U.S. patent application number 15/084711 was filed with the patent office on 2016-09-15 for gene associated with non-biological stress resistance, and transformed plant.
The applicant listed for this patent is INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY. Invention is credited to Seok Keun Cho, Woo Taek KIM, Moon Young Ryu.
Application Number | 20160264987 15/084711 |
Document ID | / |
Family ID | 47259536 |
Filed Date | 2016-09-15 |
United States Patent
Application |
20160264987 |
Kind Code |
A1 |
KIM; Woo Taek ; et
al. |
September 15, 2016 |
GENE ASSOCIATED WITH NON-BIOLOGICAL STRESS RESISTANCE, AND
TRANSFORMED PLANT
Abstract
The present invention relates to a composition for enhancing
non-biological stress resistance in plants and a composition for
accelerating germination. A nucleotide sequence of the present
invention is involved in the resistance against the drying stresses
in plants, and a transformed plant in which the nucleotide sequence
is overexpressed has prominent resistance against various kinds of
non-biological stress, including drought stress. In addition, the
nucleotide sequence of the present invention is involved in ABA
hormone sensitivity in plants, and germination is greatly improved
in a plant in which the nucleotide sequence expression is
suppressed. Therefore, the composition of the present invention can
be useful as new functional crops regardless of the climate of the
cultivation area, or as seeds for long-term storage with an
increased storage period.
Inventors: |
KIM; Woo Taek; (Goyang-si,
KR) ; Ryu; Moon Young; (Seoul, KR) ; Cho; Seok
Keun; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI
UNIVERSITY |
Seoul |
|
KR |
|
|
Family ID: |
47259536 |
Appl. No.: |
15/084711 |
Filed: |
March 30, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14122031 |
Nov 25, 2013 |
9322032 |
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PCT/KR2011/006187 |
Aug 22, 2011 |
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15084711 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/8271 20130101;
C07K 14/415 20130101; C12N 15/8293 20130101; C12N 15/8273
20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Foreign Application Data
Date |
Code |
Application Number |
May 27, 2011 |
KR |
10-2011-0050802 |
Claims
1-13. (canceled)
14. A method for improving the tolerance of a plant to an abiotic
stress, comprising: (a) introducing a nucleotide sequence encoding
the amino acid sequence of SEQ ID NO:2 into a cell of the plant;
and (b) obtaining a transgenic plant exhibiting improved tolerance
to an abiotic stress from the cell of the plant.
15. The method according to claim 14, wherein the nucleotide
sequence comprises the nucleotide sequence as set forth in SEQ ID
NO:1.
16. The method according to claim 14, wherein the abiotic stress is
selected from the group consisting of a drought stress, a
low-temperature stress and a salt stress.
17. The method according to claim 16, wherein the abiotic stress is
a drought stress.
18. The method according to claim 16, wherein the abiotic stress is
a low-temperature stress.
19. The method according to claim 16, wherein the abiotic stress is
a salt stress.
20. The method according to claim 14, wherein the nucleotide
sequence is contained in a recombinant plant expression vector; and
the recombinant plant expression vector comprises (i) the
nucleotide sequence; (ii) a promoter which is operatively linked to
the nucleotide sequence of (i) and generates a RNA molecule in
plant cells; and (iii) a poly A signal sequence inducing
polyadenylation at the 3'-end of the RNA molecule.
21. The method according to claim 14, wherein the plant is selected
from the group consisting of food crops such as rice plant, wheat,
barley, corn, bean, potato, Indian bean, oat and Indian millet;
vegetable crops such as Arabidopsis species, Chinese cabbage,
radish, red pepper, strawberry, tomato, watermelon, cucumber,
cabbage, melon, pumpkin, welsh onion, onion and carrot; crops for
special use such as ginseng, tobacco plant, cotton plant, sesame,
sugar cane, sugar beet, Perilla sp., peanut and rape; fruit trees
such as apple tree, pear tree, jujube tree, peach tree, kiwi fruit
tree, grape tree, citrus fruit tree, persimmon tree, plum tree,
apricot tree and banana tree; flowering crops such as rose,
gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; and
fodder crops such as ryegrass, red clover, orchardgrass, alfalfa,
tallfescue and perennial ryograss.
22. The method according to claim 21, wherein the plant is
vegetable crops such as Arabidopsis species, Chinese cabbage,
radish, red pepper, strawberry, tomato, watermelon, cucumber,
cabbage, melon, pumpkin, welsh onion, onion and carrot.
23. The method according to claim 22, wherein the vegetable crop is
Arabidopsis species.
24. The method according to claim 23, wherein the Arabidopsis
species is Arabidopsis thaliana.
25. A plant cell exhibiting improved tolerance to an abiotic
stress, transformed with the method according to claim 14.
26. A plant exhibiting improved tolerance to an abiotic stress,
transformed with the method according to claim 14.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a gene implicated in
abiotic stress tolerance and growth promotion and a method for
improving abiotic stress tolerance and promoting growing of
transformed plants with the same.
[0003] 2. Description of the Related Art
[0004] Due to their sessile nature, higher plants are constantly
faced with various adverse environmental factors, including
drought, high salt, heavy metals, cold, heat shock, and ozone,
during their whole life span. These abiotic stresses are a limiting
factor for the growth and development of crop plants. Water
deficiency causes dramatic reduction of crop production globally,
and the decreasing availability of fresh water may pose a future
threat to humans and higher plants. Plants have diverse defense
strategies to enhance their tolerance to transient and long-term
water shortages by triggering signaling network pathways and
inducing stress-responsive genes. The cellular and genetic defense
mechanisms in response to water stress have been widely documented
(Shinozaki and Yamaguchi-Shinozaki, 2007). However, for stress
tolerance or sensitivity, our knowledge concerning the biological
functions of stress-related genes in higher plants is still
rudimentary. Therefore, it is important to study the functions of
stress responsive genes to increase the productivity and
distribution of crop plants.
[0005] Ubiquitin is a protein consisting of 76 amino acids and it
has been found in almost all tissues of eukaryotic organisms.
Ubiquitin has a characteristic that is covalently bound to various
substrate proteins by E1-E2-E3 consecutive actions of
ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes
(E2s) and ubiquitin ligases (E3s). The substrate proteins to be
attached with ubiquitin are very diverse, affecting almost all
physiological activities. In addition, many studies have been found
that the many diseases are associated with these mechanisms. A
function of ubiquitin is firstly known to promote degradation of
protein by attaching with other proteins. However, other functions
of ubiquitin have been recently revealed one after another.
[0006] Ubiquitin is attached to substrate by consecutive actions of
three types of proteins, i.e., E1, E2 and E3. The glycine residue
at the C-terminal domain of ubiquitin binds to NH.sub.3 at R--
group of lysine residues on the substrate protein, thereby forming
a covalent bond with the substrate. In general, proteins attached
with ubiquitin are degraded by proteasome. Polyubiquitin as a chain
of several ubiquitin molecules has to be attached to the substrate
for degradation by proteasome. Until now, it has been known that
proteasome-dependent degradation of the substrate occurs only when
polyubiquitin consisting of at least four ubiquitins is attached to
the substrate; however, it would be controversial since these
results were obtained from in vitro experiments. Polyubiquitination
leading to the proteasome-dependent degradation is the linkage form
in which the 48.sup.th lysine residue of one ubiquitin is linked to
another ubiquitin.
[0007] There are 2 types of E1 enzymes in organism. There are
various types of E2s. In general, E2s catalyse the transfer of
ubiquitin from E1 to E3 or substrate. E3s which are also known as
E3 ligases catalyse the final step of the ubiquitination cascade.
E3s determine specificity of the substrate to be ubiquitinated. In
other words, the substrate being capable of interaction with
certain E3s is specifically determined. E3 enzymes may be
classified into two major types according to domains. E3 enzymes
possess one of two domains: the homologous to the E6-AP carboxyl
terminus (HECT) domain and the really interesting new gene (RING)
domain. E3 enzymes having RING domain serves to position E2 and
substrate in close proximity each other. In other words, where E2
and the substrate bind to E3, distance between ubiquitin of E2 and
the substrate is formed to close sufficiently such that ubiquitin
of E2 is chemically passed to the substrate. In contrast, E3
enzymes having HECT domain receive ubiquitin from E2, and then
transfer it to the substrate. The At5g01520 gene codes for the
protein having E3 ubiquitin ligase enzymatic activity. The
ubiquitination has been known to serve diverse functions as one of
the mechanism that all higher organisms as well as plants have.
However, the genes involved in abiotic stresses have been unknown.
The present inventors have isolated the At5g01520 genes in which
the expression is induced by abiotic stresses and ABA hormone in
Arabidopsis thaliana. Then, they have prepared
At5g01520s-overexpresors and knock-out mutants and analyzed their
physiological phenotypes.
[0008] Throughout this application, various publications and
patents are referred and citations are provided in parentheses. The
disclosures of these publications and patents in their entities are
hereby incorporated by references into this application in order to
fully describe this invention and the state of the art to which
this invention pertains.
SUMMARY OF THE INVENTION
[0009] The present inventors have made intensive studies to improve
a tolerance to abiotic stresses of a plant. As results, they have
discovered that a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO:2 was involved in the above-mentioned
characteristic of the plant. In addition, where the gene expression
was inhibited, transgenic plants having the improved tolerance to
abiotic stresses may be obtained.
[0010] Accordingly, it is an object of this invention to provide a
composition for improving the tolerance of a plant to an abiotic
stress, and a plant cell or a plant exhibiting improved tolerance
to an abiotic stress, transformed with the composition.
[0011] It is another object of this invention to provide a
composition for promoting germination of a plant.
[0012] Other objects and advantages of the present invention will
become apparent from the following detailed description together
with the appended claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGS. 1a-d represent results of analyzing the AtAIRP2 gene
expression by RT-PCR after treatments of various abiotic stresses
and ABA hormone. After treatments of ABA hormone (FIG. 1a), drought
stress (FIG. 1b), low-temperature stress (FIG. 1c) and salt stress
(FIG. 1d), each RNA was extracted to analyze the gene expression
pattern. RD29A was used as a representative control gene in
treatments of drought, salt and low-temperature, and RAB18 was used
as a representative control gene in treatment of ABA.
[0014] FIG. 2 represents results of analyzing the promoter activity
of the AtAIRP2 gene by GUS assay. When 100 .mu.M ABA (3 hours) or
drought (2 hours) condition was treated, GUS signals were markedly
induced. It could be determined that promoter activity of the
AtAIRP2 gene was increased.
[0015] FIG. 3 represents results of analyzing the enzymatic
activity of the AtAIRP2 protein. Maltose-binding protein (MBP) was
bound to the AtAIRP2 proteins. Then, MBP-AtAIRP2 was incubated with
HIS-UBA1, HIS-UBC8, ubiquitin and AtAIRP2 at 30.degree. C. for 1
hour to perform Self-Ubiquitination, and performed by Western blot
using MBP- and ubiquitin-specific antibodies to analyze changes in
the protein levels. As a result, it was determined that the
molecular weight of the AtAIRP2 protein was increased through
Western blot using anti-MBP antibody, and the increase was induced
due to ubiquitin. Based on the results, it could be demonstrated
that the AtAIRP2 protein possessed ability for enzymatic activity
of E3 ubiquitin ligase that binds ubiquitin protein.
[0016] FIGS. 4a-d represent results of measuring the AtAIRP2 gene
mutants and their tolerance to drought stress. FIG. 4a represents
the gene map that the T-DNAs were inserted to the exon (AtAIRP2-1)
and the intron (AtAIRP2-2) in genomic DNA of the AtAIRP2 gene. FIG.
4b represents that T-DNA insertions were verified by PCR
amplification using the T-DNA border primer and primers annealing
to sites upstream and downstream of the T-DNA insertion site with
the extracted genomic DNA from the knock-out mutant. FIG. 4c
represents that the expression of the gene was analyzed by RT-PCR
with the extracted RNA from the knock-out mutant. Based on the
results, it could be demonstrated that the expression of the gene
in the AtAIRP2 mutant was inhibited. FIG. 4d represents images of
comparing the tolerance to drought stress in the AtAIRP2 gene
mutants and the wild type Arabidopsis thaliana. Each of plants was
grown for 2 weeks, and subjected to drought stress by withholding
water for 13 days, respectively. The plants were then re-watered
and monitored the number of the survived plant. As a result,
mutants were less tolerant to drought stress than the wild types
(FIG. 4d).
[0017] FIGS. 5a-b represent results of the AtAIRP2-overexpressing
transgenic plants. Arabidopsis thaliana was transformed by
355:AtAIRP2-GFP recombinant vector and it was verified whether to
overexpress the gene using anti-GFP antibody (FIG. 5a). As a result
of Western blot, it could be demonstrated that the AtAIRP2-GFP
protein was well-expressed. FIG. 5b represents images of comparing
the tolerance to drought stress in the AtAIRP2-overexpressing
transgenic and the wild type Arabidopsis thaliana. Each of plants
was grown for 2 weeks, and subjected to drought stress by
withholding water for 14 days, respectively. The plants were then
re-watered and monitored the number of the survived plant. As a
result, the AtAIRP2-overexpressing transgenic plants were more
tolerant to drought stress than the wild types.
[0018] FIGS. 6a-b represent results of analyzing germination rates
according to ABA hormone in the AtAIRP2 knock-out mutant and the
AtAIRP2-overexpressing transgenic plant. FIG. 6a represents images
of the wild type, the AtAIRP2-1 mutant and the AtAIRP2-2 mutant
which were grown on medium supplemented with different
concentrations (0.1 and 0.5 .mu.M) of ABA hormone for 7 days. It
could be understood that the germination rates of mutants which
were grown on medium supplemented with ABA hormone were higher than
that of the wild types. FIG. 6b represents images of the wild type,
the AtAIRP2-2 mutant and the AtAIRP2-sGFP overexpressing transgenic
plant which were grown on medium supplemented with different
concentrations (0.2 and 0.4 .mu.M) of ABA hormone for 7 days. It
could be understood that the germination rates of the AtAIRP2
overexpressing transgenic plants which were grown on medium
supplemented with ABA hormone were significantly inhibited. In
addition, it could be demonstrated that the AtAIRP2-2 mutant showed
the tolerant under the same condition. Therefore, it could be
understood that the germination rates in the AtAIRP2-overexpressing
transgenic plants were decreased by ABA hormone and the germination
rate in mutant was increased by ABA hormone.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In one aspect of this invention, there is provided a
composition for improving the tolerance of a plant to an abiotic
stress, comprising a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO:2.
[0020] The present inventors have made intensive studies to improve
a tolerance to abiotic stresses of a plant. As results, they have
discovered that a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO:2 was involved in the above-mentioned
characteristic of the plant. In addition, where the gene expression
was inhibited, transgenic plants having the improved tolerance to
abiotic stresses may be obtained.
[0021] According to a preferred embodiment, the present nucleotide
sequence encoding the amino acid sequence of SEQ ID NO:2 comprises
the nucleotide sequence as set forth in SEQ ID NO:1. According to
the present invention, the nucleotide sequence as set forth in SEQ
ID NO:1 is the nucleotide sequence of the At5g01520 gene in
Arabidopsis thaliana, and the gene is named as the AtAIRP2
(Arabidopsis thaliana ABA Insensitive Ring Protein 2). The gene
encodes RING protein having E3 ubiquitin ligase enzymatic activity.
The present inventors have found that expressions of the gene were
increased by various abiotic stresses and ABA hormone.
[0022] It would be obvious to the skilled artisan that the
nucleotide sequences used in this invention are not limited to
those listed in the appended Sequence Listings.
[0023] For nucleotides, the variations may be purely genetic, i.e.,
ones that do not result in changes in the protein product. This
includes nucleic acids that contain functionally equivalent codons,
or codons that encode the same amino acid, such as six codons for
arginine or serine, or codons that encode biologically equivalent
amino acids.
[0024] Considering biologically equivalent variations described
hereinabove, the nucleic acid molecule of this invention may
encompass sequences having substantial identity to them. Sequences
having the substantial identity show at least 80%, more preferably
at least 90%, most preferably at least 95% similarity to the
nucleic add molecule of this invention, as measured using one of
the sequence comparison algorithms. Methods of alignment of
sequences for comparison are well-known in the art. Various
programs and alignment algorithms are described in: Smith and
Waterman, Adv. Appl. Math. 2:482(1981); Needleman and Wunsch, J.
Mol. Bio. 48:443(1970); Pearson and Lipman, Methods in Mol. Biol.
24: 307-31(1988); Higgins and Sharp, Gene 73:237-44(1988); Higgins
and Sharp, CABIOS 5:151-3(1989) Corpet et al., Nuc. Acids Res.
16:10881-90(1988) Huang et al., Comp. Appl. BioSci. 8:155-65(1992)
and Pearson et al., Meth. Mol. Biol. 24:307-31(1994). The NCBI
Basic Local Alignment Search Tool (BLAST) [Altschul et al., J. Mol.
Biol. 215:403-10(1990)] is available from several sources,
including the National Center for Biological Information (NBCI,
Bethesda, Md.) and on the Internet, for use in connection with the
sequence analysis programs blastp, blasm, blastx, tblastn and
tblastx. It can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/.
A description of how to determine sequence identity using this
program is available at
http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.
[0025] According to a preferred embodiment, the present abiotic
stress is selected from the group consisting of a drought stress, a
low-temperature stress and a salt stress.
[0026] According to the present invention, the present inventors
have demonstrated that expressions of the At5g01520 gene were
increased when plants were subjected to drought stress,
low-temperature stress or salt stress. In addition, they have
demonstrated that the tolerances to these stresses were improved
when the gene was over-expressed in plants.
[0027] More preferably, the present abiotic stress is drought
stress.
[0028] In another aspect of this invention, there is provided a
composition for improving a tolerance of a plant to an abiotic
stress, comprising a recombinant plant expression vector which
comprises: (a) the nucleotide sequence as disclosed in the present
invention; (b) a promoter which is operatively linked to the
nucleotide sequence of (a) and generates a RNA molecule in plant
cells; and (c) a poly A signal sequence inducing polyadenylation at
the 3'-end of the RNA molecule.
[0029] The term "operatively linked" as used herein refers to
functional linkage between a nucleic acid expression control
sequence (such as a promoter, signal sequence, or array of
transcription factor binding sites) and a second nucleotide
sequence, wherein the expression control sequence affects
transcription and/or translation of the nucleic acid corresponding
to the second sequence.
[0030] The vector system of this invention may be constructed in
accordance with conventional techniques described in Sambrook et
al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory Press (2001), teachings of which are incorporated herein
by reference.
[0031] The suitable promoter in the present invention includes any
one commonly used in the art, for example SP6 promoter, T7
promoter, T3 promoter, PM promoter, maize-ubiquitin promoter,
Cauliflower mosaic virus (CaMV)-35S promoter, Nopalin synthase
(nos) promoter, Figwort mosaic virus 35S promoter, Sugarcane
bacilliform virus promoter, commelina yellow mottle virus promoter,
photo-inducible promoter of small subunit of
Ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), cytosolic
triosphosphate isomerase (TPI) promoter in rice, adenine
phosphoribosyltransferase (APRT) or octopine synthase promoter in
Arabidopsis. Preferably, the promoter used in this invention is
CaMV 35S.
[0032] According to a preferred embodiment, the poly A signal
sequence inducing polyadenylation at the 3'-end includes that from
the nopaline synthase gene of Agrobacterium tumefaciens (NOS 3'
end) (Bevan et al., Nucleic Acids Research, 11(2):369-385(1983)),
that from the octopine synthase gene of Agrobacterium tumefaciens,
the 3'-end of the protease inhibitor I or II genes from potato or
tomato, the CaMV 35S terminator, and OCS (octopine synthase)
terminator. Most preferably, the poly A signal sequence inducing
polyadenylation at the 3'-end in this invention is OCS (octopine
synthase) terminator.
[0033] Optionally, the present vector for plants may further carry
a reporter molecule (e.g., genes for luciferase and
.beta.-glucuronidase). In addition, the vector may contain
antibiotic resistant genes as selective markers (e.g., neomycin
phosphotransferase gene (nptII) and hygromycin phosphotransferase
gene (hpt)).
[0034] According to a preferred embodiment, the plant expression
vector of this invention is Agrobacterium binary vectors.
[0035] The term "binary vector" as used herein, refers to a cloning
vector containing two separate vector systems harboring one plasmid
responsible for migration consisting of left border (LB) and right
border (RB), and another plasmid for target gene-transferring. Any
Agrobacterium suitable for expressing the nucleotide of this
invention may be used, and most preferably, the transformation is
carried out using Agrobacterium tumefaciens GV3101.
[0036] Introduction of the recombinant vector of this invention
into Agrobacterium can be carried out by a large number of methods
known to one skilled in the art. For example, particle bombardment,
electroporation, transfection, lithium acetate method and heat
shock method may be used. Preferably, the electroporation is
used.
[0037] In still another aspect of this invention, there is provided
a plant cell exhibiting improved tolerance to an abiotic stress,
transformed with the composition of this invention.
[0038] In further aspect of this invention, there is provided a
plant exhibiting improved tolerance to an abiotic stress,
transformed with the composition of this invention.
[0039] To introduce a foreign nucleotide sequence into plant cells
or plants may be performed by the methods (Methods of Enzymology,
Vol. 153 (1987)) known to those skilled in the art. The plant may
be transformed using the foreign nucleotide inserted into a carrier
(e.g., vectors such as plasmid or virus) or Agrobacterium
tumefaciens as a mediator (Chilton et al., Cell, 11:263:271 (1977))
and by directly inserting the foreign nucleotide into plant cells
(Lorz et al., Mol. Genet., 199: 178-182 (1985); the disclosure is
herein incorporated by reference). For example, electroporation,
microparticle bombardment, polyethylene glycol-mediated uptake may
be used in the vector containing no T-DNA region.
[0040] Generally, Agrobacterium-mediated transformation is the most
preferable (U.S. Pat. Nos. 5,004,863, 5,349,124 and 5,416,011), and
the skilled artisan can incubate or culture the transformed cells
or seeds to mature plants in appropriate conditions.
[0041] The term "plant(s)" as used herein, is understood by a
meaning including a plant cell, a plant tissue and a plant seed as
well as a mature plant.
[0042] The plants applicable of the present invention include, but
not limited to, food crops such as rice plant, wheat, barley, corn,
bean, potato, Indian bean, oat and Indian millet; vegetable crops
such as Arabidopsis sp., Chinese cabbage, radish, red pepper,
strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin,
welsh onion, onion and carrot; crops for special use such as
ginseng, tobacco plant, cotton plant, sesame, sugar cane, sugar
beet, Perilla sp., peanut and rape; fruit trees such as apple tree,
pear tree, jujube tree, peach tree, kiwi fruit tree, grape tree,
citrus fruit tree, persimmon tree, plum tree, apricot tree and
banana tree; flowering crops such as rose, gladiolus, gerbera,
carnation, chrysanthemum, lily and tulip; and fodder crops such as
ryegrass, red clover, orchardgrass, alfalfa, tallfescue and
perennial ryograss.
[0043] In still further aspect of this invention, there is provided
a composition for promoting germination of a plant comprising a
nucleic acid molecule, wherein nucleic acid molecule inhibits an
expression of a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO:2.
[0044] According to a preferred embodiment, the present nucleotide
sequence comprises the nucleotide sequence as set forth in SEQ ID
NO:1.
[0045] According to the present invention, the nucleotide sequence
as set forth in SEQ ID NO:1 is the nucleotide sequence of the
At5g01520 gene. According to the present invention, it was
determined that Where expressions of the present nucleotide
sequence were inhibited, the sensitivity to ABA hormone suppressing
immature-germination was decreased such that the germination rates
were increased. Therefore, the present nucleic acid molecule
enables to store seeds for a long time with excellent germination
rates.
[0046] According to a preferred embodiment, the nucleic acid
molecule is T-DNA, siRNA, shRNA, miRNA, ribozyme, PNA (peptide
nucleic acids) or antisense oligonucleotide. More preferably, the
present nucleic acid molecule is T-DNA.
[0047] The term "siRNA" used herein refers to a short double strand
RNA that enables to mediate RNA interference via cleavage of mRNA.
The siRNA of the present invention may consist of a sense RNA
strand having a sequence corresponding to a target gene and an
antisense RNA strand having a sequence complementary to the target
gene. The siRNA to inhibit expression of a target gene provides
effective gene knock-down method or gene therapy method.
[0048] The siRNA of this invention is not restricted to a RNA
duplex of which two strands are completely paired and may comprise
non-paired portion such as mismatched portion with
non-complementary bases and bulge with no opposite bases. The
overall length of the siRNA is 10-100 nucleotides, preferably 15-80
nucleotides, and more preferably, 20-70 nucleotides. The siRNA may
comprise either blunt or cohesive end so long as it enables to
inhibit the target gene expression via RNAi effect. The cohesive
end may be prepared in 3'-end overhanging structure or 5'-end
overhanging structure. The base number protruded is not
particularly limited, for example 1-8 bases, preferably 2-6 bases.
In addition, siRNA may comprise low molecular weight RNA (for
example, tRNA, rRNA, natural RNA molecule such as viral RNA or
artificial RNA molecule) in the protruded portion of one end to the
extent that it enables to maintain an effect on the inhibition of
target gene expression. The terminal structure of siRNA is not
demanded as cut structure at both ends, and one end portion of
double strand RNA may be stem-and-loop structure linked by a linker
RNA. The length of linker is not restricted where it has no
influence on the pair formation of the stem portion.
[0049] The term "shRNA" used herein refers to a single strand
nucleotide consisting of 50-70 bases, and forms stem-loop structure
in vivo. Long RNA of 19-29 nucleotides is complementarily
base-paired at both directions of loop consisting of 5-10
nucleotides, forming a double-stranded stem.
[0050] The term "miRNA (microRNA)" functions to regulate gene
expression and means a single strand RNA molecule composed of 20-50
nucleotides in full-length, preferably 20-45 nucleotides, more
preferably 20-40 nucleotides, much more preferably 20-30
nucleotides and most preferably, 21-23 nucleotides. The miRNA is an
oligonucleotide which is not expressed intracellularly, and forms a
short stem-loop structure. The miRNA has a whole or partial
complementarity to one or two or more mRNAs (messenger RNAs), and
the target gene expression is suppressed by the complementary
binding of miRNA to the mRNA thereof.
[0051] The term used herein "ribozyme" refers to a RNA molecule
having an activity of an enzyme in itself which recognizes and
restricts a base sequence of a specific RNA. The ribozyme consists
of a binding portion capable of specifically binding a base
sequence complementary to a transfer RNA strand and an enzymatic
portion to cut target RNA.
[0052] The term "PNA (peptide nucleic acid)" used herein refers to
a molecule having the characteristics of both nucleic acid and
protein, which is capable of complementarily binding to DNA or RNA.
PNA was first reported in 1999 as similar DNA in which nucleobases
are linked via a peptide bond (Nielsen P E, Egholm M, Berg R H,
Buchardt O, "Sequence-selective recognition of DNA by strand
displacement with a thymine-substituted polyamide", Science 1991,
Vol. 254: pp 1497-1500). PNA is absent in the natural world and
artificially synthesized through a chemical method. PNA is reacted
with a natural nucleic acid having a complementary base sequence
through hybridization response, forming double strand. In the
double strand with the same length, PNA/DNA and PNA/RNA double
strand are more stable than DNA/DNA and DNA/RNA double strand,
respectively. The form of repeating N-(2-aminoethyl)-glycine units
linked by amide bonds is commonly used as a basic peptide backbone.
In this context, the backbone of peptide nucleic acid is
electrically neutral in comparison to that of natural nucleic acids
having negative charge. Four bases of nucleic acid present in PNA
are almost the same to those of natural nucleic acid in the respect
of spatial size and distance between nucleobases. PNA has not only
a chemical stability compared with natural nucleic acid, but also a
biological stability due to no degradation by a nuclease or
protease.
[0053] The term "antisense oligonucleotide" used herein is intended
to refer to nucleic acids, preferably, DNA, RNA or its derivatives,
that are complementary to the base sequences of a target mRNA,
characterized in that they bind to the target mRNA and interfere
its translation to protein. The antisense oligonucleotide of the
present invention refers to DNA or RNA sequences which are
complementary to a target mRNA, characterized in that they bind to
the target mRNA and interfere its translation to protein,
translocation into cytoplasm, maturation or essential activities to
other biological functions. The length of antisense nucleic acids
is in a range of 6-100 nucleotides and preferably 10-40
nucleotides.
[0054] The antisense oligonucleotides may be modified at above one
or more positions of base, sugar or backbone to enhance their
functions [De Mesmaeker, et al., Curr Opin Struct Biol., 5(3):
343-55 (1995)]. The oligonucleotide backbone may be modified with
phosphothioate, phosphotriester, methyl phosphonate, single chain
alkyl, cycloalkyl, single chain heteroatomic, heterocyclic bond
between sugars, and so on. In addition, the antisense nucleic acids
may include one or more substituted sugar moieties. The antisense
oligonucleotides may include a modified base. The modified base
includes hypoxanthine, 6-methyladenine, 5-me pyrimidine
(particularly, 5-methylcytosine), 5-hydroxymethylcytosine (HMC),
glycosyl HMC, gentobiosyl HMC, 2-aminoadenine, 2-thiouracil,
2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine,
7-deazaguanine, N6(6-aminohexyl)adenine, 2,6-diaminopurine, and so
forth.
[0055] The term "T-DNA" used herein refers to a DNA fragment as a
transfer DNA in Ti (tumor-inducing) plasmid of Agrobacterium sp.,
which is transferred into a nucleus of a host plant cell. A 25 bp
repeat sequence is present in both termini of T-DNA, and DNA
transfer proceeds at the direction from a left border to a right
border. A bacterial T-DNA with about 20,000 in length destroys a
target gene by insertion, resulting in insertional muatagenesis. In
addition to mutation, inserted T-DNA sequence may label a target
gene. According to the present invention, the present inventors
have used seeds of Arabidopsis thaliana for suppressing the
expression of the At5g01520 gene by means of Ti-plasmid
transformation.
[0056] In still further aspect of this invention, there is provided
a composition for promoting germination of a plant, comprising a
recombinant plant expression vector which comprises: (a) the
nucleotide sequence as disclosed in the present invention; (b) a
promoter which is operatively linked to the nucleotide sequence of
(a) and generates a RNA molecule in plant cells; and (c) a poly A
signal sequence inducing polyadenylation at the 3'-end of the RNA
molecule.
[0057] Since the nucleic acid molecule, the plant expression
recombinant vector and the introduction method thereof are
mentioned above, they are omitted herein to avoid excessive
overlaps.
[0058] The features and advantages of the present invention will be
summarized as follows:
[0059] (a) The present invention provides a composition for
improving the tolerance of a plant to an abiotic stress and a
composition for promoting germinating of a plant.
[0060] (b) The present nucleotide sequence is involved in tolerance
to drought stress of plants. Therefore, the overexpressing
transgenic plants have excellent tolerances to various abiotic
stresses including drought stress, whereby they may be useful as
novel functional crops which are affected by climates and
environments of the cultivated areas.
[0061] (c) In addition, the present nucleotide sequence is involved
in sensitivity to ABA hormone of plants. Therefore, germination
abilities of the knock-out mutant plants in which the expression is
inhibited are remarkably enhanced, whereby they may effectively
used for cultivating the plants with novel function of storage
period increasing, and biomass.
[0062] The present invention will now be described in further
detail by examples. It would be obvious to those skilled in the art
that these examples are intended to be more concretely illustrative
and the scope of the present invention as set forth in the appended
claims is not limited to or by the examples.
EXAMPLES
Experimental Methods
Isolation of the Genes
[0063] The present inventors have isolated the AtAIRP2 genes
inducible by ABA hormone and abiotic stresses from cDNAs of
Arabidopsis thaliana. 10-day-old Arabidopsis thaliana seedlings
were grinded with liquid nitrogen in mortar. The powders were added
with 2 ml of an extraction buffer (4 M guanidine-HCl 20 mM, 10 mM
EDTA, 10 mM EGTA (USB), 0.5% Sarkosyl (SIGMA), pH 9) and
.beta.-mercaptoethanol (SIGMA-ALDRICH) per 1 g of the powder for
extraction. The extraction resultant was transferred to new conical
tube, suspended with an equal volume of PCI
(phenol:chloroform:isoamyl alcohol=25:24:1), vortexed for 5 min and
centrifuged at 3,500 rpm for 25 min (Hanil centrifuge, HA-1000-3).
After centrifugation, the upper organic solvent phase was removed.
The extract was resuspended with an equal volume of PCI, vortexed
and centrifuged twice. Then, the lower aqueous phase was undergone
twice ethanol precipitation and once LiCl precipitation to isolate
RNA. RNA was quantified. Single-strand cDNA was synthesized by
using 2 .mu.g of RNA with oligo dT primer and MMLV reverse
transcriptase (Fermentas). PCR was conducted in the final volume of
50 .mu.L containing 20 ng of cDNA as a template, 10 pmole of each
of two types of primers, 5 .mu.L of 10.times. Taq polymerase buffer
(Takara), 8 .mu.L of dNTPs (each of 1.25 mM) and 1 unit of Taq DNA
polymerase (Takara). The tube containing the reaction mixture was
placed in Perkin Elmer DNA thermal cycler. The sequences of primers
used in this Example are as follows: 5'-ATGCGAAAATCGTTCAAGGA-3'
(AtAIRP2 ORF FW: SEQ ID NO: 3) and 5'-TCACCGAGGAAGAGGAGCATAA-3'
(AtAIRP2 ORF RV: SEQ ID NO: 41). The reaction mixture was denatured
for 2 min at 94.degree. C. and subjected to 30 cycles of 30 sec at
94.degree. C., 30 sec at 52.degree. C. and 1 min at 72.degree. C.
After 30 cycles, polymerization was further performed at 72.degree.
C. for 5 min. Then, the AtAIRP2 gene amplification was verified by
using electrophoresis method. In addition, the DNA was confirmed by
sequencing.
Plant Growth Conditions and Sampling
[0064] In order to prepare the AtAIRP2-overexpressing transgenic
plants, Invitrogen gateway system was used to construct. First,
AtAIRP1-sGFP was introduced into pENTR SD Topo vector (Invitrogen,
USA) and subsequently integrated into pEarlygate 100 vector
(Arabidopsis research center, USA) of plants using LR clonase
enzyme (Invitrogen).
[0065] Seeds of the AtAIRP2 knock-out mutants (seed number:
SAIL_686_G08 (AtAIRP2-1), Salk_005082 (AtAIRP2-2)) which are T-DNA
insertion lines were purchased from SIGNAL Salk Institute Genomic
Analysis Laboratory (http://signal.salk.edu/).
[0066] The seeds of the AtAIRP2-overexpressing transgenic plants,
knock-out mutants and the wild type Arabidopsis thaliana were
soaked in 30% bleach solution (Yuhanclorox) and 0.025% Triton X-100
for 10 min, and washed 10 times with sterilized water. The treated
seeds were grown on MS (Murashige and Skoog) medium (Duchefa
Biochemie) that contained 3% sucrose, B5 vitamin (12 mg/L) and 0.8%
agar (pH 5.7) in a growth chamber for 2 weeks (under a condition of
16 hrs-light/8 hrs-dark cycle). Where green whole plants of light
condition were used as materials, seeds were grown on soil of
Sunshine MIX #5 (Sun GroHorticulture) in a growth chamber for 3
weeks (under a condition of 16 hrs-light/8 hrs-dark cycle).
Treatments of Stresses (Salt, Low-Temperature, Drought and ABA
Hormone)
[0067] In order to determine expressions of the AtAIRP2 gene to
drought stress, the wild type Arabidopsis thaliana seedlings which
were grown on medium for 2 weeks were exposed in the air, and
sampled after 1 hour and 2 hours. In order to determine expressions
of the AtAIRP2 gene to salt stress, the wild type Arabidopsis
thaliana seedlings which were grown on medium for 2 weeks were
treated with 300 mM sodium chloride, and sampled after 1.5 hour and
3 hours. In order to determine expressions of the AtAIRP2 gene to
low-temperature stress, the wild type Arabidopsis thaliana
seedlings which were grown on medium for 2 weeks were incubated at
4.degree. C. for 12 hours and 24 hours, and sampled. In order to
determine expressions of the AtAIRP2 gene to ABA hormone stress,
the wild type Arabidopsis thaliana seedlings which were grown on
medium for 2 weeks were treated with 100 .mu.M of ABA (SIGMA), and
sampled after 1.5 hour and 3 hours. The sampled tissues were
grinded with liquid nitrogen in mortar. The powders were added with
.beta.-mercaptoethanol (SIGMA-ALDRICH) and 2 ml of an extraction
buffer (4 M guanidine-HCl 20 mM, 10 mM EDTA, 10 mM EGTA (USB), 0.5%
Sarkosyl (SIGMA), pH 9) per 1 g of the powder to extract. The
extract was transferred to new conical tube, suspended with an
equal volume of PCI (phenol:chloroform:isoamyl alcohol=25:24:1),
vortexed for 5 min and centrifuged at 3,500 rpm for 25 min (Hanil
centrifuge, HA-1000-3). After centrifugation, the supernatant which
is upper organic solvent phase was removed. The extract was
resuspended with an equal volume of PCI, vortexed and centrifuged.
The extract was performed twice with the process described above.
Then, the lower aqueous phase was performed twice with ethanol
precipitation and once with LiCl precipitation to isolate RNA.
Quantitative Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR)
[0068] Total RNA was isolated from leaves of the
AtAIRP2-overexpressing transgenic plants, knock-out mutants and the
wild type Arabidopsis thaliana. Single-strand cDNA was synthesized
by using 2 .mu.g of RNA with oligo dT primer and MMLV reverse
transcriptase (Fermentas). PCR was conducted in the final volume of
50 .mu.L containing 20 ng of cDNA as a template, 10 pmole of each
of two types of primers, 5 .mu.L of 10.times. Taq polymerase buffer
(Intron), 8 .mu.L of dNTPs (each of 1.25 mM) and 1 unit of Taq DNA
polymerase (Intron). The tube containing the reaction mixture was
placed in Perkin Elmer DNA thermal cycler. The reaction mixture was
denatured for 2 min at 94.degree. C. and subjected to 25 cycles of
30 sec at 94.degree. C., 30 sec at 52.degree. C. and 1 min at
72.degree. C. After 25 cycles, polymerization was further performed
at 72.degree. C. for 5 min. Then, the PCR products were stored at
-20.degree. C. in a freezer. The sequences of primers used in this
Example are shown in Table 1.
TABLE-US-00001 TABLE 1 Primers used in RT-PCR Primer sequence
AtAIRP2 RT FW 5'-GATGGTGGCTACGTTCAGA-3' (SEQ ID: 5) AtAIRP2 RT RV
5'-AAATGTCAATAACCAATGGTTG-3' (SEQ ID: 6) Rab18 FW
5'-GCGTCTTACCAGAACCGTCC-3' (SEQ ID: 7) Rab18 RV
5'-CCCTTCTTCTCGTGGTGC-3' (SEQ ID: 8) RD29a FW
5'-CAGGTGAATCAGGAGTTGTT-3' (SEQ ID: 9) RD29a RV
5'-CCGGAAATTTATCCTCTTCT-3' (SEQ ID: 10) UBC10 FW
5'-TGGATATGGCGTCGAAGC-3' (SEQ ID: 11) UBC10 RV
5'-GTGGGATTTTCCATTTAGCC-3' (SEQ ID: 12)
Extraction of Genomic DNA of Mutants Inserted with T-DNA and
Acquisition of Homozygous Mutant
[0069] The seeds of the wild type Arabidopsis thaliana and
knock-out mutants were grown on soil for 2 weeks and their leave
were sampled. The leaves were grinded with liquid nitrogen in
mortar. The powders were added with 700 mL of CTAB buffer (2% CTAB,
100 mM Tris pH 8, 20 mM EDTA, 1.4 M NaCl, 2% PVP), mixed and heated
at 65.degree. C. for 10 min. The resultants were added to 200 mL of
chloroform, mixed and centrifuged. After centrifugation, the
supernatant was removed. The resultant was mixed with isopropanol
to precipitate DNA. The precipitate was washed with 70% ethanol,
dried. The obtained genomic DNA was dissolved in water to use.
Genotyping PCR was performed using T-DNA border primer (LB_6313R)
and primers annealing to sites upstream and downstream of the T-DNA
insertion site.
TABLE-US-00002 TABLE 2 Primers used in Genotyping PCR and RT-PCR
Primer Sequence LB_6313R 5'-GAGCTGCTATACACTGATCTGAG-3' (SEQ ID: 13)
AtAIRP2 FW1 5'-CGTGTGCTCTACGCGAATC-3' (SEQ ID: 14) AtAIRP2 RV1
5'-CCCTTAATCAGCAAATATGATATCG-3' (SEQ ID: 15)
[0070] It was revealed that the T-DNA insertions were mapped to the
second exon and the first intron in the AtAIRP2 gene knock-out
mutant, which was verified by PCR amplification using the T-DNA
border primer and primers annealing to sites upstream and
downstream of the T-DNA insertion site (FIG. 4b). In addition, the
suppression of the gene expression was analyzed by RT-PCR using
AtAIRP2 FW1 and AtAIRP2 RV1 primers with the extracted RNA from the
knock-out mutant (FIG. 4c).
Preparation of Vector Construct of the AtAIRP2 Gene
[0071] For construction of a recombinant plasmid for expressing the
fusion protein between the AtAIRP2 and maltose-binding protein
(MBP), PCR was carried out using a primer set designed to contain
XbaI and PstI restriction sites linked to 5'-direction and
3'-direction of the coding region of the AtAIRP2 gene,
respectively. PCR products and pMAL-X vector (New England Biolabs,
Beverly, Mass.) were restricted by XbaI and PstI restriction
enzymes and then ligated using T4 DNA ligase (New England Bio Lab).
The recombinant MBP-AtAIRP2 was expressed in Escherichia coli
strain BL21-CodonPlus (DE3) RIL (Stratagene) and purified using
amylose column chromatography. The protein was quantified using BSA
as a standard protein. In addition, in the present invention,
Invitrogen gateway system was used to construct for preparing
transgenic plants. First, AtAIRP2-sGFP was introduced into pENTR SD
Topo vector (Invitrogen, USA) and subsequently integrated into
pEarlygate 100 vector (Arabidopsis research center, USA) of plants
using LR clonase enzyme (Invitrogen).
AtAIRP2 Transformation with Agrobacterium tumefaciens Strain and
Preparation of the AtAIRP2 Transgenic Plants
[0072] The prepared constructs were transferred to Agrobacterium
tumefaciens strain GV3101 by electroporation. The presence of the
gene was confirmed by PCR. An aerial part of approximately
4-week-old Arabidopsis thaliana (columbia [Col-0]) was soaked on MS
medium (Duchefa Biochemie) containing 0.05% Silwet for 1.5 min to
transform (clough and Bent, 1998, Plant J 16; 735-743). The
seedlings were grown for 3 weeks in a 23.degree. C.-growth chamber
to obtain seeds (T1). The transformed seeds (T1) were selected from
medium containing 25 .mu.g/mL of BASTA (Glufosinate ammonium) and
250 .mu.g/mL of carbenicillin. The presence of the transgene was
verified by RT-PCR and western blot. Overexpression of the
transgene was observed using anti-GFP antibody (clontech).
Analysis on Enzymatic Activity of the AtAIRP2 Protein
[0073] For the enzymatic activity analysis of the AtAIRP2 protein,
the ORF of the AtAIRP2 gene was subcloned into pMAL-X vector
in-frame with MBP (maltose-binding protein). 40 mM Tris-HCl, pH
7.5, 5 mM MgCl2, 2 mM ATP, 2 mM dithiothreitol (DTT), 300 ng/.mu.L
ubiquitin (Sigma), 25 .mu.M MG132 (A.G. Scientific Inc.), 500 ng
UBA1 (ABRC, http://www.arabidopsis.org), 500 ng UBC8 (ABRC,
http://www.arabidopsis.org) and 500 ng MBP-AtAIRP2 were added to
each of tubes and incubated at 30.degree. C. After addition of
sample buffer solution, the resultant was heated at 100.degree. C.
for 5 min, and performed by Western blot using anti-MBP (New
England Bio Labs) and anti-ubiquitin (Santa Cruz) antibodies.
Comparison of Plant Growth
[0074] For comparing phenotypes to ABA hormone, seeds obtained from
the wild type, the AtAIRP2 knock-out mutant and the
AtAIRP2-overexpressing transgenic plant were grown on medium
supplemented with different concentrations (0, 0.2, 0.4 and 0.5
.mu.M) of ABA hormone for 7 days, and their germination degrees
were then measured. In order to compare phenotypes to salt, seeds
obtained from the wild type, the AtAIRP2 knock-out mutant and the
AtAIRP2-overexpressing transgenic plant were grown on medium
supplemented with different concentrations (0, 0.2, 0.4 and 0.5
.mu.M) of sodium chloride for 7 days, and their germination degrees
were then measured.
Measurement of Sensitivity of Adult Plants to Drought Stress
[0075] Seeds obtained from the wild type, the AtAIRP2 knock-out
mutant and the AtAIRP2-overexpressing transgenic plant were grown
on soil for 2 weeks, and subjected to drought stress by withholding
water for 13 or 14 days, respectively. The plants were then
re-watered and measured the degrees of the tolerance to drought
stress.
Histochemical GUS Assay
[0076] The wild type Arabidopsis thaliana was grown on medium for
10 days, subjected to 100 mM of ABA hormone and drought stress, and
fixed with 90% acetone for 5 min. After removal of the acetone, the
plant was washed 3 times with rincing solution containing 50 mM
NaPO.sub.4, 1 mM K.sub.3Fe(CN).sub.6, and 1 mM K.sub.4Fe(CN).sub.6,
and immersed in 2 mM X-Gluc (5-bromo-4-chloro-3-indolyl
glucuronide, sigma) and vacuumed for 1 min. After staining at
37.degree. C. in the dark until the color was changed, the plant
was incubated in 90% ethanol to remove chlorophyll.
Experimental Results
[0077] The AtAIRP2 Gene Expression after Stress Treatments (Salt,
Low-Temperature and Drought)
[0078] The AtAIRP2 gene expressions in various abiotic stresses
were analyzed by RT-PCR. After treatments of low-temperature (12
hours and 24 hours), drought (1 hour and 2 hours), salt (1.5 hour
and 3 hours) and ABA hormone (1.5 hour and 3 hours) stresses, RNA
from each of samples was extracted to analyze the gene expression
patterns. As a result, it was determined that the gene expression
levels of stress treatments were increased than those of non-stress
treatments, thereby the AtAIRP2 gene expression is induced by salt,
low-temperature, drought and ABA hormone stresses (FIG. 1). In
addition, the AtAIRP2 protein expressions in various abiotic
stresses were analyzed by histochemical GUS assay. After treatments
of ABA hormone (3 hours) and drought (2 hours) stresses, the degree
of the staining was analyzed. As a result, it was determined that
the degrees of the staining of stress treatments were increased
than those of before the stress treatments (FIG. 2).
Analysis on Enzymatic Activity of the AtAIRP2 Protein
[0079] Maltose-binding protein (MBP) was bound to the AtAIRP2
proteins. Then, the MBP-AtAIRP2 was incubated with UBA1, UBC8,
ubiquitin and AtAIRP2 at 30.degree. C. for 1 hour to perform
Self-Ubiquitination, and performed by Western blot using MBP- and
ubiquitin-specific antibodies to analyze changes in the protein
levels. As a result, it was determined that the molecular weight of
the AtAIRP2 protein was increased through Western blot using
anti-MBP antibody, and the increase was induced due to ubiquitin
(FIG. 3). Based on the results, it could be demonstrated that the
AtAIRP2 protein possessed ability for enzymatic activity of ligase
that binds ubiquitin protein.
Acquisition of the AtAIRP2 Mutants
[0080] As shown in FIG. 4a, the gene that the T-DNA insertions were
mapped to the second exon and the first intron was used in order to
prepare the mutants. In order to determine whether to insert and
position of T-DNA, genotyping PCR was performed using T-DNA boder
primer and primers annealing to sites upstream and downstream of
the T-DNA insertion site. As a result, it was determined that T-DNA
was inserted in the same direction as that of the gene (FIG. 4b).
In addition, in order to determine whether to express mRNA of the
full-length AtAIRP2 gene in mutants, the same primers used in
genotyping PCR were used. As a result, it was determined that the
AtAIRP2 gene was not expressed (FIG. 4c).
Measurement of Sensitivity of Adult Plants to Drought Stress
[0081] In order to measure the tolerance to drought stress in the
AtAIRP2 knock-out mutant and the wild type Arabidopsis thaliana,
each of the plants was grown for 2 weeks, and subjected to drought
stress by withholding water for 13 days, respectively. The plants
were then re-watered and monitored the number of the survived
plant. As a result, knock-out mutants survived respectively by 25%
and 35% whereas the wild types survived by 85%. Therefore, it could
be demonstrated that the mutants were less tolerant to drought
stress than the wild types (FIG. 4d). In order to measure the
tolerance to drought stress in the AtAIRP2-overexpressing
transgenic plants, homozygous plants of each of transgenic plants
obtained through Basta selection were acquired. The obtained seeds
were grown on medium. The protein expressions in transgenic plants
of #10 and #19 were verified by Western Blot using anti-GFP (FIG.
5a). In order to compare the tolerance to drought stress on the
AtAIRP2-overexpressing transgenic plant and the wild type
Arabidopsis thaliana, each of the plants was grown for 2 weeks, and
subjected to drought stress by withholding water for 14 days,
respectively. The plants were then re-watered and monitored the
number of the survived plant. As a result, the
AtAIRP2-overexpressing transgenic plant #10 and #19 survived
respectively by 87.5% and 84.2% whereas the wild types survived by
10%. Therefore, it could be demonstrated that the transgenic plants
were more tolerant to drought stress than the wild types (FIG.
5b).
Comparison of Plant Germination Rate
[0082] As a result of comparison on germination rate in the wild
type and the AtAIRP2 knock-out mutant which were grown on medium
supplemented with different concentrations (0, 0.1 and 0.5 .mu.M)
of ABA hormone for 7 days, it could be understood that the mutant
showed the tolerant (FIG. 6a). As a result of comparison on
germination rate in the wild type, the AtAIRP2 knock-out mutant and
the AtAIRP2-overexpressing transgenic plant which were grown on
medium supplemented with different concentrations (0, 0.2 and 0.4
.mu.M) of ABA hormone for 7 days, it could be understood that the
germination rates in the AtAIRP2-overexpressing transgenic plants
were decreased by ABA hormone and the germination rate in the
AtAIRP2 knock-out mutant was increased by ABA hormone (FIG.
6b).
[0083] Those skilled in the art will appreciate that the
conceptions and specific embodiments disclosed in the foregoing
description may be readily utilized as a basis for modifying or
designing other embodiments for carrying out the same purposes of
the present disclosure. Those skilled in the art will also
appreciate that such equivalent embodiments do not depart from the
spirit and scope of the disclosure as set forth in the appended
claims.
Sequence CWU 1
1
41729DNAArabidopsis thaliana 1atgcgaaaat cgttcaagga ttcactcaag
gctcttgaag ctgatatcca gttcgccaac 60actctggcgt cagagtaccc agaggagtat
gatggtggct acgttcagat gagattatct 120tacagcccgg cggctcatct
ctttctcttc cttcttcagt ggactgattg tcatttcgct 180ggcgctttgg
gcttgcttag gatccttatt tataaggcat atgttgatgg gaagaccaca
240atgtcgctac atgaacgcaa aactagtatc agagaattct atgatgtgtt
gtttccttcg 300ctattgcaac ttcatggagg gatcaccgat gtagaagaaa
ggaaacagaa ggagatatgc 360gacaaaagat accgtaaaaa ggacagaaca
gataaaggaa agatgtcgga gatcgatttg 420gagagggaag aagagtgtgg
aatctgcttg gagattcgaa acaaagttgt tcttcctacg 480tgcaatcact
ccatgtgtat aaactgctac agaaactggc gtgcacggtc acagtcgtgc
540ccgttctgtc gaggcagctt gaaaagagtg aattctggtg atctatggat
atacacttgt 600agcgccgaga ttgcagattt accagcgatt tacaaggaga
atctgaagag gttgttgata 660tacattgaca agttgcctct cgttacttct
gatccaaatc ttgtccctta tgctcctctt 720cctcggtga 7292242PRTArabidopsis
thaliana 2Met Arg Lys Ser Phe Lys Asp Ser Leu Lys Ala Leu Glu Ala
Asp Ile 1 5 10 15 Gln Phe Ala Asn Thr Leu Ala Ser Glu Tyr Pro Glu
Glu Tyr Asp Gly 20 25 30 Gly Tyr Val Gln Met Arg Leu Ser Tyr Ser
Pro Ala Ala His Leu Phe 35 40 45 Leu Phe Leu Leu Gln Trp Thr Asp
Cys His Phe Ala Gly Ala Leu Gly 50 55 60 Leu Leu Arg Ile Leu Ile
Tyr Lys Ala Tyr Val Asp Gly Lys Thr Thr 65 70 75 80 Met Ser Leu His
Glu Arg Lys Thr Ser Ile Arg Glu Phe Tyr Asp Val 85 90 95 Leu Phe
Pro Ser Leu Leu Gln Leu His Gly Gly Ile Thr Asp Val Glu 100 105 110
Glu Arg Lys Gln Lys Glu Ile Cys Asp Lys Arg Tyr Arg Lys Lys Asp 115
120 125 Arg Thr Asp Lys Gly Lys Met Ser Glu Ile Asp Leu Glu Arg Glu
Glu 130 135 140 Glu Cys Gly Ile Cys Leu Glu Ile Arg Asn Lys Val Val
Leu Pro Thr 145 150 155 160 Cys Asn His Ser Met Cys Ile Asn Cys Tyr
Arg Asn Trp Arg Ala Arg 165 170 175 Ser Gln Ser Cys Pro Phe Cys Arg
Gly Ser Leu Lys Arg Val Asn Ser 180 185 190 Gly Asp Leu Trp Ile Tyr
Thr Cys Ser Ala Glu Ile Ala Asp Leu Pro 195 200 205 Ala Ile Tyr Lys
Glu Asn Leu Lys Arg Leu Leu Ile Tyr Ile Asp Lys 210 215 220 Leu Pro
Leu Val Thr Ser Asp Pro Asn Leu Val Pro Tyr Ala Pro Leu 225 230 235
240 Pro Arg 320DNAArtificial SequenceAtAIRP2 ORF FW primer
3atgcgaaaat cgttcaagga 20422DNAArtificial SequenceAtAIRP2 ORF RV
primer 4tcaccgagga agaggagcat aa 22
* * * * *
References