U.S. patent application number 10/388269 was filed with the patent office on 2003-11-27 for plants with modified growth.
This patent application is currently assigned to Cambridge University Technical Services, Ltd.. Invention is credited to Murray, James Augustus Henry.
Application Number | 20030221221 10/388269 |
Document ID | / |
Family ID | 8229264 |
Filed Date | 2003-11-27 |
United States Patent
Application |
20030221221 |
Kind Code |
A1 |
Murray, James Augustus
Henry |
November 27, 2003 |
Plants with modified growth
Abstract
A process is provided for modifying growth or architecture of
plants by altering the level or the functional level of a cell
division controlling protein, preferably a cell-division
controlling protein that binds or phosphorylates retinoblasoma-like
proteins, more preferably a cyclin, particularly a D-type cyclin
within cells of a plant. Also provided are chimeric genes
comprising a transcribed DNA region encoding an RNA or a protein,
which when expressed either increases or decreases the level or
functional level of a cell-division controlling protein, and plant
cells and plants expressing such chimeric genes.
Inventors: |
Murray, James Augustus Henry;
(Cambridge, GB) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Cambridge University Technical
Services, Ltd.
The Old Schools Trinity Lane
Cambridge
GB
CB2 1TS
|
Family ID: |
8229264 |
Appl. No.: |
10/388269 |
Filed: |
March 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10388269 |
Mar 12, 2003 |
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09404296 |
Sep 24, 1999 |
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6559358 |
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09404296 |
Sep 24, 1999 |
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PCT/EP98/01701 |
Mar 24, 1998 |
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Current U.S.
Class: |
800/287 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8261 20130101; Y02A 40/146 20180101; C12N 15/827
20130101 |
Class at
Publication: |
800/287 |
International
Class: |
A01H 001/00; C12N
015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 1997 |
GB |
97302096.9 |
Claims
1. A process to obtain a plant which, when compared to a wild type
plant, exhibits a phenotype selected from increased growth rate,
increased number of flowers or increased seed yield per plant, said
process comprising the step of transforming cells of said plant
with a chimeric gene comprising the following operably linked DNA
fragments: a) a plant expressible promoter region; b) a transcribed
DNA region encoding a D-type cyclin, said D-type cyclin comprising
the amino acid sequence of SEQ ID No. 30; and c) a 3' end formation
and polyadenylation signal functional in plant cells.
2. The process of claim 1, wherein said transcribed DNA region
comprises a nucleotide sequence of SEQ ID No. 29 from the
nucleotide position 195 to the nucleotide position 1280.
3. The process of claim 1, wherein said plant expressible promoter
region is a CaMV35S promoter region.
4. A chimeric gene comprising the following operably linked DNA
fragments: a) a plant expressible promoter region; b) a transcribed
DNA region encoding a D-type cyclin, said D-type cyclin comprising
the amino acid sequence of SEQ ID No. 30; and c) a 3' end formation
and polyadenylation signal functional in plant cells.
5. A plant cell comprising the chimeric gene of claim 4.
6. A plant, consisting essentially of the plant cells of claim
5.
7. The plant of claim 6, which is a greenhouse-grown plant.
8. A seed of the plant of claim 7, said seed comprising the
chimeric gene of claim 4.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending application
Ser. No. 09/404,296, filed on Sep. 24, 1999 and for which priority
is claimed under 35 U.S.C. .sctn.120. application Ser. No.
09/404,296 is a continuation of PCT International Application No.
PCT/EP98/01701 filed on Mar. 24, 1998 under 35 U.S.C. .sctn.120.
The entire contents of each of the above-identified applications
are hereby incorporated by reference. This application also claims
priority of application Ser. No. 97302096.9 filed in Great Britain
on Mar. 26,1997 under 35 U.S.C. .sctn. 119.
BRIEF SUMMARY OF THE INVENTION
[0002] This invention relates to the use of cell-division
controlling proteins or parts thereof, preferably cell-division
controlling proteins that bind retinoblasoma-like proteins, more
preferably cyclins, particularly D-type cyclins and genes encoding
same, for producing plants with modified phenotypes, particularly
plants with modified growth rates or plants comprising parts with
modified growth rates and/or modified relative sizes or plants with
modified architecture. This invention also relates to plant cells
and plants expressing such DNAs.
BACKGROUND TO THE INVENTION
[0003] All eukaryotic cells undergo the same sequential series of
events when they divide, and the term "cell cycle" reflects the
ordered nature and universality of these events. In the eukaryotic
cell cycle DNA replication (S) and cell division (M) are normally
temporally separated by "gap" phases (G1 and G2) in the sequence
G1-S-G2-M. This arrangement allows entry to the critical processes
of DNA replication and mitosis to be precisely controlled.
Underlying the cytological events of the cell cycle is an ordered
series of temporally and spatially organised molecular and cellular
processes which define the direction and order of the cycle. Cell
cycle progression appears to be regulated in all eukaryotes by
major controls operating at the G1-to-S phase and G2-to-M phase
boundaries. Passage through these control points requires the
activation of cyclin-dependent kinases (CDKs), whose catalytic
activity and substrate specificity are determined by specific
regulatory subunits known as cyclins and by interactions with other
proteins that regulate the phosphorylation state of the complex
(reviewed in Atherton-Fessier et al., 1993; Solomon, 1993). In
budding and fission yeasts, both the G1-to-S and G2-to-M phase
transitions are controlled by a single CDK, encoded by the cdc2+
gene in Schizosaccharomyces pombe and by CDC28 in Saccharomyces
cerevisiae. The association of p34.sup.cdc2 (p34.sup.CDC28 in
budding yeast) with different cyclin partners distinguishes the two
control points (reviewed in Nasmyth, 1993). In mammalian cells, a
more complex situation prevails, with at least six related but
distinct CDKs (encoded by cdc2/cdk1, cdk2, cdk3, cdkain 4, cdk5,
and cdk6) having distinct roles, each in conjunction with one or
more cognate cyclin partners (Fang and Newport, 1991; Meyerson et
al., 1991, 1992; Xiong et al., 1992b; Tsai et al., 1993a; van den
Heuvel and Harlow, 1993; Meyerson and Harlow, 1994). B-type cyclins
are the major class involved in the G2-to-M transition and
associate with p34.sup.cdc2 or its direct homologs (reviewed in
Nurse, 1990). Cyclin B is one of two cyclins originally described
as accumulating in invertebrate eggs during interphase and rapidly
destroyed in mitosis (Evans et al., 1983), and it is a component of
Xenopus maturation-promoting factor (Murray et al., 1989).
Subsequently, cyclin B homologs have been identified from many
eukaryotic species. Cyclin A is also of widespread occurrence in
multicellular organisms, and its precise role is unclear, although
its peak of abundance suggests a function in S phase (reviewed in
Pines, 1993).
[0004] The G1-to-S phase transition is best understood in S.
cerevisiae. Genetic studies define a point late in G1 called START.
After passing START, cells are committed to enter S phase and to
complete a full additional round of division, which will result in
two daughter cells again in G1 phase (Hartwell, 1974; reviewed in
Nasmyth, 1993). The products of three S. cerevisiae G1 cyclin genes
called CLN1, CLN2, and CLN3 are the principal limiting components
for passage through START (Richardson et al., 1989; Wiftenberg et
al., 1990; Tyers et al., 1993). Transcription of CLN1 and CLN2 is
activated in G1, and accumulation of their protein products to a
critical threshold level by a positive feedback mechanism leads to
activation of the p34.sup.CDC28 kinase and transition through START
(Dirick and Nasmyth, 1991). The G1 cyclins are then degraded as a
consequence of PEST motifs in their primary sequence that appear to
result in rapid protein turnover (Rogers et al., 1986; Lew et al.,
1991; reviewed in Reed,1991).
[0005] The S. cerevisiae G1 cyclins are at least partially
redundant, because yeast strains in which two of the three G1
cyclin genes are deleted and the third placed under the control of
a galactose-regulated promoter show a galactose-dependent growth
phenotype. Such strains have been used to identity Drosophila and
human cDNA clones that rescue this conditional cln-deficient
phenotype on glucose plates when the single yeast CLN gene present
is repressed (Koff et al., 1991; Lahue et al., 1991; Lopold and
O'Farrell, 1991; Lew et al., 1991; Xiong et al., 1991). Human cDNAs
encoding three new classes of cyclins, C, D, and E, were identified
by this means. Although these cyclins show only limited homology
with the yeast CLN proteins, they have proved important for
understanding controls that operate in mammalian cells during G1
and at the restriction point at the G1-to-S phase boundary (Pardee,
1989; Matsushime et al., 1992; Koff et al., 1992, 1993; Ando et
al., 1993; Quelle et al., 1993; Tsai et al., 1993b). Cyclin E may
act as a rate-limiting component at the G1-to-S phase boundary
(Ohtsubo and Roberts, 1993; Wimmel et al., 1994), whereas the
dependency of cyclin D levels on serum growth factors (Matsushime
et al., 1991; Baldin et al., 1993; Sewing et al., 1993) suggests
that cyclins of the D-type may form a link between these signals
and cell cycle progression.
[0006] An important factor involved in the regulation of cell cycle
progression in mammals is the retinoblastoma susceptibility gene
encoding the retinoblastoma protein (Rb). Rb binds and inactivates
the E2F family of transcription factors, and it is through this
ability that Rb exerts most of its potential to restrain cell
division in the G1-phase. E2F transcription factors are known to
switch on cyclin E and S-phase genes and the rising levels off
cyclin E and/or E2F lead to the onset of replication (Nevins, 1992,
Johnson et al., 1993). The ability of Rb to inactivate E2F depends
on its phosphorylation state. During most of G1, Rb is in a
hypophosphorylated state, but in late G1 phase, phosphorylation of
Rb is carried out by cyclin-dependent kinases particularly CDK4
complexed to its essential regulatory subunit, cyclin D (Pines
,1995) and CDK2 complexed to cyclin E (at the G1/S boundary) or
cyclin A (in S phase). These multiple phosphorylations of Rb cause
it to release E2F, which can then, ultimately promote transcription
of the S-phase genes.
[0007] Plant cells were used in early studies of cell growth and
division to define the discrete phases of the eukaryotic cell cycle
(Howard and Pelc, 1953), but there is a paucity of data on
molecular cell cycle control in plant systems. Plant cells that
cease dividing in vivo due to dormancy, or in vitro due to nutrient
starvation, arrest at principal control points in G1 and G2 (van't
Hof and Kovacs, 1972; Gould et al., 1981; reviewed in van't Hof,
1985); this is in general agreement with the controls operating in
other eukaryotic systems. Although mature plant cells may be found
with either a G1 or a G2 DNA content (Evans and van't Hof, 1974;
Gould et al., 1981), the G1 population generally predominates. The
G1 control point is found to be more stringent in cultured plant
cells subjected to nitrogen starvation; these cells arrest
exclusively in G1 phase (Gould et al., 1981). Strong analogies thus
exist between the principal control point in G1 of the plant cell
cycle, the START control in yeasts, and the restriction point of
mammalian cells.
[0008] Antibodies or histone HI kinase assays have been used to
indicate the presence and localization of active CDC2-related
kinases in plant cells (John et al., 1989,1990, 1991; Mineyuki et
al., 1991; Chiatante et al., 1993; Colasanti et al., 1993; reviewed
in John et al., 1993), and cDNAs encoding functional homologs of
CDC2 kinase have been isolated by reduced stringency hybridization
or redundant polymerase chain reaction from a number of plant
species, including pea (Feiler and Jacobs, 1990), alfalfa (Hirt et
al., 1991, 1993), Arabidopsis (Ferreira et al., 1991; Hirayama et
al., 1991), soybean (Miao et al., 1993), Antirrhinum (Fobert et
al., 1994), and maize (Colasanti et al., 1991). A number of cDNA
sequences encoding plant mitotic cyclins with A- or B-type
characteristics or having mixed A- and B-type features have also
been isolated from various species, including carrot (Hata et al.,
1991), soybean (Hata et al., 1991), Arabidopsis (Hemerly et al.,
1992; Day and Reddy, 1994), alfalfa (Hirt et al., 1992),
Antirrhinum (Fobert et al., 1994), and maize (Renaudin et al.,
1994).
[0009] Soni et al. (1995) identified a new family of three related
cyclins in Arabidopsis by complementation of a yeast strain
deficient in G1 cyclins. Individual members of this family showed
tissue-specific expression and are conserved in other plant
species. They form a distinctive group of plant cyclins and were
named -type cyclins to indicate their similarities with mammalian
D-type cyclins. The sequence relationships between and D cyclins
include the N-terminal sequence LxCxE. The leucine is preceded at
position -1 or -2 by an amino acid with an acidic side chain (D,
E). This motif was originally identified in certain viral
oncoproteins and is strongly implicated in binding to the
retinoblastoma protein. By analogy to mammalian cyclin D, these
plant homologs may mediate growth and phytohormonal signals into
the plant cell cycle. In this respect it was shown that, on
restimulation of suspension-cultured cells, cyclin 3 was rapidly
induced by the plant growth regulator cytokinin and cyclin 2 was
induced by carbon source. Renaudin et al. (1996) defined the groups
and nomenclature of plant cyclins and -cyclins are now called CycD
cyclins.
[0010] Dahl et al. (1995) identified in alfalfa a cyclin (cycMs4)
related to 3 in alfalfa.
[0011] Recently, Rb-like proteins were identified in plant. Both
Xie et al. (1996) and Grafi et al. (1996) describe the isolation
and preliminary characterization of an Rb homologue from maize.
[0012] Doerner et al. (1996) describe the ectopic expression of a
B-type cyclin (cyc1At from Arabidopsis) under control of a promoter
from the cdc2a gene, in Arabidopsis. The "cdc2a" transgenic plants
expressing the transgene strongly had a markedly increased root
growth rate. Moreover, growth and development of lateral roots was
accelerated following induction with indoleacetic acid in the
transgenic plants relative to the control plants.
[0013] Hemerly et al. (1995) describe transgenic tobacco and
Arabidopsis plants expressing wild type or dominant mutations of a
kinase operating at mitosis (CDC2a). Plants constitutively
overproducing the wild-type CDC2a or a mutant form predicted to
accelerate the cell cycle did not exhibit a significantly altered
development. A mutant CDC2a, expected to arrest the cell cycle,
abolished cell division when expressed in Arabidopsis. Some tobacco
plants constitutively producing the latter mutant kinase, were
recovered. These plants contained considerably fewer but larger
cells.
[0014] PCT patent publication "WO" 92/09685 describes a method for
controlling plant cell growth comprising modulating the level of a
cell cycle protein in a plant for a time and under conditions
sufficient to control cell division. The preferred protein,
identified in the examples, is a p34.sup.cdc2 kinase or the like
operating at mitosis.
[0015] WO93/12239 describes plants with altered stature and other
phenotypic effects, particularly precocious flowering and increased
numbers of flowers by transformation of the plant genome with a
cdc25 gene from a yeast such as Schizosaccharomyces pombe.
[0016] WO97/47647 relates to the isolation and characterization of
a plant DNA sequence coding for a retinoblastoma protein, the use
thereof for the control of the growth in plant cells, plants and/or
plant viruses as well as the use of vectors, plants, or animals or
animal cells modified through manipulation of the control route
based on the retinoblastoma protein of plants.
[0017] US Patent "U.S." Pat. No. 5,514,571 discloses the use of
cyclin D1 as a negative regulator of mammalian cell proliferation.
Overexpression of cyclin D1 blocks mammalian cell growth, while
blocking cyclin D1 expression promotes cell proliferation.
SUMMARY OF THE INVENTION
[0018] The invention provides a process to obtain a plant with
altered growth characteristics or altered architecture,
particularly plants with reduced or increased growth rate, plants
which require less time to flower or plants with an increased
number of flowers per plant, or plant with an increased size of an
organ comprising the step of altering the level or the functional
level of a cell-division controlling protein, capable of binding
and/or phosphorylating an Rb-like protein, preferably a
cell-division controlling protein comprising an LxCxE binding motif
or related motif, preferably in the N-terminal part of the protein,
particularly a D-type cyclin, within the cells of a plant.
[0019] Also provided is a process to obtain a plant with altered
growth characteristics or altered architecture comprising the step
of altering the level or functional level of the cell-division
controlling protein by integrating a chimeric gene into the genome
of the cells of the plant, comprising the following operably linked
DNA fragments:
[0020] a) a plant expressible promoter region, particularly a
CaMV35S promoter region,
[0021] b) a transcribed DNA region encoding an RNA or a protein,
which when expressed either increases or decreases the level or the
functional level of the cell-division controlling protein; and
optionally
[0022] c) a 3' end formation and polyadenylation signal functional
in plant cells.
[0023] In accordance with the invention, the transcribed DNA region
encodes an antisense RNA, a ribozyme, or a sense RNA strand which
when expressed reduces, inhibits or prevents the expression of a
cell-division controlling protein, particularly an endogenous
D-type cyclin.
[0024] Further in accordance with the invention the transcribed DNA
encodes a cell-division controlling protein capable of binding the
pocket domain of an Rb-like protein, preferably a cell-division
controlling protein comprising an LxCxE binding motif, more
preferably a D-type cyclin, particularly a D-type cyclin from
plants, more particularly a D-type cyclin is selected from group of
Arabidopsis thaliana CYCD1, Arabidopsis thaliana CYCD2, Arabidopsis
thaliana CYCD3, Nicotiana tabacum CYCD2;1, Nicotiana tabacum
CYCD3;1, Nicotiana tabacum CYCD3;2, Helianthus tuberosus CYCD1;1,
Zea mays CYCD2 and Helianthus tuberosus CYCD3;1.
[0025] Also in accordance with the invention the transcribed RNA
encodes a protein or peptide which when expressed increases said
functional level of said cell division controlling protein,
particularly a protein or peptide selected from: a mutant D-type
cyclin, a part of a D-type cyclin, a D-type cyclin which has a
mutation in the cyclin box, a D2-type cyclin which has a
substitution of amino acid 185 or amino acid 155, a D2-type cyclin
which has mutation E185A or K155A, a D-type cyclin wherein the PEST
sequences are removed, a D-type cyclin wherein the LxCxE binding
motif has been changed or deleted, or a D-type cyclin wherein the
C-residue from the LxCxE binding motif has been deleted.
[0026] It is also an object of the invention to provide such
chimeric genes.
[0027] Further provided are plant cells, plants and seed thereof,
comprising the chimeric genes of the invention and having altered
growth characteristics and/or altered architecture.
[0028] Another object of the invention is to provide the use of a
cell-division controlling protein, capable of binding the pocket
domain of an Rb-like protein and/or capable of phosphorylating an
Rb-like protein, particularly a cell-division controlling protein
comprising an LxCxE binding motif in the N-terminal part of the
protein, more particularly a D-type cyclin and genes encoding same,
to alter the growth characteristics or architecture of a plant. The
cell-division controlling protein is preferably encoded by a
chimeric gene, integrated in the genome of the cells of a
plant.
DETAILED DESCRIPTION OF THE INVENTION
[0029] As used herein "architecture" of a plant refers to the
general morphology as defined by the relative sizes, positions and
number of the several parts of a plant (i.e. organs such as but not
limited to leaves, inflorescences, storage organs such as tubers,
roots, stems, flowers, or parts of organs such as petals, sepals,
anthers, stigma, style, petiole and the like). "Altering the
architecture of a plant" thus refers to changes in the general
morphology as the result of changing e.g. the number, size and
position of organs or parts of organs. It is clear that altering
either one organ or part of an organ or several organs or parts of
organs, as described, will result in an altered plant architecture.
This can be achieved by altering (i.e. enhancing or reducing) cell
division activity in existing meristems and/or organ primordia or
by creating de novo meristems.
[0030] As used herein, "co-suppression" refers to the process of
transcriptional and/or post-transcriptional suppression of RNA
accumulation in a sequence specific manner, resulting in the
suppresion of expression of homologous endogenous genes or
transgenes. Suppressing the expression of a endogenous gene can be
achieved by introduction of a transgene comprising a strong
promoter operably linked to a DNA region whereby the resulting
transcribed RNA is a sense RNA comprising a nucleotide sequence
which is has at least 75%, preferably at least 80%, particularly at
least 85%, more particularly at least 90%, especially at least 95%
to the coding or transcribed DNA sequence (sense) of the gene whose
expression is to be suppressed. Preferably, the transcribed DNA
region does not code for a functional protein. Particularly, the
transcribed region does not code for a protein.
[0031] As used herein, the term "plant-expressible promoter" means
a promoter which is capable of driving transcription in a plant
cell. This includes any promoter of plant origin, but also any
promoter of non-plant origin which is capable of directing
transcription in a plant cell, e.g., certain promoters of viral or
bacterial origin such as the CaMV35S or the T-DNA gene
promoters.
[0032] The term "expression of a gene" refers to the process
wherein a DNA region under control of regulatory regions,
particularly the promoter, is transcribed into an RNA which is
biologically active i.e., which is either capable of interaction
with another nucleic acid or protein or which is capable of being
translated into a biologically active polypeptide or protein. A
gene is said to encode an RNA when the end product of the
expression of the gene is biologically active RNA, such as e.g. an
antisense RNA or a ribozyme. A gene is said to encode a protein
when the end product of the expression of the gene is a
biologically active protein or polypeptide.
[0033] The term "gene" means any DNA fragment comprising a DNA
region (the "transcribed DNA region") that is transcribed into a
RNA molecule (e.g., a mRNA) in a cell under control of suitable
regulatory regions, e.g., a plant-expressible promoter. A gene may
thus comprise several operably linked DNA fragments such as a
promoter, a 5' leader sequence, a coding region, and a 3' region
comprising a polyadenylation site. An endogenous plant gene is a
gene which is naturally found in a plant species. A chimeric gene
is any gene which is not normally found in a plant species or,
alternatively, any gene in which the promoter is not associated in
nature with part or all of the transcribed DNA region or with at
least one other regulatory regions of the gene.
[0034] This invention is based on the unexpected finding that
chimeric genes comprising DNA encoding a cell-division controlling
protein capable of binding an Rb-like protein, particularly a plant
cyclin of the D-type, under control of a plant-expressible promoter
could be stably integrated in the genome of plant cells, without
deleterious effects, and furthermore that the increased expression
of such a cell-division controlling protein, particularly a cyclin
of the D-type, in the plant cells led to specific alterations in
the growth rate and architecture of the resulting transformed
plants.
[0035] Thus, the invention relates to modulating the level of
expression or activity of functional cell-division controlling
proteins, preferably in a stable manner, within plant cells of a
plant to alter the architecture or the growth rate or both of the
transformed plant and its progeny. Conveniently, the level or
functional level of cell-division controlling proteins is
controlled genetically by altering the expression of genes encoding
these cell-division controlling proteins. Increasing the level or
functional level of a cell-division controlling protein genetically
can be achieved e.g. by manipulating the copy number of the
encoding gene(s), by altering the promoter region of the encoding
genes or by manipulation of the genes regulating directly or
indirectly the level of the expression of a cell-division
controlling protein. Alternatively, the level of a cell-division
controlling protein can be increased by stabilizing the mRNA
encoding the cell-division controlling protein, or by stabilizing
the cell-division controlling protein e.g. by removal of
destruction motifs or so-called PEST sequences.
[0036] The functional level or activity of cell-division
controlling protein can be increased by the decreasing the level of
an antagonist or an inhibitor of the cell-division promoting
protein, through techniques such as, but not limited to, providing
the cell with a protein, such as an inactive cell-division
controlling protein similar to the one whose functional level is to
be increased, or part of a such a cell-division controlling
protein, which is still capable of binding an inhibitor or other
regulatory protein, or is still capable of binding to
cyclin-dependent kinases.The functional level or activity of
cell-division controlling protein can also be increased by
alteration or mutation of the cell-division controlling protein to
reduce or elimate binding of an antagonist or inhibitor of the
activity of the cell division related protein.
[0037] Reducing the functional level of a cell-division controlling
protein can be achieved e.g. by decreasing the mRNA pool encoding
the cell-division controlling protein that is available for
translation, through techniques such as, but not limited to,
antisense RNA, ribozyme action or co-suppresion. Alternatively, the
functional level of of cell-division controlling protein can be
decreased by the increasing the level of an antagonist or an
inhibitor of the cell-division promoting protein.
[0038] For the purpose of this invention, a "cell-division
controlling protein" is a polypeptide or protein which is required
for the regulation of the progression through the cell cycle of a
eukaryotic cell, preferably a plant cell, or a protein which can
effect the entry of cells into the cell cycle or affect progression
of cells through the cell cycle by direct interaction with a
protein required for the regulation of progression through the cell
cycle, or a polypeptide or protein which can assume an equivalent
function but is not required for the regulation of the cell
cycle.
[0039] Suitable cell-division controlling proteins are proteins
capable of phosphorylating either alone or in combination with
other proteins an Rb-like protein, preferably capable of
phosphorylating an Rb-like protein in a plant cell in the G1-S
transition phase, or are capable of binding the pocket domain of
retinoblastoma-like (Rb-like) proteins, preferably proteins having
an LxCxE binding motif comprised within the amino-acid sequence or
a related motif such as LxSxE or FxCxE (binding motifs are
represented in the one-letter amino acid code wherein x represents
any amino-acid). Particularly preferred are cyclins which comprise
the LxCxE binding motif (and/or related motif) in the N-terminal
half of the protein, preferably within the first 50 amino acid
residues, particularly within the first 30 amino acid residues,
such as the cyclins of the D-type, particularly plant cyclins of
the D-type, especially a cyclin from the group of Arabidopsis
thaliana CYCD1, Arabidopsis thaliana CYCD2, Arabidopsis thaliana
CYCD3, Nicotiana tabacum CYCD3;1, Nicotiana tabacum CYCD2;1,
Nicotiana tabacum CYCD3;2, Helianthus tuberosus CYCD1;1, Zea mays
CYCD2 and Helianthus tuberosus CYCD3;1 or a cyclin with essentially
similar protein sequences.
[0040] The mentioned plant cyclins of the D-type are fully
characterized by the amino acid sequence encoded by the DNA
sequence of EMBL Accession N.degree. X83369 (hereinafter, may be
referred to as SEQ ID NO: 27) from the nucleotide position 104 to
the nucleotide position 1108 for Arabidopsis thaliana CYCD1, EMBL
Accession N.degree. X83370 (hereinafter, may be referred to as SEQ
ID NO:29) from the nucleotide position 195 to the nucleotide
position 1346 for Arabidopsis thalina CYCD2, EMBL Accession
N.degree. X83371 (hereinafter, may be referred to as SEQ ID NO:31)
from the nucleotide position 266 to the nucleotide position 1396
for Arabidopsis thaliana CYCD3, the nucleotide sequence of SEQ ID
NO: 1 from nucleotide position 182 to nucleotide position 1243 for
Nicotiana tabacum CYCD2;1, the nucleotide sequence of SEQ ID NO:3
from nucleotide position 181 to nucleotide position 1299 for
Nicotiana tabacum CYCD3;1, the nucleotide sequence of SEQ ID NO:5
from nucleotide position 198 to nucleotide position 1298 for
Nicotiana tabacum CYCD3;2, the nucleotide sequence of SEQ ID NO:7
from nucleotide position 165 to nucleotide position 1109 for
Helianthus tuberosus CYCD1;1, the nucleotide sequence of SEQ ID
NO:9 from nucleotide position 48 to nucleotide position 1118 for
Helianthus tuberosus CYCD3;1 and the nucleotide sequence of SEQ ID
NO:26 from nucleotide position 316 to nucleotide position 1389 for
Zea mays CYCD2.
[0041] It is thought that increasing, respectively decreasing, the
level or the functional level or the activity of these
cell-division controlling proteins accelerates, respectively
delays, the transition of G1 to the S-phase in plant cells, or
increases, respectively decreases, the proportion of actively
dividing cells, by their interaction with Rb-like proteins
affecting the ability of the Rb-like protein to inactivate certain
transcription factors. It is further thought that expression of
these cell-division controlling proteins interacting with Rb-like
proteins effectively allows the cells to initiate division
processes, whereas (over)expression of G2/mitotic cyclins (such as
cyclins of the B-type or the cdc25 gene product) is in contrast
expected to lead to faster progression through the G2/mitotic
phases of cell cycles already started.
[0042] For the purpose of this invention "Rb-like proteins" are
defined as proteins from the group of human Rb-1 protein (Lee et
al. 1987; Accession n.degree. P06400), human p107 (Ewen et al.,
1991; Accession n.degree. L14812) and human p130 (Hannon et al.
1993; Accession A49370), Drosophila RBF (Du et al., 1996; Accession
n.degree. for DNA entry of the encoding gene X96975), mouse RB
(Bernards et al. 1989; Accession n.degree. P13405) chicken RB
(Boehmelt et al., 1994; Accession n.degree. X72218), Xenopus Rb
(Destree et al. 1992; Accession A44879), ZmRb and Rb1 from Zea mays
(Xie et al., 1996; Grafi et al. 1996; Accession numbers for DNA
entry of the encoding genes: X98923; GenBank U52099) as well as any
protein that has simultaneously at least 25-30% amino acid sequence
similarity (identity) to at least three members of the
above-mentioned group, and comprises the conserved cysteine residue
located at position 706 of human Rb-1 or at equivalent positions in
the other Rb-like proteins (see e.g. Xie et al. 1996).
[0043] Rb-like proteins are members of a small family known as
"pocket proteins". This term is derived from a conserved bipartite
domain, the so-called "pocket domain", which is the binding site
for several growth control proteins such as E2F family of
transcription factors, D-type cyclins and viral oncoproteins. The A
and B subdomains of the pocket domain are more conserved than the
rest of the protein (.about.50-64% for the A and B subdomains) and
are separated by a non-conserved spacer. Pocket domains are located
between amino acids at positions 451 and 766 for human Rb, 321 to
811 for human p107, 438 to 962 for human p130, 445 to 758 for mouse
RB, 441 to 758 for chicken RB, 440 to 767 for Xenopus Rb, 11 to 382
for corn ZmRb, 89 to 540 for corn Rb1.
[0044] For the purpose of the invention "binding to an Rb-like
protein" or "binding to the pocket domain of an Rb-like protein"
can be analyzed by either an in vitro assay or one of the in vivo
assays, or a combination thereof. In the in vitro assay, the
binding is analyzed between the protein in question which has been
labelled by .sup.35S-methionine, and a fusion protein of
glutathione-S-transferase (GST) and the pocket domain of an Rb-like
protein, such as the human Rb. The fusion to GST allows easy
purification and fixation of the fusion protein on glutathione
sepharose beads. The interaction between the assayed protein and
the Rb-like protein is compared to the binding between the same
protein and a fusion protein of GST and an Rb-like protein with a
mutation in the conserved cysteine at a position equivalent to
cysteine 706 in human Rb, such as human Rb C706F. Such an assay has
been described e.g. by Dowdy et al. (1993) and Ewen et al. (1993).
In a variant of this assay, the Rb-like protein can be expressed in
baculovirus-infected insect cells (Dowdy et al. ,1993). In a
further variant, both the Rb-like protein and Rb-binding protein
can be co-expressed in insect cells, and association detected by
gel-filtration or co-immunoprecipitation (O'Reilly et al.,
1992).
[0045] An in vivo assay which can be used to determine the binding
of a protein to the pocket domain of Rb-like proteins, is the yeast
two-hybrid system (Fields and Song, 1989). This analysis relies on
the ability to reconstitute a functional GAL4 activity from two
separated GAL4 fusion proteins containing the DNA binding domain
(GAL4.sup.BD) and the activation domain (GAL4.sup.AD) fused to a
pocket domain of an Rb-like protein and the protein to be assayed
respectively. Expression plasmids comprising chimeric genes
encoding these fusion proteins are introduced into a yeast strain
encoding appropriate GAL4 inducible markers, such as strain HF7c
(Feilloter et al., 1994) containing GAL4-inducible HIS3 and LacZ
markers, or strain Y190 (Harper et al., 1993). Proteins binding to
the pocket domain of the Rb-like protein will allow growth in the
absence of histidine. An example of a two-hybrid assay to
demonstrate interaction of a protein with an Rb-like protein has
been described by Durfee et al. (1993).
[0046] Preferably, suitable control experiments should be included,
such as separate introduction into the same yeast strain of the
expression plasmids, or introduction of expression plasmids
encoding fusion proteins containing the DNA binding domain
(GAL4.sup.BD) and the activation domain (GAL4.sup.AD) fused to a
mutated pocket domain of an Rb-like protein, preferably mutated at
the C706 or equivalent positions and the protein to be assayed
respectively.
[0047] An alternative in vitro assay to determine the binding of a
protein to the pocket domain of Rb-like proteins comprises
transient expression of both proteins in plant cells, preferably
tobacco protoplasts, and immunoprecipitation using an antibody
directed against one of the two proteins to measure
co-precipitation of the other protein.
[0048] For the purpose of the invention "phosphorylating an Rb-like
protein" can be analyzed by an in vitro assay relying on the use of
gamma .sup.32P-labeled adenosine-triphosphate to monitor the
capacity of a protein (or a combination of proteins such as cyclins
and cyclin dependent kinases) to transfer the labeled phosphate
group to a target protein, as known in the art.
[0049] For the purpose of the invention "cyclin" can be defined as
a regulatory protein, comprising a protein domain of about 100
amino acids known as the "cyclin box". The cyclin box is the
binding site for cyclin-dependent kinases, allowing the cyclin to
exert its regulatory effect on the kinase activity of the CDKs.
[0050] A cyclin box can be identified by comparing the amino acid
sequence of the protein with known cyclin boxes, such as the amino
acid sequence between positions 81-186 of CYCD1 from Arabidopsis
thaliana, between positions 96-201 of CYCD2 from Arabidopsis
thaliana, between positions 86-191 of CYCD3 from Arabidopsis
thaliana, the cyclin boxes described by Renaudin et al. (1994;
1996), by Soni et al. (1995), and by Hemerly et al. (1992). An
amino acid sequence identified as a cyclin box on the basis of
sequence comparison should posses at least the five conserved
residues required for cyclin activity R(97), D(126), L (144),
K(155), E(185) (indicated positions are from the sequence of CYCD2
from Arabidopsis thaliana) at equivalent positions. (see e.g. Soni
et al. (1995) and Renaudin et al. (1996).
[0051] D-type cyclins (cyclin D or CycD) are cyclins that are
characterized by the presence of additional characteristic
sequences, such as the LxCxE motif or related motifs for binding
Rb-like proteins, which is found within the N-terminal part of the
protein, preferably located between the N-terminus and the cyclin
box, particularly within the first 50 amino acids, more
particularly within the first 30 amino acids of the initiating
methionine-residue. Preferably, the leucine of the binding motif is
preceded at position -1 or -2 by an amino acid with an acidic side
chain (D, E). Alternative binding motifs such as LxSxE or FxCxE can
be found. Indeed, Phelps et al. (1992) have identified that
mutating the binding motif LxCxE in human papillomavirus E7 to
LxSxE does not affect the ability of the protein to bind Rb-like
proteins. Three groups of D-type cyclins have been identified on
the basis of sequence homology: CycD1 (comprising Arabidopsis
thaliana CycD1 and Helianthus tuberosus CYCD1;1) CycD2 (comprising
Arabidopsis thaliana CYCD2, Nicotiana tabacum CYCD2;1, Zea mays
CYCD2), CycD3 (comprising Arabidopsis thaliana CYCD3, Nicotiana
tabacum CYCD3;1,, Nicotiana tabacum CYCD3;2, and Helianthus
tuberosus CYCD3;1).
[0052] Nomenclature and consensus sequences for the different types
and groups of plant cyclins, including cyclins of the D-type, have
been described by Renaudin et al. (1996) and can be used to
classify new cyclins based on their amino acid sequence.
[0053] For the purpose of the invention, the cell-division
controlling proteins can be provided to the cells either directly,
e.g. by electroporation of the protoplasts in the presence of the
cell-division controlling proteins, or indirectly, by transforming
the plant cells with a plant-expressible chimeric gene encoding the
protein to be tested either transiently, or stably integrated in
the genome of the protoplasts.
[0054] In one aspect of the invention the level or the functional
level of the cell-division controlling protein, capable of
phosphorylating an RB-like protein or binding the pocket domain of
an Rb-like proteins, is increased, to obtain a plant with altered
growth rate or architecture, by integrating a chimeric gene into
the genome of the cells of the plant, comprising the following
operably linked DNA fragments:
[0055] a) a plant-expressible promoter region, particularly a
CaMV35S promoter region,
[0056] b) a transcribed DNA region encoding a protein, which when
expressed increases the level or the functional level of the
cell-division controlling protein; and optionally
[0057] c) a 3' end formation and polyadenylation signal functional
in plant cells.
[0058] In a preferred embodiment of the invention, the expression
level of cyclin D is increased by introduction into the genome of a
plant cell, a chimeric gene comprising a transcribed DNA region
encoding a cyclin D, under control of a plant-expressible promoter.
The transcribed DNA region preferably comprises a nucleotide
sequence selected from the nucleotide sequence of EMBL Accession
N.degree. X83369 (SEQ ID NO:27) from the nucleotide position 104 to
the nucleotide position 1108, the nucleotide sequence of EMBL
Accession N.degree. X83370 (SEQ ID NO:29) from the nucleotide
position 195 to the nucleotide position 1346, the nucleotide
sequence of EMBL Accession N.degree. X83371 (SEQ ID NO:31) from the
nucleotide position 266 to the nucleotide position 1396, the
nucleotide sequence of SEQ ID NO:1 from nucleotide position 182 to
nucleotide position 1243, the nucleotide sequence of SEQ ID NO:3
from nucleotide position 181 to nucleotide position 1299, the
nucleotide sequence of SEQ ID NO:5 from nucleotide position 198 to
nucleotide position 1298, the nucleotide sequence of SEQ ID NO:7
from nucleotide position 165 to nucleotide position 1109, the
nucleotide sequence of SEQ ID NO:9 from nucleotide position 48 to
nucleotide position 1118 or the nucleotide sequence of SEQ ID NO:26
from nucleotide position 316 to nucleotide position 1389 for Zea
mays CYCD2.
[0059] In a particularly preferred embodiment the expression level
of a cyclin of the CycD2 type is altered (i.e. increased) by
introduction into the genome of a plant cell, of a "chimeric cycD2
gene" comprising a transcribed DNA region encoding a cyclin of the
CycD2 type, under control of a plant-expressible promoter,
preferably a constitutive promoter, particularly a CaMV35S
promoter, such as the chimeric cycD2 gene of plasmid pCEC1, in
order to alter the morphology, architecture and growth
characteristics of the transgenic plant, particularly to increase
the vegetative growth of the transgenic plant, more particularly to
alter the growth rate of the transgenic plant.
[0060] For the purpose of the invention, "increase" or "decrease"
of a measurable phenotypic trait is quantified as the difference
between the mean of the measurements pertinent to the description
of that trait in different plants of one transgenic plant line, and
the mean of the measurements of that trait in wild type plants,
divided by the mean of the measurements of that trait in wild type
plants, expressed in percentage, whereby transgenic and control
(wild type) plants are grown under the same conditions of nutrient
supply, light, moisture, temperature and the like, preferably under
standardized conditions. Prefered levels of increase or decrease
are statistically significant, preferably at the 0.05 confidence
level, particularly at the 0.01 confidence level, e.g. by one way
variance analysis (e.g. as described in Statistical Methods by
Snedecor and Cochran).
[0061] Increase of the vegetative growth of a transgenic plant is
preferably monitored by measuring the increase in dry weight during
the growth period. The mean increase of dry weight is defined as
the difference in mean dry weight of transgenic plants and wild
type plants multiplied by 100 and divided by the mean dry weight of
wild type plants. Typical increases in dry weight, particularly
early in growth period, by introduction of the chimeric cycD2 genes
of the invention range from at least about 39% to about 350%,
particularly from about 68% to about 150%.
[0062] It is clear that increases in dry weight resulting from
introduction of the chimeric genes of the invention may vary,
depending on the plant species or chimeric genes used, and any
significant increase in dry weight in transgenic plants is
encompassed by the invention, particularly a dry weight of at least
about 1.4 times to at least about 4.5 times the dry weight in
untransformed control plants, particularly of at least about 1.8
times to at least about 2.7 times the dry weight in untransformed
control plants. In any case, the mean dry weight of the transgenic
plants is statistically significantly different from the mean dry
weight of the untransformed plants.
[0063] Increase in the vegetative growth of a transgenic plant can
also be determined by comparing the number of leaves visible on the
transgenic plants and the control wild-type plants at any given
point in time. The difference in number of leaves of transgenic
plants in the middle of the growth period is expected to be at
least about 1.1 to at least about 3 times, particularly at least
about 1.5 to at least about 2 times the leaf number in
untransformed plants.
[0064] Increase of the vegetative growth of a transgenic plant can
also be monitored by measuring the height of the stem (measured
from soil level to the top of growing point) during the growth
period. The mean increase of the stem height is defined as the
difference in mean stem height of transgenic plants and wild type
plants multiplied by 100 and divided by the mean height of wild
type plants. Typical increases in stem height by introduction of
the chimeric cycD2 genes of the invention range from at least about
65% early during growth, over at least about 20-30% in the middle
of the growing period, to at least about 10% by the time of
flowering, but may be as high as about 120% to about 190% early
during growth, as high as about 40-50% to about 75% in the middle
of the growing period, and as high as about 15-20% at the end of
the flowering stage.
[0065] It is clear that increases in stem height resulting from
introduction of the chimeric genes of the invention may vary,
depending on the plant species or chimeric genes used, and any
significant increase in stem height in transgenic plants is
encompassed by the invention, particularly stem height of at least
about 1.1 times to at least about 3 times the stem height in
untransformed control plants, particularly of at least about 1.5
times to at least about 2 times the stem height in untransformed
control plants.
[0066] The difference in stem height between transgenic and control
plants diminishes as growth progresses, because the growth rate
slows down in plants that are flowering. The terminal height of a
transgenic plant, may thus be similar to the terminal height of a
non-transgenic plant.
[0067] The transgenic plants comprising the chimeric cycD2 genes of
the invention have an increased growth rate, when compared with
untransformed plants, resulting in a reduced time required to reach
a given dry weight or stem height. "Growth rate" as used herein,
refers to the increase in size of a plant or part of plant per day,
particularly to increase in stem height per day, and can be
calculated as the difference between the size of a plant or part of
a plant at the start and end of a period comprising a number of
days, particularly 6 to 8 days, divided by the number of days.
Increase in growth rate is preferably expressed according to the
general definition of increase of a measurable phenotype, but can
also be expressed as the ratio between the growth rate of the
transgenic plants, versus the growth rate of the untransformed
control plants, during the same period, under the same
conditions.
[0068] As mentioned before the increase in growth rate resulting
from introduction of the chimeric genes, particularly the chimeric
cycD2 genes of the invention may vary, depending on the plant
species or the chimeric genes used, and any significant increase in
growth rate in transgenic plants is encompassed by the invention,
particularly increase in growth rate ranging from about 4% to about
85%, more particularly from about 20% to about 60%, especially from
about 30% to about 50%.
[0069] Increase of the vegetative growth of a transgenic plant can
also be monitored by measuring the length or the size of the
largest leaf at different time points during the growth period
whilst the leaves are still expanding. This measurable phenotype is
a measure of the increased maturity of the transgenic plants. The
mean increase of the length of the largest leaf (defined as the
difference between mean length of the largest leaf of transgenic
plants and wild type plants multiplied by 100 and divided by the
mean length of the largest leaf of wild type plants) obtained by
introduction of the chimeric genes of the invention ranges from
about 7 to 31% (mean about 17%) early during growth, to about 3-14%
(mean about 7%) in the middle of the growing period. Again, these
increases in the size of the largest leaf, resulting from
introduction of the chimeric genes of the invention, may vary,
depending on the plant species or chimeric gene used, and any
significant increase in leaf growth or size in transgenic plants is
encompassed by the invention.
[0070] As another object of the invention, the chimeric
cell-division controlling gene, particularly the chimeric cycD2
genes, can also be introduced in plants to increase the root
development, particularly to increase the mean root length. In
general, the increase in root development, is parallel to the
increase in the vegetative part above the ground (stem, leaves,
flowers) and may range from about 40% to about 70%, but again these
increases may vary depending on the plant species or chimeric gene
used, and any significant increase, particularly statistically
significant increase in root development is encompassed by the
invention.
[0071] As yet another object of the invention, the chimeric
cell-division controlling gene, particularly the chimeric cycD2
genes, can also be introduced in plants to increase the size as
well as the number of flowers, particularly the number of
fertilised flowers, and the number of fertilised ovules in each
flower . As a result of the increase in the number of fertilized
flowers, and the number of fertilised ovules in each flower
(generally leading to a greater number of seeds per plant), it is
clear that also an increase in seed yield per plant can be
obtained. It is clear that the increase in the number of flowers
and ovules per flower, as well as the increase in seed yield can
vary, depending on the plant species transformed with the chimeric
cell-division controlling genes of the invention or the chimeric
genes used. Typical increases in flower size resulting from the
introduction of a chimeric gene comprising a CycD2 encoding DNA
region under control of a CaMV35S promoter range from at least
about 4% to at least about 30%, particularly at least about 10% to
at least about 20%. Typical increases in the number of flowers
range from about at least 20% to at least about 50%, particularly
from about 24% to about 45% while increases in the number of
seeds/plants (expressed on a weight basis) are in a range from at
least about 5% to at least about 55%, particularly from at least
about 10% to at least about 30%, more particularly about 25%.
[0072] In still another embodiment of the invention, the chimeric
cell-division controlling gene, particularly the chimeric cycD2
genes, can also be introduced in plants or their seeds to
accelerate germination. It has been found that transgenic seeds
comprising the chimeric cycD2 genes of the invention can germinate
at least between about 8 to about 16 hrs faster than wild type
controls.
[0073] Moreover, the mentioned chimeric genes can also be
introduced in plants to decrease the mean number of days required
to reach the development of an inflorescence, thus effectively
reducing the time required to start flowering. Transgenic plants
comprising the chimeric cycD2 genes of the invention thus reach
maturity, particularly the flowering stage, earlier, but have the
normal size of a flowering plant. The actual reduction in time
required to reach the flowering stage may depend on the plant
species or chimeric genes used. Typically, transgenic plants
harboring the chimeric gene comprising a CycD2 encoding DNA region
under control of a CaMV35S promoter exhibit a reduction in the time
required to flower of at least about 3% to 11-12%, particularly at
least about 4% to 7%.
[0074] In another particularly preferred embodiment, a chimeric
gene comprising a CycD3 encoding transcribed DNA region under
control of a plant-expressible promoter, preferably a constitutive
promoter, particularly a CaMV35S promoter, such as a chimeric gene
comprising the nucleotide sequence of the chimeric cycD3 gene of
pCRK9, is introduced into a plant cell to obtain transgenic plants
with altered morphological traits or architecture, particularly
with altered size of specific plant parts or organs, more
particularly with altered flower size and morphology such as
flowers with elongated and/or enlarged petals. Transgenic plants
transformed with a chimeric gene comprising a CycD3 encoding DNA
region under control of a plant-expressible promoter (and the
progeny thereof) exhibit an increase in the flower size of about
31% to about 44%. Moreover these transgenic plants also flower
later than wild type plants, corresponding to an increase in
flowering time of about 5% to about 20%, particularly about 8% to
about 16%.
[0075] In another embodiment of the invention the functional level
of the cell-division controlling protein, capable of
phosphorylating an RB-like protein or binding the pocket domain of
an Rb-like proteins, particularly of the D-type cyclin is
increased, to obtain a plant with altered growth rate or
architecture, by integrating a chimeric gene into the genome of the
cells of the plant, comprising the following operably linked DNA
fragments:
[0076] a) a plant-expressible promoter region, particularly a
CaMV35S promoter region,
[0077] b) a transcribed DNA region encoding a protein, which when
expressed increases the functional level of a cell-division
controlling protein, preferably encoding a mutant cell-division
controlling protein or part of a mutant cell-division controlling
protein, more preferably encoding a mutant D-type cyclin or part of
a D-type cyclin, particularly encoding a D-type cyclin which has a
mutation in cyclin box (quite particularly a substitution of amino
acid 185 or amino acid 155 of a D2-type cyclin, especially E185A or
K155A), or a D-type cyclin wherein the PEST sequences are removed,
particularly which has been C-terminally deleted to remove the PEST
sequences, or a D-type cyclin wherein the LxCxE binding motif has
been changed or deleted, particularly wherein the C-residue from
the LxCxE binding motif has been deleted; and optionally
[0078] c) a 3' end formation and polyadenylation signal functional
in plant cells.
[0079] Although not intending to limit the invention to a mode of
action, it is thought that the mutant cell-division controlling
proteins exert their effects by sequestering of inhibitors or
antagonist of the normal functional cell-division controlling
proteins.
[0080] It is clear from this description that chimeric genes
comprising a transcribed DNA region encoding other cyclins of the
D-type, particularly plant-derived cyclins of the CycD group, may
be used to obtain similar effects. These genes can be obtained from
other plant species or varieties, by different methods including
hybridization using the available CycD1, CycD2 or CycD3 encoding
DNAs as probes and hybridization conditions with reduced
stringency, or polymerase chain reaction based methods using
oligonucleotides based on the available nucleotide sequences of
D-type cyclins, preferably oligonucleotides having a nucleotide
sequence corresponding to the sequences encoding the consensus
amino acid sequences, particularly oligonucleotides having a
nucleotide sequence corresponding to the sequences encoding
conserved amino acid sequences within the cyclin box for each group
of cyclins. These conserved amino acid sequences can be deduced
from available aligned DNA encoding such amino acid sequences. A
particularly preferred combination of oligonucleotides for PCR
amplification of plant cyclins of the D1 type is an oligonucleotide
selected from the group of oligonucleotides having the DNA sequence
of SEQ ID N.degree. 12, SEQ ID N.degree. 13 or SEQ ID N.degree. 14
and an oligonucleotide selected from the group of oligonucleotides
having the DNA sequence of SEQ ID N.degree. 15 or SEQ ID N.degree.
16.
[0081] A particularly preferred combination of oligonucleotides for
PCR amplification of plant cyclins of the D2 type is an
oligonucleotide selected from the group of oligonucleotides having
the DNA sequence of SEQ ID N.degree. 17 or SEQ ID N.degree. 18 and
an oligonucleotide selected from the group of oligonucleotides
having the DNA sequence of SEQ ID N.degree. 19 or SEQ ID N.degree.
20. A particularly preferred combination of oligonucleotides for
PCR amplification of plant cyclins of the D3 type is an
oligonucleotide selected from the group of oligonucleotides having
the DNA sequence of SEQ ID N.degree. 21, SEQ ID N.degree. 22 or SEQ
ID N.degree. 23 and an oligonucleotide selected from the group of
oligonucleotides having the DNA sequence of SEQ ID N.degree. 24 or
SEQ ID N.degree. 25. The amplified DNA fragment is then used to
screen a cDNA or genomic library (under stringent conditions) to
isolate full length clones.
[0082] Alternatively, additional genes encoding plant-derived
cyclins can be obtained by techniques such as, but not limited to,
functional complementation of conditional G1-S cyclin deficient
yeast strains, as described by Soni et al. (1995) and Dahl et al.
(1995) or by using the yeast two-hybrid system (Fields and Song,
1989) to isolate DNA sequences encoding cyclins binding to the
pocket domain of Rb-like proteins as described supra.
[0083] It is further known that some plants contain more than one
gene encoding a D-type cyclin of the same subgroup (e.g. tobacco
contains at least two genes of the CycD3 subgroup) and it is clear
that these variants can be used within the scope of the
invention.
[0084] Moreover D-type cyclins which have an amino acid sequence
which is essentially similar to the ones disclosed in this
invention, such as mutant D-type cyclins, can be used to the same
effect. With regard to "amino acid sequences", essentially similar
means that when the two relevant sequences are aligned, the percent
sequence identity--i.e., the number of positions with identical
amino acid residues divided by the number of residues in the
shorter of the two sequences--is higher than 80%, preferably higher
than 90%. The alignment of the two amino acid sequences is
performed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann
1983) using a window-size of 20 amino acids, a word length of 2
amino acids, and a gap penalty of 4. Computer-assisted analysis and
interpretation of sequence data, including sequence alignment as
described above, can be conveniently performed using the programs
of the Intelligenetics.RTM. Suite (Intelligenetics Inc.,
Calif.).
[0085] It is clear that any DNA sequence encoding a cell-division
controlling protein, particularly a D-type cyclin, can be used to
construct the chimeric cell-division controlling genes of the
invention, especially DNA sequences which are partly or completely
synthesized by man.
[0086] It is also clear that other plant-expressible promoters,
particularly constitutive promoters, such as the the opine synthase
promoters of the Agrobacterium Ti- or Ri-plasmids, particularly a
nopaline synthase promoter can be used to obtain similar effects.
Moreover, in the light of the existence of variant forms of the
CaMV35S promoter, as known by the skilled artisan, the object of
the invention can be equally be achieved by employing these
alternative CaMV35S promoters.
[0087] It is a further object of the invention to provide plants
with altered morphology or architecture, restricted to specific
organs or tissues by using tissue-specific or organ-specific
promoters to control the expression of the DNA encoding a
cell-division controlling protein, particularly a cyclin of the
D-type. Such tissue-specific or organ-specific promoters are well
known in the art and include but are not limited to seed-specific
promoters (e.g. WO89/03887), organ-primordia specific promoters (An
et al., 1996), stem-specific promoters (Keller et al., 1988), leaf
specific promoters (Hudspeth et al., 1989), mesophyl-specific
promoters (such as the light-inducible Rubisco promoters),
root-specific promoters (Keller et al., 1989), tuber-specific
promoters (Keil et al., 1989), vascular tissue specific promoters
(Peleman et al,. 1989), meristem specific promoters (such as the
promoter of the SHOOTMERISTEMLESS (STM) gene, Long et al., 1996),
primordia specific promoter (such as the promoter of the
Antirrhinum CycD3a gene, Doonan et al. 1998) and the like.
[0088] In another embodiment of the invention, the expression of a
chimeric gene encoding a cell-division controlling protein can be
controlled at will by the application of an appropriate chemical
inducer, by operably linking the DNA region coding for the
cell-division controlling protein to a promoter whose expression is
induced by a chemical compound, such as the promoter of the gene
disclosed in European Patent publication "EP" 0332104, or the
promoter of the gene disclosed in WO 90/08826.
[0089] In yet another embodiment of the invention, the expression
of a chimeric gene encoding a cell-division controlling protein can
be controlled by use of site-specific recombinases and their
corresponding cis-acting sequences, e.g. by inserting between the
plant-expressible promoter and the transcribed region encoding the
cell-division controlling protein, a unrelated nucleotide sequence
(preferably with transcriptional and/or translational termination
signals) flanked by the cis-acting sequences recognized by a
site-specific recombinase (e.g. lox or FRT sites); providing the
plant cells comprising this chimeric gene with the site-specific
recombinase (e.g. Cre or FLP) so that the inserted unrelated
nucleotide sequence is eliminated by recombination, thus allowing
the chimeric cell division controlling gene to be expressed.
[0090] It is thought that the morphological alterations obtained by
increased expression of cell-division controlling proteins,
particularly D-type cyclins in plants due to the introduction of a
chimeric gene comprising a DNA region encoding a cell-division
controlling protein, particularly a D-type cyclin under control of
a plant expressible promoter, can be enhanced, by removal,
adaptation or inactivation of PEST sequences. PEST sequences are
amino acid sequences which are rich in proline, glutamate or
aspartate and serine or threonine, located between positively
charged flanking residues, which are involved in rapid turnover of
the protein comprising such sequences (Tyers et al., 1992; Cross,
1988; Wittenberg and Reed, 1988; Salama et al., 1994). Removal of
these PEST sequences in yeast cyclins stabilizes the cyclins in
vivo (Pines, 1995). PEST regions can be identified by computer
analysis, using software packages such as PESTFIND (Rogers et al.,
1986; Rechsteiner, 1990). Mutation of a DNA encoding cell-division
controlling protein with altered PEST sequences is well within the
reach of the skilled artisan using methods such as described e.g.
by Sambrook et al. (1989)
[0091] It is further expected that the quantitative effects of
phenotypic alterations can be modulated--ie enhanced or
repressed--by expression of endogenous cell-division controlling
encoding chimeric genes, particularly endogenous CycD encoding
chimeric genes as an alternative to using heterologous genes
encoding similar proteins from other plants. Preferably,
heterologous genes are used, particularly heterologous genes
encoding similar proteins with less than about 65%, preferably less
than about 75%, more preferably less than about 65% amino acid
sequence identity to the endogenous cell division controlling
protein.
[0092] In another aspect of this invention, the morphology of
plants can be altered by decreasing expression of a functional
cell-division controlling protein, particularly a D-type cyclin.
This can be achieved using e.g. antisense-RNA, ribozyme, or
co-suppresion techniques. To this end, a chimeric gene comprising a
transcribed DNA region which is transcribed into an RNA, the
production of which reduces, inhibits or prevents the expression of
a cell-division controlling protein, particularly a D-type cyclin
within the plant cells is introduced in the plant cells,
particularly stably integrated in the genome of the plant
cells.
[0093] In one embodiment of this aspect, the transcribed DNA region
of the chimeric gene encodes an antisense RNA which is
complementary to at least part of a sense mRNA encoding a
cell-division controlling protein, particularly a D-type cyclin.
The antisense RNA thus comprises a region which is complementary to
a part of the sense mRNA preferably to a continuous stretch thereof
of at least 50 bases in length, particularly of at least between
100 and 1000 bases in length. The antisense RNA can be
complementary to any part of the mRNA sequence: it may be
complementary to the sequence proximal to the 5' end or capping
site, to part or all of the leader region, to an intron or exon
region (or to a region bridging an exon and intron) of the sense
pre-mRNA, to the region bridging the noncoding and coding region,
to all or part of the coding region including the 3' end of the
coding region, and/or to all or part of the 3' or trailer region.
The sequence similarity between the antisense RNA and the
complement of the sense RNA encoding a cell-division controlling
protein, should be in the range of at least about 75% to about
100%.
[0094] In another embodiment of this aspect, the transcribed DNA
region of the chimeric gene encodes a specific RNA enzyme or
so-called ribozyme (see e.g. WO 89/05852) capable of highly
specific cleavage of the sense mRNA encoding a cell-division
controlling protein, particularly a D-type cyclin.
[0095] In yet another embodiment, the level of a functional
cell-division controlling protein, particularly a D-type cyclin can
be decreased by the expression of chimeric gene comprising a DNA
region encoding a protein or polypeptide which when expressed
reduces the level of a cell-division controlling protein,
particularly a D-type cyclin, or inhibits the cell division
controlling protein, particularly the D-type cyclin, to exert its
function within the plant cells. Preferably, the chimeric gene
encodes an antibody that binds to a cell-division controlling
protein, particularly a D-type cyclin.
[0096] Decreasing the level or the functional level of a
cell-division controlling protein, particularly a D-type cyclin
within the cells of a transgenic plant, comprising the chimeric
genes of this embodiment of the invention, results in altered
architecture, particularly in a decreased stem height, a decrease
of the growth rate or a delaying in the flowering of the transgenic
plants when compared to untransformed plants, grown under the same
conditions. The effect obtained might vary, depending on the plant
species or chimeric genes used, and any effect on architecture
and/or growth rate, particularly a decrease in stem height or
growth rate, or an increase in the time required to develop an
inflorescence, is encompassed by the invention.
[0097] The decrease in growth rate due to decreasing the level of a
cell-division controlling protein, preferably a D-type cyclin,
particularly a CYCD2 type cyclin, ranges from about 30% to about
60%, particularly from about 35% to about 50%.
[0098] The-decrease in stem height due to decreasing the level of a
cell-division controlling protein, preferably a D-type cyclin,
particularly a CYCD2 type cyclin, ranges from about 10% to about
60%, particularly from about 30% to about 50%, more particularly
around 40%.
[0099] The increase in flowering time due to decreasing the level
of a cell-division controlling protein, preferably a D-type cyclin,
particularly a D2 type cyclin, ranges from about 10% to about 40%,
particularly from about 15% to about 38%.
[0100] The chimeric cell-division controlling gene may include
further regulatory or other sequences, such as leader sequences
[e.g. cab22L leader from Petunia or the omega leader from TMV
(Gallie et al., 1987)], 3' transcription termination and
polyadenylation signals (e.g. of the octopine synthase gene [De
Greve et al., 1982)], of the nopaline synthase gene [Depicker et
al., 1982] or of the T-DNA gene 7 [Velten and Schell, 1985] and the
like [Guerineau et al., 1991; Proudfoot, 1991; Safacon et al.,
1991; Mogen et al., 1990; Munroe et al., 1990; Ballas et al., 1989;
Joshi et al., 1987], plant translation initiation consensus
sequences [Joshi, 1987], introns [Luehrsen and Walbot, 1991] and
the like, operably linked to the nucleotide sequence of the
chimeric cell-division controlling gene.
[0101] Preferably, the recombinant DNA comprising the chimeric
cell-division controlling gene is accompanied by a chimeric marker
gene. The chimeric marker gene can comprise a marker DNA that is
operably linked at its 5' end to a plant-expressible promoter,
preferably a constitutive promoter, such as the CaMV 35S promoter,
or a light inducible promoter such as the promoter of the gene
encoding the small subunit of Rubisco; and operably linked at its
3' end to suitable plant transcription 3' end formation and
polyadenylation signals. It is expected that the choice of the
marker DNA is not critical, and any suitable marker DNA can be
used. For example, a marker DNA can encode a protein that provides
a distinguishable color to the transformed plant cell, such as the
A1 gene (Meyer et al., 1987), can provide herbicide resistance to
the transformed plant cell, such as the bar gene, encoding
resistance to phosphinothricin (EP 0,242,246), or can provided
antibiotic resistance to the transformed cells, such as the aac(6')
gene, encoding resistance to gentamycin (WO94/01560).
[0102] Although it is clear that the invention can be applied
essentially to all plant species and varieties, the invention will
be especially suited to alter the architecture or to increase the
growth rate of plants with a commercial value. It is expected that
the enhancements in vegetative growth will be most pronounced in
plants which have not undergone extensive breeding and selection
for fast vegetative growth. The invention will be particularly
relevant for plants which are grown in greenhouses, particularly to
reduce the time required for greenhouse plants to reach the desired
developmental stage, such as but not limited to flowering, fruit
setting or seed setting. The invention will further be relevant to
enhance the growth rate of trees, particularly softwood trees such
as pine, poplar, Eucalyptus trees and the like. Another important
application of the invention encompasses the expansion of effective
area wherein plants can be cultivated by reduction of the time
required to reach the economically important developmental stage.
Particularly preferred plants to which the invention can be applied
are corn, oil seed rape, linseed, wheat, grasses, alfalfa, legumes,
a brassica vegetable, tomato, lettuce, rice, barley, potato,
tobacco, sugar beet, sunflower, and ornamental plants such as
carnation, chrysanthemum, roses, tulips.
[0103] A recombinant DNA comprising a chimeric cell-division
controlling gene can be stably incorporated in the nuclear genome
of a cell of a plant. Gene transfer can be carried out with a
vector that is a disarmed Ti-plasmid, comprising a chimeric gene of
the invention, and carried by Agrobacterium. This transformation
can be carried out using the procedures described, for example, in
EP 0,116,718.
[0104] Alternatively, any type of vector can be used to transform
the plant cell, applying methods such as direct gene transfer (as
described, for example, in EP 0,233,247), pollen-mediated
transformation (as described, for example, in EP 0,270,356,
WO85/01856 and U.S. Pat. No. 4,684,611), plant RNA virus-mediated
transformation (as described, for example, in EP 0,067,553 and U.S.
Pat. No. 4,407,956), liposome-mediated transformation (as
described, for example, in U.S. Pat. No. 4,536,475), and the
like.
[0105] Other methods, such as microprojectile bombardment as
described, for corn by Fromm et al. (1990) and Gordon-Kamm et al.
(1990), are suitable as well. Cells of monocotyledonous plants,
such as the major cereals, can also be transformed using wounded
and/or enzyme-degraded compact embryogenic tissue capable of
forming compact embryogenic callus, or wounded and/or degraded
immature embryos as described in WO92/09696. The resulting
transformed plant cell can then be used to regenerate a transformed
plant in a conventional manner.
[0106] The obtained transformed plant can be used in a conventional
breeding scheme to produce more transformed plants with the same
characteristics or to introduce the chimeric cell-division
controlling gene of the invention in other varieties of the same or
related plant species. Seeds obtained from the transformed plants
contain the chimeric cell-division controlling gene of the
invention as a stable genomic insert.
[0107] The following non-limiting Examples describe the
construction of chimeric cell-division controlling genes and the
use of such genes for the modification of the architecture and
growth rate of plants. Unless stated otherwise in the Examples, all
recombinant DNA techniques are carried out according to standard
protocols as described in Sambrook et al. (1989) Molecular Cloning:
A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current
Protocols in Molecular Biology, Current Protocols, USA. Standard
materials and methods for plant molecular work are described in
Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly
published by BIOS Scientific Publications Ltd (UK) and Blackwell
Scientific Publications, UK.
[0108] Throughout the description and Examples, reference is made
to the following sequences:
[0109] SEQ ID N.degree. 1: cDNA encoding Nicotiana tabacum
CYCD2;1
[0110] SEQ ID N.degree. 3: cDNA encoding Nicotiana tabacum
CYCD3;1
[0111] SEQ ID N.degree. 5: cDNA encoding Nicotiana tabacum
CYCD3;2
[0112] SEQ ID N.degree. 7: cDNA encoding Helianthus tuberosus
CYCD1;1
[0113] SEQ ID N.degree. 9: cDNA encoding Helianthus tuberosus
CYCD3;1
[0114] SEQ ID N.degree. 11: T-DNA of pGSV5
[0115] SEQ ID N.degree. 12: PCR primer 1
[0116] SEQ ID N.degree. 13: PCR primer2
[0117] SEQ ID N.degree. 14: PCR primer 3
[0118] SEQ ID N.degree. 15: PCR primer 4
[0119] SEQ ID N.degree. 16: PCR primer 5
[0120] SEQ ID N.degree. 17: PCR primer 6
[0121] SEQ ID N.degree. 18: PCR primer 7
[0122] SEQ ID N.degree. 19: PCR primer 8
[0123] SEQ ID N.degree. 20: PCR primer 9
[0124] SEQ ID N.degree. 21: PCR primer 10
[0125] SEQ ID N.degree. 22: PCR primer 11
[0126] SEQ ID N.degree. 23: PCR primer 12
[0127] SEQ ID N.degree. 24: PCR primer 13
[0128] SEQ ID N.degree. 25: PCR primer 14
[0129] SEQ ID N.degree. 26: cDNA encoding Zea mays CYCD2
[0130] Plasmids pCEC1 and pCRK9 have been deposited at the Belgian
Coordinated Collections of Microorganisms (BCCM)
[0131] Laboratorium voor Moleculaire Biologie-Plasmidecollectie
(LMBP)
[0132] Universiteit Gent
[0133] K. L. Ledeganckstraat 35
[0134] B-9000 Gent, Belgium on Mar. 11, 1997 and have been
attributed the following deposition numbers:
[0135] MC1061(pCEC1): BCCM/LMBP3657
[0136] DH5 (pCRK9): BCCM/LMBP3656
[0137] Plasmids pBlueScript-ZM18 has been deposited at the Belgian
Coordinated Collections of Microorganisms (BCCM)
[0138] Laboratorium voor Moleculaire Biologie-Plasmidecollectie
(LMBP)
[0139] Universiteit Gent
[0140] K. L. Ledeganckstraat 35
[0141] B-9000 Gent, Belgium. on Mar. 19, 1998.
EXAMPLES
Example 1
[0142] Construction of the Chimeric Genes
[0143] 1.1 Construction of the CaMV35S-AthcycD2 Chimeric Gene and
inclusion in a T-DNA Vector.
[0144] A 1298 bp NcoI-SacI fragment comprising the DNA encoding
CYCD2 from A. thaliana (having the nucleotide sequence of EMBL
Accesion N.degree. X83370 from nucleotide position 194 to nuceotide
position 1332) was treated with Klenow polymerase to render the
protruding termini blunt, and ligated to Smal linearized pART7
(Gleave, 1992), yielding plasmid pCEC1. In this way, a chimeric
gene flanked by NotI sites was constructed, wherein the DNA
encoding the CYCD2 was operably linked to a CaMV35S promoter of the
CabbB-J1 isolate (Harpster et a., 1988) and a 3'ocs region
(MacDonald et al., 1991). The chimeric gene was then inserted
between the T-DNA border of a T-DNA vector, comprising also a
selectable chimeric marker gene.
[0145] To this end, the chimeric cycD2 gene was excised from pCEC1,
using NotI, and ligated to NotI linearized pART27 (Gleave, 1992) to
create pCEC5. pART27 comprises a chimeric selectable marker gene
consisting of the following operably linked fragments: a nopaline
synthase gene promoter, a neo coding region and 3' end of a
nopaline synthase gene (An et al., 1988).
[0146] Alternatively, the chimeric cycD2 gene is excised from pCEC1
using an appropriate restriction enzyme (e.g. NotI) and introduced
in the polylinker between the T-DNA border sequences of the T-DNA
vector pGSV5, together with a selectable chimeric marker gene
(pSSU-bar-3'ocs; De Almeida et al., 1989) yielding pCEC5b.
[0147] pGSV5 was derived from plasmid pGSC1700 (Cornelissen and
Vandewiele, 1989) but differs from the latter in that it does not
contain a beta-lactamase gene and that its T-DNA is characterized
by the sequence of SEQ ID No 11.
[0148] 1.2 Construction of the CaMV35S-AthcycD3 Chimeric Gene and
Inclusion in a T-DNA Vector.
[0149] The cycD3 cDNA was isolated as a 1335 bp BslI-Dral fragment,
rendered blunt-ended by treatment with Klenow polymerase (having
the nucleotide sequence of EMBL Accesion N.degree. X83371 from
nucleotide position 104 to nucleotide position 1439) and inserted
into the Smal site of pUC18, to create pRS14a. This clone carries
the full coding sequence of cycD3, with the translation initiation
codon located immediately adjacent to the cleaved Smal site of
pUC18 in such an orientation that the SacI site of pUC18 is at the
5' end of the cycD3 cDNA and the BamHI site is at the 3' end. The
1.35 kb SacI-BamHI fragment of pRS14a was isolated and ligated to
the about 26.6 kb SacI-BamHI fragment of pSLJ94 (Jones et al.,
1992), generating pCRK9. In this way a chimeric gene was
constructed wherein the DNA encoding the cycD3 coding region from
A. thaliana was operably linked to a CaMV35S promoter and the 3'ocs
region. In pCRK9 the chimeric gene is located between T-DNA
borders, accompanied by a chimeric selectable neo gene (Jones et
al., 1992)
[0150] Alternatively, the chimeric cycD3 gene is excised from
pRS14a using appropriate restriction enzymes and introduced in the
polylinker between the T-DNA border sequences of the T-DNA vector
pGSV5, together with a selectable chimeric marker gene
(pSSU-bar-3'ocs; De Almeida et al., 1989) yielding pCRK9b.
Example 2
[0151] Agrobacterium-mediated Transformation of Tobacco Plants with
the T-DNA Vectors of Example 1.
[0152] T-DNA vectors pCEC5 and pCRK9 were introduced in
Agrobacterium tumefaciens LBA4404 (Klapwijk et al., 1980) by
electroporation as described by Walkerpeach and Velten.(1995) and
transformants were selected using spectinomycin and tetracycline
respectively.
[0153] T-DNA vectors pCEC5b and pCRK9b are introduced in A.
tumefaciens C58C1 Rif.sup.R by triparental mating (Diffa et al.,
1980).
[0154] The resulting Agrobacterium strains were used to transform
Nicotiana tabacum var Xanthi, applying the leaf disc transformation
method as described in An et al. (1985).
[0155] Eight tobacco plants transformed with pCRK9 (designated 1
K9, 2K9, 3K9, 4K9, 8K9, 10K9, 17K9, 19K9 and 28K9) were generated
and eleven tobacco plants transformed with pCEC5 (designated C8
lines 1 to 3 and 5 to 12).
[0156] Plants transformed by pCRK9 T-DNA were analyzed for the copy
number of the inserted transgenes by Southern hybridization using
the labelled cDNA insert of pRS14a as probe. Lines 2K9, 3K9 and 4K9
each had obtained 1 copy of the transgene, while line 1K9 contained
three copies of the transgene.
[0157] Plants transformed by pCEC5 T-DNA were analyzed for the copy
number of the inserted transgenes by Southern hybridization using
BamHI digested DNA prepared from these plants and labelled 0.7 kb
NcoI-EcoRI fragment from J22 cDNA (comprising part of the cycD2
coding region; Soni et al., 1995). Lines C8-2, C8-3, C8-5, C8-8,
C8-1, C8-9, C8-10, C8-11, C8-12 all had one copy of the transgene,
line C8-7 had two copies, line C8-6 had three copies and line C8-1
had four copies of the transgene.
[0158] The T0 (primary transformants) were self-fertilized and
allowed to set seeds (T1 seeds).
[0159] Plants grown from T1 seeds are designated C8-T1-X, where X
stands for the line number of the original transformant. Seeds from
T1 plants are referred to as T2 seed; plants grown from such seed
are named C8-T2-X, where X is again the line number of the original
transformant. Whenever the generation is not mentioned, the plants
are grown from T1 seed.
[0160] Northern analysis confirmed transcription of the transgenes
in at least lines C8-1, C8-3, C8-7, 3K9, 4K9 and 8K9.
Example 3
[0161] Phenotypic Analysis of the Transformed Tobacco Plants.
[0162] 3.1. Tobacco Plants Comprising the CaMV35S-AthCycD2 Chimeric
Gene.
[0163] Seeds from primary transformants (T0 plants) were surface
sterilized in 10% bleach for 15 minutes and thoroughly washed in
sterile water. The surface-sterilized seeds were germinated on GM
medium containing kanamycin to a final concentration of 100
.mu.g/ml. Seeds on plates were placed for 5 days at 4.degree. C.
(vernalization) and then moved to 23.degree. C. in a growth
chamber. All time points refer to the day of placing in the growth
chamber. Eighteen days after moving to the growth chamber (ie after
23 days in total), the kanamycine-resistant seedlings were
transplanted into seed trays containing soil, and grown under 18 hr
photoperiod in a growth room. After a further 10 days these plants
were transferred to 3 inch plant pots and after an additional 15
days to 8 inch plant pots where they remained for the rest of the
experiment. The 3 inch and 8 inch plant pots were incubated in a
greenhouse supplemented with additional lighting to achieve an 18
hour photoperiod. Plants were placed in randomised design within
the greenhouse.
[0164] Measurements were started two days later (i.e. after 45 days
or after 27 days in soil; referred to as week 1), and repeated
every week for seven weeks, when appropriate. The following number
of plants were analyzed for each line: 22 plants for line C8-1, 7
plants for line C8-2, 22 plants for line C8-3, 8 plants for line
C8-5, 6 plants for line C8-6, 22 plants for line C8-7, 5 plants for
line C8-8, 6 plants for line C8-9, 4 plants for line C8-10, 6
plants for line C8-11, 5 plants for line C8-12, 34 plants for
untransformed control (wild type).
[0165] The following parameters were analyzed:height of the plants
from the soil surface to the highest point (i.e. growing tip;
summarized in Table 1 as mean height.+-.standard deviation in cm);
length of the largest leaf at defined times (summarized in Table 2
as mean length.+-.standard deviation in cm); time (summarized in
Table 3 as mean time.+-.standard deviation in days) at which an
infloresence meristem is visible with the naked eye (inflorescences
of 0.25 cm and 1 cm); height at which an infloresence meristem is
visible (summarized in Table 3 as mean length.+-.standard deviation
in cm); length of the petal tube of the flowers; width of the
collar of the petal tube (summarized in Table 3 as mean length and
width.+-.standard deviation in mm); total number of seed pods per
plant; and average seed yield (on a weight basis) per plant.
[0166] The transgenic plants exhibited an increased growth rate,
apparent from the seedling stage, resulting in a larger average
stem height (Table 1). At time point week 3, all populations of
transgenic lines are significantly larger than the untransformed
controls (t-test; at confidence level 95%), while lines C8-1, C8-2,
C8-3, C8-5, C8-11 are significantly larger than the untransformed
controls at a confidence level of 99%. The increased growth rate
also resulted on average in larger leaves at the indicated times,
which correspond to a period when leaf expansion is continuing
(Table 2) and larger flowers, wherein the petal tube of transgenic
plants is on average longer than the petal tube from flowers on
untransformed plants.
[0167] Also the number of flowers is increased in transgenic
plants, as well as the number of fertilized flowers, resulting in a
larger number of seed pods, and a greater seed yield per plant
(data summarized in Table 4). Moreover, the number of seeds per pod
is larger in the transgenic plants than in the wild-type control
plants. The aberrant seed yield in line C8-T1-6, is due to
excessive high percentage of flower abscission.
[0168] It can thus be concluded that constitutive expression of
AthCycD2 encoding DNA, leads to an increase both in number of seed
pods and total yield of seeds on a per plant basis.
[0169] Finally, the root development in wild-type seedlings and
transgenic seedlings was compared (Table 4B). Seeds were
sterilised, sown on GM media plates without selection, vernalised
and then stored in the vertical position in the growth room. Root
length was measured 9 days and 13 days after vernalisation and the
presence of lateral roots recorded. Seeds from line C8-T1-7 and
C8-T2-2 (homozygous) were used. Line C8-T1-7 possesses two inserts
which segregate approximately 15:1 on kanamycin plates. 35
seedlings were grown from this line and of these, three appeared to
represent the rate of growth observed in wild type seedlings. Data
from these seedlings are recorded separately nine days after
vernalisation. The t-test was applied to determine the significance
of the mean difference and the level of significance is indicated
in the table. ns denotes no significant difference between the
samples. It thus seems that the increase in vegative growth in the
apical parts is balanced by an equal increase in the root
development.
1TABLE 1 Mean height (in cm) of transformed tobacco plants
comprising CaMV35S-AtcycD2 Week 1 Week 2 Week 3 Week 4 Week 5 Week
6 Week 7 Line (45 days) (51 days) (59 days) (65 days) (73 days) (81
days) (89 days) C8-T1-1 5.86 .+-. 2.45 13.71 .+-. 3.43 38.91 .+-.
6.61 63.09 .+-. 9.69 99.42 .+-. 10.25 123.30 .+-. 24.60 137.09 .+-.
31.90 C8-T1-2 8.50 .+-. 1.23 18.29 .+-. 2.21 49.86 .+-. 4.73 77.64
.+-. 4.73 117.14 .+-. 10.71 147.71 .+-. 17.75 168.00 .+-. 9.93
C8-T1-3 8.61 .+-. 2.59 17.41 .+-. 4.94 43.14 .+-. 9.08 66.61 .+-.
11.04 100.41 .+-. 15.57 134.95 .+-. 21.17 145.73 .+-. 22.15 C8-T1-5
6.81 .+-. 1.16 16.31 .+-. 1.89 44.38 .+-. 3.66 70.69 .+-. 5.30
106.50 .+-. 6.12 143.63 .+-. 12.33 159.88 .+-. 9.88 C8-T1-6 4.83
.+-. 1.75 10.75 .+-. 2.51 33.10 .+-. 6.47 52.67 .+-. 5.83 84.83
.+-. 8.08 120.50 .+-. 13.73 141.80 .+-. 5.63 C8-T1-7 8.64 .+-. 3.04
18.82 .+-. 4.56 48.32 .+-. 6.12 74.66 .+-. 7.12 111.50 .+-. 8.38
149.59 .+-. 11.05 169.14 .+-. 11.99 C8-T1-8 5.50 .+-. 1.41 13.2
.+-. 2.41 41.2 .+-. 2.17 62.40 .+-. 3.98 97.40 .+-. 8.08 134.20
.+-. 5.63 156.80 .+-. 11.86 C8-T1-9 3.75 .+-. 1.44 10.58 .+-. 3.32
35.67 .+-. 6.80 62.17 .+-. 9.72 100.67 .+-. 12.24 137.83 .+-. 20.34
162.17 .+-. 18.67 C8-T1-10 9.88 .+-. 1.89 21.00 .+-. 4.08 47.75
.+-. 8.02 75.38 .+-. 7.11 113.75 .+-. 9.21 152.75 .+-. 11.99 164.50
.+-. 177.21 C8-T1-11 10.00 .+-. 2.30 19.51 .+-. 3.82 45.17 .+-.
5.63 72.25 .+-. 5.50 103.33 .+-. 11.52 144.58 .+-. 5.63 152.83 .+-.
18.28 C8-T1-12 9.20 .+-. 2.66 17.9 .+-. 5.15 42.8 .+-. 11.01 68.6
.+-. 13.32 103.40 .+-. 14.40 140.80 .+-. 12.16 161.8 .+-. 10.76
wild-type 4.48 .+-. 1.63 10.50 .+-. 3.33 31.82 .+-. 6.62 54.00 .+-.
7.89 86.56 .+-. 10.91 121.81 .+-. 18.28 145.18 .+-. 19.44
[0170]
2TABLE 2 Mean leaf length (in cm) of the largest leaf of
transformed tobacco plants comprising CaMV35S-AtcycD2 Line Week 1
Week 2 Week 3 C8-T1-1 13.500 .+-. 1.846 19.909 .+-. 2.004 27.114
.+-. 2.182 C8-T1-2 16.643 .+-. 1.282 23.500 .+-. 1.354 29.643 .+-.
1.842 C8-T1-3 15.955 .+-. 2.400 20.951 .+-. 3.737 27.341 .+-. 3.095
C8-T1-5 15.062 .+-. 1.635 21.563 .+-. 1.741 28.875 .+-. 2.372
C8-T1-6 14.667 .+-. 1.402 20.833 .+-. 1.807 29.927 .+-. 1.201
C8-T1-7 15.886 .+-. 1.718 22.000 .+-. 1.498 28.909 .+-. 1.974
C8-T1-8 14.167 .+-. 2.229 20.500 .+-. 1.871 27.583 .+-. 1.856
C8-T1-9 12.417 .+-. 2.035 18.917 .+-. 2.010 26.333 .+-. 2.113
C8-T1-10 14.750 .+-. 2.693 20.167 .+-. 2.825 27.583 .+-. 2.635
C8-T1-11 15.833 .+-. 2.113 21.333 .+-. 1.602 28.333 .+-. 1.722
C8-T1-12 14.600 .+-. 1.432 21.400 .+-. 1.475 28.100 .+-. 2.608
wild-type 12.676 .+-. 1.846 18.691 .+-. 2.280 26.352 .+-. 1.960
[0171]
3TABLE 3A Floral development [mean height to infloresence of 0.25
cm or 1 cm (in cm), mean time required to reach the development of
an infloresence of 0.25 or 1 cm (in days after vernalization)] in
tobacco transformed with CaMV35SAthCycD2 Mean time to Mean time to
Mean height at inflorescence of inflorescence of infloresence of
Line 0.25 cm (days) 1 cm (days) 1 cm (cm) C8-T1-1 67.35 .+-. 4.580
74.75 .+-. 4.541 105.5 .+-. 21.670 C8-T1-2 65.42 .+-. 2.573 72.00
.+-. 3.546 110.6 .+-. 21.439 C8-T1-3 68.77 .+-. 3.436 74.32 .+-.
3.414 106.5 .+-. 14.134 C8-T1-5 70.25 .+-. 2.712 76.63 .+-. 2.387
122.4 .+-. 6.737 C8-T1-6 68.00 .+-. 2.828 73.33 .+-. 2.944 117.0
.+-. 5.550 C8-T1-7 70.95 .+-. 3.034 77.15 .+-. 2.852 133.4 .+-.
14.497 C8-T1-8 72.60 .+-. 3.286 77.60 .+-. 2.793 116.1 .+-. 5.482
C8-T1-9 73.17 .+-. 3.251 79.50 .+-. 2.429 129.25 .+-. 10.324
C8-T1-10 72.50 .+-. 2.517 77.75 .+-. 2.986 127.7 .+-. 19.202
C8-T1-11 66.67 .+-. 1.033 73.17 .+-. 2.137 104.6 .+-. 4.924
C8-T1-12 70.00 .+-. 4.000 76.40 .+-. 3.715 121.6 .+-. 7.893 mean
value 69.61 .+-. 2.587 75.69 .+-. 2.329 117.70 .+-. 10.082
wild-type 74.90 .+-. 3.222 79.09 .+-. 2.342 111 .+-. 10.020
[0172]
4TABLE 3B Floral development [mean flower size i.e. length and
width (mm)] in tobacco transformed with CaMV35SAthCycD2. The length
and width of five flowers from each plant was measured and the mean
flower length or width for each transgenic line was calculated. The
values for each independent transgenic line were compared to wild
type using the t-test. The table reveals the level of probability
that the results are statistically significant compared to wild
type. ns means not significant. Level of Mean flower Level of Mean
flower signifi- Line length (mm) significance width (mm) cance
C8-T1-1 47.22 .+-. 2.261 P < 0.001 33.45 .+-. 1.668 P < 0.001
C8-T1-2 44.19 .+-. 1.848 P < 0.01 31.14 .+-. 1.486 P < 0.001
C8-T1-3 41.30 .+-. 1.720 ns 31.04 .+-. 1.360 P < 0.001 C8-T1-5
47.30 .+-. 2.822 P < 0.002 33.30 .+-. 1.945 P < 0.001 C8-T1-6
50.78 .+-. 1.990 P < 0.001 34.25 .+-. 1.467 P < 0.001 C8-T1-7
48.04 .+-. 2.604 P < 0.001 33.19 .+-. 1.391 P < 0.001 C8-T1-8
42.90 .+-. 1.252 ns 30.13 .+-. 2.270 ns C8-T1-9 45.12 .+-. 1.906 P
< 0.01 30.56 .+-. 1.333 P < 0.002 C8-T1-10 44.60 .+-. 1.627 P
< 0.01 30.93 .+-. 2.002 P < 0.01 C8-T1-11 42.40 .+-. 1.891 ns
29.10 .+-. 2.998 ns C8-T1-12 42.57 .+-. 0.978 P < 0.05 28.55
.+-. 1.190 P < 0.05 mean 45.093 .+-. 2.877 P < 0.002 31.43
.+-. 1.886 P < 0.001 value wild-type 41.22 .+-. 1.005 -- 26.76
.+-. 1.099
[0173]
5TABLE 4A Mean number of seed pods per plant, mean weight of the
seed content of six pods (g), mean seed yield per plant (g), in
tobacco transformed with CaMV35SAthCycD2 mean weight Mean number of
of seed content Mean seed yield per Line seed pods of six pods (g)
plant (g) C8-T1-1 105.15 .+-. 14.96 1.085 .+-. 0.174 19.015 C8-T1-2
127.29 .+-. 7.82 0.824 .+-. 0.137 17.481 C8-T1-3 110.46 .+-. 16.30
1.106 .+-. 0.179 20.361 C8-T1-5 97.86 .+-. 10.81 1.105 .+-. 0.178
18.023 C8-T1-6 78.60 .+-. 12.97 1.078 .+-. 0.150 14.123 C8-T1-7
118.17 .+-. 15.64 1.131 .+-. 0.253 22.275 C8-T1-8 123.75 .+-. 4.78
1.123 .+-. 0.165 23.162 C8-T1-9 110.83 .+-. 20.91 1.090 .+-. 0.218
20.134 C8-T1-10 104.20 .+-. 10.99 1.122 .+-. 0.311 19.485 C8-T1-11
138.20 .+-. 8.35 1.116 .+-. 0.222 25.705 C8-T1-12 106.20 .+-. 12.62
1.134 .+-. 0.056 20.072
[0174]
6TABLE 4B Comparison of root development in wild-type and
transgenic seedlings Mean root Number of % Lateral length Level of
Line plants roots (mm) significance 9 days after vernalization WT
23 36 15.326 .+-.1.893 C8-T1-7 25 100 26.520 .+-.1.971 0.001 3 33
14.000 .+-.3.464 ns C8-T2-2 28 100 26.911 .+-.2.064 0.001 13 days
after vernalization WT 16 100 28.188 .+-.1.893 C8-T1-7 13 100
53.846 .+-.1.971 0.001 C8-T2-2 15 100 51.267 .+-.3.464 0.001
[0175] 3.2. Tobacco Plants Comprising the CaMV35S-AthCycD3 Chimeric
Gene.
[0176] Plants comprising the CaMV35S-AthCycD3 chimeric genes, were
grown from T1 seeds and treated as described under 3.1.
Measurements were started at 49 days after germination, with
intervals of about 7 days. The following number of plant lines were
analyzed for each line: 11 plants for line 1K9; 19 plants for line
3K9, 20 plants for line 4K9 and 18 plants for the untransformed
control.
[0177] The following parameters were analyzed: the petal tube
length and width (in cm) and the time (in days) at which at least
75% of the plants have reached at least the stage wherein an
infloresence is clearly developed, summarized in Table 5.
7TABLE 5 Summary of the measurements on tobacco plants comprising
the CaMV35S-AthCycD3 chimeric gene. mean time required to reach
Mean petal tube mean petal tube inflorescence of 1 Line length (cm)
width (cm) cm (days) 1K9 5.66 .+-. 0.46 3.44 .+-. 0.27 100 3K9 5.18
.+-. 0.37 3.20 .+-. 0.35 100 4K9 5.48 .+-. 0.38 2.90 .+-. 0.35 93
wt 3.96 .+-. 0.12 2.39 .+-. 0.10 84
[0178] These transgenic plants had larger flowers, wherein the
petal tube of transgenic plants was on average longer than the
petal tube from flowers on untransformed plants, and also required
more time to reach the stage wherein an infloresence is clearly
developed.
Example 4
[0179] Isolation of cycD-homologous Genes from Other Plants
[0180] A c-DNA library, made from exponentially growing tobacco
BY-2 cells was constructed in a Lambda Zap Express vector
(Stratagene). Approximately 7.5.times.10.sup.5 library clones were
plated out, and replica blots made from each plate using Hybond
N.sup.+ nylon membranes (Amersham Int.) which were then fixed by
baking at 80.degree. C. for two hours. The membranes were
hybridized with cycD2 or cycD3 heterologous probes labelled with
-32P dCTP by random priming. The cycD3 probe comprised a cycD3
fragment from A. thaliana (405 bp HincII-KpnI fragment; having the
nucleotide sequence of EMBL Accesion N.degree. X83371 from
nucleotide position 557 to nuceotide position 962). The cycD2 probe
consisted of an 1298 bp NcoI-SacI fragment of cycD2 from A.
thaliana (having the nucleotide sequence of EMBL Accesion N.degree.
X83370 from nucleotide position 194 to nuceotide position 1332).
cycD3 hybridizations were carried out at 55.degree. C. and the
membranes were washed for 10 min in 2.times.SSC/0.1% SDS twice,
followed by a single 10 min wash in 0.1 SSC/0.1% SDS prior to
autoradiography. The cycD2 hybridizations were carried out at
48.degree. C.; the membranes were washed for 10 min in
2.times.SSC/0.1% SDS three times. All washes were carried out at
room temperature. Isolated library clones were excised in vivo
(according to the manufacturer's protocol) to generate subclones in
the pBK-CMV phagemid (Stratagene) and DNA sequence was determined
according to standard methods. Sequence information was analyzed
using the GCG (Genetics Computer Group) Software (1994). The
sequences of cycD2;1, cycD3;1 and cycD3;2 cDNAs from tobacco are
represented in respectively, SEQ ID No. 1, SEQ ID No. 2, SEQ ID No.
3.
[0181] Another cDNA library was made from polyadenylated RNA
isolated from tubers, roots and leaves of Helianthus tuberosus. The
cDNA was synthesized from an oligo (dT) primer and ligated into
lambda ZAPII vector at the EcoRI site.
[0182] Approximately 1.25.times.106 clones were plated out, replica
plaque blots were made as described above and hybridized using the
labelled probes mentioned above. In addition the blots were
screened with a cycD1 probe, comprising the 401 bp Xbal-Aval
fragment of cycD1 gene of A. thaliana (having the nucleotide
sequence of EMBL Accesion N.degree. X83369 from nucleotide position
312 to nuceotide position 713). Isolated clones were analysed as
above. The sequence of cycD1;1 and cycD3;1 genes from Helianthus
tuberosus is represented in (SEQ ID NO:7) and (SEQ ID NO:9),
respectively.
[0183] Yet another cDNA library was made from polyadenylated RNA
isolated from callus material of Zea mays
(Pa91.times.H99).times.H99. The cDNA was synthesized from an oligo
(dT) primer and ligated into lambda ZAPII vector at the EcoRI site.
Approximately 1.25.times.10.sup.6 clones were plated out, replica
plaque blots were made as described above and hybridized using the
labelled probes mentioned above. Isolated clones were analysed as
above. The sequence of the cycD2 cDNA from Zea mays is represented
in SEQ ID NO:26.
Example 5
[0184] Construction of the Antisense Chimeric Genes and
Transformation of Tobacco.
[0185] A 1298 bp NcoI-SacI fragment comprising the DNA encoding
CYCD2 from A. thaliana (having the nucleotide sequence of EMBL
Accesion N.degree. X83370 from nucleotide position 194 to nuceotide
position 1332) was treated with Klenow polymerase to render the
protruding termini blunt, and ligated to Smal linearized pART7
(Gleave, 1992). A plasmid was selected wherein the inserted DNA
fragment was in such an orientation that the DNA encoding the CYCD2
was introduced in the reverse way between a CaMV35S promoter of the
CabbB-J1 isolate (Harpster et al., 1988) and a 3'ocs region
(MacDonald et al., 1991), so that upon expression an antisense RNA
is produced.
[0186] The chimeric antisense gene was then inserted between the
T-DNA border of a T-DNA vector, comprising also a selectable
chimeric marker gene. To this end, the chimeric cycD2 gene was
excised from pCEC2, using NotI, and ligated to NotI linearized
pART27 (Gleave et al, 1992) to create pCEC6.
[0187] Tobacco plants were transformed with this chimeric genes as
described in Example 2.
Example 6
[0188] Analysis of the Transformants
[0189] Plants transformed with the chimeric genes of Example 5 were
treated as described in Example 3.1 and the following number of
plants were analyzed: 7 plants for line C9-2, and 6 plants for line
C9-7.
[0190] The following parameters were analyzed:height of the plants
from the soil surface to the highest point (summarized in Table 6
as mean height.+-.standard deviation in cm); length of the largest
leaf at defined times (summarized in Table 7 as mean
length.+-.standard deviation in cm); time (summarized in Table 8 as
mean time.+-.standard deviation in days) at which an infloresence
merisitem is visible with the naked eye; height at which an
infloresence meristem is visible (summarized in Table 8 as mean
length.+-.standard deviation in cm).
[0191] The transgenic plants exhibited an decreased growth rate,
apparent from the seedling stage, resulting in a smaller average
stem height (Table 6). The decreased growth rate also resulted on
average in smaller leaves at the indicated times, which correspond
to a period when leaf expansion is continuing (Table 7)
8TABLE 6 Mean height (in cm) of transformed tobacco plants
comprising CaMV35Santisense cycD2 untransformed Line C9-2 C9-7
control Week 1 2.64 .+-. 1.22 2.75 .+-. 0.89 4.48 .+-. 1.63 Week 2
6.64 .+-. 1.68 6.07 .+-. 1.43 10.50 .+-. 3.33 Week 3 20.00 .+-.
3.74 17.21 .+-. 6.47 31.82 .+-. 7.89 Week 4 34.07 .+-. 6.13 28.50
.+-. 5.83 54.00 .+-. 7.89 Week 5 54.00 .+-. 8.87 45.14 .+-. 8.46
86.56 .+-. 10.91 Week 6 74.29 .+-. 9.97 61.29 .+-. 5.11 121.80 .+-.
18.28 Week 7 85.92 .+-. 12.03 71.50 .+-. 23.19 145.18 .+-.
19.44
[0192]
9TABLE 7 Difference in mean leaf length of the largest leaf of
transformed tobacco plants comprising CaMV35Santisense cycD2 and
the mean leaf length of the largest leaf of untransformed tobacco
plants (in cm). untransformed Line C9-2 C9-7 control Week 1 -2.31
-4.30 0 Week 2 -3.26 -6.44 0 Week 3 -4.35 -8.91 0
[0193]
10TABLE 8A Mean flower size (mm), mean height to infloresence(cm),
mean time required to reach the development of an infloresence
(days) in tobacco transformed with CaMV35S antisense cycD2. Mean
time to Mean height to Mean flower Line infloresence (days)
infloresence (cm) length (mm) C9-2 102.sup.a 95 NA C9-7 89 .+-.
7.95 68.57 .+-. 9.62 38.31 untransformed 79 .+-. 2.39 111 .+-.
10.02 41.22 control .sup.aOnly one plant developed an infloresence
during the monitoring period.
[0194] Table 8B. The effect of antisense CycD2 expression on flower
length of transgenic tobacco was analyzed in other lines (T1
generation) and statistically compared to wild type using the
student t-test. The length of five flowers from each plant was
measured and the mean flower length for each transgenic line was
calculated. The values for each independent transgenic line were
compared to wild type using the t-test. The table reveals the level
of probability that the results are statistically significant
compared to wild type.
11 Mean flower Line length (mm) Level of significance C9-T1-1 41.05
.+-. 1.558 ns C9-T1-3 40.68 .+-. 1.574 ns C9-T1-7 38.68 .+-. 1.991
ns C9-T1-10 39.78 .+-. 1.024 P < 0.05 C9-T1-12 39.55 .+-. 1.568
P < 0.05 Mean value 40 .+-. 1.301 P < 0.05 wild type 41.22
.+-. 1.005 --
Example 7
[0195] Transformation of Oil Seed Rape with the T-DNAs of Example 1
and Similar Vectors and Analysis of Transformed Plants.
[0196] Hypocotyl explants of Brassica napus are obtained, cultured
and transformed essentially as described by De Block et al. (1989),
except for the following modifications:
[0197] hypocotyl explants are precultured for 1 day on A2 medium
[MS, 0.5 g/l Mes (pH5.7), 1.2% glucose, 0.5% agarose, 1 mg/l 2,4-D,
0.25 mg/l naphthalene acetic acid (NAA)and 1 mg/l
6-benzylaminopurine (BAP)].
[0198] infection medium A3 is MS, 0.5 g/l Mes (pH5.7), 1.2%
glucose, 0.1 mg/l NAA, 0.75 mg/l BAP and 0.01 mg/l gibberellinic
acid (GA3).
[0199] selection medium A5G is MS, 0.5 g/l Mes (pH5.7), 1.2%
glucose, 40 mg/l adenine.SO.sub.4, 0.5 g/l polyvinylpyrrolidone
(PVP), 0.5% agarose, 0.1 mg/l NM, 0.75 mg/l BAP, 0.01 mg/l GA3, 250
mg/l carbenicillin, 250 mg/l triacillin, 5 mg/l AgNO.sub.3 for
three weeks. After this period selection is continued on A5J medium
(similar a A5G but with 3% sucrose)
[0200] regeneration medium A6 is MS, 0.5 g/l Mes (pH5.7), 2%
sucrose, 40 mg/l adenine.SO.sub.4, 0.5 g/l PVP, 0.5% agarose,
0.0025mg/l BAP and 250 mg/l triacillin.
[0201] healthy shoots are transferred to rooting medium which was
A9: half concentrated MS, 1,5% sucrose (pH5.8), 100 mg/l
triacillin, 0.6% agar in 1 liter vessels. MS stands for Murashige
and Skoog medium (Murashige and Skoog, 1962) Hypocotyl explants are
infected with Agrobacterium tumefaciens strain C58C1Rif.sup.R
carrying a helper Ti-plasmid such as pGV4000 which is a derivative
of pMP90 (Koncz and Schell, 1986) obtained by insertion of a
bacterial chloramphenicol resistance gene linked to a 2.5 kb
fragment having homology with the T-DNA vector pGSV5, into pMP90;
and a T-DNA vector derived from pGSV5 comprising between the T-DNA
borders the chimeric genes of Example 1 and the chimeric marker
gene (pCEC5b and pCRK9b).
[0202] Transgenic oilseed rape plants comprising the chimeric genes
of the invention, exhibit an accelerated vegetative program
(increased growth rate), a reduction in the time required to reach
the flowering stage, an increased number of flowers and an
increased seed yield per plant.
Example 8
[0203] Transformation of Corn Plants with the Vectors of Example 1
and Similar Vectors and Analysis of the Transformed Plants.
[0204] Corn plants are transformed with the vectors of Example 1,
according to WO92/09696. Transgenic corn plants comprising the
chimeric genes of the invention exhibit an accelerated vegetative
program (increased growth rate), a reduction in the time required
to reach the flowering stage, an increased number of flowers and an
increased seed yield per plant.
Example 9
[0205] Transformation of Tomato Plants with the Vectors of Example
1 and Similar Vectors and Analysis of the Transformed Plants.
[0206] Tomato plants are transformed with the vectors of Example 1,
according to De Block et al. (1987) Transgenic tomato plants
comprising the chimeric genes of the invention exhibit an
accelerated vegetative program (increased growth rate), a reduction
in the time required to reach the flowering stage, an increased
number of flowers and an increased fruit yield per plant.
Example 10
[0207] Transformation of Lettuce Plants with the Vectors of Example
1 and Similar Vectors and Analysis of the Transformed Plants.
[0208] Lettuce plants are transformed with the vectors of Example
1, according to Micheimore et al. (1987). Transgenic lettuce plants
comprising the chimeric genes of the invention exhibit an
accelerated vegetative program (increased growth rate), a reduction
in the time required to reach the flowering stage, an increased
number of flowers and an increased seed yield per plant.
Example 11
[0209] Further Phenotypic Analysis of the Progeny of the Transgenic
Tobacco Lines Transformed with the CaMV35SAthCycD2 Constructs of
Example 3 in Segregating and Non-segregating Populations.
[0210] Progeny populations (either segregating or non-segregating)
of plants from two transgenic tobacco lines transformed with the
CaMV35SAthCycD2 constructs (line 2 and line 5 of Example 3) were
analyzed for length of time to flowering and increase in vegetative
growth by measuring the mean height of the stem or the mean dry
weight of the plants.
[0211] Seggregation of the transgenes was monitored by establishing
their resistance to kanamycine. For segregating populations, 32
plants were analyzed, while for non-segregating populations, 12
plants were analyzed. The non-transformed population consisted also
of 12 plants.
[0212] The following populations were used:
[0213] Segregating populations:
[0214] Line 2
[0215] C8-T1-2 [T1 seed from C8-2 primary transformant; segregates
3:1 for T-DNA]
[0216] C8-T2-2 [T2 seed from C8-T1-2 plant #3 selfed, which was
hemizygous and thus seed segregates 3:1 for T-DNA]
[0217] C8-T2-2 [T2 seed from a cross of C8-T1-2 plant #3 to wild
type plant using wild type as pollen parent. This seed segregates
1:1 for T-DNA, and all T-DNA containing plants are hemizygous]
[0218] Line 5
[0219] C8-T1-5 [T1 seed from C8-5 primary transformant; segregates
3:1 for T-DNA]
[0220] C8-T2-5 [T2 seed from C8-T1-5 plant #304 selfed, which was
hemizygous and thus seed segregates 3:1 for T-DNA]
[0221] Non-segregating populations
[0222] Line 2
[0223] C8-T2-2 [T2 seed from C8-T1-2 plant #302 selfed, which was
homozygous for T-DNA]
[0224] Line 5
[0225] C8-T2-5 [T2 seed from C8-T1-5 plant #121 crossed to wild
type plant using wild type as pollen parent. Plant #121 was
homozygous for T-DNA and all T2 seed is hemizygous for the
T-DNA]
[0226] C8-T2-5 [T2 seed from C8-T1-5 plant #121 selfed. Plant #121
was homozygous for T-DNA and all T2 seed is homozygous for the
T-DNA].
[0227] The effect of CycD2 overexpression on the length of time to
initiatiate inflorescence development in transgenic tobacco was
measured and statistically compared to values for the same
parameter measured for a wild type control population, using a
non-parametric t-test in which the variances of the wt and
transgenic populations are not assumed to be equal. The length of
the time for each plant to develop an inflorescence of 0.5 cm was
recorded and the mean number of days , post-vernalization was
calculated. The values for each transgenic population was compared
to the value for the wild-population using the t-test. The data for
the segregating lines are separated in data for the kanamycin
resistant population and the kanamycin sensitive population. The
data for the kanamycin resistant population are also indicated
separately for the homozygous kanamycin resistant subpopulation
(not further segregating) and the hemizygous kanamycin resistant
subpopulation (further segregating 3:1). In Table 9 these data are
summarized. Table 10 summarizes the mean values of the stem heights
in transgenic non-segregating lines at different timepoints
post-vernilization, in comparison with a wild type population
(statiscally analyzed). A significance level of less than 0.05 is
considered a highly significant difference between the mean heigth
of each transgenic line and the mean heigth of the controls. ns
indicates there is no significant difference between the
populations. In addition, the biomass of seedlings from the
mentioned non-segregating populations was compared to wild type
seedlings during early vegetative growth. Seedlings were harvested
at the days indicated after vernalisation and weighed before drying
at 70.degree. C. for 2 days. The mean dry weight of the seedlings
and standard deviation was calculated and the results are presented
in Table 11.
12TABLE 9 The effect of CycD2 overexpression on the length of time
to initiatiate inflorescence development in transgenic tobacco Mean
time to inflorescence of Standard Level of Population 0.5 cm (days)
deviation significance Non-segregating lines WT 72.125 2.258 --
C8-T2-2 (302 selfed) 63.62 3.863 0.001 C8-T1-5 (121 selfed) 67.78
2.438 0.02 C8-T1-5 (121 .times. WT) 64.18 1.991 0.001 Mean value
65.19 2.258 0.001 Segregating lines C8-T1-2 selfed all Kan R 59.04
2.973 0.001 hemizygous 59.32 3.110 0.001 homozygous 58.50 2.507
0.001 Kanamycin sensitive 73.00 5.944 ns C8-T2-2 pl 3 .times. WT
Kanamycin resistant all 59.44 2.756 0.001 Kanamycin sensitive 69.10
2.846 ns C8-T2-2 pl 3 selfed all Kan 59.20 2.141 0.001 R hemizygous
59.25 1.653 0.001 homozygous 59.38 3.021 0.001 Kanamycin sensitive
73.50 5.431 ns C8-T1-5 All Kan R 62.67 3.367 0.001 Kanamycin
sensitive 71.50 5.782 ns C8-T2-5 pl 304 selfed hemizygous 64.50
3.030 0.001 homozygous 65.83 2.483 0.002 Kanamycin sensitive 74.50
7.764 ns
[0228]
13TABLE 10 Staticstical comparison of stem height of transgenic
tobacco comprising CaMV35SAthCycD2 with wild type controls terminal
Population 34 days 37 days 41 days 45 days 49 days 55 days 63 days
70 days 77 days height wild type 1.48 .+-. 3.50 .+-. 7.59 .+-.
14.59 .+-. 25.81 .+-. 58.24 .+-. 107.06 .+-. 136.75 .+-. 153.94
.+-. 177.71 .+-. 0.238 0.831 1.932 3.816 6.030 8.423 8.306 7.105
7.430 13.129 C8-T2-2 3.55 .+-. 6.02 .+-. 11.11 .+-. 19.35 .+-.
32.81 .+-. 67.50 .+-. 120.12 .+-. 151.46 .+-. 165.23 .+-. 177.23
.+-. (302 selfed) 0.451 1.662 3.823 6.528 9.181 12.281 12.829
15.253 14.696 13.935 level of 0.002 0.001 0.01 0.05 0.05 0.05 0.01
0.01 0.05 ns significance C8-T2-5 4.25 .+-. 6.02 .+-. 11.11 .+-.
19.35 .+-. 32.81 .+-. 71.82 .+-. 119.86 .+-. 152.82 .+-. 170.73
.+-. 192.18 .+-. (121 selfed) 0.507 1.662 3.823 6.528 9.181 7.604
10.675 11.297 11.130 7.846 level of 0.001 0.001 0.001 0.001 0.001
0.001 0.01 0.001 0.001 0.01 significance C8-T2-5 4.37 .+-. 8.14
.+-. 14.76 .+-. 25.18 .+-. 39.91 .+-. 75.04 .+-. 117.83 .+-. 146.54
.+-. 160.71 .+-. 174.1 .+-. (121 .times. WT) 0.378 1.914 2.550
3.314 4.898 6.258 9.808 13.422 15.183 14.963 level of 0.001 0.001
0.001 0.001 0.001 0.001 0.01 0.05 ns ns significance
[0229]
14TABLE 11 Summary of dry weight measurements (in mg) obtained from
non-segregating populations of transgenic seedlings overexpressing
CycD2 and wild type (WT) seedlings at different time points
post-vernilization. For all cases, the t-test indicates that there
is a highly significant difference between the mean biomass of each
transgenic line and the mean biomass of the controls. Population 17
days 23 days 28 days 34 days 38 days C8-T2-2 3.75 .+-. 1.462 22.11
.+-. 6.59 53.19 .+-. 9.97 337 .+-. 58.3 530 .+-. 60.0 (302 selfed)
C8-T2-5 4.30 .+-. 1.623 29.05 .+-. 10.50 49.14 .+-. 8.51 351 .+-.
67.6 547 .+-. 24.9 (121 selfed C8-T2-5 5.48 .+-. 1.130 39.51 .+-.
10.13 79.81 .+-. 20.36 476 .+-. 120.2 946 .+-. 154 (121 .times. WT)
Wild type 1.2 .+-. 0.510 13.16 .+-. 3.09 29.88 .+-. 14.89 135 .+-.
60.72 382 .+-. 90.3
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Sequence CWU 1
1
26 1 1284 DNA Nicotiana tabacum CDS (182)..(1243) 1 caaatttttc
tcccttctat agtctctttc ctgttctctt aaaaatcctt aaaaatttat 60
tttttttaac aatctcatgt aaatgggatt aaattttgta aaaatataag attttgataa
120 agggggttta attataacat agtaaattaa gatttttttt ttgctttgct
agtttgcttt 180 a atg gca gct gat aac att tat gat ttt gta gcc tca
aat ctt tta tgt 229 Met Ala Ala Asp Asn Ile Tyr Asp Phe Val Ala Ser
Asn Leu Leu Cys 1 5 10 15 aca gaa aca aaa agt ctt tgt ttt gat gat
gtt gat tct ttg act ata 277 Thr Glu Thr Lys Ser Leu Cys Phe Asp Asp
Val Asp Ser Leu Thr Ile 20 25 30 agt caa cag aac att gaa act aag
agt aaa gac ttg agc ttt aac aat 325 Ser Gln Gln Asn Ile Glu Thr Lys
Ser Lys Asp Leu Ser Phe Asn Asn 35 40 45 ggt att aga tca gag cca
ttg att gat ttg cca agt tta agt gaa gaa 373 Gly Ile Arg Ser Glu Pro
Leu Ile Asp Leu Pro Ser Leu Ser Glu Glu 50 55 60 tgc ttg agt ttt
atg gtg caa agg gaa atg gag ttt ttg cct aaa gat 421 Cys Leu Ser Phe
Met Val Gln Arg Glu Met Glu Phe Leu Pro Lys Asp 65 70 75 80 gat tat
gtc gag aga ttg aga agt gga gat ttg gat ttg agt gtg aga 469 Asp Tyr
Val Glu Arg Leu Arg Ser Gly Asp Leu Asp Leu Ser Val Arg 85 90 95
aaa gag gct ctt gat tgg att ttg aag gct cat atg cac tat gga ttt 517
Lys Glu Ala Leu Asp Trp Ile Leu Lys Ala His Met His Tyr Gly Phe 100
105 110 gga gag ctg agt ttt tgt ttg tcg ata aat tac ttg gat cga ttt
cta 565 Gly Glu Leu Ser Phe Cys Leu Ser Ile Asn Tyr Leu Asp Arg Phe
Leu 115 120 125 tct ctg tat gaa ttg cca aga agt aaa act tgg aca gtg
caa ttg tta 613 Ser Leu Tyr Glu Leu Pro Arg Ser Lys Thr Trp Thr Val
Gln Leu Leu 130 135 140 gct gtg gcc tgt cta tca ctt gca gcc aaa atg
gaa gaa att aat gtt 661 Ala Val Ala Cys Leu Ser Leu Ala Ala Lys Met
Glu Glu Ile Asn Val 145 150 155 160 cct ttg act gtt gat tta cag gta
ggg gat ccc aaa ttt gta ttt gaa 709 Pro Leu Thr Val Asp Leu Gln Val
Gly Asp Pro Lys Phe Val Phe Glu 165 170 175 ggc aaa act ata caa aga
atg gaa ctt ttg gta tta agc aca ttg aag 757 Gly Lys Thr Ile Gln Arg
Met Glu Leu Leu Val Leu Ser Thr Leu Lys 180 185 190 tgg aga atg caa
gct tat aca cct tac aca ttc ata gat tat ttt atg 805 Trp Arg Met Gln
Ala Tyr Thr Pro Tyr Thr Phe Ile Asp Tyr Phe Met 195 200 205 aga aag
atg aat ggt gat caa atc cca tct cgg ccg ttg att tct gga 853 Arg Lys
Met Asn Gly Asp Gln Ile Pro Ser Arg Pro Leu Ile Ser Gly 210 215 220
tca atg caa ctg ata tta agc ata ata aga agt att gat ttc ttg gaa 901
Ser Met Gln Leu Ile Leu Ser Ile Ile Arg Ser Ile Asp Phe Leu Glu 225
230 235 240 ttc agg tct tct gaa att gca gca tca gtg gca atg tct gtt
tca ggg 949 Phe Arg Ser Ser Glu Ile Ala Ala Ser Val Ala Met Ser Val
Ser Gly 245 250 255 gaa ata caa gca aaa gac att gat aag gca atg cct
tgc ttc ttc ata 997 Glu Ile Gln Ala Lys Asp Ile Asp Lys Ala Met Pro
Cys Phe Phe Ile 260 265 270 cac tta gac aag ggt aga gtg cag aag tgt
gtt gaa ctg att caa gat 1045 His Leu Asp Lys Gly Arg Val Gln Lys
Cys Val Glu Leu Ile Gln Asp 275 280 285 ttg aca act gct act att act
act gct gct gct gcc tca tta gta cct 1093 Leu Thr Thr Ala Thr Ile
Thr Thr Ala Ala Ala Ala Ser Leu Val Pro 290 295 300 caa agt cct att
gga gtg ttg gaa gca gca gca tgc ttg agc tac aaa 1141 Gln Ser Pro
Ile Gly Val Leu Glu Ala Ala Ala Cys Leu Ser Tyr Lys 305 310 315 320
agt ggt gat gag aga aca gtt gga tca tgt aca act tct tca cat act
1189 Ser Gly Asp Glu Arg Thr Val Gly Ser Cys Thr Thr Ser Ser His
Thr 325 330 335 aaa agg aga aaa ctt gac aca tca tct tta gag cat ggg
act tca gaa 1237 Lys Arg Arg Lys Leu Asp Thr Ser Ser Leu Glu His
Gly Thr Ser Glu 340 345 350 aag ttg tgaatctgaa ttttcccttt
ttaaaaaaaa aaaaaaaaaa a 1284 Lys Leu 2 354 PRT Nicotiana tabacum 2
Met Ala Ala Asp Asn Ile Tyr Asp Phe Val Ala Ser Asn Leu Leu Cys 1 5
10 15 Thr Glu Thr Lys Ser Leu Cys Phe Asp Asp Val Asp Ser Leu Thr
Ile 20 25 30 Ser Gln Gln Asn Ile Glu Thr Lys Ser Lys Asp Leu Ser
Phe Asn Asn 35 40 45 Gly Ile Arg Ser Glu Pro Leu Ile Asp Leu Pro
Ser Leu Ser Glu Glu 50 55 60 Cys Leu Ser Phe Met Val Gln Arg Glu
Met Glu Phe Leu Pro Lys Asp 65 70 75 80 Asp Tyr Val Glu Arg Leu Arg
Ser Gly Asp Leu Asp Leu Ser Val Arg 85 90 95 Lys Glu Ala Leu Asp
Trp Ile Leu Lys Ala His Met His Tyr Gly Phe 100 105 110 Gly Glu Leu
Ser Phe Cys Leu Ser Ile Asn Tyr Leu Asp Arg Phe Leu 115 120 125 Ser
Leu Tyr Glu Leu Pro Arg Ser Lys Thr Trp Thr Val Gln Leu Leu 130 135
140 Ala Val Ala Cys Leu Ser Leu Ala Ala Lys Met Glu Glu Ile Asn Val
145 150 155 160 Pro Leu Thr Val Asp Leu Gln Val Gly Asp Pro Lys Phe
Val Phe Glu 165 170 175 Gly Lys Thr Ile Gln Arg Met Glu Leu Leu Val
Leu Ser Thr Leu Lys 180 185 190 Trp Arg Met Gln Ala Tyr Thr Pro Tyr
Thr Phe Ile Asp Tyr Phe Met 195 200 205 Arg Lys Met Asn Gly Asp Gln
Ile Pro Ser Arg Pro Leu Ile Ser Gly 210 215 220 Ser Met Gln Leu Ile
Leu Ser Ile Ile Arg Ser Ile Asp Phe Leu Glu 225 230 235 240 Phe Arg
Ser Ser Glu Ile Ala Ala Ser Val Ala Met Ser Val Ser Gly 245 250 255
Glu Ile Gln Ala Lys Asp Ile Asp Lys Ala Met Pro Cys Phe Phe Ile 260
265 270 His Leu Asp Lys Gly Arg Val Gln Lys Cys Val Glu Leu Ile Gln
Asp 275 280 285 Leu Thr Thr Ala Thr Ile Thr Thr Ala Ala Ala Ala Ser
Leu Val Pro 290 295 300 Gln Ser Pro Ile Gly Val Leu Glu Ala Ala Ala
Cys Leu Ser Tyr Lys 305 310 315 320 Ser Gly Asp Glu Arg Thr Val Gly
Ser Cys Thr Thr Ser Ser His Thr 325 330 335 Lys Arg Arg Lys Leu Asp
Thr Ser Ser Leu Glu His Gly Thr Ser Glu 340 345 350 Lys Leu 3 1679
DNA Nicotiana tabacum CDS (181)..(1299) cDNA encoding cyclin
CYCD3;1 3 aaacgagtct ctgtgtactc ctcctcctat agcttttctc tcttcttctc
ttcacacctc 60 ccacaacaca caatcagaca aaatagagag gaaaatgagt
atggtgaaaa agctttgttt 120 tgtataatga gaaaaagaga tttatataca
tctcttcttc tacttccttc ttactagaag 180 atg gca ata gaa cac aat gag
caa caa gaa cta tct caa tct ttt ctt 228 Met Ala Ile Glu His Asn Glu
Gln Gln Glu Leu Ser Gln Ser Phe Leu 1 5 10 15 tta gat gct ctt tac
tgt gaa gaa gaa gaa gaa aaa tgg gga gat tta 276 Leu Asp Ala Leu Tyr
Cys Glu Glu Glu Glu Glu Lys Trp Gly Asp Leu 20 25 30 gta gat gat
gag act att att aca cca ctc tct tca gaa gta aca aca 324 Val Asp Asp
Glu Thr Ile Ile Thr Pro Leu Ser Ser Glu Val Thr Thr 35 40 45 aca
aca aca aca aca aca aag cct aat tct tta tta cct ttg ctt ttg 372 Thr
Thr Thr Thr Thr Thr Lys Pro Asn Ser Leu Leu Pro Leu Leu Leu 50 55
60 ttg gaa caa gat tta ttt tgg gaa gat gaa gag ctt ctt tca ctt ttc
420 Leu Glu Gln Asp Leu Phe Trp Glu Asp Glu Glu Leu Leu Ser Leu Phe
65 70 75 80 tct aaa gaa aaa gaa acc cat tgt tgg ttt aac agt ttt caa
gat gac 468 Ser Lys Glu Lys Glu Thr His Cys Trp Phe Asn Ser Phe Gln
Asp Asp 85 90 95 tct tta ctc tgt tct gcc cgt gtt gat tct gtg gaa
tgg att tta aaa 516 Ser Leu Leu Cys Ser Ala Arg Val Asp Ser Val Glu
Trp Ile Leu Lys 100 105 110 gtg aat ggt tat tat ggt ttc tct gct ttg
act gcc gtt tta gcc ata 564 Val Asn Gly Tyr Tyr Gly Phe Ser Ala Leu
Thr Ala Val Leu Ala Ile 115 120 125 aat tac ttt gac agg ttt ctg act
agt ctt cat tat cag aaa gat aaa 612 Asn Tyr Phe Asp Arg Phe Leu Thr
Ser Leu His Tyr Gln Lys Asp Lys 130 135 140 cct tgg atg att caa ctt
gct gct gtt act tgt ctt tct tta gct gct 660 Pro Trp Met Ile Gln Leu
Ala Ala Val Thr Cys Leu Ser Leu Ala Ala 145 150 155 160 aaa gtt gaa
gaa act caa gtt cct ctt ctt tta gat ttt caa gtg gag 708 Lys Val Glu
Glu Thr Gln Val Pro Leu Leu Leu Asp Phe Gln Val Glu 165 170 175 gat
gct aaa tat gtg ttt gag gca aaa act att caa aga atg gag ctt 756 Asp
Ala Lys Tyr Val Phe Glu Ala Lys Thr Ile Gln Arg Met Glu Leu 180 185
190 tta gtg ttg tct tca cta aaa tgg agg atg aat cca gtg acc cca ctt
804 Leu Val Leu Ser Ser Leu Lys Trp Arg Met Asn Pro Val Thr Pro Leu
195 200 205 tca ttt ctt gat cat att ata agg agg ctt ggg cta aga aat
aat att 852 Ser Phe Leu Asp His Ile Ile Arg Arg Leu Gly Leu Arg Asn
Asn Ile 210 215 220 cac tgg gaa ttt ctt aga aga tgt gaa aat ctc ctc
ctc tct att atg 900 His Trp Glu Phe Leu Arg Arg Cys Glu Asn Leu Leu
Leu Ser Ile Met 225 230 235 240 gct gat tgt aga ttc gta cgt tat atg
ccg tct gta ttg gcc act gca 948 Ala Asp Cys Arg Phe Val Arg Tyr Met
Pro Ser Val Leu Ala Thr Ala 245 250 255 att atg ctt cac gtt att cat
caa gtt gag cct tgt aat tct gtt gac 996 Ile Met Leu His Val Ile His
Gln Val Glu Pro Cys Asn Ser Val Asp 260 265 270 tac caa aat caa ctt
ctt ggg gtt ctc aaa att aac aag gag aaa gtg 1044 Tyr Gln Asn Gln
Leu Leu Gly Val Leu Lys Ile Asn Lys Glu Lys Val 275 280 285 aat aat
tgc ttt gaa ctc ata tca gaa gtg tgt tct aag ccc att tca 1092 Asn
Asn Cys Phe Glu Leu Ile Ser Glu Val Cys Ser Lys Pro Ile Ser 290 295
300 cac aaa cgc aaa tat gag aat cct agt cat agc cca agt ggt gta att
1140 His Lys Arg Lys Tyr Glu Asn Pro Ser His Ser Pro Ser Gly Val
Ile 305 310 315 320 gat cca att tac agt tca gaa agt tca aat gat tca
tgg gat ttg gag 1188 Asp Pro Ile Tyr Ser Ser Glu Ser Ser Asn Asp
Ser Trp Asp Leu Glu 325 330 335 tca aca tct tca tat ttt cct gtt ttc
aag aaa agc aga gta caa gaa 1236 Ser Thr Ser Ser Tyr Phe Pro Val
Phe Lys Lys Ser Arg Val Gln Glu 340 345 350 cag caa atg aaa ttg gca
tct tca att agc aga gtt ttt gtg gaa gct 1284 Gln Gln Met Lys Leu
Ala Ser Ser Ile Ser Arg Val Phe Val Glu Ala 355 360 365 gtt ggt agt
cct cat taaaatcaat cacctgattt atctcttttc tttcttatta 1339 Val Gly
Ser Pro His 370 ccaactatgg tggtaataat atttattgat attcagaagt
atttaccttt aatgtcattt 1399 tcaaaaatta catgaaaatg gaaaaaaaga
aaagaagagc ttagctggtg gttgcagttg 1459 gcagagaaga ggactggctt
ttttttgcag gagtgtagtc tactactact ggaaagcaga 1519 gatagagaga
ggagaaaaga cagaaaatct gcactatttg ttttttctct attcatatca 1579
attctctctt aggtcctttt catgcatgca tacttttgat ggacatattt tatatattta
1639 ctataatcat aaattcttga ataaaaaaaa aaaaaaaaaa 1679 4 373 PRT
Nicotiana tabacum 4 Met Ala Ile Glu His Asn Glu Gln Gln Glu Leu Ser
Gln Ser Phe Leu 1 5 10 15 Leu Asp Ala Leu Tyr Cys Glu Glu Glu Glu
Glu Lys Trp Gly Asp Leu 20 25 30 Val Asp Asp Glu Thr Ile Ile Thr
Pro Leu Ser Ser Glu Val Thr Thr 35 40 45 Thr Thr Thr Thr Thr Thr
Lys Pro Asn Ser Leu Leu Pro Leu Leu Leu 50 55 60 Leu Glu Gln Asp
Leu Phe Trp Glu Asp Glu Glu Leu Leu Ser Leu Phe 65 70 75 80 Ser Lys
Glu Lys Glu Thr His Cys Trp Phe Asn Ser Phe Gln Asp Asp 85 90 95
Ser Leu Leu Cys Ser Ala Arg Val Asp Ser Val Glu Trp Ile Leu Lys 100
105 110 Val Asn Gly Tyr Tyr Gly Phe Ser Ala Leu Thr Ala Val Leu Ala
Ile 115 120 125 Asn Tyr Phe Asp Arg Phe Leu Thr Ser Leu His Tyr Gln
Lys Asp Lys 130 135 140 Pro Trp Met Ile Gln Leu Ala Ala Val Thr Cys
Leu Ser Leu Ala Ala 145 150 155 160 Lys Val Glu Glu Thr Gln Val Pro
Leu Leu Leu Asp Phe Gln Val Glu 165 170 175 Asp Ala Lys Tyr Val Phe
Glu Ala Lys Thr Ile Gln Arg Met Glu Leu 180 185 190 Leu Val Leu Ser
Ser Leu Lys Trp Arg Met Asn Pro Val Thr Pro Leu 195 200 205 Ser Phe
Leu Asp His Ile Ile Arg Arg Leu Gly Leu Arg Asn Asn Ile 210 215 220
His Trp Glu Phe Leu Arg Arg Cys Glu Asn Leu Leu Leu Ser Ile Met 225
230 235 240 Ala Asp Cys Arg Phe Val Arg Tyr Met Pro Ser Val Leu Ala
Thr Ala 245 250 255 Ile Met Leu His Val Ile His Gln Val Glu Pro Cys
Asn Ser Val Asp 260 265 270 Tyr Gln Asn Gln Leu Leu Gly Val Leu Lys
Ile Asn Lys Glu Lys Val 275 280 285 Asn Asn Cys Phe Glu Leu Ile Ser
Glu Val Cys Ser Lys Pro Ile Ser 290 295 300 His Lys Arg Lys Tyr Glu
Asn Pro Ser His Ser Pro Ser Gly Val Ile 305 310 315 320 Asp Pro Ile
Tyr Ser Ser Glu Ser Ser Asn Asp Ser Trp Asp Leu Glu 325 330 335 Ser
Thr Ser Ser Tyr Phe Pro Val Phe Lys Lys Ser Arg Val Gln Glu 340 345
350 Gln Gln Met Lys Leu Ala Ser Ser Ile Ser Arg Val Phe Val Glu Ala
355 360 365 Val Gly Ser Pro His 370 5 1431 DNA Nicotiana tabacum
CDS (198)..(1298) cDNA encoding cyclin CYCD3;2 5 cacctttact
ctcttctcct ttttggctct tcccattctc tccttctctt tctttatttt 60
ctgtcctgta gagagagaga gaaagtataa gcaaagcagc agatatgtta ctgggtccaa
120 gattgagttt tggcttacct tgaagataat gagtagagcc tccattgtct
tcttccgtca 180 agaagaagaa gaagaag atg gtt ttc cct tta gat act cag
ctc cta aat 230 Met Val Phe Pro Leu Asp Thr Gln Leu Leu Asn 1 5 10
cca atc ttt gat gtc ctt tac tgt gag gaa gat cga ttc ttg gac gat 278
Pro Ile Phe Asp Val Leu Tyr Cys Glu Glu Asp Arg Phe Leu Asp Asp 15
20 25 gat gat tta gga gaa tgg tct agt act tta gaa caa gta gga aat
aat 326 Asp Asp Leu Gly Glu Trp Ser Ser Thr Leu Glu Gln Val Gly Asn
Asn 30 35 40 gtg aaa aag act cta cct tta tta gaa tgt gac atg ttt
tgg gaa gat 374 Val Lys Lys Thr Leu Pro Leu Leu Glu Cys Asp Met Phe
Trp Glu Asp 45 50 55 gac cag ctt gtc act ctt tta act aag gaa aaa
gag tct cat ttg ggt 422 Asp Gln Leu Val Thr Leu Leu Thr Lys Glu Lys
Glu Ser His Leu Gly 60 65 70 75 ttt gat tgt tta atc tca gat gga gat
ggg ttt tta gtg gag gtt aga 470 Phe Asp Cys Leu Ile Ser Asp Gly Asp
Gly Phe Leu Val Glu Val Arg 80 85 90 aaa gag gca ttg gat tgg atg
ttg aga gtc att gct cac tat ggt ttc 518 Lys Glu Ala Leu Asp Trp Met
Leu Arg Val Ile Ala His Tyr Gly Phe 95 100 105 act gct atg act gct
gtt tta gct gtg aat tat ttt gat agg ttt gta 566 Thr Ala Met Thr Ala
Val Leu Ala Val Asn Tyr Phe Asp Arg Phe Val 110 115 120 tct gga ctc
tgc ttt cag aaa gat aag cct tgg atg agt caa ctt gct 614 Ser Gly Leu
Cys Phe Gln Lys Asp Lys Pro Trp Met Ser Gln Leu Ala 125 130 135 gct
gtg gct tgt ctt tct att gct gct aaa gtg gaa gag acc caa gtc 662 Ala
Val Ala Cys Leu Ser Ile Ala Ala Lys Val Glu Glu Thr Gln Val 140 145
150 155 ccc ctt ctc tta gac ctc caa gtg gct gat tca aga ttt gtg ttt
gag 710 Pro Leu Leu Leu Asp Leu Gln Val Ala Asp Ser Arg Phe Val Phe
Glu 160 165 170 gca aag act att cag aga atg gaa ctc ttg gtg ctc tcc
act ctt aag 758 Ala Lys Thr Ile Gln Arg Met Glu Leu Leu Val Leu Ser
Thr Leu Lys 175 180 185 tgg aaa atg aat cca gtg aca cca cta tct ttc
att gat cat atc atg 806 Trp Lys Met Asn Pro Val Thr Pro Leu Ser Phe
Ile Asp His Ile Met 190 195 200 agg aga ttt gga ttc atg acc aat cta
cat ttg gat ttt ctt agg aga 854 Arg Arg Phe Gly Phe Met Thr Asn Leu
His Leu Asp Phe Leu Arg Arg 205
210 215 tgt gaa cgc ctc att ctt ggt att atc act gat tct agg ctc ttg
cat 902 Cys Glu Arg Leu Ile Leu Gly Ile Ile Thr Asp Ser Arg Leu Leu
His 220 225 230 235 tat cct cca tct gtt att gca act gca gta gtg tat
ttc gtg atc aat 950 Tyr Pro Pro Ser Val Ile Ala Thr Ala Val Val Tyr
Phe Val Ile Asn 240 245 250 gag att gag cct tgc aat gca atg gaa tac
cag aat cag ctc atg act 998 Glu Ile Glu Pro Cys Asn Ala Met Glu Tyr
Gln Asn Gln Leu Met Thr 255 260 265 gtt ctt aaa gtc aaa cag gat agt
ttt gaa gaa tgc cat gat ctt att 1046 Val Leu Lys Val Lys Gln Asp
Ser Phe Glu Glu Cys His Asp Leu Ile 270 275 280 cta gag cta atg ggc
act tct ggc tac aat atc tgc caa agc ctc aag 1094 Leu Glu Leu Met
Gly Thr Ser Gly Tyr Asn Ile Cys Gln Ser Leu Lys 285 290 295 cgc aaa
cat caa tct gta cct ggc agt cca agt gga gtt atc gat gca 1142 Arg
Lys His Gln Ser Val Pro Gly Ser Pro Ser Gly Val Ile Asp Ala 300 305
310 315 tat ttt agt tgc gac agc tct aat gat tcg tgg tcg gta gca tct
tca 1190 Tyr Phe Ser Cys Asp Ser Ser Asn Asp Ser Trp Ser Val Ala
Ser Ser 320 325 330 att tca tcg tca cca gaa cct cag tat aag agg atc
aaa act cag gat 1238 Ile Ser Ser Ser Pro Glu Pro Gln Tyr Lys Arg
Ile Lys Thr Gln Asp 335 340 345 cag aca atg aca ctg gct cca ctg agt
tct gtt tct gtc gtt gtg ggc 1286 Gln Thr Met Thr Leu Ala Pro Leu
Ser Ser Val Ser Val Val Val Gly 350 355 360 agt agt cct cgt
tgatcagtat ctcattctct agattatcta gtattacggc 1338 Ser Ser Pro Arg
365 tatggttact atatgatctc tcttttttgg tatgttctct taaactgcag
ttgcacaatg 1398 ctctgatgtt ccattaaaaa aaaaaaaaaa aaa 1431 6 367 PRT
Nicotiana tabacum 6 Met Val Phe Pro Leu Asp Thr Gln Leu Leu Asn Pro
Ile Phe Asp Val 1 5 10 15 Leu Tyr Cys Glu Glu Asp Arg Phe Leu Asp
Asp Asp Asp Leu Gly Glu 20 25 30 Trp Ser Ser Thr Leu Glu Gln Val
Gly Asn Asn Val Lys Lys Thr Leu 35 40 45 Pro Leu Leu Glu Cys Asp
Met Phe Trp Glu Asp Asp Gln Leu Val Thr 50 55 60 Leu Leu Thr Lys
Glu Lys Glu Ser His Leu Gly Phe Asp Cys Leu Ile 65 70 75 80 Ser Asp
Gly Asp Gly Phe Leu Val Glu Val Arg Lys Glu Ala Leu Asp 85 90 95
Trp Met Leu Arg Val Ile Ala His Tyr Gly Phe Thr Ala Met Thr Ala 100
105 110 Val Leu Ala Val Asn Tyr Phe Asp Arg Phe Val Ser Gly Leu Cys
Phe 115 120 125 Gln Lys Asp Lys Pro Trp Met Ser Gln Leu Ala Ala Val
Ala Cys Leu 130 135 140 Ser Ile Ala Ala Lys Val Glu Glu Thr Gln Val
Pro Leu Leu Leu Asp 145 150 155 160 Leu Gln Val Ala Asp Ser Arg Phe
Val Phe Glu Ala Lys Thr Ile Gln 165 170 175 Arg Met Glu Leu Leu Val
Leu Ser Thr Leu Lys Trp Lys Met Asn Pro 180 185 190 Val Thr Pro Leu
Ser Phe Ile Asp His Ile Met Arg Arg Phe Gly Phe 195 200 205 Met Thr
Asn Leu His Leu Asp Phe Leu Arg Arg Cys Glu Arg Leu Ile 210 215 220
Leu Gly Ile Ile Thr Asp Ser Arg Leu Leu His Tyr Pro Pro Ser Val 225
230 235 240 Ile Ala Thr Ala Val Val Tyr Phe Val Ile Asn Glu Ile Glu
Pro Cys 245 250 255 Asn Ala Met Glu Tyr Gln Asn Gln Leu Met Thr Val
Leu Lys Val Lys 260 265 270 Gln Asp Ser Phe Glu Glu Cys His Asp Leu
Ile Leu Glu Leu Met Gly 275 280 285 Thr Ser Gly Tyr Asn Ile Cys Gln
Ser Leu Lys Arg Lys His Gln Ser 290 295 300 Val Pro Gly Ser Pro Ser
Gly Val Ile Asp Ala Tyr Phe Ser Cys Asp 305 310 315 320 Ser Ser Asn
Asp Ser Trp Ser Val Ala Ser Ser Ile Ser Ser Ser Pro 325 330 335 Glu
Pro Gln Tyr Lys Arg Ile Lys Thr Gln Asp Gln Thr Met Thr Leu 340 345
350 Ala Pro Leu Ser Ser Val Ser Val Val Val Gly Ser Ser Pro Arg 355
360 365 7 1788 DNA Helianthus tuberosus CDS (165)..(1109) cDNA
encoding cyclin CYCD1;1 7 cacaacaatc acttctactc actattcact
acttactaat cactgcaact tctccggcca 60 cttttcacct caaaccgccg
gaactccgcc gctccggtcg acggtgaatc actgaatctt 120 agcaattatg
ttcacaacag tatgaacaat caacaccggt catc atg tca atc tcg 176 Met Ser
Ile Ser 1 tgc tct gac tgc ttc tcc gac tta ctc tgc tgc gag gac tcc
ggc ata 224 Cys Ser Asp Cys Phe Ser Asp Leu Leu Cys Cys Glu Asp Ser
Gly Ile 5 10 15 20 tta tcc ggc gac gac cgg ccg gag tgc tcc tat gat
ttc gaa tat tcc 272 Leu Ser Gly Asp Asp Arg Pro Glu Cys Ser Tyr Asp
Phe Glu Tyr Ser 25 30 35 ggc gac ttt gat gat tcg atc gcg gag ttt
ata gaa cag gag aga aag 320 Gly Asp Phe Asp Asp Ser Ile Ala Glu Phe
Ile Glu Gln Glu Arg Lys 40 45 50 ttc gtt cca gga atc gat tac gtc
gag cga ttt caa tcg caa gtt ctc 368 Phe Val Pro Gly Ile Asp Tyr Val
Glu Arg Phe Gln Ser Gln Val Leu 55 60 65 gat gct tct gct aga gaa
gaa tcg gtt gcc tgg atc ctt aag gtg caa 416 Asp Ala Ser Ala Arg Glu
Glu Ser Val Ala Trp Ile Leu Lys Val Gln 70 75 80 cgg ttt tac gga
ttt cag ccg ttg acg gcg tac ctc tcc gtt aac tat 464 Arg Phe Tyr Gly
Phe Gln Pro Leu Thr Ala Tyr Leu Ser Val Asn Tyr 85 90 95 100 ctg
gat cgt ttc atc tat tgc cgt ggc ttc ccg gtg gca aat ggg tgg 512 Leu
Asp Arg Phe Ile Tyr Cys Arg Gly Phe Pro Val Ala Asn Gly Trp 105 110
115 ccc ttg caa ctc tta tct gta gca tgc ttg tct tta gct gct aaa atg
560 Pro Leu Gln Leu Leu Ser Val Ala Cys Leu Ser Leu Ala Ala Lys Met
120 125 130 gag gaa acc ctt att cct tct att ctt gat ctc cag gtt gaa
ggt gca 608 Glu Glu Thr Leu Ile Pro Ser Ile Leu Asp Leu Gln Val Glu
Gly Ala 135 140 145 aaa tat att ttc gag ccg aaa aca atc cga aga atg
gag ttt ctt gtg 656 Lys Tyr Ile Phe Glu Pro Lys Thr Ile Arg Arg Met
Glu Phe Leu Val 150 155 160 ctt agt gtt ttg gat tgg aga cta aga tcc
gtt aca ccg ttt agc ttt 704 Leu Ser Val Leu Asp Trp Arg Leu Arg Ser
Val Thr Pro Phe Ser Phe 165 170 175 180 atc ggc ttc ttt tcg cac aaa
atc gat cca tct gga atg tat acg ggt 752 Ile Gly Phe Phe Ser His Lys
Ile Asp Pro Ser Gly Met Tyr Thr Gly 185 190 195 ttc ctt atc tca agg
gca aca caa att atc ctc tca aat att caa gaa 800 Phe Leu Ile Ser Arg
Ala Thr Gln Ile Ile Leu Ser Asn Ile Gln Glu 200 205 210 gct agt tta
ctt gag tat tgg cca tca tgt att gct gct gca aca ata 848 Ala Ser Leu
Leu Glu Tyr Trp Pro Ser Cys Ile Ala Ala Ala Thr Ile 215 220 225 ctt
tgt gca gca agt gat ctt tct aaa ttc tca ctt atc aat gct gat 896 Leu
Cys Ala Ala Ser Asp Leu Ser Lys Phe Ser Leu Ile Asn Ala Asp 230 235
240 cat gct gaa tca tgg tgt gat ggc ctt agc aaa gag aag atc aca aaa
944 His Ala Glu Ser Trp Cys Asp Gly Leu Ser Lys Glu Lys Ile Thr Lys
245 250 255 260 tgt tac aga ctt gta caa tct cca aag ata ttg ccg gta
cat gtt cga 992 Cys Tyr Arg Leu Val Gln Ser Pro Lys Ile Leu Pro Val
His Val Arg 265 270 275 gtc atg acg gct cga gtg agt act gag tca ggt
gac tca tcg tcg tcg 1040 Val Met Thr Ala Arg Val Ser Thr Glu Ser
Gly Asp Ser Ser Ser Ser 280 285 290 tct tct tcg cca tcg cct tac aaa
aag agg aaa cta aat aac tac tca 1088 Ser Ser Ser Pro Ser Pro Tyr
Lys Lys Arg Lys Leu Asn Asn Tyr Ser 295 300 305 tgg ata gag gag gac
aaa aga tgaaaataag gagacaaaat aaataaataa 1139 Trp Ile Glu Glu Asp
Lys Arg 310 315 atccggattc ctctctatat tttttaaagg aatcaacaaa
tatatataaa aaaaaaaaat 1199 ggagtcagga aaagcaacga aagccgccgg
aggaagaaaa ggcgccggag cgaggaagaa 1259 gtccgtcaca aagtccgtca
aagccggtct ccagttcccc gtcggaagaa tcgctaggtt 1319 tctaaaaaaa
ggccgatacg ctcaacgtac cggatccgga gctccgatct accttgctgc 1379
tgttctagaa taccttgctg ctgaggtttt ggagttggcg ggaaatgcag cgagagataa
1439 caagaagaca aggataaacc ctaggcactt gctattggct gttaggaacg
atgaggaatt 1499 ggggaaattg cttgctggtg ttactattgc tagtggaggt
gtgttgccca atatcaatcc 1559 ggttcttttg cccaagaagt cttcttcttc
ttctgctgct gagaagaccc ccaaatctaa 1619 aaagtcgcct aaaaaggctg
cttagataga tgtttctggt tatagttggt tagattaagt 1679 tgaagcaaaa
cagtctcttt tgttcaatta gtcgtctggc aatgtaacta ttttggtcgt 1739
cttcaaaatg ttaattggat actatcttct ttaaaaaaaa aaaaaaaaa 1788 8 315
PRT Helianthus tuberosus 8 Met Ser Ile Ser Cys Ser Asp Cys Phe Ser
Asp Leu Leu Cys Cys Glu 1 5 10 15 Asp Ser Gly Ile Leu Ser Gly Asp
Asp Arg Pro Glu Cys Ser Tyr Asp 20 25 30 Phe Glu Tyr Ser Gly Asp
Phe Asp Asp Ser Ile Ala Glu Phe Ile Glu 35 40 45 Gln Glu Arg Lys
Phe Val Pro Gly Ile Asp Tyr Val Glu Arg Phe Gln 50 55 60 Ser Gln
Val Leu Asp Ala Ser Ala Arg Glu Glu Ser Val Ala Trp Ile 65 70 75 80
Leu Lys Val Gln Arg Phe Tyr Gly Phe Gln Pro Leu Thr Ala Tyr Leu 85
90 95 Ser Val Asn Tyr Leu Asp Arg Phe Ile Tyr Cys Arg Gly Phe Pro
Val 100 105 110 Ala Asn Gly Trp Pro Leu Gln Leu Leu Ser Val Ala Cys
Leu Ser Leu 115 120 125 Ala Ala Lys Met Glu Glu Thr Leu Ile Pro Ser
Ile Leu Asp Leu Gln 130 135 140 Val Glu Gly Ala Lys Tyr Ile Phe Glu
Pro Lys Thr Ile Arg Arg Met 145 150 155 160 Glu Phe Leu Val Leu Ser
Val Leu Asp Trp Arg Leu Arg Ser Val Thr 165 170 175 Pro Phe Ser Phe
Ile Gly Phe Phe Ser His Lys Ile Asp Pro Ser Gly 180 185 190 Met Tyr
Thr Gly Phe Leu Ile Ser Arg Ala Thr Gln Ile Ile Leu Ser 195 200 205
Asn Ile Gln Glu Ala Ser Leu Leu Glu Tyr Trp Pro Ser Cys Ile Ala 210
215 220 Ala Ala Thr Ile Leu Cys Ala Ala Ser Asp Leu Ser Lys Phe Ser
Leu 225 230 235 240 Ile Asn Ala Asp His Ala Glu Ser Trp Cys Asp Gly
Leu Ser Lys Glu 245 250 255 Lys Ile Thr Lys Cys Tyr Arg Leu Val Gln
Ser Pro Lys Ile Leu Pro 260 265 270 Val His Val Arg Val Met Thr Ala
Arg Val Ser Thr Glu Ser Gly Asp 275 280 285 Ser Ser Ser Ser Ser Ser
Ser Pro Ser Pro Tyr Lys Lys Arg Lys Leu 290 295 300 Asn Asn Tyr Ser
Trp Ile Glu Glu Asp Lys Arg 305 310 315 9 1414 DNA Helianthus
tuberosus CDS (48)..(1118) cDNA encoding CYCD3;1 9 ttgaaccttc
atttcttttc ttttcttctt tctaatcacc aacccca atg gcc att 56 Met Ala Ile
1 tta tca cca tat tca tct tct ttc tta gac aca ctc ttt tgc aat gaa
104 Leu Ser Pro Tyr Ser Ser Ser Phe Leu Asp Thr Leu Phe Cys Asn Glu
5 10 15 caa caa gat cat gaa tat cat gaa tat gag tat gaa gat gaa ttt
aca 152 Gln Gln Asp His Glu Tyr His Glu Tyr Glu Tyr Glu Asp Glu Phe
Thr 20 25 30 35 caa acc acc ctc aca gat tca tct gat ctc cat ctt ccc
ccc ctg gac 200 Gln Thr Thr Leu Thr Asp Ser Ser Asp Leu His Leu Pro
Pro Leu Asp 40 45 50 caa cta gat ttg tca tgg gaa cat gaa gag ctt
gtg tcc ttg ttc aca 248 Gln Leu Asp Leu Ser Trp Glu His Glu Glu Leu
Val Ser Leu Phe Thr 55 60 65 aaa gaa caa gag cag caa aaa caa acc
cct tgt act ctc tct ttt ggc 296 Lys Glu Gln Glu Gln Gln Lys Gln Thr
Pro Cys Thr Leu Ser Phe Gly 70 75 80 aaa act agt ccc tca gtt ttt
gct gct cgt aaa gag gct gta gat tgg 344 Lys Thr Ser Pro Ser Val Phe
Ala Ala Arg Lys Glu Ala Val Asp Trp 85 90 95 atc ctt aag gtc aaa
agt tgt tat gga ttc aca cct ctt aca gcc att 392 Ile Leu Lys Val Lys
Ser Cys Tyr Gly Phe Thr Pro Leu Thr Ala Ile 100 105 110 115 tta gcc
atc aat tat ctt gat agg ttt ctt tct agc ctc cat ttt caa 440 Leu Ala
Ile Asn Tyr Leu Asp Arg Phe Leu Ser Ser Leu His Phe Gln 120 125 130
gaa gat aaa cct tgg atg att caa ctt gtt gct gtt agt tgt ctc tct 488
Glu Asp Lys Pro Trp Met Ile Gln Leu Val Ala Val Ser Cys Leu Ser 135
140 145 tta gct gct aaa gtt gaa gaa act caa gtg cca ctc tta cta gat
ctt 536 Leu Ala Ala Lys Val Glu Glu Thr Gln Val Pro Leu Leu Leu Asp
Leu 150 155 160 caa gta gag gac act aag tac ttg ttt gag gct aaa aac
ata caa aaa 584 Gln Val Glu Asp Thr Lys Tyr Leu Phe Glu Ala Lys Asn
Ile Gln Lys 165 170 175 atg gag ctt ttg gtg atg tca act ttg aaa tgg
agg atg aac cca gtg 632 Met Glu Leu Leu Val Met Ser Thr Leu Lys Trp
Arg Met Asn Pro Val 180 185 190 195 aca cca atc tca ttt ctt gat cac
att gta aga agg ctt gga tta act 680 Thr Pro Ile Ser Phe Leu Asp His
Ile Val Arg Arg Leu Gly Leu Thr 200 205 210 gat cat gtt cat tgg gat
ttt ttc aag aaa tgt gaa gct atg atc ctt 728 Asp His Val His Trp Asp
Phe Phe Lys Lys Cys Glu Ala Met Ile Leu 215 220 225 tgt tta gtt tca
gat tca aga ttc gtg tgt tat aaa cca tcc gtg ttg 776 Cys Leu Val Ser
Asp Ser Arg Phe Val Cys Tyr Lys Pro Ser Val Leu 230 235 240 gcc aca
gct aca atg ctt cac gtt gta gat gaa att gat cct ccc aat 824 Ala Thr
Ala Thr Met Leu His Val Val Asp Glu Ile Asp Pro Pro Asn 245 250 255
tgt att gac tac aaa agt caa ctt ctg gat ctt ctc aaa acc act aag 872
Cys Ile Asp Tyr Lys Ser Gln Leu Leu Asp Leu Leu Lys Thr Thr Lys 260
265 270 275 gac gac ata aac gag tgt tac gag ctc att gtc gag cta gct
tac gat 920 Asp Asp Ile Asn Glu Cys Tyr Glu Leu Ile Val Glu Leu Ala
Tyr Asp 280 285 290 cat cac aac aaa cga aaa cat gat gca aac gag aca
aca acc aat ccg 968 His His Asn Lys Arg Lys His Asp Ala Asn Glu Thr
Thr Thr Asn Pro 295 300 305 gtt agt cca gct ggc gtg atc gat ttc act
tgt gat gaa agt tca aat 1016 Val Ser Pro Ala Gly Val Ile Asp Phe
Thr Cys Asp Glu Ser Ser Asn 310 315 320 gag tca tgg gaa ctt aat gct
cat cat ttc cgc gag cct tca ttc aag 1064 Glu Ser Trp Glu Leu Asn
Ala His His Phe Arg Glu Pro Ser Phe Lys 325 330 335 aaa aca aga atg
gat tca aca att cgg gtt cgg gtt tgg ttc act tat 1112 Lys Thr Arg
Met Asp Ser Thr Ile Arg Val Arg Val Trp Phe Thr Tyr 340 345 350 355
aag ctt taatcgaggg tagttgtaaa catgtaatcc gcatgcacgc tattaatcct 1168
Lys Leu acggtccact actacatata atcggcctat aaaattatag gttaagatga
ccagtcgtag 1228 gcgtcgagat gtccttatgg ttggtcaatt tctctatggt
tttaggtcgt ttttaatgtg 1288 agataaatta aattcggtat gttaagtctt
tatcaagcaa tggacgttat atttattgtt 1348 tgatattgag aattaaattc
catgggaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1408 aaaaaa 1414 10 357
PRT Helianthus tuberosus 10 Met Ala Ile Leu Ser Pro Tyr Ser Ser Ser
Phe Leu Asp Thr Leu Phe 1 5 10 15 Cys Asn Glu Gln Gln Asp His Glu
Tyr His Glu Tyr Glu Tyr Glu Asp 20 25 30 Glu Phe Thr Gln Thr Thr
Leu Thr Asp Ser Ser Asp Leu His Leu Pro 35 40 45 Pro Leu Asp Gln
Leu Asp Leu Ser Trp Glu His Glu Glu Leu Val Ser 50 55 60 Leu Phe
Thr Lys Glu Gln Glu Gln Gln Lys Gln Thr Pro Cys Thr Leu 65 70 75 80
Ser Phe Gly Lys Thr Ser Pro Ser Val Phe Ala Ala Arg Lys Glu Ala 85
90 95 Val Asp Trp Ile Leu Lys Val Lys Ser Cys Tyr Gly Phe Thr Pro
Leu 100 105 110 Thr Ala Ile Leu Ala Ile Asn Tyr Leu Asp Arg Phe Leu
Ser Ser Leu 115 120 125 His Phe Gln Glu Asp Lys Pro Trp Met Ile Gln
Leu Val Ala Val Ser 130 135 140 Cys Leu Ser Leu
Ala Ala Lys Val Glu Glu Thr Gln Val Pro Leu Leu 145 150 155 160 Leu
Asp Leu Gln Val Glu Asp Thr Lys Tyr Leu Phe Glu Ala Lys Asn 165 170
175 Ile Gln Lys Met Glu Leu Leu Val Met Ser Thr Leu Lys Trp Arg Met
180 185 190 Asn Pro Val Thr Pro Ile Ser Phe Leu Asp His Ile Val Arg
Arg Leu 195 200 205 Gly Leu Thr Asp His Val His Trp Asp Phe Phe Lys
Lys Cys Glu Ala 210 215 220 Met Ile Leu Cys Leu Val Ser Asp Ser Arg
Phe Val Cys Tyr Lys Pro 225 230 235 240 Ser Val Leu Ala Thr Ala Thr
Met Leu His Val Val Asp Glu Ile Asp 245 250 255 Pro Pro Asn Cys Ile
Asp Tyr Lys Ser Gln Leu Leu Asp Leu Leu Lys 260 265 270 Thr Thr Lys
Asp Asp Ile Asn Glu Cys Tyr Glu Leu Ile Val Glu Leu 275 280 285 Ala
Tyr Asp His His Asn Lys Arg Lys His Asp Ala Asn Glu Thr Thr 290 295
300 Thr Asn Pro Val Ser Pro Ala Gly Val Ile Asp Phe Thr Cys Asp Glu
305 310 315 320 Ser Ser Asn Glu Ser Trp Glu Leu Asn Ala His His Phe
Arg Glu Pro 325 330 335 Ser Phe Lys Lys Thr Arg Met Asp Ser Thr Ile
Arg Val Arg Val Trp 340 345 350 Phe Thr Tyr Lys Leu 355 11 100 DNA
Artificial Sequence T-DNA of pGSV5 11 aattacaacg gtatatatcc
tgccagtact cggccgtcga ccgcggtacc cggggaagct 60 tagatccatg
gagccattta caattgaata tatcctgccg 100 12 17 DNA Artificial Sequence
derived from Arabidopsis thaliana 12 gcmtggatyc tyaaggt 17 13 17
DNA Artificial Sequence derived from Arabidopsis thaliana 13
tgcttgtcwt tagctgc 17 14 18 DNA Artificial Sequence derived from
Arabidopsis thaliana 14 aagaatggar yttcttgt 18 15 19 DNA Artificial
Sequence derived from Arabidopsis thaliana 15 aragnatycy kgcwgcagc
19 16 19 DNA Artificial Sequence derived from Arabidopsis thaliana
16 ccrtcacacc awgnytcag 19 17 16 DNA Artificial Sequence derived
from Arabidopsis thaliana 17 tggwgatttg gatttg 16 18 17 DNA
Artificial Sequence derived from Arabidopsis thaliana 18 atnaantact
tggatcg 17 19 19 DNA Artificial Sequence derived from Arabidopsis
thaliana 19 agcttgcant ctccanttc 19 20 17 DNA Artificial Sequence
derived from Arabidopsis thaliana 20 tcagaagncc tgaantc 17 21 17
DNA Artificial Sequence derived from Arabidopsis thaliana 21
gantggatny tnaargt 17 22 17 DNA Artificial Sequence derived from
Arabidopsis thaliana 22 aagabaarcc wtggatg 17 23 20 DNA Artificial
Sequence derived from Arabidopsis thaliana 23 gtkgaagara ctcaagtbcc
20 24 24 DNA Artificial Sequence derived from Arabidopsis thaliana
24 tggngtnacw ggntkcatyy tcca 24 25 20 DNA Artificial Sequence
derived from Arabidopsis thaliana 25 gcwgnngcna nnncagangg 20 26
1846 DNA Zea mays 26 ctgcagtggc ctagccggcg tcgtcctccc cctctchcgc
tcctctgtcc tcccctctcc 60 acttgagaag aacacaatta ggaaaaaaag
gcaaaaaaca tttacctttt ttctatctgt 120 atattatctg aataaatcaa
gaggaggaag aggggaggga gcgagggagg gggaggagta 180 gcaaatccag
actccatagc aaccagctcg cgagaagggg aaaaggggga ggaagagctt 240
cgcttgtgta ttgattgctc gctgctccag tccctgcatt cgtgccgttt ttggcaagta
300 ggtggcgtgg caagcatggt gccgggctat gactgcgccg cctccgtgct
gctgtgcgcg 360 gaggacaacg ctgctattct cggcctggac gacgatgggg
aggagtcctc ctgggcggcc 420 gccgctacgc cgccacgtga caccgtcgcc
gccgccgccg ccaccggggt cgccgtcgat 480 gggattttga cggagttccc
cttgctctcg gatgactgcg ttgcgacgct cgtggagaag 540 gaggtggagc
acatgcccgc ggaggggtac ctccagaagc tgcagcgacg gcatggggac 600
ctggatttgg ccgccgtcag gaaggacgcc atcgattgga tttggaaggt cattgagcat
660 tacaatttcg caccgttgac tgccgttttg tctgtgaact acctcgatag
attcctctcc 720 acgtatgagt tccctgaagg cagagcttgg atgactcagc
tcttggcagt ggcttgcttg 780 tctttggctt cgaaaatcga agagactttt
gtgccactcc ccttggattt gcaggtagcg 840 gaggcaaagt ttgtttttga
gggaaggacc ataaaaagga tggagcttct ggtgctaagc 900 accttaaagt
ggaggatgca tgctgttact gcttgctcat ttgttgaata ctttcttcat 960
aaattgagtg atcatggtgc accctccttg cttgcacgct ctcgctcttc ggaccttgtc
1020 ttgagcaccg ctaaaggtgc tgaattcgtg gtattcagac cctccgagat
tgctgccagt 1080 gttgcacttg ctgctatcgg cgaatgcagg agttctgtaa
ttgagagagc tgctagtagc 1140 tgcaaatatt tggacaagga gagggtttta
agatgccatg aaatgattca agagaagatt 1200 actgcgggaa gcattgtcct
aaagtctgct ggatcatcaa tctcctctgt gccacaaagc 1260 ccaataggtg
tcctggacgc tgcagcctgt ctgagtcaac aaagcgatga cgctactgtc 1320
gggtctcctg cagtatgtta ccatagttct tccacaagca agaggagaag gatcactaga
1380 cgtctactct aattgtggta cgcttcaggt gtgctcctca ccgctctagg
agtttttgat 1440 tggttcaaac atcttaaatt tagtttggcc gctggaggat
tatggtttag tcaagtagtt 1500 gctgaatgga caacaaaaca cgcacactac
ttggtccata aagacaagaa aataactggc 1560 agcgtcccgc gagccagcgc
tgcaatccag ttcatgcaag accctagagt ccaggggggg 1620 tgctggtgta
ggtagagagg gaacaaggca ttcacatacg ccgtagagat gagagagcct 1680
ctcgtatgtt ttgtactttt gctccttcag tttgcaatga actatataaa caaggattgc
1740 cttggggcag tgaacatttg tcggatgaaa agaatcaaaa aggatggggg
tcggcagagg 1800 aatagaacaa tttgatatat ttccataaac taaaaaaaaa aaaaaa
1846
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