U.S. patent application number 10/551696 was filed with the patent office on 2007-03-22 for plants having improved growth characteristics and a method for making the same.
This patent application is currently assigned to CROPDESIGN N.V.. Invention is credited to Valerie Frankard, Gabor Horvath, Eva Kondorosi, Sylvie Tarayre.
Application Number | 20070067875 10/551696 |
Document ID | / |
Family ID | 33104196 |
Filed Date | 2007-03-22 |
United States Patent
Application |
20070067875 |
Kind Code |
A1 |
Horvath; Gabor ; et
al. |
March 22, 2007 |
Plants having improved growth characteristics and a method for
making the same
Abstract
The present invention concerns a method for improving plant
growth characteristics by increasing expression in a plant of a
nucleic acid encoding a CCS52 protein and/or by increasing level
and/or activity in a plant of a CCS52 protein. The invention also
relates to transgenic plants having improved growth
characteristics, such as increased plant size, increased organ size
or increased number of organs, which plants have increased
expression of a nucleic acid encoding a CCS52 protein.
Inventors: |
Horvath; Gabor; (Szeged,
HU) ; Tarayre; Sylvie; (Arpajon, FR) ;
Kondorosi; Eva; (Bures sur Yvette, FR) ; Frankard;
Valerie; (Rhodes-Saint-Genese, BE) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
CROPDESIGN N.V.
Zwijnaarde
BE
B-9052
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (C.N.R.S.)
Paris
FR
F-75016
|
Family ID: |
33104196 |
Appl. No.: |
10/551696 |
Filed: |
March 31, 2004 |
PCT Filed: |
March 31, 2004 |
PCT NO: |
PCT/IB04/00970 |
371 Date: |
August 8, 2006 |
Current U.S.
Class: |
800/287 ;
435/419; 435/468 |
Current CPC
Class: |
Y02A 40/146 20180101;
C12N 15/8261 20130101 |
Class at
Publication: |
800/287 ;
435/419; 435/468 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C12N 15/82 20060101 C12N015/82; C12N 5/04 20060101
C12N005/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2003 |
EP |
03290812.1 |
Claims
1. Method to improve plant growth characteristics relative to
corresponding wild-type plants, comprising introduction into a
plant of a nucleic acid encoding a CCS52 protein under the control
of a medium-strength promoter.
2. Method according to claim 1, wherein said growth characteristic
comprises increased yield/biomass.
3. Method according to claim 2, wherein said increased
yield/biomass comprises increased plant size, increased organ size
or increased number of organs.
4. Method according to claim 3, wherein said increased organ size
is selected from increased leaf size, increased seed size or
increased stem diameter.
5. Method according to claim 3, wherein said increased number of
organs is selected from increased number of leaves, increased
number of branches, increased number of flowers or increased number
of seeds.
6. Method according to claim 1, wherein said CCS52 protein is a
CCS52A protein.
7. Method according to claim 1, wherein said nucleic acid encoding
a CCS52 protein is as represented by SEQ ID NO 1, 3 or 5, or a
variant of any of SEQ ID NO 1, 3 or 5 and/or wherein said CCS52
protein is a protein as represented by SEQ ID NO 2, 4 or 6, or a
variant of any of SEQ ID NO 2, 4 or 6.
8. Method according to claim 1, wherein said medium-strength
promoter is a medium-strength constitutive promoter.
9. Method according to claim 8, wherein said promoter is a
ubiquitin promoter or a promoter with a similar expression
pattern.
10. Genetic construct comprising: (a) a CCS52 nucleic acid or a
variant thereof, encoding a CCS52 protein or a variant thereof;
operably linked to (b) a medium-strength promoter; and optionally
(c) a transcription termination sequence.
11. Genetic construct according to claim 10, wherein said promoter
is a medium-strength constitutive promoter.
12. Genetic construct according to claim 10, wherein said promoter
is a ubiquitin promoter or a promoter with a similar expression
pattern.
13. Method for the production of a transgenic plant having improved
growth characteristics relative to corresponding wild-type plants,
comprising: a) introducing into a plant cell a genetic construct
according claim 10; b) cultivating said plant cell under conditions
promoting plant growth.
14. Host cell containing a genetic construct as defined in claim
10.
15. Plant obtainable by a method according to claim 1, which plant
has improved growth characteristics relative to corresponding
wild-type plants.
16. Transgenic plant containing a genetic construct as defined in
claim 10, which plant has improved growth characteristics relative
to corresponding wild-type plants.
17. Transgenic plant according to claim 16, wherein said plant is a
monocotyledonous plant, preferably a cereal such as rice or
maize.
18. Transgenic plant according to claim 16, wherein said plant is a
dicotyledoneous plant, preferably a dicotyledoneous crop plant or
ornamental, such as azalea.
19. Plant part, preferably a harvestable part, such as a seed, or a
propagule of a plant as defined in claim 15.
20. Progeny of a plant as defined in claim 15.
21. Use of a nucleic acid encoding a CCS52 protein under control of
a medium-strength promoter for improving plant growth
characteristics.
Description
[0001] The present invention concerns a method for improving plant
growth characteristics. More specifically, the present invention
concerns a method for improving plant growth characteristics by
increasing, in a plant, expression of a cell cycle switch gene
encoding a 52 kDa protein (CCS52 protein) and/or by increasing
activity of the CCS52 protein itself. The present invention also
concerns plants having increased expression of a nucleic acid
encoding a CCS52 protein and/or increased activity of a CCS52
protein, which plants have improved growth characteristics relative
to corresponding wild-type plants.
[0002] Given the ever-increasing world population, it remains a
major goal of agricultural research to improve the efficiency of
agriculture. Conventional means for crop and horticultural
improvements utilise selective breeding techniques to identify
plants having desirable characteristics. However, such selective
breeding techniques have several drawbacks, namely that these
techniques are typically labour intensive and result in plants that
often contain heterogenous genetic complements that may not always
result in the desirable trait being passed on from parent plants.
In contrast, advances in molecular biology have allowed mankind to
more precisely manipulate the germplasm of plants. Genetic
engineering of plants entails the isolation and manipulation of
genetic material (typically in the form of DNA or RNA) and the
subsequent introduction of that genetic material into a plant. Such
technology has led to the development of plants having various
improved economic, agronomic or horticultural traits. A trait of
particular economic interest is high yield.
[0003] The ability to improve one or more plant growth
characteristics, would have many applications in areas such as crop
enhancement, plant breeding, production of ornamental plants,
arboriculture, horticulture, forestry, production of algae or
plants (for use as bioreactors for example, for the production of
pharmaceuticals, such as antibodies or vaccines, or for the
bioconversion of organic waste, or for use as fuel, in the case of
high-yielding algae and plants).
[0004] CCS52 belongs to a small group of proteins containing
several WD repeat motifs and is the plant homologue of animal APC
activators involved in mitotic cyclin degradation (WO99/64451). In
Cebolla et al. (EMBO J., 1999, 18: 4476-84), the isolation of CCS52
clones from Medicago sativa root nodules was reported and CCS52 was
described to be part of a small gene family that appears to be
conserved in plants. Furthermore, the functional domains and
regulation mechanisms of CCS52 proteins have been described in
detail by Tarayre et al. (The plant Cell, 2004, vol 16,
422-434).
[0005] In document WO99/64451 it was suggested that downregulation
of CCS52 expression pushes the cells towards proliferation and that
overproduction of CCS52 pushes the cells towards differentiation.
Also, in the document in the name of Kondorosi et al. (1999, The
EMBO J. 18 (16), p. 4476-4484), it is stated that expression of
CCS52 may switch proliferating cells to differentiation programs.
For some cells differentiation means endoreduplication. This switch
to differentiation (or endoreduplication) clearly involves an
arrest in proliferation, thus an arrest in cell division. These
data were in line with earlier findings in yeast that teach when
CCS52 is used to increase differentiation (or endoreduplication), a
cell cycle arrest is inevitably triggered. Therefore, the effect on
endoreduplication on the one hand, namely the increased cell size,
is inherently linked to a reduction of cell number due to cell
division arrest. The results obtained in Medicago and Arabidopsis,
for CCS52 overexpression driven by the CaMV35S promoter
corroborated this view.
[0006] The examples in document WO99/64451 show that Medicago
plants expressing an anti-sense version of a Medicago CCS52 gene
form fewer seeds and fewer lateral branches. Furthermore,
constructs for overexpression of a Medicago CCS52 gene, under
control of a strong constitutive promoter (CaMV35S), have been
disclosed and were used to transform Medicago plants. Although it
was indicated that overexpression of a CCS52 gene under the control
of a CaMV35S promoter resulted in a positive effect on somatic
embryogenesis, no plants were regenerated and no further positive
effects were observed. To the contrary, evidence has been presented
that overexpression of CCS52 under the control of a CaMV35S
promoter is detrimental. This detrimental effect was first observed
in Medicago transgenic plants. Later, this detrimental effect was
also observed in Arabidopsis thaliana transformed with the
Arabidopsis CCS52 gene under control of a CaMV35S promoter.
[0007] Therefore, the prior art does not teach how the CCS52 gene
can be used to improve plant growth characteristics, and so far
only negative results with respect to the use of CCS52 for growth
improvement have been obtained.
[0008] Unexpectedly, it has now been found that, in contrast to
earlier observations, overexpression of a CCS52 gene does not cause
a detrimental effect. Moreover, it has now been found that plant
growth characteristics may even be improved by the methods of the
present invention. These improved growth characteristics are
obtained when overexpression of a CCS52 gene in a plant is
controlled by an medium-strength promoter.
[0009] Further surprisingly, it has also been found that plants
made by the methods of the present invention have specific
characteristics such as increased plant size, increased organ size
and/or increased number of organs, compared to corresponding
wild-type plants.
[0010] Therefore, the present invention teaches how to improve
plant growth characteristics, such as plant size, organ size and/or
organ number by increased expression in a plant of a nucleic acid
encoding a CCS52 protein.
[0011] According to a first embodiment of the present invention,
there is provided a method to improve plant growth characteristics
relative to corresponding wild-type plants, comprising the
introduction into a plant of a nucleic acid encoding a CCS52
protein, under control of a medium-strength promoter.
[0012] The introduction into a plant of a nucleic acid encoding a
CCS52 protein under control of a medium-strength promoter, may
result in an increased expression of the nucleic acid encoding a
CCS52 protein. Additionally, this introduction may result in an
increased level and/or activity of the CCS52 protein.
[0013] Advantageously, and according to a preferred embodiment of
the present invention, increased expression of a nucleic acid
encoding a CCS52 protein and/or increased level and/or activity of
the CCS52 protein itself may be effected by a direct recombinant
approach, for example, by transforming the plant with a nucleic
acid encoding a CCS52 protein or a variant thereof.
[0014] Alternatively, increased expression of a nucleic acid
encoding a CCS52 protein and/or increased level and/or activity of
the CCS52 protein itself may be effected by an indirect recombinant
approach, for example, by transforming a plant to modify the
expression of a CCS52 gene already in that plant, which CCS52 gene
may be endogenous or a transgene (previously) introduced into the
plant. This may be effected by the inhibition or stimulation of
regulatory sequences that drive expression of the endogenous gene
or transgene. Such regulatory sequences may be introduced into a
plant. For example, a medium-strength promoter may be introduced
into a plant to drive the endogenous CCS52 gene, which
medium-strength promoter may be heterologous to the endogenous
CCS52 gene; Heterologous being not naturally occurring in the
nucleic acid sequences flanking the CCS52 coding region when it is
in its biological genomic environment.
[0015] The term "CCS52 protein" as used herein encompasses a cell
cycle switch gene encoding a 52 kDa protein and this term also
encompasses variants thereof. Examples of CCS52 proteins are herein
represented by SEQ ID NO 2, 4 or 6. Other examples of CCS52
proteins are described in Cebolla et al. (EMBO 1999, vol. 18(16)
4476-4484) and in Tarayre et al. (The plant cell, 2004, vol. 16:
422-434). The terms "CCS52 nucleic acid" or "CCS52 gene" or
"nucleic acid encoding a CCS52 protein" are used interchangeably
herein and encompass, for example, nucleic acids as represented by
SEQ ID NO 1, 3 or 5, or variants thereof. A variant CCS52 protein
or a variant nucleic acid encoding a CCS52 protein include:
[0016] (i) Functional portions of a CCS52 nucleic acid, for example
of SEQ ID NO 1, 3 or 5;
[0017] (ii) Nucleic acids capable of hybridising with a CCS52
nucleic acid, for example with SEQ ID NO 1, 3 or 5;
[0018] (iii) Alternative splice variants of a CCS52 nucleic acid,
for example of SEQ ID NO 1, 3 or 5;
[0019] (iv) Allelic variants of a CCS52 nucleic acid, for example
of SEQ ID NO1, 3 or5;
[0020] (v) Homologues of a CCS52 protein, for example of SEQ ID NO
2, 4 or 6;
[0021] (vi) Derivatives of a CCS52 protein, for example of SEQ ID
NO 2, 4 or 6; and
[0022] (vii) Active fragments of a CCS52 protein, for example of
SEQ ID NO 2, 4 or 6.
[0023] According to a preferred embodiment, such variants are (or
encode) proteins having at least one of the conserved CCS52 motifs
as described hereinafter.
[0024] According to a preferred embodiment, such variants are (or
encode) proteins having CCS52 activity, or are (or encode) proteins
that retain similar biological activity or at least part of the
biological activity of a CCS52 protein. The biological activity of
a CCS52 protein may be tested as described in Cebolla et al., 1999.
This test involves overexpressing the CCS52 or variant in
Saccharomyces pombe. The phenotypes of the transformed yeast cells
are compared with the phenotypes of yeast cells transformed with
the empty vector pREP1 as negative control, and with the phenotypes
of the yeast cells transformed with the pREP1-srw1.sup.+ as
positive control. Expression of either srw1.sup.+ or CCS52 should
result in growth arrest of the cells.
[0025] Advantageously, the methods according to the invention may
be practised using variant CCS52 proteins and variant CCS52 nucleic
acids. Suitable variants include variants of SEQ ID NO 2, 4 or 6
and/or variants of SEQ ID NO 1, 3 or 5.
[0026] The term "variant" includes variants in the form of a
complement, DNA, RNA, cDNA or genomic DNA. The variant nucleic acid
may be synthesized in whole or in part, it may be a double-stranded
nucleic acid or a single-stranded nucleic acid. Also, the term
"variant" encompasses a variant due to the degeneracy of the
genetic code, a family member of the gene or protein and variants
that are interrupted by one or more intervening sequences, such as
introns, spacer sequences or transposons.
[0027] One variant nucleic acid encoding a CCS52 protein is a
functional portion of a nucleic acid encoding a CCS52 protein.
Advantageously, the method of the present invention may also be
practised using a portion of a nucleic acid encoding a CCS52
protein. A functional portion refers to a piece of DNA derived from
an original (larger) DNA molecule, which portion, retains at least
part of the functionality of the original DNA, which functional
portion, when expressed in a plant, gives plants having improved
growth characteristics. The portion may be made by one or more
deletions and/or truncations of the nucleic acid. Techniques for
making such deletions and/or truncations are well known in the art.
Portions suitable for use in the methods according to the invention
may readily be determined by following the methods described in the
Examples section by simply substituting the sequence used in the
actual Example with the portion.
[0028] Another variant of a nucleic acid encoding a CCS52 protein
is a nucleic acid capable of hybridising with a nucleic acid
encoding a CCS52 protein, for example with any of the nucleic acids
as represented by SEQ ID NO 1, 3 or 5. Hybridising sequences
suitable for use in the methods according to the invention may
readily be determined, for example by following the methods
described in the Examples section by simply substituting the
sequence used in the actual Example with the hybridising
sequence.
[0029] The term "hybridising" as used herein means annealing to a
substantially homologous complementary nucleotide sequences in a
hybridization process. The hybridisation process may occur entirely
in solution, i.e. both complementary nucleic acids are in solution.
Tools in molecular biology relying on such a process include the
polymerase chain reaction (PCR; and all methods based thereon),
subtractive hybridisation, random primer extension, nuclease S1
mapping, primer extension, reverse transcription, cDNA synthesis,
differential display of RNAs, and DNA sequence determination. The
hybridisation process may also occur with one of the complementary
nucleic acids immobilised to a matrix such as magnetic beads,
Sepharose beads or any other resin. Tools in molecular biology
relying on such a process include the isolation of poly (A+) mRNA.
The hybridisation process may furthermore occur with one of the
complementary nucleic acids immobilised to a solid support such as
a nitro-cellulose or nylon membrane or immobilised by e.g.
photolithography to e.g. a siliceous glass support (the latter
known as nucleic acid arrays or microarrays or as nucleic acid
chips). Tools in molecular biology relying on such a process
include RNA and DNA gel blot analysis, colony hybridisation, plaque
hybridisation, in situ hybridisation and microarray hybridisation.
In order to allow hybridisation to occur, the nucleic acid
molecules are generally thermally or chemically denatured to melt a
double strand into two single strands and/or to remove hairpins or
other secondary structures from single stranded nucleic acids. The
stringency of hybridisation is influenced by conditions such as
temperature, sodium/salt concentration and hybridisation buffer
composition. High stringency conditions for hybridisation include
high temperature and/or low salt concentration (salts include NaCl
and Na.sub.3-citrate) and/or the inclusion of formamide in the
hybridisation buffer and/or lowering the concentration of compounds
such as SDS (sodium dodecyl sulphate detergent) in the
hybridisation buffer and/or exclusion of compounds, such as dextran
sulphate or polyethylene glycol (promoting molecular crowding) from
the hybridisation buffer. Conventional hybridisation conditions are
described in, for example, Sambrook (2001) Molecular Cloning: a
laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press,
CSH, New York, but the skilled craftsman will appreciate that
numerous different hybridisation conditions may be designed in
function of the known or the expected sequence identity and/or
length of the nucleic acids. Sufficiently low stringency
hybridisation conditions are particularly preferred (at least in
the first instance) to isolate nucleic acids heterologous to the
DNA sequences of the invention defined supra. An example of low
stringency conditions is 4-6.times.SSC/0.1-0.5% w/v SDS at
37-45.degree. C. for 2-3 hours. Depending on the source and
concentration of the nucleic acid involved in the hybridisation,
alternative conditions of stringency may be employed, such as
medium stringency conditions. Examples of medium stringency
conditions include 1-4.times.SSC/0.25% w/v SDS at
.gtoreq.45.degree. C. for 2-3 hours. Preferably, the variants
capable of hybridizing with a CCS52 gene are capable of
specifically hybridizing. With "specifically hybridizing" is meant
hybridising under stringent conditions. An example of high
stringency conditions includes 0.1-2.times.SSC, 0.1.times.SDS, and
1.times.SSC, 0.1.times.SDS at 60.degree. C. for 2-3 hours.
[0030] The methods according to the present invention may also be
practised using an alternative splice variant of a nucleic acid
encoding a CCS52 protein, for example, an alternative splice
variant of SEQ ID NO 1, 3 or 5. The term "alternative splice
variant" as used herein encompasses variants of a nucleic acid in
which selected introns and/or exons have been excised, replaced or
added. Such splice variants may be found in nature or may be
manmade. Methods for making such splice variants are well known in
the art. Splice variants suitable for use in the methods according
to the invention may readily be determined, for example, by
following the methods described in the Examples section by simply
substituting the sequence used in the actual Example with the
splice variant.
[0031] Another variant CCS52 nucleic acid useful in practising the
method for improving plant growth characteristics, is an allelic
variant of a CCS52 gene, for example, an allelic variant of SEQ ID
NO 1, 3 or 5. Alielic variants exist in nature and encompassed
within the methods of the present invention is the use of these
natural alleles. Allelic variants also encompass Single Nucleotide
Polymorphisms (SNPs) as well as Small Insertion/Deletion
Polymorphisms (INDELs). The size of INDELs is usually less than 100
bp. SNPs and INDELs form the largest set of sequence variants in
naturally occurring polymorphic strains of most organisms. Allelic
variants suitable for use in the methods according to the invention
may readily be determined, for example, by following the methods
described in the Examples section by simply substituting the
sequence used in the actual Example with the allelic variant.
[0032] The present invention provides a method for improving plant
growth characteristics, comprising increasing expression in a plant
of an alternative splice variant or of an allelic variant of a
nucleic acid encoding a CCS52 protein and/or by increasing the
level and/or activity in a plant of a CCS52 protein encoded by an
alternative splice variant or allelic variant.
[0033] One example of a variant CCS52 protein useful in practising
the methods of the present invention is a homologue of a CCS52
protein. "Homologues" of a CCS52 protein encompass peptides,
oligopeptides, polypeptides, proteins and enzymes having an amino
acid substitution, deletion and/or insertion relative to the CCS52
protein in question and having similar biological and functional
activity as the CCS52. Homologues of a CCS52 protein may be manmade
via the techniques of genetic engineering and/or protein
engineering. To produce such homologues, amino acids of the protein
may be replaced by other amino acids having similar properties
(such as similar hydrophobicity, hydrophilicity, antigenicity,
propensity to form or break .alpha.-helical structures or
.beta.-sheet structures). Conservative substitution tables are well
known in the art (see for example Creighton (1984) Proteins. W.H.
Freeman and Company).
[0034] Homologues of a particular CCS52 protein may exist in nature
and may be found in the same or different species or organism from
which the particular CCS52 protein is derived. Two special forms of
homologues, orthologues and paralogues, are evolutionary concepts
used to describe ancestral relationships of genes. The term
"orthologues" relates to genes in different organisms that are
homologous due to ancestral relationship. The term "paralogues"
relates to gene-duplications within the genome of a species leading
to paralogous genes. The term "homologues" as used herein also
encompasses paralogues and orthologues of a CCS52 protein, which
are also useful in practising the methods of the present
invention.
[0035] Another special form of a CCS52 homologue is a member of the
same gene family of CCS52 proteins. It is known that AtCCS52A1
belongs to a multigene family, and therefore a person skilled in
the art will recognize that the methods according to the present
invention may also be practised using the encoding sequence of a
family member of a CCS52 protein, such as a family member of SEQ ID
NO 2, 4 or 6.
[0036] The homologues useful in the method according to the
invention have in increasing order of preference, at least 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to
a CCS52 protein, for example, to any one of SEQ ID NO 2, 4 or 6.
Alternatively, the nucleic acid sequence encoding any one of the
above-mentioned homologue may have at least 51%, 52%, 53%, 54%,
55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% sequence identity to a CCS52 nucleic
acid, for example, to any one of SEQ ID NO 1, 3 or 5.
[0037] The percentage of sequence identity as mentioned above,
between proteins or nucleic acids, may be calculated using a
pairwise global alignment program implementing the algorithm of
Needleman-Wunsch (J. Mol. Biol. 48: 443-453, 1970), which maximizes
the number of matches and keeps the number of gaps to a minimum.
For calculation of the above-mentioned percentages, the program
needle (EMBOSS package) may be used with a gap opening penalty of
10 and gap extension penalty of 0.1. For proteins, the blosum62
matrix with a word length of 3 is preferably used. For nucleic
acids, the program needle uses the matrix "DNA-full", with a
word-length of 11, as provided by the EMBOSS package. The
Needleman-Wunsch algorithm is best suited for analysing related
protein sequences over their full length.
[0038] The homologues useful in the methods according to the
invention (the proteins or their encoding nucleic acid sequences)
may be derived (either directly or indirectly (if subsequently
modified) from any source as described hereinafter, provided that
the sequence, when expressed in a plant, leads to improved plant
growth characteristics. The nucleic acid (or protein) may be
isolated from yeast, fungi, plants, algae, insects or animals
(including humans). This nucleic acid may be substantially modified
from its native form in composition and/or genomic environment
through deliberate human manipulation.
[0039] The nucleic acid encoding a CCS52 homologue is preferably
isolated from a plant. Examples of CCS52 proteins are Arabidopsis
thaliana CCS52A1 (SEQ ID NO 2 and corresponding encoding sequence
SEQ ID NO 1), Oryza sativa CCS52A (SEQ ID NO 4 and corresponding
encoding sequence SEQ ID NO 3), and Oryza sativa CCS52B (SEQ ID NO
6 and corresponding genomic sequence SEQ ID NO 5).
[0040] CCS52 proteins of Arabidopsis thaliana and Medicago sativa
have been subdivided into different classes (Cebolla et al., 1999,
EMBO J. 18: p 4476-4484). Class CCS52A (with A1 and A2 isoforms)
and class CCS52B (with the B1 isoform). These classes and isoforms
are also encompassed by the term "homologue" as used herein.
Advantageously, these different classes and isoforms of CCS52
proteins, or their encoding nucleic acids, may be used in the
methods of the present invention. Accordingly, the present
invention provides a method as described hereinabove, wherein the
CCS52 nucleic acid or CCS52 protein is obtained from a plant,
preferably from a dicotyledoneous plant, further preferably from
the family Brassicaceae, more preferably from Arabidopsis thaliana.
According to a further embodiment, CCS52 is CCS52A or CCS52B.
According to a further embodiment of the invention, CCS52 is a
CCS52A1 protein. A person skilled in the art will recognize that a
"CCS52A1" is a protein being closer related to AtCCS52A1, than to
AtCCS52A2 or AtCCS52B. This closer relationship may be determined
by calculating percentage of sequence identity, or by comparing the
presence of conserved motifs as described hereinafter.
[0041] Still other suitable CCS52 homologues and their encoding
sequences may be found in (public) sequence databases. Methods for
the search and identification of CCS52 protein homologues in
sequence databases would be well within the realm of a person
skilled in the art. Such methods, involve screening sequence
databases with the sequences provided by the present invention, for
example, SEQ ID NO 2, 4 or 6 (or SEQ ID NO 1, 3 or 5), preferably
in a computer readable form. Useful sequence databases include, but
are not limited, to Genbank
(http:/www.ncbi.nlm.nih.gov/web/Genbank), the European Molecular
Biology Laboratory Nucleic acid Database (EMBL)
(http:/w.ebi.ac.uk/ebi-docs/embl-db.html) or versions thereof or
the MIPS database (http://mips.gsf.de/). Different search
algorithms and software for the alignment and comparison of
sequences are well known in the art. Such software includes for
example, GAP, BESTFIT, BLAST, FASTA and TFASTA. Preferably the
BLAST software is used, which calculates percent sequence identity
and performs a statistical analysis of the similarity between the
sequences. The suite of programs referred to as BLAST programs has
5 different implementations: three designed for nucleotide sequence
queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein
sequence queries (BLASTP and TBLASTN) (Coulson, Trends in
Biotechnology: 76-80, 1994; Birren et al., GenomeAnalysis, 1: 543,
1997). The software for performing BLAST analysis is publicly
available through the National Centre for Biotechnology
Information.
[0042] Orthologues of a CCS52 protein in other plant species may
easily be found by performing a reciprocal Blast search. This
method comprises searching one or more sequence databases with a
query gene or protein (for example, any one of SEQ ID NO 1 to 6),
using for example, the BLAST program. The highest-ranking subject
genes that result from this search are then used as a query
sequence in a similar BLAST search. Only those genes that have as a
highest match again the original query sequence are considered to
be orthologous genes. For example, to find a rice orthologue of an
Arabidopsis thaliana gene, one may perform a BLASTN or TBLASTX
analysis on a rice database such as the Oryza sativa Nipponbare
database available at the NCBI website
(http://www.ncbi.nlm.nih.gov). In a next step, the highest ranking
rice sequences are used in a reverse BLAST search on an Arabidopsis
thaliana sequence database. The method may be used to identify
orthologues from many different species, for example, from
corn.
[0043] Paralogues of a CCS52 protein in the same species may easily
be found by performing a Blast search on sequences of the same
species from which the CCS52 protein is derived. From the sequences
that are selected by the Blast search, the true paralogues may be
identified by looking for the highest sequence identity or for the
highest conservation of typical CCS52 motifs as described
hereinafter.
[0044] Homologues of a AtCCS52A1 protein, as represented by SEQ ID
NO 2, and their encoding sequences, may be found in many different
species. Examples of such homologues are presented in the
phylogenetic tree in FIG. 12. The homologues are presented by their
Genbank accession number. Preferred homologues to be used in the
present invention are the homologues that group close to
AtCCS52A1_At4g22910, for example, those homologues that group
between OsAP003298.3 and Hs19_NP.sub.--057347.1. These homologues
include but are not limited to Hs19_NP.sub.--057347.1,
Mm_NP.sub.--062731, XL-CAA74576.1, Ggcdh1c_AAL31949,
Ggcdh1b_AAL31948.1, Ggcdh1d_AAL31950, Ggcdh1a_AAL31947,
Dm_NP.sub.--726941, Ag_agCP12792, Ce_NP.sub.--496075.1,
Dm_NP.sub.--611854, and the homologues grouping closest to
AtCCS52A1_At4g22910, including Le_AW0030735, AtCCS52A2_At4g11920,
MtCCS52A_AF134835, Gm_BG044933, Os_AK070642, Zm_AY112458,
AtCCS52B_At5g13840, MsCCSB, Gm_A1736659 and Zm_A1861254. The genome
sequences of Arabidopsis thaliana and Oryza sativa are now
available in public databases such as Genbank and other genomes are
currently being sequenced. Therefore, it is expected that further
homologues will readily be identifiable by sequence alignment with
any one of SEQ ID NO 1 to 6 using the programs BLASTX or BLASTP or
other programs.
[0045] The above-mentioned software analyses for comparing
sequences, for the calculation of sequence identity, for the search
of homologues, orthologues or paralogues or for the making of a
phylogenetic tree, is preferentially done with full-length
sequences. Alternatively, these software analyses may be carried
out with a conserved region of the CCS52 protein or nucleic acid
sequence, as described hereinafter. Accordingly, these analyses may
be based on the comparison and calculation of sequence identity
between conserved regions, functional domains, motifs or boxes.
[0046] The identification of protein domains, motifs and boxes,
would also be well within the realm of a person skilled in the art
by using protein domain information as available in the PRODOM
(http://www.biochem.ucl.ac.uk/bsm/dbbrowser/jj/prodomsrchjj.html),
PIR (http://pir.georgetown.edu/), PROSITE
(http://au.expasy.org/PROSITE/) or pFAM (http://pFAM.wustl.edu/)
databases. Software programs designed for such domain searching
include, but are not limited to, MotifScan, MEME, SIGNALSCAN, and
GENESCAN. MotifScan is a preferred software program and is
available at (http://hits.isb-sib.ch/cgi-bin/PFSCAN, which program
uses the protein domain information of PROSITE and pFAM. A MEME
algorithm (Version 3.0) may be found in the GCG package; or at
http://www.sdsc.edu/MEME/meme. SIGNALSCAN version 4.0 information
is available at http://biosci.cbs.umn.edu/software/sigscan.html.
GENESCAN may be found at
http://gnomic.stanford.edu/GENESCANW.html.
[0047] Ten conserved motifs have been identified in CCS52 proteins
and the consensus sequences for these motifs are represented herein
by SEQ ID NO 7 to 16 (see FIG. 13). Preferably, these motifs are
used to search databases and to identify homologous CCS52
sequences. The presence of these motifs (for example, as
represented by SEQ ID NO 7 to 16), may be determined by screening
proteins sequences for sequence identity with these consensus
motifs. Another aspect of the present invention is the use of
conserved CCS52 motifs as represented by ant one of SEQ ID NO 7 to
15, to identify, or to manufacture (via protein engineering or
grafting of such motifs into a target protein), homologues of a
CCS52 gene or protein which are capable of improving plant growth
characteristics. The N-terminal conserved motif, the C-box (SEQ ID
NO 16) is further described in Tarayre et al. 2004.
[0048] Preferred CCS52 homologues useful in the methods of the
present invention are plant CCS52 proteins that comprise at least 4
of the aforementioned consensus motifs. Motif number 2, as
represented by SEQ ID NO 8 has also been described as a N-terminal
"CSM" motif in Tarayre et al., 2004. Motif number 9, as represented
by SEQ ID NO 15, is presumably involved in the interaction with
other proteins; it is a C-terminal IR motif, which has been
described as necessary for the functionality of CCS52 in the APC
complex. Furthermore, the presence of multiple conserved motifs
(SEQ ID NO 7 to 16) strongly suggests that CCS52 proteins are
involved in multiple interactions and that several CCS52 target
genes/proteins exist. Further details on the relationship between
the IR motif and the CCS52 functionality are described in Tarayre
et al. (2004, Plant Cell., 16(2): 422-34), which document is herein
incorporated by reference as if fully set forth.
[0049] FIG. 13 shows the individual conserved motifs of different
CCS52 proteins as well as the consensus sequences thereof, which
are herein represented by SEQ ID NO 7 to 16. A person skilled in
the art will recognize that a CCS52 motif may deviate, by for
example 1 or 2 mismatches, from the abovementioned consensus CCS52
motifs, without losing its functionality. One example of such a
deviation is number of "X" amino acids in motif 3.
[0050] As may be deducted from FIG. 13, the consensus sequences may
be more defined when only taking CCS52A proteins into account. For
example, for CCS52A proteins, Motif number 1 has G on position 1, N
at position 3, F or L at position 4, A at position 5, L at position
6 and L or I at position 9. This consensus Motif 1 for CCS52A
proteins is represented herein by SEQ ID NO 17. For CCS52A
proteins, Motif number 7 has T at position 5 and H at position 8.
Also, for CCS52A proteins, Motif number 9 has "I" at position 2 and
"R" at position 9.
[0051] Some of the variants as mentioned hereinabove may occur in
nature and may be isolated from nature. Once the sequence of a
variant is known, and its corresponding encoding sequence, the
person skilled in the art will be able to isolate the corresponding
CCS52 gene or variant from biological material such as genomic
libraries, for example, by the technique of PCR. One example of
such an experiment is outlined in Example 1. Alternatively, when
the exact sequence is not known, new CCS52 proteins may be isolated
from biological material via hybridization techniques based on
probes from known CCS52 proteins.
[0052] Alternatively and/or additionally, some variants as
mentioned above may be manmade via techniques involving, for
example, mutation (substitution, insertion or deletion) or
derivation. These variants are herein referred to as "derivatives",
which derivatives are also useful in the methods of the present
invention. Derivatives of a protein may readily be made using
peptide synthesis techniques well known in the art, such as solid
phase peptide synthesis and the like, or by protein engineering via
recombinant DNA manipulations. The manipulation of DNA sequences to
produce substitution, insertion or deletion variants of a protein
are well known in the art. For example, techniques for making
substitution mutations at predetermined sites in DNA are well known
to those skilled in the art and include M13 mutagenesis, T7-Gen in
vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed
mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated
site-directed mutagenesis or other site-directed mutagenesis
protocols.
[0053] One example of a derivative is a substitutional variant. The
term "substitutional variants" of a CCS52 protein refers to those
variants in which at least one residue in an amino acid sequence
has been removed and a different amino acid inserted in its place.
Amino acid substitutions are typically of single residues, but may
be clustered depending upon functional constraints placed upon the
polypeptide; insertions usually are of the order of about 1-10
amino acids, and deletions can range from about 1-20 amino acids.
Preferably, amino acid substitutions comprise conservative amino
acid substitutions.
[0054] Other derivatives are "insertional variants" in which one or
more amino acids are introduced into a predetermined site in the
CCS52 protein. Insertions may comprise amino-terminal and/or
carboxy-terminal fusion as well as intra-sequence insertion of
single or multiple amino acids. Generally, insertions within the
amino acid sequence are of the order of about 1 to 10 amino acids.
Examples of amino- or carboxy-terminal fusions include fusion of
the binding domain or activation domain of a transcriptional
activator as used in the yeast two-hybrid system, phage coat
proteins, (histidine)6-tag, glutathione S-transferase-tag, protein
A, maltose-binding protein, dihydrofolate reductase, Tag-100
epitope, c-myc epitope, FLAGa-epitope, lacZ, CMP
(calmodulin-binding peptide), HA epitope, protein C epitope and VSV
epitope.
[0055] Other derivatives of a CCS52 protein are "deletion
variants", characterised by the removal of one or more amino acids
from the protein.
[0056] Another derivative of a CCS52 protein is characterised by
substitutions, and/or deletions and/or additions of naturally and
non-naturally occurring amino acids compared to the amino acids of
a naturally-occurring CCS52 protein. A derivative may also comprise
one or more non-amino acid substituents compared to the amino acid
sequence from which it is derived. Such non-amino acid substituents
include for example, non-naturally occurring amino acids, a
reporter molecule or other ligand, covalently or non-covalently
bound to the amino acid sequence. Such a reporter molecule may be
bound to facilitate the detection of the CCS52 protein.
[0057] Another variant of a CCS52 protein useful in the methods of
the present invention is an active fragment of a CCS52 protein.
"Active fragments" of a CCS52 protein encompass at least five
contiguous amino acid residues of a CCS52 protein, which residues
retain similar biological and/or functional activity to a naturally
occurring protein or a part thereof. Suitable fragments include
fragments of a CCS52 protein starting at the second or third or
further internal methionine residues. These fragments originate
from protein translation, starting at internal ATG codons, whilst
retaining its functionality in the methods of the present
invention. Suitable functional fragments of a CCS52 protein, or
suitable portions of nucleic acids that correspond to such
fragments, useful in the methods of the present invention, may have
one or more of the conserved motifs of CCS52 proteins as
represented by SEQ ID NO 7 to 16, whilst retaining its
functionality in the methods of the present invention. One
particular example of a functional fragment is a fragment of a rice
CCS52 protein, for example of SEQ ID NO 6, which ends with the IR
motif.
[0058] According to a preferred embodiment of the present
invention, a method to improve plant growth characteristics
comprises increased expression of a nucleic acid encoding a CCS52
protein. Methods for obtaining increased expression of genes or
gene products (proteins) are well documented in the art and
include, for example, overexpression driven by an operably linked
promoter, or the use of transcription enhancers or translation
enhancers. The term overexpression as used herein means any form of
expression that is additional to the original wild-type expression
level. Preferably the nucleic acid to be introduced into the plant
and/or the nucleic acid that is to be overexpressed in the plant is
in the sense direction with respect to the promoter to which it is
operably linked. Preferably, in the methods of the present
invention a nucleic acid encoding a CCS52 protein is overexpressed
in a plant, such as a CCS52 nucleic acid of SEQ ID NO 1.
[0059] Alternatively and/or additionally, increased expression of a
CCS52 gene or increased level, and/or activity of a CCS52 protein
in a plant cell, may be achieved by mutagenesis. For example, the
mutations may be responsible for altered control of an endogenous
CCS52 gene, resulting in more expression of the gene, relative to
the wild-type gene. Mutations can also cause conformational changes
in a protein, resulting in higher levels and/or more activity of
the CCS52 protein. Such mutations or such mutant genes may be
selected, or isolated and/or introduced into the same or different
plant species in order to obtain plants having improved growth
characteristics. Examples of such mutants include dominant positive
mutants of a CCS52 gene.
[0060] According to a further aspect of the present invention,
there is provided genetic constructs and vectors to facilitate
introduction and/or to facilitate expression and/or to facilitate
maintenance in a host cell of the nucleic acids useful in the
methods according to the invention. Therefore, according to a
further embodiment of the present invention, there is provided a
genetic construct comprising:
[0061] (a) a nucleic acid encoding a CCS52 protein or a variant
thereof; operably linked to
[0062] (b) a medium-strength promoter; and optionally
[0063] (c) a transcription termination sequence.
[0064] Constructs useful in the methods according to the present
invention may be constructed using recombinant DNA technology well
known to persons skilled in the art. The gene constructs may be
inserted into vectors, which may be commercially available,
suitable for transforming into plants and suitable for maintenance
and expression of the gene of interest in the transformed cells.
Preferably, the genetic construct according to the present
invention is a plant expression vector, suitable for introduction
and/or maintenance and/or expression of a nucleic acid in a plant
cell, tissue, organ or whole plant.
[0065] The nucleic acid according to (a) is advantageously any of
the nucleic acids described hereinbefore. A preferred nucleic acid
is a nucleic acid represented by SEQ ID NO 1, 3 or 5, or a variant
thereof as hereinbefore defined, or is a nucleic acid encoding a
protein as represented by SEQ ID NO 2, 4 or 6, or a variant thereof
as hereinbefore defined.
[0066] With the term "promoter" it meant a transcription control
sequence. The promoter of (b) is operable in a plant, most
preferably the promoter is derived from a plant sequence.
[0067] The terms "transcription control sequence" or "promoter" are
used interchangeably herein and are to be taken in a broad context
to refer to regulatory nucleic acids capable of effecting
expression of the sequences to which they are operably linked.
Encompassed by the aforementioned terms are transcriptional
regulatory sequences derived from a classical eukaryotic genomic
gene (including the TATA box which is required for accurate
transcription initiation, with or without a CCAAT box sequence) and
additional regulatory elements (i.e. upstream activating sequences,
enhancers and silencers) which alter gene expression in response to
developmental and/or external stimuli, or in a tissue-specific
manner. Also included within the term is a transcriptional
regulatory sequence of a classical prokaryotic gene, in which case
it may include a -35 box sequence and/or -10 box transcriptional
regulatory sequences. The term "regulatory element" also
encompasses a synthetic fusion molecule or derivative, which
confers, activates or enhances expression of a nucleic acid
molecule in a cell, tissue or organ.
[0068] The term "operably linked" as used herein refers to a
functional linkage between the promoter sequence and the gene of
interest, such that the promoter sequence is able to initiate
transcription of the gene of interest. Preferably, the gene of
interest is operably linked in the sense orientation to the
promoter.
[0069] The term "medium-strength promoter" means a promoter other
than a strong promoter and refers to the expression level in green
vegetative tissues.
[0070] Advantageously, any promoter may be used for the methods of
the invention, provided that it has a medium-strength expression
pattern in green vegetative tissues. These promoters have, when
compared to a strong constitutive promoter (such as the strong
constitutive/ubiquitous CaMV35S promoter), a lower expression level
at least in green vegetative tissues. Promoters useful in the
methods of the present invention do not reach the same strong
expression level in green vegetative tissue of a plant as the
CaMV35S promoter.
[0071] Preferably, the medium-strength promoter is of overall
medium-strength during vegetative growth of the plant. One example
of such a promoter is the sunflower ubiquitin promoter.
[0072] The term "medium-strength promoter" clearly does not include
a CaMV35S promoter, which is known to be a very strong promoter. To
the contrary, a medium-strength promoter has an expression level in
green vegetative tissue that is at least 10-fold lower than the
CaMV35S promoter. A person skilled in the art will recognize that
for many plant species the CaMV35S promoter activity has been
measured and that in many different plant species, such as rice and
corn, the level of activity of the CaMV35S promoter is very
high.
[0073] One method to measure the promoter strength is through the
use of promoter-beta-glucuronidase fusions. The promoter if hereby
fused to the Escherichia coli uidA gene encoding beta-glucuronidase
and the chimeric construct is transformed into a plant. Proteins
are extracted from the plant material and GUS activity is measured
(Jefferson et al., 1987, EMBO J. 20;6(13):3901-7). Promoter
activity is then calculated as the optical density in units per mg
of extracted protein.
[0074] Examples of measurements of CaMV35S expression levels have
been described previously, for example for rice (Battraw and Hall,
1990, Plant Mol Biol. 15(4): 527-38), for tobacco (Jefferson et al.
,1987, EMBO J., 20-6(13): 3901-7) and for Arabidopisis (S.
Planchais, PhD. thesis University of Ghent, 2000).
[0075] In the context of this invention, GUS activity is measured
from vegetative tissues after germination. Preferably, these
measurements are performed during vegetative growth of the plant,
for example after 2, preferably after 4 weeks post germination.
[0076] According to one embodiment of the present invention, the
medium-strength promoter is a constitutive promoter. The term
"constitutive" as defined herein refers to a promoter that is
expressed substantially continuously and substantially in all
tissues of a plant. Examples of useful constitutive promoters are
ubiquitin promoters (in case of monocots intron-less ubiquitin
promoters), such as rice or maize ubiquitin promoters.
[0077] According to one particular embodiment of invention, the
medium-strength promoter is the sunflower ubiquitin promoter
(without intron). The term "medium-strength promoter" as used
herein therefore also means a promoter that has the same or similar
activity, as the sunflower ubiquitin promoter in Arabidopsis
thaliana. Similar activity in this context means an activity that
is at most 20-fold higher or lower than the sunflower ubiquitin
promoter, preferably at most 10-fold higher or lower or 5-fold
higher or lower or 3-fold higher or lower.
[0078] Alternatively and according to another embodiment of the
invention, the medium-strength promoter is a tissue-preferred
promoter, characterized by the fact that it shows medium-strength
expression in green vegetative tissue. The term "tissue-specific"
promoter is used interchangeably herein with a "tissue-preferred"
promoter. A promoter useful in the methods of the present invention
may have a strong expression level, in other parts of the plant but
the green vegetative tissue. For example, the Arabidopsis thaliana
2S2 promoter, which confers strong expression in seeds, may be used
for the methods of the present invention. Besides the 2S2 promoter,
other suitable tissue-preferred promoters include pPROLAMIN or
pOLEOSIN, or promoters that show strong expression in aleurone,
embryo, scutellum or endosperm. One example of a useful
young-tissue preferred promoter is the beta-expansin promoter.
[0079] In document WO99/64451, it was suggested to clone a CCS52
gene under control of the endod12Ams promoter or the Srglb3
promoter in order to have a positive effect on differentiation and
somatic embryogenesis. These positive effects have never been
shown. These promoters are disclaimed from the constructs of the
present invention.
[0080] Optionally, in the genetic construct according to the
invention, one or more terminator sequences may also be
incorporated. The term "transcription termination sequence"
encompasses a control sequence at the end of a transcriptional
unit, which signals 3' processing and polyadenylation of a primary
transcript and termination of transcription. Additional regulatory
elements, such as transcriptional or translational enhancers, may
be incorporated in the genetic construct. Those skilled in the art
will be aware of terminator and enhancer sequences, which may be
suitable for use in performing the invention. Such sequences would
be known or may readily be obtained by a person skilled in the
art.
[0081] The genetic constructs of the invention may further include
an origin of replication, which is required for maintenance and/or
replication in a specific cell type. One example is when a genetic
construct is required to be maintained in a bacterial cell as an
episomal genetic element (e.g. plasmid or cosmid). Preferred
origins of replication include, but are not limited to, the f1-ori
and colE1.
[0082] The genetic construct may optionally comprise a selectable
marker gene. As used herein, the term "selectable marker gene"
includes any gene, which confers a phenotype on a cell in which it
is expressed to facilitate the identification and/or selection of
cells, which are transfected or transformed with a genetic
construct of the invention. Suitable markers may be selected from
markers that confer antibiotic or herbicide resistance. Cells
containing the recombinant DNA will thus be able to survive in the
presence of antibiotic or herbicide concentrations that kill
untransformed cells. Examples of selectable marker genes include
genes conferring resistance to antibiotics (such as nptll encoding
neomycin phosphotransferase capable of phosphorylating neomycin and
kanamycin, or hpt encoding hygromycin phosphotransferase capable of
phosphorylating hygromycin), to herbicides (for example, bar which
provides resistance to Basta; aroA or gox providing resistance
against glyphosate), or genes that provide a metabolic trait (such
as manA that allows plants to use mannose as sole carbon source).
Visual marker genes result in the formation of colour (for example,
beta-glucuronidase, GUS), luminescence (such as luciferase) or
fluorescence (Green Fluorescent Protein, GFP, and derivatives
thereof. Further examples of suitable selectable marker genes
include the ampicillin resistance gene (Ampr), tetracycline
resistance gene (Tcr), bacterial kanamycin resistance gene (Kanr),
phosphinothricin resistance gene, and the chloramphenicol
acetyltransferase (CAT) gene, amongst others
[0083] According to a further embodiment of the present invention,
there is provided a method for the production of transgenic plants
having improved growth characteristics relative to corresponding
wild-type plants, comprising:
[0084] (a) introducing into a plant cell a CCS52 nucleic acid or a
variant thereof, preferably introducing a genetic construct as
described hereinabove;
[0085] (b) cultivating said plant cell under conditions promoting
plant growth.
[0086] "Introducing" the CCS52 nucleic acid or the genetic
construct into the plant cell is preferably achieved by
transformation. The term "transformation" as used herein
encompasses the transfer of an exogenous polynucleotide into a host
cell, irrespective of the method used for transfer. Plant tissue
capable of subsequent clonal propagation, whether by organogenesis
or embryogenesis, may be transformed with a genetic construct of
the present invention. The choice of tissue depends on the
particular plant species being transformed. Exemplary tissue
targets include leaf disks, pollen, embryos, cotyledons,
hypocotyls, megagametophytes, callus tissue, existing meristematic
tissue (e.g., apical meristem, axillary buds, and root meristems),
and induced meristem tissue (e.g., cotyledon meristem and hypocotyl
meristem). The polynucleotide may be transiently or stably
introduced into a host cell and may be maintained non-integrated,
for example, as a plasmid. Alternatively, it may be integrated into
the host genome. Preferably, the CCS52 nucleic acid is stably
integrated in the genome of the plant cell, which may be achieved,
for example, by using a plant transformation vector or a plant
expression vector having T-DNA borders, which flank the nucleic
acid to be introduced into the genome.
[0087] Transformation of a plant species is now a fairly routine
technique. Advantageously, any of several transformation methods
may be used to introduce the gene of interest into a suitable
ancestor cell. Transformation methods include the use of liposomes,
electroporation, chemicals that increase free DNA uptake, injection
of the DNA directly into the plant, particle gun bombardment,
transformation using viruses or pollen and microprojection. Methods
may be selected from the calcium/polyethylene glycol method for
protoplasts (Krens, F.A. et al., 1882, Nature 296, 72-74; Negrutiu
I. et al, June 1987, Plant Mol. Biol. 8, 363-373); electroporation
of protoplasts (Shillito R. D. et al., 1985 Bio/Technol 3,
1099-1102); microinjection into plant material (Crossway A. et al.,
1986, Mol. Gen Genet 202, 179-185); DNA or RNA-coated particle
bombardment (Klein T. M. et al., 1987, Nature 327, 70) infection
with (non-integrative) viruses and the like. A preferred method for
the production of transgenic plants according to the invention, is
an Agrobactiedum-mediated transformation method.
[0088] Transgenic rice plants are preferably produced via
Agrobacterium-mediated transformation using any of the well-known
methods for rice transformation, such as the ones described in any
of the following: published European patent application EP1198985,
Aldemita and Hodges (Planta, 1996, 199: 612-617,); Chan et al.
(Plant Mol. Biol., 1993, 22 (3): 491-506,); Hiei et al. (Plant J.,
1994, 6 (2): 271-282,); which disclosures are incorporated by
reference herein as if fully set forth. In the case of corn
transformation, the preferred method is as described in either
Ishida et al. (Nat. Biotechnol., 1996, 14(6): 745-50) or Frame et
al. (Plant Physiol., 2002, 129(1): 13-22), which disclosures are
incorporated by reference herein as if fully set forth.
[0089] Generally after transformation, plant cells or cell
groupings are selected for the presence of one or more markers,
which are co-transformed with the CCS52 gene.
[0090] The resulting transformed plant cell, cell grouping, or
plant tissue, may then be used to regenerate a whole transformed
plant via regeneration techniques well known to persons skilled in
the art. Therefore, cultivating the plant cell under conditions
promoting plant growth, may encompass the steps of selecting and/or
regenerating and/or growing to reach maturity.
[0091] Following DNA transfer and regeneration, putatively
transformed plants may be evaluated, for instance using Southern
analysis, for the presence of the gene of interest, copy number
and/or genomic organisation. Alternatively or additionally,
expression levels of the newly introduced DNA may be monitored
using Northern and/or Western analysis, both techniques being well
known to persons having ordinary skill in the art.
[0092] The generated transformed plants may be propagated by a
variety of means, such as by clonal propagation or classical
breeding techniques. For example, a first generation (or T1)
transformed plant may be selfed to give homozygous second
generation (or T2) transformants, and the T2 plants further
propagated through classical breeding techniques.
[0093] The generated transformed organisms may take a variety of
forms. For example, they may be chimeras of transformed cells and
non-transformed cells; clonal transformants (e.g., all cells
transformed to contain the expression cassette); grafts of
transformed and untransformed tissues (e.g., in plants, a
transformed rootstock grafted to an untransformed scion).
[0094] The invention also includes host cells containing an
isolated nucleic acid molecule encoding a CCS52 or a genetic
construct as mentioned hereinbefore. Preferred host cells according
to the invention are plant cells. Accordingly, there is provided
plant cells, tissues, organs and whole plants that have been
transformed with a genetic construct of the invention.
[0095] The present invention clearly extends to plants obtainable
by any of the methods as described hereinbefore, which plants have
improved growth characteristics relative to corresponding wild-type
plants. The present invention extends to plants, which have
increased expression levels of a nucleic acid encoding a CCS52
protein and/or increased level and/or activity od a CCS52 protein.
The present invention extends to plants containing a genetic
construct as described hereinabove, which plants have improved
growth characteristics.
[0096] The present invention clearly also extends to any plant cell
or plant produced by any of the methods described herein, and to
all plant parts and propagules thereof. The present invention
extends further to encompass the progeny of a primary transformed
cell, tissue, organ or whole plant that has been produced by any of
the aforementioned methods, the only requirement being that progeny
exhibit the same genotypic and/or phenotypic characteristic(s) as
those produced in the parent by the methods according to the
invention.
[0097] The invention also extends to any part of the plant
according to the invention, preferably a harvestable part of a
plant, such as, but not limited to, a seed, leaf, fruit, flower,
stem culture, stem, rhizome, root, tuber, bulb and cotton
fiber.
[0098] The term "plant" as used herein encompasses whole plants,
ancestors and progeny of the plants and plant parts, including
seeds, shoots, stems, roots (including tubers), and plant cells,
tissues and organs. The term "plant" also therefore encompasses
suspension cultures, embryos, meristematic regions, callus tissue,
leaves, seeds, roots, shoots, gametophytes, sporophytes, pollen,
and microspores. Plants that are particularly useful in the methods
of the invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous
plants including a fodder or forage legume, ornamental plant, food
crop, tree, or shrub selected from the list comprising Acacia spp.,
Acer spp., Actinidia spp., Aesculus spp., Agathis australis,
Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp,
Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea
plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza,
Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp,
Camellia sinensis, Canna indica, Capsicum spp., Cassia spp.,
Centroema pubescens, Chaenomeles spp., Cinnamomum cassia, Coffea
arabica, Colophospermum mopane, Coronillia varia, Cotoneaster
serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea
dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp.,
Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia
divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon
amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum,
Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana,
Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi,
Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp.,
Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo
biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum,
Grevillea spp., Guibourtia coleospenma, Hedysarum spp., Hemarthia
altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia
rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incamata,
Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp.,
Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus
spp., Macmtyloma axillare, Malus spp., Manihot esculenta, Medicago
sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum
spp., Onobrychis spp., Omithopus spp., Oryza spp., Peltophorum
africanum, Pennisetum spp., Persea gratissima, Petunia spp.,
Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia
spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara,
Pogonarthria fleckii, Pogonarthna squarrosa, Populus spp., Prosopis
cineraria, Pseudotsuga menziesli, Pterolobium stellatum, Pyrus
communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis
sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia
pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium
sanguineum, Sciadopitys verticillata, Sequoia sempervirens,
Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp.,
Spormbolus fimbriatus, Stiburus alopecuroides, Stylosanthos
humilis, Tadehagi spp, Taxodium distichum, Themeda triandra,
Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp.,
Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschia
aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli,
brussel sprout, cabbage, canola, carrot, cauliflower, celery,
collard greens, flax, kale, lentil, oilseed rape, okra, onion,
potato, rice, soybean, straw, sugarbeet, sugar cane, sunflower,
tomato, squash tea, trees, grasses (including forage grass) and
algae, amongst others.
[0099] According to a preferred feature of the present invention,
the plant is a crop plant, such as soybean, sunflower, canola,
rapeseed, cotton, alfalfa, tomato, potato, tobacco, papaya, squash,
poplar, eucalyptus, pine, leguminosa, flax, lupinus and sorghum.
According to a further preferred embodiment of the present
invention, the plant is a monocotyledonous plant, such as
sugarcane, further preferably the plant is a cereal, such as rice,
maize (including forage corn), wheat, barley, millet, oats and
rye.
[0100] Accordingly, the present invention provides any of the
methods as described hereinabove, or a transgenic plant as
described hereinabove, wherein the plant is a monocotyledonous crop
plant, preferably a cereal, more preferably wherein the plant is
rice or corn.
[0101] According to a particular embodiment of the invention, the
plant is a dicotyledonous crop plant, or a dicotyledonous
ornamental, such as azalea.
[0102] Advantageously, performance of the method according to the
present invention leads to plants having a variety of improved
growth characteristics relative to corresponding wild-type
plants.
[0103] The term "growth characteristic" as used herein, preferably
refers to, but is not limited to, increased yield/biomass or to any
other growth characteristic as described hereinafter.
[0104] The term "yield" refers to the amount of produced biological
material and is used interchangeably with "biomass". For crop
plants, "yield" also means the amount of harvested material per
acre or unit of production. Yield may be defined in terms of
quantity or quality. The harvested material may vary from crop to
crop, for example, it may be seeds (e.g. for rice, sorghum or corn
when grown for seed); above-ground biomass (e.g. for com, when used
as silage), roots (e.g. for sugar beet, tumip, potato), fruits
(e.g. for tomato, papaya), cotton fibers, or any other part of the
plant which is of economic value. "Yield" also encompasses yield
stability of the plants. High yield stability means that yield is
not strongly affected by changes in environmental conditions, such
as suboptimal conditions caused by drought, chilling, freezing,
heat, salinity or nutrient deficiency. "Yield" also encompasses
yield potential, which is the maximum obtainable yield under
optimal growth conditions. Yield may be dependent on a number of
yield components, which may be monitored by certain parameters.
These parameters are well known to persons skilled in the art and
vary from crop to crop. For example, breeders are well aware of the
specific yield components and the corresponding parameters for the
crop they are aiming to improve. For example, key yield parameters
for corn include number of plants per hectare or acre, number of
ears per plant, number of rows (of seeds) per ear, number of
kernels per row, and thousand kernel weight. For silage corn,
typical parameters are the above-ground biomass and energy content.
Key yield parameters for rice include number of plants per hectare
or acre, number of panicles per plant, number of flowers
(spikelets) per panicle, seed filling rate (number of filled seeds
per spikelet) and thousand kernel weight.
[0105] Generally, the term "increased yield" means an increase in
biomass in one or more parts of a plant relative to the biomass of
corresponding reference plants, for example relative to
corresponding wild-type plants. The plants of the present invention
exhibit increased plant size, manifested in taller plants and
increased rosette diameter. Accordingly, the term "yield/biomass"
as used herein encompasses increased plant size.
[0106] The plants of the present invention also exhibit increased
organ size, and therefore, the term "increased yield/biomass" as
used herein encompasses increased organ size. For example, the
plants according to the present invention are characterized by
increased size of the leaves, which is particularly important for
forage and feed crops (and omamentals). Furthermore, the plants
exhibit increased size of the stem. Besides the contribution to
increased yield, for example, in trees, an increase in stem
thickness contributes to improved wind/rain resistance, for example
in cereals. Furthermore, the plants according to the invention
exhibit increased seed size.
[0107] The plants of the present invention exhibit an increased
number of organs, and therefore, the term "increased yield/biomass"
as used herein encompasses increased number of organs. For example,
the plants according to the present invention exhibit an increased
number of the leaves, which is particularly important for forage
crops and ornamentals. Furthermore, the plants according to the
present invention exhibit an increased number of the branches
(lateral branches, rosette branches), which contributes to
increased bushiness of the plant. Also, the plants according to the
invention have increased number of trichome branches. An increased
biomass of specialised epidermal outgrowth structures is
advantageous in the production of cotton fibres or glandular
trichomes. Specialised trichomes may also be used for the
production of useful metabolites, pharmaceutical compounds,
nutraceuticals and food additives. Furthermore, the plants
according to the invention exhibit increased number of flowers,
which is important for ornamentals and seed crops.
[0108] Also encompassed within the term "increased yield/biomass"
is increased seed yield. Seed-yield may be manifested by increased
total seed weight, increased number of total seeds, increased
number of filled seeds, and/or increased seed size. An increase in
seed size and/or volume may also influence the composition of
seeds.
[0109] The term "growth characteristic" as used herein, also
encompasses plant architecture. For example, the plants of the
invention exhibit altered leaf shape, which may be advantageous for
ornamental plant, and altered vascularization, which is important
for wood and/or paper and pulp producing trees. The term
"architecture" as used herein encompasses the appearance or
morphology of a plant, including any one or more structural
features or combination of structural features thereof. Such
structural features include the shape, size, number, position,
texture, arrangement, and pattern of any cell, tissue or organ or
groups of cells, tissues or organs of a plant, including the root,
leaf, shoot, stem, petiole, trichome, flower, inflorescence (for
monocots and dicots), panicles, petal, stigma, style, stamen,
pollen, ovule, seed, embryo, endosperm, seed coat, aleurone, fibre,
cambium, wood, heartwood, parenchyma, aerenchyma, sieve element,
phloem or vascular tissue, amongst others. The term "architecture"
therefore encompasses leaf area, leaf thickness, arrangement of
lateral stems, stem shape and arrangement of flowers (and
fruits).
[0110] The present invention also relates to use of a nucleic acid
encoding a CCS52 protein or a variant thereof for improving plant
growth characteristics, preferably for increasing yield, further
preferably seed yield. Preferably, the nucleic acid is under the
control of a medium-strength promoter.
[0111] Alternatively, increasing expression of a CCS52 nucleic
acid, or introducing a CCS52 nucleic acid or the genetic construct
into the plant cell, may be achieved by crossing or by
breeding.
[0112] Furthermore, classical breeding techniques, aimed at
improving plant growth characteristics, may be based on the
selection of better performing allelic variants of a CCS52 gene,
which better performing alleles may have an expression level that
is higher than the wild-type level. Allelic variation may occur in
nature, or may be created by mutagenic treatment of biological
material, for example, by EMS mutagenesis. Therefore, the use of
CCS52 allelic variants in breeding programmes, aimed at improving
any of the growth characteristics as mentioned above, is also
encompassed by the present invention; this may be in addition to
their use in the methods according to the present invention. One
example of a breeding program is a conventional marker-assisted
breeding program.
[0113] Further information concerning the function of a CCS52 gene
and related genes may be discovered by the use of reverse genetics,
such a TILLING (Targeted Induced Local Lesions IN Genomes) in
combination with the discovery of sites and motifs crucial for the
gene and protein function (McCAllum et al., 2000, Plant Physiol
123(2):439-42; Perry et al., 2003 Plant Physiol 131(3):866-71).
Plants having mutant or dominant negative, or dominant positive
phenotypes may be analysed and compared to identify the most
effective mutations. Phenotypes may be compared with phenotypes
identified in, for example, QTL (Quantitative Trait Loci) analysis
and sequence information may be compared with the gene mapping
included in a QTL. Both methods may be useful when combined in
identifying new phenotypes of interest for crop breeding.
[0114] The present invention will now be described with reference
to the following figures in which:
[0115] FIG. 1 is a map of the entry clone, p1627, containing the
gene of interest, CCS52A1, (CDS0198) within the AttLl and AttL2
sites for Gateway.RTM. cloning in the pDONR201 backbone. This
vector also contains a bacterial kanamycin-resistance cassette and
a bacterial origin of replication.
[0116] FIG. 2 is a map of the binary vector for expression in
Arabidopsis thaliana of the Arabidopsis thaliana CCS52A1 gene
(CDS0198) under the control of a sunflower ubiquitin promoter
(pUBIdeltaT). The CCS52A1 expression cassette further comprises the
T-zein and T-rbcS-deltaGA double terminator sequence. This
expression cassette is located within the left border (LB repeat,
LB Ti C58) and a right border (RB repeat, RB Ti C58) of the
nopaline Ti plasmid. Cloned within these borders is also a
selectable marker and a screenable marker, both under control of a
constitutive promoter and followed by a nopaline (tNOS) or octopine
(tOCS) transcription termination sequence. Furthermore, this vector
also contains an origin of replication (pBR322 ori+bom) for
bacterial replication and a bacterial selectable marker (Spe/SmeR)
for bacterial selection.
[0117] FIG. 3 shows an aerial view of a wild-type Arabidopsis
thaliana plant (left) and a transgenic Arabidopsis thaliana plant
expressing a CCS52A1 transgene under control of an ubiquitin
promoter (right). Both plants are 4 weeks old.
[0118] FIG. 4 shows a first cauline leaf of a wild-type Arabidopsis
thaliana plant (left) and of a transgenic Arabidopsis thaliana
plant expressing a CCS52A1 gene under control of an ubiquitin
promoter (right).
[0119] FIG. 5 shows a first rosette leaf of a wild-type Arabidopsis
thaliana plant (left) and of a transgenic Arabidopsis thaliana
plant expressing a CCS52A1 gene under control of an ubiquitin
promoter (right).
[0120] FIG. 6 shows leaf tissue of a wild-type Arabidopsis thaliana
plant (left) and of a transgenic Arabidopsis thaliana plant
expressing a CCS52A1 gene under control of an ubiquitin
promoter.
[0121] FIG. 7 shows epidermis and trichomes of a wild-type
Arabidopsis thaliana plant (A) and of a transgenic Arabidopsis
thaliana plant expressing a CCS52A1 gene under control of an
ubiquitin promoter (B).
[0122] FIG. 8 shows a wild-type Arabidopsis thaliana plant (left)
and a transgenic Arabidposis thaliana plant expressing a CCS52A1
gene under the control of a 2S2 promoter (right), which are more
bushier.
[0123] FIG. 9 shows a wild-type Arabidopsis thaliana plant (left)
and a transgenic Arabidposis thaliana plant expressing a CCS52A1
gene under the control of an ubiquitin promoter (right).
[0124] FIG. 10 shows transversal sections of the main stem of a
wild-type Arabidopsis thaliana plant (left) and of a transgenic
Arabidopsis thaliana plant expressing a CCS52A1 gene under control
of an ubiquitin promoter (right).
[0125] FIG. 11 shows seeds produced by a wild-type Arabidopsis
thaliana plant (left) and by a transgenic Arabidopsis thaliana
plant expressing a CCS52A1 gene under the control of an ubiquitin
promoter (right).
[0126] FIG. 12 shows a phylogentic tree of CCS52 related proteins
in plants and animals. The sequences are presented by their Genbank
accession number. Multiple sequence alignment across the entire
sequences was done using CLUSTAL W (Higgins et al., (1994) Nucleic
Acids Res. 22:4673-4680), with the BLOSSUM 62 matrix and with the
parameters GAPOPEN 10, GAPEXT 0.05 and GAPDIST 8. The Phylogram
view gives an estimate of phylogeny, i.e. branch lengths are
proportional to evolutionary change.
[0127] FIG. 13 shows the conserved consensus motifs in plant CCS52
related proteins.
[0128] FIG. 14 shows the sequences of the present invention with
their respective SEQ ID numbers.
[0129] FIG. 15 is a map of the binary vector p35S::AtCCS52A1 for
expression in Arabidopsis thaliana of the Arabidopsis thaliana
CCS52A1 gene (internal reference CDS0198) under control of the
CaMV35S promoter. The CCS52A1 expression cassette further comprises
a T-zein and T-rbcS-deltaGA double transcription termination
sequence. This expression cassette is located within the left
border (LB repeat, LB Ti C58) and the right border (RB repeat, RB
Ti C58) of the nopaline Ti plasmid. Within the T-DNA there is
further provided a selectable and a screenable marker, both under
control of a constitutive promoter and followed by a T-NOS or a
T-OCS transcription terminator sequence. This vector further
comprises an origin of replication (pBR322 ori+bom) for bacterial
replication and a bacterial selectable marker (Spe/SmeR) for
bacterial selection.
[0130] FIG. 16 shows wild-type Arabidopsis thaliana plants and
transgenic Arabidopsis thaliana plants transformed with the vector
carrying the p35S::AtCCS52A1 expression cassette.
[0131] FIG. 17 is a map of the binary vector pEXP::AtCCS52A1 for
expression in Oryza sativa of the Arabidopsis thaliana CCS52A1 gene
(internal reference CDS0198) under the control of the rice
beta-expansin promoter. The CCS52A1 expression cassette further
comprises a T-zein and T-rbcS-deltaGA double transcription
termination sequence. This expression cassette is located within
the left border (LB repeat, LB Ti C58) and the right border (RB
repeat, RB Ti C58) of the nopaline Ti plasmid. Within the T-DNA
there is further provided a selectable and a screenable marker,
both under control of a constitutive promoter and followed by polyA
or a T-NOS transcription terminator sequence. This vector further
comprises an origin of replication (pBR322 ori+bom) for bacterial
replication and a bacterial selectable marker (Spe/SmeR) for
bacterial selection.
EXAMPLES
[0132] The present invention will now be described with reference
to the following examples, which are by way of illustration
alone.
[0133] DNA Manipulation
[0134] Unless otherwise stated, recombinant DNA techniques are
performed according to standard protocols described in Sambrook
(2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold
Spring Harbor Laboratory Press, CSH, New York or in Volumes 1 and 2
of Ausubel et al. (1998), Current Protocols in Molecular Biology.
Standard materials and methods for plant molecular work are
described in Plant Molecular Biology Labfase (1993) by R. D. D.
Croy, published by BIOS Scientific Publications Ltd (UK) and
Blackwell Scientific Publications (UK).
Example 1
Cloning of Arabidopsis thaliana CCS52A1
[0135] The Arabidopsis CCS52A1 gene (internal reference CDS0198)
was amplified by PCR using as template an Arabidopsis thaliana
seedling cDNA library (Invitrogen, Paisley, UK). After reverse
transcription of RNA extracted from seedlings, the cDNA fragments
were cloned into pCMV Sport 6.0. Average insert size of the cDNA
library was 1.5 kb, and original number of clones was about
1.59.times.10.sup.7 cfu. The original titer of 9.6.times.10.sup.5
cfu/ml was brought to 6.times.10.sup.11 cfu/ml after amplification
of the library. After plasmid extraction of the clones, 200 ng of
plasmid template was used in a 50 .mu.l PCR mix. The primers used
for PCR amplification, prm01391 with the sequence
5'GGGGACCAAGTTTGTACAAAAAAGCAGGCTTCACAATGGAAGAAGAAGATCCTACAGC 3'
(SEQ ID NO 18) and prm01392 with the sequence
5'GGGGACCACTTTGTACAAGAAAGCTGGGTTTCTCACCGAATTGTTGTTCTAC 3' (SEQ ID
NO 19) an AttB site for Gateway recombination cloning (italics).
PCR was performed using Hifi Taq DNA polymerase in standard
conditions. A PCR fragment of the expected length was amplified and
purified also using standard methods. The first step of the Gateway
procedure, the BP reaction, was then performed, during which the
PCR fragment recombines in vivo with the pDONR201 plasmid to
produce the "entry clone", p1627 (FIG. 1). Plasmid pDONR201 was
purchased from Invitrogen, as part of the Gateway.RTM.
technology.
Example 2
Vector construction (pUBI::AtCCS52A1)
[0136] The entry clone p1627 was subsequently used in an LR
reaction with p0712, a destination vector used for Arabidopsis
thaliana transformation. This vector contains as functional
elements within the T-DNA borders, a plant selectable marker, a
screenable marker and a Gateway cassette intended for LR in vivo
recombination with the sequence of interest already cloned in the
entry clone. Upstream of this Gateway cassette lies the sunflower
ubiquitin promoter (internal reference PRO155) for constitutive
expression of the gene of interest. After the LR recombination
step, the resulting expression vector pUBI::AtCCS52A1 (FIG. 2) was
transformed into Agrobacterium strain LBA4044 and subsequently into
Arabidopsis thaliana plants as described in Example 3.
Example 3
Arabidopsis Transformation
[0137] Sowing and Growing of Parental Plants
[0138] For the parental plants, approximately 12 mg of wild-type
seeds from Arabidopsis thaliana (ecotype Columbia) was suspended in
27.5 ml of 0.2% agar solution. The seeds were incubated for 2 to 3
days at a temperature of 4.degree. C. and were then sown. The seeds
were then allowed to germinate under the following standard
conditions: 22.degree. C. during the day, 18.degree. C. at night,
65-70% relative humidity, 12 hours of photoperiod, sub-irrigation
with water for 15 min every 2 to 3 days. The developed seedlings
were planted in pots of 5.5 cm diameter, containing a mixture of
sand and peat (ratio 1:3). The plants were allowed to grow under
the same standard conditions as mentioned above.
[0139] Agrobacterum Growth Conditions and Preparation
[0140] Agrobactedum strain C58C1RIF with helper plasmid pMP90
containing vector pUBI::AtCCS52A1 was inoculated in a 50 ml plastic
tube containing 1 ml Luria Broth (LB) without antibiotics. The
culture was shaken at 28.degree. C. for 8-9 h. After addition of 10
ml of LB without antibiotic, the plastic tube was shaken overnight
at 28.degree. C. The OD at 600 nm was monitored. At an optical
density of approximately 2.0, 40 ml of 10% sucrose and 0.05% Silwet
L-77 (a chemical mixture of polyalkyleneoxide modified
heptamethyltrisiloxane (84%) and allyloxypolyethyleneglycol methyl
ether (16%), OSI Specialties Inc.) was added to the culture. The
Agrobacterum culture obtained was labelled CD2175 and used to
transform the parental Arabidopsis plants.
[0141] Flower Dip
[0142] When each parental plant had one inflorescence of 7-10 cm in
height, the inflorescences were inverted into the Agrobacterum
culture and agitated gently for 2-3 seconds. 2 plants per
transformation were used. Subsequently, the plants were returned to
normal growing conditions as described above.
[0143] Seed Collection
[0144] 5 weeks after the flowers were dipped in the Agrobactedum
culture, watering of the plants was stopped. The plants were
incubated at 25.degree. C. with a photoperiod of 20 hours. One week
later, the seeds were harvested and placed in a seed drier for one
week. The seeds were then cleaned and collected in 15 ml plastic
tubes. The seeds were stored at 4.degree. C. until further
processing.
Example 4
Evaluation of Transformed Arabidopsis Plants
[0145] Selection of the First Generation of Transgenic Plants
[0146] 100 mg of seeds were placed in a 50 ml plastic tube and
suspended in 27 ml of a 0.2% agar solution. The tubes were stored
at 4.degree. C. for 3 days to release the seeds from dormancy.
Following this period, the seed suspension was examined under blue
light to determine the presence of transformed seeds. 20 bright
fluorescent seeds (expressing the selectable marker) were aspirated
with a Pasteur pipette, transferred to a 15 ml plastic tube, and
the suspension volume was adjusted to 15 ml with a 0.2% agar
solution. The same amount of non-fluorescent seed was transferred
to a separate 15 ml plastic tube and the suspension volume adjusted
to 15 ml with a 0.2% agar solution. The suspension of expressing
seeds was evenly dispensed as drops of 50 .mu.l on one half of a
50.times.30 cm tray containing a mixture of sand and soil in a
ratio of 1 to 2. The non-expressing seeds were dispensed in the
same way on the other half of the tray. The tray was placed in a
greenhouse under the following conditions: 22.degree. C. during the
day, 18.degree. C. at night, 60% relative humidity, 20 hour
photoperiod, sub-irrigation once a day with water for 15 min. On
the 14th day after sowing, 5 expressing and 5 non-expressing
seedlings were transplanted into individual pots of 10 cm diameter
filled with a mixture of sand and peat (ratio 1:3).
[0147] Cultivation and Imaging of the First Generation of
Transgenic Plants
[0148] The pots were then placed in a greenhouse under the same
conditions as described for the trays. The pots were sub-irrigated
for 15 minutes, once a week, or more if needed. On the 21.sup.st,
28.sup.th, 35.sup.th, 42.sup.nd and 49.sup.th day after sowing, the
rosettes of each plant were photographed using a digital camera. On
the 35.sup.th, 42.sup.nd, 49.sup.th and 56.sup.th day after sowing,
the inflorescence of each plant was photographed, using a digital
camera. The number of pixels corresponding to plant tissues was
recorded on each picture, converted to cm.sup.2 and used as a
measurement of plant size. On the 57.sup.th day after sowing, when
the first siliques were ripening, a breathable plastic bag was
placed on each plant and tightly attached at the base of the plants
to collect the shedding seeds. On the 90.sup.th day after sowing,
when all the siliques were ripe, the seeds were collected and
placed in a seed drier for 1 week before storage in a sealed
container at 4.degree. C.
[0149] Seed Vield of the First Generation of Transgenic Plants
[0150] Harvested inflorescences of the T1 plants were taken and
gently rubbed to release seeds from the siliques. The mixture of
seeds and chaff was then passed over a mesh to remove large
fragments of stems, leaves, siliques, etc. The seeds were then
poured onto a vibrating gutter equipped with a vacuum cleaner
allowing the lighter fragments, such as petals and small fibers, to
be aspirated whilst retaining the heavier seeds. Data on the seed
parameters were measured using an automated system.
[0151] A similar procedure was followed to evaluate the phenotypic
characteristics of Arabidopsis T2 lines. At least 15 expressing and
at least 15 non-expressing seedlings were transplanted into
individual pots with a diameter of 10 cm (containing a mixture of
sand and peat in a ratio of 1 to 3) and processed as described
above. The phenotypic characteristics, as described above, were
inherited to further generations.
Example 5
Phenotypic Characteristics of pUBI:AtCCS52A1 Transgenic Plants
[0152] Increased Biomass
[0153] CCS52 transgenic plants showed increased biomass relative to
control plants. This was manifested by increased leaf size (see
FIGS. 4, 5 and 6).
[0154] Increased leaf biomass was also manifested by increased
number of rosette leaves (see FIG. 3) and increased number of
cauline leaves (FIG. 8).
[0155] Increased biomass was further manifested by increased stem
thickness and more branching, which leads to a bushy phenotype. As
illustrated in FIG. 9 and FIG. 10, pUBI::CCS52 transgenic plants
have an increased rosette diameter as well as an increased (main)
stem diameter and an increased diameter of the lateral branches. As
a consequence it is estimated that overall plant biomass is
multiplied by 3 to 4 in CCS52 transgenic Arabidopsis plants.
[0156] Modified Trichomes
[0157] As shown in FIG. 7, transgenic plants have trichomes with
increased number of branches relative to the wild-type
trichomes.
[0158] Modified Plant and Organ Shape
[0159] As shown in FIG. 4, the cauline leaf of the transgenic plant
was of a different shape and of a larger size than the
corresponding wild-type plant. As shown in FIG. 5, the rosette leaf
of the transgenic plant had increased width and a larger area than
the corresponding wild-type leaf. Further, this figure illustrates
that a substantial increase of the vascularisation system was
visible in the transgenic leaf.
[0160] Increase Yield--Seed Yield
[0161] As shown in FIG. 11, seed size was enlarged in the
pUBI::CCS52 transgenic plant.
Example 6
Overexpression of AtCCS52A1 Under Control of the 2S2 Promoter
Resulted in Bushier Plants
[0162] Starting from the entry clone p1627, an expression vector
was made in a similar way as described in Examples 1 and 2, except
that the promoter upstream of the AtCCS52A1 gene was the
Arabidopsis 2S2 seed-preferred promoter. This expression vector was
transformed into Arabidopsis as described in Example 3 and plant
evaluation was carried out as described in Example 4.
[0163] The phenotypic characteristics of the p2S2::CCS52
transformed plants was similar as the pUBI::CCS52 transformed
plants described in Example 5. It was observed that p2S2::CCS52
transformed plants had increased biomass of leaves, increased
number of branches and/or increased biomass of stems. As further
illustrated in FIG. 8, the p2S2::CCS52 transgenic plant had an
increased number of leaves, at least 2 times more rosette branches,
thicker stems and more lateral branches, which gave rise to a
bushier phenotype. Furthermore, these plants showed more
flowers.
Example 7
Overexpression of CCS52 Under Control of the CaMV35S Promoter in
Arabidopsis Resulted in Small, Aberrant Plants
[0164] Starting from the entry clone p1627, an expression vector
was made in a similar way as described in Examples 1 and 2, except
that the promoter upstream of the AtCCS52A1 gene was the CaMV35S
promoter. The resulting expression vector p35S::AtCCS52A1 (FIG. 15)
was transformed into Arabidopsis as described in Example 3 and
plant evaluation was carried out as described in Example 4.
[0165] Arabidopsis plants were regenerated and grown under optimal
growth conditions as mentioned in Example 4. Nullizygote plant
without the transgene were alternated with transgenic plant
comprising the transgene in a growing tray (FIG. 16). During
growth, in optimal conditions, a significant difference between
transgenic and wild-type plant was observed. After 5 to 6 weeks the
plants were photographed (FIG. 16). At this stage the transgenic
plants showed a small and aberrant phenotype compared with the
mature and healthy wild-type plant. The transgenic plant clearly
had smaller leaves, smaller or no stems, smaller rosette diameter,
fewer leaves and fewer flowers compared to the wild-type plant.
Clearly the p35S::CCS52 transgenic plants suffered from an early
growth arrest. These transgenic plants are small and have aberrant
organ formation. In transgenic plants the leaves were reddish,
indicating that these plant suffered from stress and the aberrant
plants produced significantly reduced amounts of siliques and
seeds, compared to wild-type plants.
Example 8
Overexpression of AtCCS52 Under Control of Different
Medium-Strength Promoters in Rice
[0166] Starting from the entry clone p1627, different expression
vectors are made in a similar way as described in Examples 1 and 2,
except that the destination vector for the LR recombination
reaction is a destination vector useful for transformation of Oryza
sativa. This destination vector carries as functional elements
within the T-DNA borders, a plant selectable marker, a screenable
marker and a Gateway cassette intended for LR in vivo recombination
with the CCS52 sequence already cloned in the entry clone.
Different versions of this destination vector have different
medium-strength promoters upstream of this Gateway cassette. The
different resulting expression vectors therefore have different
promoters upstream of the CCS52 gene.
[0167] One example of such an expression vector, pEXP::AtCCS52A1
carrying the rice beta-expansin promoter (PRO0061) upstream of the
AtCCS52A1 gene, is represented in FIG. 17. Other examples of
expression vectors are CD02376, carrying the rice prolamin promoter
(PRO090); or CD05509, carrying the rice Oleosin 18 kDa promoter
(PRO0218); or CD13390, carrying the rice putative
protochlorophyllide reductase promoter (PRO0123), or a vector
carrying the methallothionein promoter upstream of the AtCCS52A1
gene.
[0168] Similar vectors are made, for the expression of other CCS52A
genes or CCS52B genes under control of the promoters as mentioned
hereinabove.
[0169] All these expression vectors are suitable for the
transformation of rice following the protocols as mentioned
hereinabove.
[0170] AtCCS52A1 transgenic rice plants, overexpressing AtCCS52A1
under control of a medium-strength promoter, have improved growth
characteristics. Especially, the transgenic rice plants have
increased yield/biomass, manifested by increased plant size
(increased plant area and/or increased plant height) or increased
harvest index, which is the ratio of the total biomass over the
harvested biomass. Increased biomass is also manifested by
increased organ size such as increased leaf size, increased seed
size (increased thousand kernel weight (TKW)), increased seed
yield/seed biomass or increased stem diameter. Increased biomass is
also manifested by increased number of organs such as increased
number of leaves, increased number of branches, increased number of
tillers, increased number of panicles, increased number of flowers,
increased number of seeds or increased number of filled seeds or
increased filling rate. Further these transgenic rice plants show
early flowering (shorter life cycle), compared to the corresponding
nullizygotes.
Example 9
Overexpression of CCS52 Under Control of a Medium-Strength Promoter
in Corn
[0171] Similar constructs as described in Example 7 are made for
the transformation of corn and the methods of the invention
described herein are also used in corn (Zea mays). To this aim, a
CCS52 gene, for example, a corn orthologue, is cloned under control
of a promoter operable in corn, in a plant transformation vector
suitable for Agrobacterium-mediated corn transformation. The
promoter operable in corn may for example, be a medium-strength
promoter, which is constitutive, for example, an ubiquitin promoter
or any of the useful promoters as mentioned hereinabove. Methods to
use for corn transformation have been described in literature
(Ishida et al., Nat Biotechnol. 1996 Jun; 14(6):745-50; Frame et
al., Plant Physiol. 2002 May; 129(1):13-22).
[0172] Transgenic (inbred) lines made by these methods may be
crossed with another non-transgenic or transgenic (inbred) line or
be self/sib-pollinated. Importantly, transgenic (inbred) lines may
be used as a female or male parent. Inheritability and copy number
of the transgene are checked by quantitative real-time PCR and
Southern blot analysis and expression levels of the transgene are
determined by reverse PCR and Northern analysis. Transgenic events
with single copy insertions of the transgene and with varying
levels of transgene expression are selected for further evaluations
in subsequent generations.
[0173] Progeny seeds obtained as described hereinabove are
germinated and grown in the greenhouse in conditions well adapted
for corn (16:8 photoperiod, 26-28.degree. C. daytime temperature
and 20-24.degree. C. night time temperature) as well under
water-deficient, nitrogen-deficient, and excess NaCl conditions.
Null segregants from the same parental line (inbred line or
hybrids), as well as wild-type plants of the same inbred line or
hybrids are used as controls. The progeny plants are evaluated on
different biomass and developmental parameters, including but not
limited to plant height, stalk width, nodes below ear, nodes above
ear, brace roots, number of leaves, leaf greenness, leaf angle,
total above-ground area time to tassel, time to silk, time to
maturity, ear height, ear number, ear length, ear weight, row
number, kernel number, grain moisture. Kernel traits include but
are not limited to kernel size, kernel weight, starch content,
protein content, and oil content are also monitored. Corn yield is
calculated according to well-known methods. Corn plants transformed
with a CCS52 protein show improved growth characteristics. More
particularly they show an improvement in any one or more of the
abovementioned biomass and developmental parameters.
[0174] Transgenic events that are most significantly improved
compared to corresponding control lines are selected for further
field-testing and marker-assisted breeding, with the objective of
transferring the field-validated transgenic traits into another
germplasm. The phenotyping of maize for growth and yield-related
parameters in the field is conducted using well-established
protocols. The corn plants are particularly evaluated on yield
components at different plant densities and under different
environmental conditions. Subsequent improvements for introgressing
specific loci (such as transgene containing loci) from one
germplasm into another is also conducted using well-established
protocols including but not limited to MAS.
Example 10
Overexpression of AtCCS52A2, AtCCS52B or Orthologues from Other
Plants, Such as OsCCS52A
[0175] The experiments as described in Examples 7 to 9 are repeated
with other CCS52 genes.
[0176] The AtCCS52A2 (internal reference CDS0199) is cloned under
control of the rice Oleosin 18 kDa promoter (PRO0128) in vector
CD04769, the rice Prolamin promoter (PRO0090) in vector CD04778,
the rice beta-expansin promoter (PRO0061) in vector CD13386 or the
rice putative protochlorophyllide reductase promoter (PRO0123) in
vector CD13522.
[0177] AtCCS52B (CDS0390) is cloned under control of the rice
prolamin promoter (PRO0090) in the vector CD02164, the rice
beta-expansin promoter (PRO0061) in vector CD13388 or the rice
metallothionein promoter (PRO0126) in the vector CD13530.
[0178] Plants transformed with a CCS52 gene under the control of a
medium-strength promoter, for example, transformed with one of the
constructs as mentioned above, show improved growth
characteristics, such as increased plant size, increased organ size
and/or increased number of organs.
Sequence CWU 1
1
19 1 1905 DNA Arabidopsis thaliana misc_feature CCS52A1 cDNA 1
atggaagaag aagatcctac agcaagcaat gtgataacga attcgaattc ttcatctatg
60 agaaacctat cgccggcgat gaatactccg gtggtttcac ttgagtcacg
aatcaatcga 120 ttaatcaatg ctaatcaatc tcaatcacca tcaccatcat
cactatcaag gtctatatac 180 tctgatagat ttatccccag tagatccgga
tccaatttcg ctcttttcga tctatctcct 240 tctcctagta aagatggtaa
ggaagatgga gctggctctt acgctactct gttgcgtgcg 300 gcgatgtttg
gtcctgagac gccggagaag agagatatta ctgggttttc ttcttccagg 360
aatattttta ggtttaagac ggagactcat cggtctttga attcgttttc tccttttggt
420 gttgatgatg attctcctgg tgtttctcat agtggtcctg ttaaagctcc
caggaaagtg 480 ccgcgatcgc cgtataagat tcttgatctc gttgacttta
gatctttggt ttcgataatg 540 catgaaacaa tttgtgatct ttgtgatgtt
ttggtctctg agggtctaga atttgagtct 600 gaggtattgg atgcaccggc
cttgcaagat gatttttatc tgaatcttgt ggattggtct 660 gcacaaaatg
ttctagcagt gggactaggg aactgtgtgt atttatggaa tgcttgtagc 720
agcaaggtta ctaagttatg tgatctcgga gctgaggata gtgtttgctc agtgggttgg
780 gcgttacgtg gaactcatct ggctgttgga actagtaccg ggaaagttca
gatatgggat 840 gcgtcacgct gcaagagaac aagaacaatg gaaggtcatc
gtctaagagt tggagccctg 900 gcatggggtt catcggttct gtcatctggt
agcagagaca agagtattct tcagagagac 960 ataaggtgtc aagaagatca
tgtcagtaaa ttggcaggtc ataaatctga ggtatgcgga 1020 ctcaagtggt
cttatgacaa cagagagcta gcatctggtg gaaacgacaa taggcttttt 1080
gtatggaacc aacattcaac acaaccggtt ttgaaatata gtgaacacac tgcagctgtt
1140 aaagccattg cttggtctcc tcatgttcat gggcttcttg cttctggtgg
tggtactgct 1200 gatagatgca tacgtttttg gaatacaacc acgaatactc
atttaagttc catagatact 1260 tgcagtcagg tatgcaatct agcttggtct
aagaacgtaa acgagcttgt tagcacacac 1320 ggatactctc agaaccaaat
cattgtctgg aaatacccaa ccatgtccaa aattgctact 1380 ctaaccggtc
acacataccg agtcttatac cttgcggttt cacccgatgg acagacgatt 1440
gtaacaggag caggagatga aaccttaagg ttctggaatg ttttcccttc cccaaaatct
1500 cagaacacgg atagtgaaat cgggtcgtct ttctttggta gaacaacaat
tcggtgagaa 1560 gttactttca aaacacacag aaaaagtcat aaattcttga
tttcttcagc agcagccagc 1620 ttgagttggt cgtctcaacc aacttttttc
acacgggagc agagagtcat taaattcttt 1680 tacacacgga tgcaacaaga
tctaaccctt ttgatttaat cacgatcttt gggtttccat 1740 caagatgcac
aacattttcc cccaaaattt tccaaagtgt atatctttat tcaatttttc 1800
ttcatatatc aaaatatagt ttcttttgta tttatttact tacgaacaca acattttata
1860 aaataagccc atgataataa tgcaataatt cgttaccatt ctctt 1905 2 518
PRT Arabidopsis thaliana MISC_FEATURE CCS52A1 protein 2 Met Glu Glu
Glu Asp Pro Thr Ala Ser Asn Val Ile Thr Asn Ser Asn 1 5 10 15 Ser
Ser Ser Met Arg Asn Leu Ser Pro Ala Met Asn Thr Pro Val Val 20 25
30 Ser Leu Glu Ser Arg Ile Asn Arg Leu Ile Asn Ala Asn Gln Ser Gln
35 40 45 Ser Pro Ser Pro Ser Ser Leu Ser Arg Ser Ile Tyr Ser Asp
Arg Phe 50 55 60 Ile Pro Ser Arg Ser Gly Ser Asn Phe Ala Leu Phe
Asp Leu Ser Pro 65 70 75 80 Ser Pro Ser Lys Asp Gly Lys Glu Asp Gly
Ala Gly Ser Tyr Ala Thr 85 90 95 Leu Leu Arg Ala Ala Met Phe Gly
Pro Glu Thr Pro Glu Lys Arg Asp 100 105 110 Ile Thr Gly Phe Ser Ser
Ser Arg Asn Ile Phe Arg Phe Lys Thr Glu 115 120 125 Thr His Arg Ser
Leu Asn Ser Phe Ser Pro Phe Gly Val Asp Asp Asp 130 135 140 Ser Pro
Gly Val Ser His Ser Gly Pro Val Lys Ala Pro Arg Lys Val 145 150 155
160 Pro Arg Ser Pro Tyr Lys Ile Leu Asp Leu Val Asp Phe Arg Ser Leu
165 170 175 Val Ser Ile Met His Glu Thr Ile Cys Asp Leu Cys Asp Val
Leu Val 180 185 190 Ser Glu Gly Leu Glu Phe Glu Ser Glu Val Leu Asp
Ala Pro Ala Leu 195 200 205 Gln Asp Asp Phe Tyr Leu Asn Leu Val Asp
Trp Ser Ala Gln Asn Val 210 215 220 Leu Ala Val Gly Leu Gly Asn Cys
Val Tyr Leu Trp Asn Ala Cys Ser 225 230 235 240 Ser Lys Val Thr Lys
Leu Cys Asp Leu Gly Ala Glu Asp Ser Val Cys 245 250 255 Ser Val Gly
Trp Ala Leu Arg Gly Thr His Leu Ala Val Gly Thr Ser 260 265 270 Thr
Gly Lys Val Gln Ile Trp Asp Ala Ser Arg Cys Lys Arg Thr Arg 275 280
285 Thr Met Glu Gly His Arg Leu Arg Val Gly Ala Leu Ala Trp Gly Ser
290 295 300 Ser Val Leu Ser Ser Gly Ser Arg Asp Lys Ser Ile Leu Gln
Arg Asp 305 310 315 320 Ile Arg Cys Gln Glu Asp His Val Ser Lys Leu
Ala Gly His Lys Ser 325 330 335 Glu Val Cys Gly Leu Lys Trp Ser Tyr
Asp Asn Arg Glu Leu Ala Ser 340 345 350 Gly Gly Asn Asp Asn Arg Leu
Phe Val Trp Asn Gln His Ser Thr Gln 355 360 365 Pro Val Leu Lys Tyr
Ser Glu His Thr Ala Ala Val Lys Ala Ile Ala 370 375 380 Trp Ser Pro
His Val His Gly Leu Leu Ala Ser Gly Gly Gly Thr Ala 385 390 395 400
Asp Arg Cys Ile Arg Phe Trp Asn Thr Thr Thr Asn Thr His Leu Ser 405
410 415 Ser Ile Asp Thr Cys Ser Gln Val Cys Asn Leu Ala Trp Ser Lys
Asn 420 425 430 Val Asn Glu Leu Val Ser Thr His Gly Tyr Ser Gln Asn
Gln Ile Ile 435 440 445 Val Trp Lys Tyr Pro Thr Met Ser Lys Ile Ala
Thr Leu Thr Gly His 450 455 460 Thr Tyr Arg Val Leu Tyr Leu Ala Val
Ser Pro Asp Gly Gln Thr Ile 465 470 475 480 Val Thr Gly Ala Gly Asp
Glu Thr Leu Arg Phe Trp Asn Val Phe Pro 485 490 495 Ser Pro Lys Ser
Gln Asn Thr Asp Ser Glu Ile Gly Ser Ser Phe Phe 500 505 510 Gly Arg
Thr Thr Ile Arg 515 3 2028 DNA Oryza sativa misc_feature CCS52A
cDNA 3 atccccaaat ctctcgcccc cacccatgga tcaccaccac caccacctgc
cgccgccgcc 60 gccgcggtcg ccgatggaga actccgcgtc ctccaagccg
cccaccccgg cgtccacccc 120 gtcgtcgcgc ctcgccgccg cgccgtcctc
ccgcgtctcc tccgcggcgc cgcacccctc 180 cccgtcctcc tccgcgccca
cgccggcctc gcggacggtc tacagcgacc gcttcatccc 240 cagccgcgcc
ggatccaacc tcgcgctctt cgacctcgcc ccgtcgccgt cccaccacga 300
cgccgccgcc gccgccgcct cccccggcgc gccgcccccc tccggatcta ccccggcctc
360 gtcgccctac tgcgcgctcc tccgcgccgc gctcttcggc cccaccacgc
ccgaccgggt 420 ggcgtcgtcg gcgtccgcgt gctcctcctc ctcctccgcc
ggggcgtcgc ccgtgggctc 480 acccgccacc ggcaacatat tcaggttcaa
ggcggaggtg ccccggaatg ctaagcgcgc 540 ccttttctcc gacggggacg
acgagggcgt gctcttcccc ggggtgttca cgacgagggg 600 cactggcccc
aggaagatcc ctaggtcacc ttataaggtg ctggatgctc ccgcattgca 660
ggatgacttc tacctgaacc ttgtggattg gtcttcgcat aatatccttg cagttggatt
720 ggggaattgt gtctacttat ggaatgcatg cagcagcaag gtcaccaagc
tatgtgattt 780 gggggtggat gacaatgtct gttcagtggg ttgggcacag
cgtggcactc accttgctgt 840 agggacaaac caaggcaaag ttcaggtatg
ggatgccact cgttgtaaga gaataagaac 900 catggaaagc catcggatgc
gagtaggtgc tcttgcatgg aattcatcat tgctttcgtc 960 aggcagtcgt
gacaagagca tccttcacca tgatatccgt gcccaggatg attatattag 1020
tagacttgct gggcataaat cggaggtctg tgggctcaag tggtcttatg ataaccgtca
1080 gcttgcatct ggtggtaatg acaacagact ttatgtatgg aatcaacact
cggcgcaccc 1140 ggtactgaag tatactgagc atacagcagc tgtcaaagct
attgcgtggt cacctcatct 1200 tcatgggctg cttgcatctg gtggaggaac
tgcagataga tgcatacgat tttggaatac 1260 caccacgaat atgcacttaa
attgcgtcga cacaggcagt caggtctgta atcttgtatg 1320 gtcaaagaat
gttaatgagc ttgttagcac tcatggatat tctcaaaatc agataattgt 1380
ttggcgatac ccaacaatgt caaagctcgc cacattgaca ggccatacat atagggtatt
1440 atatttagcc atctccccag atggacagac tatagtaact ggcgctggtg
atgaaacgct 1500 tcggttttgg aacgtgtttc catctcccaa gtcccagagt
tctgacagcc taagtagcat 1560 cggggccaca tcatttgtta ggagctacat
ccggtgacac tgagatgtgg taatctaata 1620 acacttggct cataagtcat
aacactactg cagcagagtg ttgatgatca tcaatatcat 1680 tccatttgta
ccacttgcat caccagttca tgaaccatca aacctagcca aattttagag 1740
atagtaggat gcagaatggt gaaactggct cgcagacctc ggagtggctc atttgctgaa
1800 tgctgtatat atttattcat tggctttgta ggagcgaaga tggcaaacac
tgaccatccg 1860 caatgtacca ttgataagtt cacggcctcc tgtttttgtt
tttgctgagt caacttggag 1920 ctggagctct tatgtatacc atgctagggc
ttaacaacat tggccaactc atgatgctca 1980 ttgcatccaa gttggaatat
gctaaggaag ctggagaatt tctggtgc 2028 4 507 PRT Oryza sativa
MISC_FEATURE CCS52A protein 4 Met Glu Asn Ser Ala Ser Ser Lys Pro
Pro Thr Pro Ala Ser Thr Pro 1 5 10 15 Ser Ser Arg Leu Ala Ala Ala
Pro Ser Ser Arg Val Ser Ser Ala Ala 20 25 30 Pro His Pro Ser Pro
Ser Ser Ser Ala Pro Thr Pro Ala Ser Arg Thr 35 40 45 Val Tyr Ser
Asp Arg Phe Ile Pro Ser Arg Ala Gly Ser Asn Leu Ala 50 55 60 Leu
Phe Asp Leu Ala Pro Ser Pro Ser His His Asp Ala Ala Ala Ala 65 70
75 80 Ala Ala Ser Pro Gly Ala Pro Pro Pro Ser Gly Ser Thr Pro Ala
Ser 85 90 95 Ser Pro Tyr Cys Ala Leu Leu Arg Ala Ala Leu Phe Gly
Pro Thr Thr 100 105 110 Pro Asp Arg Val Ala Ser Ser Ala Ser Ala Cys
Ser Ser Ser Ser Ser 115 120 125 Ala Gly Ala Ser Pro Val Gly Ser Pro
Ala Thr Gly Asn Ile Phe Arg 130 135 140 Phe Lys Ala Glu Val Pro Arg
Asn Ala Lys Arg Ala Leu Phe Ser Asp 145 150 155 160 Gly Asp Asp Glu
Gly Val Leu Phe Pro Gly Val Phe Thr Thr Arg Gly 165 170 175 Thr Gly
Pro Arg Lys Ile Pro Arg Ser Pro Tyr Lys Val Leu Asp Ala 180 185 190
Pro Ala Leu Gln Asp Asp Phe Tyr Leu Asn Leu Val Asp Trp Ser Ser 195
200 205 His Asn Ile Leu Ala Val Gly Leu Gly Asn Cys Val Tyr Leu Trp
Asn 210 215 220 Ala Cys Ser Ser Lys Val Thr Lys Leu Cys Asp Leu Gly
Val Asp Asp 225 230 235 240 Asn Val Cys Ser Val Gly Trp Ala Gln Arg
Gly Thr His Leu Ala Val 245 250 255 Gly Thr Asn Gln Gly Lys Val Gln
Val Trp Asp Ala Thr Arg Cys Lys 260 265 270 Arg Ile Arg Thr Met Glu
Ser His Arg Met Arg Val Gly Ala Leu Ala 275 280 285 Trp Asn Ser Ser
Leu Leu Ser Ser Gly Ser Arg Asp Lys Ser Ile Leu 290 295 300 His His
Asp Ile Arg Ala Gln Asp Asp Tyr Ile Ser Arg Leu Ala Gly 305 310 315
320 His Lys Ser Glu Val Cys Gly Leu Lys Trp Ser Tyr Asp Asn Arg Gln
325 330 335 Leu Ala Ser Gly Gly Asn Asp Asn Arg Leu Tyr Val Trp Asn
Gln His 340 345 350 Ser Ala His Pro Val Leu Lys Tyr Thr Glu His Thr
Ala Ala Val Lys 355 360 365 Ala Ile Ala Trp Ser Pro His Leu His Gly
Leu Leu Ala Ser Gly Gly 370 375 380 Gly Thr Ala Asp Arg Cys Ile Arg
Phe Trp Asn Thr Thr Thr Asn Met 385 390 395 400 His Leu Asn Cys Val
Asp Thr Gly Ser Gln Val Cys Asn Leu Val Trp 405 410 415 Ser Lys Asn
Val Asn Glu Leu Val Ser Thr His Gly Tyr Ser Gln Asn 420 425 430 Gln
Ile Ile Val Trp Arg Tyr Pro Thr Met Ser Lys Leu Ala Thr Leu 435 440
445 Thr Gly His Thr Tyr Arg Val Leu Tyr Leu Ala Ile Ser Pro Asp Gly
450 455 460 Gln Thr Ile Val Thr Gly Ala Gly Asp Glu Thr Leu Arg Phe
Trp Asn 465 470 475 480 Val Phe Pro Ser Pro Lys Ser Gln Ser Ser Asp
Ser Leu Ser Ser Ile 485 490 495 Gly Ala Thr Ser Phe Val Arg Ser Tyr
Ile Arg 500 505 5 1587 DNA Oryza sativa misc_feature DNA encoding
CCS52B protein 5 atgctaatgg gccggcccgc atggcagaga gagtacaacg
gctactcggg tggggggccc 60 acagtcagag ggagacagct cgtgctagaa
aaagtaggcg acttgcccac tccaaccaaa 120 gtgaccgttg caacctcatc
tccgctcctc ttcctcctcc tcgtcgtcgt tgtcgtcgtc 180 ggcggcgcat
ccagcctcga cgtgccggcg gcgccggcgc cgccgcgcct caacgtgccg 240
ccggcgatgg cgggggggct ccgcctcgat cccgccgtcg cctccccggc ccgcctcctc
300 ctcgacgtcc ccaagacgcc atccccttcc aagaccacgt acagcgaccg
cttcatcccc 360 tgccgctcct cctcccgcct ccacaacttc gccctcctcg
accgcgaccg cgcctccccc 420 tcctccacca ccgacgacgc cccctactcc
cgcctcctcc gcgccgagat cttcggcccg 480 gactccccct ccccggctcc
ctcctccccc aacaccaacc tcttccgctt caagaccgac 540 cacccctcgc
ccaaatcgcc cttcgccgcc tccgccgccg ccaccgccgg ccactacgac 600
tgcaccgccg gctccgctga atcctccacg ccgcgcaagc cgcccaggaa ggtccccaag
660 accccgcaca aggtcctgga cgcgccgtcg ctgcaggacg acttctacct
caatcttgtc 720 gactggtcgt cgcagaacac gctcgccgtc ggcctcggga
attgcgtcta cctctggtcg 780 gcttccaatt gcaaggtcac caagctctgc
gatttggggc ccagggacag cgtctgcgct 840 gtgcactgga cccgagaagg
ctcctatctt gccatcggca ccagccttgg cgatgtccag 900 atttgggata
gctctcgctg taaacggatt aggaacatgg gaggacacca aacacggact 960
ggtgtattag catggagctc ccgaatcttg tcctccggta gcagggacaa gaacatattg
1020 cagcatgaca tccgtgtccc aagtgactat atcagcaagt tctcagggca
cagatcagag 1080 aaccatgtat gtgcatcaag tgacagtttt tttggtcagg
tctgtggact gaaatggtcg 1140 cacgacgacc gtgagcttgc atccggtgga
aatgataatc agctgctagt atggaaccaa 1200 cgttcgcagc agccgatatt
gaggctgaca gaacacacag ctgcagttaa agcaatagca 1260 tggtcaccac
atcagcaagg cctcctggca tcaggtggtg gaaccgctga taggtgtatc 1320
aggttctgga acacggttaa tggaaacatg ctgaattcag tggacacagg cagccaggcg
1380 acttgtgagc actcatgggt attcccaaaa ccaaatcatg gtgtggaagt
acccatctat 1440 gtcaaaggtt gctactctaa ctggacacac gctgcgagtg
ctttaccttg caatgtcacc 1500 acaatagtaa caggagccgg ggatgaaacc
ctcagatttt ggaatatttt tccttcaatg 1560 aagacacagg taggcatcta ttgttga
1587 6 528 PRT Oryza sativa MISC_FEATURE CCS52B protein 6 Met Leu
Met Gly Arg Pro Ala Trp Gln Arg Glu Tyr Asn Gly Tyr Ser 1 5 10 15
Gly Gly Gly Pro Thr Val Arg Gly Arg Gln Leu Val Leu Glu Lys Val 20
25 30 Gly Asp Leu Pro Thr Pro Thr Lys Val Thr Val Ala Thr Ser Ser
Pro 35 40 45 Leu Leu Phe Leu Leu Leu Val Val Val Val Val Val Gly
Gly Ala Ser 50 55 60 Ser Leu Asp Val Pro Ala Ala Pro Ala Pro Pro
Arg Leu Asn Val Pro 65 70 75 80 Pro Ala Met Ala Gly Gly Leu Arg Leu
Asp Pro Ala Val Ala Ser Pro 85 90 95 Ala Arg Leu Leu Leu Asp Val
Pro Lys Thr Pro Ser Pro Ser Lys Thr 100 105 110 Thr Tyr Ser Asp Arg
Phe Ile Pro Cys Arg Ser Ser Ser Arg Leu His 115 120 125 Asn Phe Ala
Leu Leu Asp Arg Asp Arg Ala Ser Pro Ser Ser Thr Thr 130 135 140 Asp
Asp Ala Pro Tyr Ser Arg Leu Leu Arg Ala Glu Ile Phe Gly Pro 145 150
155 160 Asp Ser Pro Ser Pro Ala Pro Ser Ser Pro Asn Thr Asn Leu Phe
Arg 165 170 175 Phe Lys Thr Asp His Pro Ser Pro Lys Ser Pro Phe Ala
Ala Ser Ala 180 185 190 Ala Ala Thr Ala Gly His Tyr Asp Cys Thr Ala
Gly Ser Ala Glu Ser 195 200 205 Ser Thr Pro Arg Lys Pro Pro Arg Lys
Val Pro Lys Thr Pro His Lys 210 215 220 Val Leu Asp Ala Pro Ser Leu
Gln Asp Asp Phe Tyr Leu Asn Leu Val 225 230 235 240 Asp Trp Ser Ser
Gln Asn Thr Leu Ala Val Gly Leu Gly Asn Cys Val 245 250 255 Tyr Leu
Trp Ser Ala Ser Asn Cys Lys Val Thr Lys Leu Cys Asp Leu 260 265 270
Gly Pro Arg Asp Ser Val Cys Ala Val His Trp Thr Arg Glu Gly Ser 275
280 285 Tyr Leu Ala Ile Gly Thr Ser Leu Gly Asp Val Gln Ile Trp Asp
Ser 290 295 300 Ser Arg Cys Lys Arg Ile Arg Asn Met Gly Gly His Gln
Thr Arg Thr 305 310 315 320 Gly Val Leu Ala Trp Ser Ser Arg Ile Leu
Ser Ser Gly Ser Arg Asp 325 330 335 Lys Asn Ile Leu Gln His Asp Ile
Arg Val Pro Ser Asp Tyr Ile Ser 340 345 350 Lys Phe Ser Gly His Arg
Ser Glu Asn His Val Cys Ala Ser Ser Asp 355 360 365 Ser Phe Phe Gly
Gln Val Cys Gly Leu Lys Trp Ser His Asp Asp Arg 370 375 380 Glu Leu
Ala Ser Gly Gly Asn Asp Asn Gln Leu Leu Val Trp Asn Gln 385 390 395
400 Arg Ser Gln Gln Pro Ile Leu Arg Leu Thr Glu His Thr Ala Ala Val
405 410 415 Lys Ala Ile Ala Trp Ser Pro His Gln Gln Gly Leu Leu Ala
Ser Gly 420 425 430 Gly Gly Thr Ala Asp Arg Cys Ile Arg Phe Trp Asn
Thr Val Asn Gly 435 440 445 Asn Met Leu Asn Ser Val Asp Thr Gly Ser
Gln Ala Thr Cys Glu His 450 455 460 Ser Trp Val Phe Pro Lys Pro Asn
His
Gly Val Glu Val Pro Ile Tyr 465 470 475 480 Val Lys Gly Cys Tyr Ser
Asn Trp Thr His Ala Ala Ser Ala Leu Pro 485 490 495 Cys Asn Val Thr
Thr Ile Val Thr Gly Ala Gly Asp Glu Thr Leu Arg 500 505 510 Phe Trp
Asn Ile Phe Pro Ser Met Lys Thr Gln Val Gly Ile Tyr Cys 515 520 525
7 9 PRT Artificial Sequence consensus motif 1 of CCS52 protein
MISC_FEATURE (1)..(6) Xaa = unknown 7 Xaa Ser Xaa Xaa Xaa Xaa Phe
Asp Leu 1 5 8 17 PRT Artificial Sequence consensus motif 2 of CCS52
protein MISC_FEATURE (1)..(15) Xaa = unknwon 8 Xaa Xaa Xaa Xaa Xaa
Xaa Tyr Xaa Xaa Leu Leu Xaa Xaa Xaa Xaa Phe 1 5 10 15 Gly 9 18 PRT
Artificial Sequence consensus motif 3 of CCS52 protein MISC_FEATURE
(13)..(14) Xaa = unknown or absent MISC_FEATURE (1)..(12) Xaa =
unknown MISC_FEATURE (17)..(18) Xaa = unknown 9 Xaa Xaa Xaa Xaa Asn
Xaa Xaa Arg Phe Lys Xaa Xaa Xaa Xaa Arg Arg 1 5 10 15 Xaa Xaa 10 6
PRT Artificial Sequence consensus motif 4 of CCS52 protein 10 Ser
Lys Val Thr Lys Leu 1 5 11 11 PRT Artificial Sequence consensus
motif 5 of CCS52 protein MISC_FEATURE (2)..(7) Xaa = unknown 11 Asp
Xaa Xaa Ser Xaa Leu Xaa Gly His Lys Ser 1 5 10 12 12 PRT Artificial
Sequence consensus motif 6 of CCS52 protein MISC_FEATURE (3)..(10)
Xaa = unknown 12 His Ser Xaa Xaa Pro Xaa Leu Xaa Xaa Xaa Glu His 1
5 10 13 14 PRT Artificial Sequence consensus motif 7 of CCS52
protein MISC_FEATURE (5)..(12) Xaa = unknown 13 Trp Asn Thr Thr Xaa
Xaa Xaa Xaa Leu Xaa Xaa Xaa Asp Thr 1 5 10 14 14 PRT Artificial
Sequence consensus motif 8 of CCS52 protein MISC_FEATURE (5)..(5)
Xaa = unknown 14 Leu Tyr Leu Ala Xaa Ser Pro Asp Gly Gln Thr Ile
Val Thr 1 5 10 15 13 PRT Artificial Sequence consensus motif 9 of
CCS52 protein MISC_FEATURE (1)..(11) Xaa = unknown 15 Xaa Xaa Gly
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Arg 1 5 10 16 7 PRT Artificial
Sequence consensus C box MISC_FEATURE (6)..(6) Xaa = unknown 16 Asp
Arg Phe Ile Pro Xaa Arg 1 5 17 9 PRT Artificial Sequence consensus
motif 1 of CCS52A proteins MISC_FEATURE (4)..(4) Xaa = F or L
MISC_FEATURE (9)..(9) Xaa = L or I 17 Gly Ser Asn Xaa Ala Leu Phe
Asp Xaa 1 5 18 57 DNA Artificial Sequence primer 18 ggggacaagt
ttgtacaaaa aagcaggctt cacaatggaa gaagaagatc ctacagc 57 19 52 DNA
Artificial Sequence primer 19 ggggaccact ttgtacaaga aagctgggtt
tctcaccgaa ttgttgttct ac 52
* * * * *
References