His Leu Trp Asp Asn Ser 290 295 300Phe Lys Lys
Glu Gly His Ser Arg Lys Gly Arg Gln Val Tyr Leu Gly305 310 315
320Gly Tyr Asp Met Glu Glu Lys Ala Ala Arg Ala Tyr Asp Leu Ala Ala
325 330 335Leu Lys Tyr Trp Gly Pro Ser Thr His Thr Asn Phe Ser Ala
Glu Asn 340 345 350Tyr Gln Lys Glu Ile Glu Asp Met Lys Asn Met Thr
Arg Gln Glu Tyr 355 360 365Val Ala His Leu Arg Arg Lys Ser Ser Gly
Phe Ser Arg Gly Ala Ser 370 375 380Ile Tyr Arg Gly Val Thr Arg His
His Gln His Gly Arg Trp Gln Ala385 390 395 400Arg Ile Gly Arg Val
Ala Gly Asn Lys Asp Leu Tyr Leu Gly Thr Phe 405 410 415Gly Thr Gln
Glu Glu Ala Ala Glu Ala Tyr Asp Val Ala Ala Ile Lys 420 425 430Phe
Arg Gly Thr Asn Ala Val Thr Asn Phe Asp Ile Thr Arg Tyr Asp 435 440
445Val Asp Arg Ile Met Ser Ser Asn Thr Leu Leu Ser Gly Glu Leu Ala
450 455 460Arg Arg Asn Asn Asn Ser Ile Val Val Arg Asn Thr Glu Asp
Gln Thr465 470 475 480Ala Leu Asn Ala Val Val Glu Gly Gly Ser Asn
Lys Glu Val Ser Thr 485 490 495Pro Glu Arg Leu Leu Ser Phe Pro Ala
Ile Phe Ala Leu Pro Gln Val 500 505 510Asn Gln Lys Met Phe Gly Ser
Asn Met Gly Gly Asn Met Ser Pro Trp 515 520 525Thr Ser Asn Pro Asn
Ala Glu Leu Lys Thr Val Ala Leu Thr Leu Pro 530 535 540Gln Met Pro
Val Phe Ala Ala Trp Ala Asp Ser545 550 55534228DNAArabidopsis
thalianaANT gene 5' promoter 3gtcgactcta ggcctcactg gcctaatacg
actcactata gggagctcga ggatccttta 60gttagaaaaa actttctttg tacgtgtgtg
tgtgtgtttt aagttcaatt ataactagtc 120acatgtgata tcacaatata
tatattgaaa ttggaattat tcatattaat gagttagcat 180taatatatat
acgctgacat taccaaccaa atgtttctgc ttttatggat agttctatat
240gttgcacttg attatagata ctatataaaa ctgggtttat ttaaaatccg
tacccataac 300aaaagtggac caaaacgaga tccatggttt tgtgtttact
ttgttggtta accagataat 360atgattatgg aagattaaat ctttactaaa
ttataaaata atttggaaaa acaaacttaa 420atatgttgag tgtcttcagt
gctcactgtt caagaataat ctcgtgttat cctacttgaa 480ctagaagttg
atatacataa acacgtgaat attttaacga ccgtacataa acacatgtat
540cgatcaaata caaattatta tgagactaga atccaagatg aggatgactc
tagcagaata 600tacacagcta agaatttgta caagagagtc gaaaaataga
ttctaatcat ttaaaaaaga 660tatggatttc agttacggat tgatattacc
attacgcagt agtacataca cataattttt 720tgtttttgtt ttaccgataa
tagaatgaaa atgttgtgtt aaaaatattg gttttactaa 780aactcgtttt
atgttaacta tataatgtct ttccgcatgt aaattgaaac aaaactgtaa
840tacaaattat gttaagccat tgcaattaaa aaatccacgg gtagtaaatc
ctcagaagat 900tatgttaagt ctacaaattt tctctttaga ttagtaaggt
ttgagacaaa attatgtata 960ccttgcaggg gtataaaggt cactgcatag
tcagactcag catgaagcca aagagtcgtc 1020tctgtcctaa agatatctac
agctgcttcg cctgtgaata gagaagaaat tgaatgatga 1080gagatcccat
ctagcgtttc acgtttgcgt tctccgtcgc aactttggcg gttgttgact
1140ttttttctta tgtcgttgtt tgactaattt tctcagagtg agagtgtaat
caagaaaact 1200aatattcgaa aagaaagaaa aaaaaggcaa gaaaactatt
gtcgaaaaga cataaatgac 1260actaaaattg gattattaaa aatggtatat
atgtttggtg gaatttataa tcattaccaa 1320aatcaaagga aggagagagg
gacctcttcg tgcttatgat ttccctccta aacaactgct 1380cccactatcc
ttttttactt ccaacaaaat cattcacacg agaaaatctg tctcgtgatc
1440actttcatgc aaaattaaac taaattttgg tattttttgt caagttcttg
ctgttttaag 1500tcgattattt ggtaatacta tatgtgtgga tatacacatc
caagctaatc aataattgat 1560ctccttctgc ttatcaataa attacaccac
attagctaat caagctaata aattacacca 1620cattctctta tcaattttta
tatggtataa ataaaacaac cgactatagg ctacagagtt 1680ggtattaagg
cattattgcc ttctagtcga aggaattttt ttgttatgat aacactcgtg
1740ggaaaaaaat ccagcctaat atgctcattt aaaggataat tgatttaaat
gctttaatca 1800ttaaaataaa aggtttttgc ttttaaaggt taccaccgct
taattcatca ttaggagaat 1860attaactttg atcgaaattc caaaatactt
ttttaacaca taagaaaatt ttcagcattt 1920ttaaataaag ggtacattta
ttgggttcaa taaatatgtt tccacgtaaa gtttggaggt 1980ttaaccacat
gaatgttttt tgatttaaaa aacacataaa ttttctagta attacacatt
2040tttaaccgtc catccagatt gtaataagtg acaaatctga aaacattttt
ttttttcttg 2100aatcttgttt aaattctctc tgctgcatac ttgcaggcat
ttgaccaacg actatacata 2160ttgaaagcaa aatatccacc agggatgata
gggttagatc ccacattcaa tatcttttgt 2220ctttgttatt tatgaaaaac
aaatatttat caggaaaaaa acgtttcttc tctagtggta 2280taagtataag
ataataacaa aatttaatac ttagttaatg tatttactat cttcaaactt
2340accatccttc aacattaata ttgatcaatt tttatttttt ttactaaact
acttccacta 2400aaaaaatgca aaagaagaga tatatattta agtcaaagta
attaaagatg gatgggtgat 2460tcttcagcaa aacggcgccg tagaggtgtc
ttatcctaca ttacagctgg gttgtggcag 2520acatcatagg gcctacgtat
atttgagctt tactgtacgt aaagctttaa catatctagt 2580tagttctcac
tgtacaaaca aaacaaaatc caattcgtaa catatataca aatactacta
2640gtactagatt acgctacgta tacatcgctt tttcgcaaat ttctaaacta
atctatacaa 2700caaacttgaa tgtttgtttt gtaatttatc ttaaaccaaa
gttttgaatt gtgcattggg 2760agctacactc tagtcccctt ttttccccaa
aataatctcc ttacatcgac cggttaaagt 2820atttaaacca acaaatttta
atttgttgct gaaggtacaa acatgtcaca tatatagaga 2880cagcatcgtt
tatacaaata atgttcgatg ttattggaaa tcaaatataa atacgaatta
2940gcgactcact tggtttaata gtttggaaga taatgaaata aaaaatgaat
tcaaaggata 3000cagagctata tatgtcgggt catttagagc cgtgaccaaa
agtttcgtcg taatttctac 3060ggtcggtcat aagaaatttt ggacttttct
tcaccctttt atgaacttct gtatagtttt 3120tgtcggatta tatatttgta
ttcgtatatt ttttgtttct aataatgata cgtaaattca 3180cgataagaaa
gacttctttt tatttaattt gatttaaaac ttttgttttt ggaaatgact
3240catacacaag gttaaagttt gatggtatcc aatttacaaa aatgtttcga
gagtgcgttc 3300gagtgtccta ccaccatcgt accaactcgt atgggtttat
tattaggttt ttttcttctt 3360tttccaatgt ctttataatt gaaccactct
aaatttcttt ttttaaatta ggttaagaat 3420cttgaatttt ctgttgattt
taaaccaagg ttttcaattc ttcttagcac aaaaaaaaaa 3480aaaaggtttt
caattattaa agaatctaaa ttttttgagt tcaagagttt aatgatagct
3540gaaaagttat gaatgattgc aagtttgcaa cagaatggtc gatgtagtac
atatcaaaaa 3600catgcatcaa aataaatatt cgtgcttagc aagagaaacg
attgaaataa acagaacaat 3660cgttaaccac ttaaaaatct tagaataatt
ttgtagtgat aattttctgt aagagagagg 3720tatcatatct tacaaaaaaa
aactcatttc agataaaata atgttgtcca atcgttacca 3780agtatgtttt
tgctgtcatc agttgtattg taactcgtct cttagccata tagttctaag
3840ttttaaatgt tttcaaagac tttacaaaaa taaaataata ataaggtgga
atttgtaggg 3900ctaaaagcga aaaataaaaa taaaataaaa gtaaagaaac
gtctttctca ataagaacac 3960agatcccaac ggattcaaac agcaaatttg
tgctttgctc ttctctctta ttataatatc 4020ctctcaaaaa ccctctccta
tatcctccta aagcccccct tccttgtttc tctaccgcaa 4080caaagaaaaa
acaaaagttt gagaaaaatg gtgtgttcgt tgtgtaacca atgattgggt
4140tttagcttac tacttcgaga gattataaga aagaaagagt gaagatacat
tatagaaaga 4200agagaagcag aaaccaaaaa aagaaacc 422841738DNABrassica
napuscanola AINTEGUMENTA (ANT) partial cDNA including coding region
4atg aag tct ttt tgt gat aat gat gat agt aat acg act aat ttg cta
48Met Lys Ser Phe Cys Asp Asn Asp Asp Ser Asn Thr Thr Asn Leu Leu1
5 10 15ggg ttc tcg ttg tct tca aat atg ttg aaa atg ggt ggt gga gaa
gct 96Gly Phe Ser Leu Ser Ser Asn Met Leu Lys Met Gly Gly Gly Glu
Ala 20 25 30ctt tac tca tct tcg tcg tct tca gtt gca act tct tct gtt
cca cca 144Leu Tyr Ser Ser Ser Ser Ser Ser Val Ala Thr Ser Ser Val
Pro Pro 35 40 45cag ctt gtt gtt ggc gac aac agt agc aac tat gga gtt
tgc tac ggt 192Gln Leu Val Val Gly Asp Asn Ser Ser Asn Tyr Gly Val
Cys Tyr Gly 50 55 60tct aac tta gca gct agg gaa atg tat tct caa atg
tct gtg atg ccc 240Ser Asn Leu Ala Ala Arg Glu Met Tyr Ser Gln Met
Ser Val Met Pro65 70 75 80ctc aga tct gac ggt tct ctt tgc tta atg
gaa gct ctc aac aga tct 288Leu Arg Ser Asp Gly Ser Leu Cys Leu Met
Glu Ala Leu Asn Arg Ser 85 90 95tct cac tcg aat aat cat cac cat agt
caa gtt tca tct cca aag atg 336Ser His Ser Asn Asn His His His Ser
Gln Val Ser Ser Pro Lys Met 100 105 110gaa gat ttc ttt ggg acc cat
cat cac aac aca agt cac aaa gaa gcc 384Glu Asp Phe Phe Gly Thr His
His His Asn Thr Ser His Lys Glu Ala 115 120
125atg gat ctt agc tta gat agt tta ttc tac aat acc act cat gcg cca
432Met Asp Leu Ser Leu Asp Ser Leu Phe Tyr Asn Thr Thr His Ala Pro
130 135 140aac aac aac acc aac ttt caa gag ttc ttt agc ttc cct caa
act aga 480Asn Asn Asn Thr Asn Phe Gln Glu Phe Phe Ser Phe Pro Gln
Thr Arg145 150 155 160aac cac cat gag gaa gaa aca aga aac tac gag
aat gac cct ggt ttg 528Asn His His Glu Glu Glu Thr Arg Asn Tyr Glu
Asn Asp Pro Gly Leu 165 170 175aca cat gga gga ggg tct ttt aat gta
ggg gta tat ggg gaa ttt caa 576Thr His Gly Gly Gly Ser Phe Asn Val
Gly Val Tyr Gly Glu Phe Gln 180 185 190cag tca ctg agc ttg tcc atg
agc cct ggg tca caa tct agc tgc atc 624Gln Ser Leu Ser Leu Ser Met
Ser Pro Gly Ser Gln Ser Ser Cys Ile 195 200 205act gcc tct cat cac
cac caa aac caa act caa aac cac cag cag atc 672Thr Ala Ser His His
His Gln Asn Gln Thr Gln Asn His Gln Gln Ile 210 215 220tct gaa gct
ttg gtc gag aca agt gct gga ttt gag aca aca aca atg 720Ser Glu Ala
Leu Val Glu Thr Ser Ala Gly Phe Glu Thr Thr Thr Met225 230 235
240gcg gct gct gct gca aag aag aag aga gga caa gaa gtt gtc gtt gga
768Ala Ala Ala Ala Ala Lys Lys Lys Arg Gly Gln Glu Val Val Val Gly
245 250 255cag aaa cag att gtt cat aga aaa tct att gat act ttt gga
caa cga 816Gln Lys Gln Ile Val His Arg Lys Ser Ile Asp Thr Phe Gly
Gln Arg 260 265 270act tcg caa tac cga ggc gtt aca aga cat aga tgg
act ggt agg tat 864Thr Ser Gln Tyr Arg Gly Val Thr Arg His Arg Trp
Thr Gly Arg Tyr 275 280 285gaa gct cat cta tgg gac aat agt ttc aag
aag gaa ggt cat agc aga 912Glu Ala His Leu Trp Asp Asn Ser Phe Lys
Lys Glu Gly His Ser Arg 290 295 300aaa gga aga caa gtt tat ctg ggg
ggt tat gat atg gag gag aaa gct 960Lys Gly Arg Gln Val Tyr Leu Gly
Gly Tyr Asp Met Glu Glu Lys Ala305 310 315 320gct cga gca tat gat
ctt gct gca ctc aag tac tgg ggt ccc tct act 1008Ala Arg Ala Tyr Asp
Leu Ala Ala Leu Lys Tyr Trp Gly Pro Ser Thr 325 330 335cac act aat
ttc tct gtg gag aat tat cag aaa gag att gat gac atg 1056His Thr Asn
Phe Ser Val Glu Asn Tyr Gln Lys Glu Ile Asp Asp Met 340 345 350aag
aac atg act cga caa gaa tat gtt gct cac ttg aga aga aaa acc 1104Lys
Asn Met Thr Arg Gln Glu Tyr Val Ala His Leu Arg Arg Lys Thr 355 360
365agt ggt ttc tct agg ggt gct tcc atc tat aga gga gtc acc aga cat
1152Ser Gly Phe Ser Arg Gly Ala Ser Ile Tyr Arg Gly Val Thr Arg His
370 375 380cac cag cat gga agg tgg caa gct cgg atc ggt aga gtc gct
gga aac 1200His Gln His Gly Arg Trp Gln Ala Arg Ile Gly Arg Val Ala
Gly Asn385 390 395 400aaa gat ctc tac ctt gga act ttc gga act caa
gaa gaa gcg gcg gaa 1248Lys Asp Leu Tyr Leu Gly Thr Phe Gly Thr Gln
Glu Glu Ala Ala Glu 405 410 415gcc tat gat gta gca gct atc aag ttc
cgt ggc aca aac gcg gtg act 1296Ala Tyr Asp Val Ala Ala Ile Lys Phe
Arg Gly Thr Asn Ala Val Thr 420 425 430aac ttt gac ata aca agg tac
gat gtt gat cgc ata atg gct agt aac 1344Asn Phe Asp Ile Thr Arg Tyr
Asp Val Asp Arg Ile Met Ala Ser Asn 435 440 445act ctc ttg tct gga
gag atg gct cga agg aac agc aac agc atc gtg 1392Thr Leu Leu Ser Gly
Glu Met Ala Arg Arg Asn Ser Asn Ser Ile Val 450 455 460gtc cgc aac
att agc gac gag gaa gcc gct tta acc gct gtc gtg aac 1440Val Arg Asn
Ile Ser Asp Glu Glu Ala Ala Leu Thr Ala Val Val Asn465 470 475
480ggt ggt tcc aat aag gaa gtg ggt agc ccg gag agg gtt ttg agt ttt
1488Gly Gly Ser Asn Lys Glu Val Gly Ser Pro Glu Arg Val Leu Ser Phe
485 490 495ccg acg ata ttt gcg ttg cct caa gtt ggt ccg aag atg ttc
gga gca 1536Pro Thr Ile Phe Ala Leu Pro Gln Val Gly Pro Lys Met Phe
Gly Ala 500 505 510aat gtg gtc gga aat atg agt tct tgg act acg aac
cct aat gct gat 1584Asn Val Val Gly Asn Met Ser Ser Trp Thr Thr Asn
Pro Asn Ala Asp 515 520 525ctc aag acc gtt tct ctt act ctg ccg cag
atg ccg gtt ttc gct gcg 1632Leu Lys Thr Val Ser Leu Thr Leu Pro Gln
Met Pro Val Phe Ala Ala 530 535 540tgg gct gat tct taa ttcaatctaa
tggctaactc tggttttctt ggtttagggt 1687Trp Ala Asp Ser545ccaagtgttt
aagtttatct ccgggtttat ccggtttgaa ctacaattcg g 17385548PRTBrassica
napuscanola AINTEGUMENTA (ANT) 5Met Lys Ser Phe Cys Asp Asn Asp Asp
Ser Asn Thr Thr Asn Leu Leu1 5 10 15Gly Phe Ser Leu Ser Ser Asn Met
Leu Lys Met Gly Gly Gly Glu Ala 20 25 30Leu Tyr Ser Ser Ser Ser Ser
Ser Val Ala Thr Ser Ser Val Pro Pro 35 40 45Gln Leu Val Val Gly Asp
Asn Ser Ser Asn Tyr Gly Val Cys Tyr Gly 50 55 60Ser Asn Leu Ala Ala
Arg Glu Met Tyr Ser Gln Met Ser Val Met Pro65 70 75 80Leu Arg Ser
Asp Gly Ser Leu Cys Leu Met Glu Ala Leu Asn Arg Ser 85 90 95Ser His
Ser Asn Asn His His His Ser Gln Val Ser Ser Pro Lys Met 100 105
110Glu Asp Phe Phe Gly Thr His His His Asn Thr Ser His Lys Glu Ala
115 120 125Met Asp Leu Ser Leu Asp Ser Leu Phe Tyr Asn Thr Thr His
Ala Pro 130 135 140Asn Asn Asn Thr Asn Phe Gln Glu Phe Phe Ser Phe
Pro Gln Thr Arg145 150 155 160Asn His His Glu Glu Glu Thr Arg Asn
Tyr Glu Asn Asp Pro Gly Leu 165 170 175Thr His Gly Gly Gly Ser Phe
Asn Val Gly Val Tyr Gly Glu Phe Gln 180 185 190Gln Ser Leu Ser Leu
Ser Met Ser Pro Gly Ser Gln Ser Ser Cys Ile 195 200 205Thr Ala Ser
His His His Gln Asn Gln Thr Gln Asn His Gln Gln Ile 210 215 220Ser
Glu Ala Leu Val Glu Thr Ser Ala Gly Phe Glu Thr Thr Thr Met225 230
235 240Ala Ala Ala Ala Ala Lys Lys Lys Arg Gly Gln Glu Val Val Val
Gly 245 250 255Gln Lys Gln Ile Val His Arg Lys Ser Ile Asp Thr Phe
Gly Gln Arg 260 265 270Thr Ser Gln Tyr Arg Gly Val Thr Arg His Arg
Trp Thr Gly Arg Tyr 275 280 285Glu Ala His Leu Trp Asp Asn Ser Phe
Lys Lys Glu Gly His Ser Arg 290 295 300Lys Gly Arg Gln Val Tyr Leu
Gly Gly Tyr Asp Met Glu Glu Lys Ala305 310 315 320Ala Arg Ala Tyr
Asp Leu Ala Ala Leu Lys Tyr Trp Gly Pro Ser Thr 325 330 335His Thr
Asn Phe Ser Val Glu Asn Tyr Gln Lys Glu Ile Asp Asp Met 340 345
350Lys Asn Met Thr Arg Gln Glu Tyr Val Ala His Leu Arg Arg Lys Thr
355 360 365Ser Gly Phe Ser Arg Gly Ala Ser Ile Tyr Arg Gly Val Thr
Arg His 370 375 380His Gln His Gly Arg Trp Gln Ala Arg Ile Gly Arg
Val Ala Gly Asn385 390 395 400Lys Asp Leu Tyr Leu Gly Thr Phe Gly
Thr Gln Glu Glu Ala Ala Glu 405 410 415Ala Tyr Asp Val Ala Ala Ile
Lys Phe Arg Gly Thr Asn Ala Val Thr 420 425 430Asn Phe Asp Ile Thr
Arg Tyr Asp Val Asp Arg Ile Met Ala Ser Asn 435 440 445Thr Leu Leu
Ser Gly Glu Met Ala Arg Arg Asn Ser Asn Ser Ile Val 450 455 460Val
Arg Asn Ile Ser Asp Glu Glu Ala Ala Leu Thr Ala Val Val Asn465 470
475 480Gly Gly Ser Asn Lys Glu Val Gly Ser Pro Glu Arg Val Leu Ser
Phe 485 490 495Pro Thr Ile Phe Ala Leu Pro Gln Val Gly Pro Lys Met
Phe Gly Ala 500 505 510Asn Val Val Gly Asn Met Ser Ser Trp Thr Thr
Asn Pro Asn Ala Asp 515 520 525Leu Lys Thr Val Ser Leu Thr Leu Pro
Gln Met Pro Val Phe Ala Ala 530 535 540Trp Ala Asp
Ser545633DNAArtificial SequenceDescription of Artificial
Sequenceconsensus ANT polynucleotide sequence-1 6atgaagtctt
tttgtgataa tgatgatagt aat 33739DNAArtificial SequenceDescription of
Artificial Sequenceconsensus ANT polynucleotide sequence-2
7acgactaatt tgttagggtt ctcattgtct tcaaatatg 39838DNAArtificial
SequenceDescription of Artificial Sequenceconsensus ANT
polynucleotide sequence-3 8agaatcagcc caagcagcga aaaccggcat
ctgcggca 38
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References