JP2011239684A - Improvement of material productivity of plant using transcription factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for improving desiccation resistance of a plant, increasing nutritious growth and also increasing chlorophyll and carotenoid contents by using a transcription factor gene.SOLUTION: This method includes improving the desiccation resistance, increasing the nutrient growth and the contents of chlorophyll and carotenoid of a plant by introducing a gene of the RGM1 transcription factor belonging to the GARP family out of "MYBL transcription factors" of Arabidopsis thaliana into the plant.

Description

  The present invention relates to a method for producing a plant highly resistant to drought using a plant transcription factor gene and / or a method for producing a plant having increased vegetative growth, and / or production of a plant having a high chlorophyll and carotenoid content. The method, the plant obtained by using the method, and use thereof belong mainly to the field of plant breeding.

  Plants are useful in a variety of applications such as the production and greening of useful substances such as food, energy, raw materials, bioactive substances, and pharmaceutical raw materials, and the collection of pollutants. It is being advanced. High expression of transcription factors by gene recombination technology is highly expected as a powerful tool for molecular breeding, and there are various types of transcription factor gene identification methods based on genomic information and advances in functional analysis technology of transcription factors. There are reports on the use of transcription factor genes related to functions (Non-patent Documents 1 and 2).

  Drought and irrigation water shortages drastically reduce plant biomass productivity and contribute to serious food shortages and economic losses. Plants have a mechanism to protect themselves against drought. When a drought condition is detected, the signal transduction system is activated, and the expression of genes related to drought tolerance or genes that regulate their expression is induced. Have been adapted. However, when the drying conditions are harsh or for a long time, the development and growth of the plant are significantly reduced. Therefore, by improving the drought tolerance of plants, it becomes possible to overcome these limitations and improve the biomass productivity of plants, increasing yields, saving labor and resources, and cultivating various cultivated plants This is useful for expanding the region.

  Until now, attempts have been made to improve drought tolerance of plants by enhancing the expression of drought responsive genes in plants using techniques such as genetic recombination and enhancing the ability of drought response and dehydration tolerance ( Non-patent documents 3 to 6). In recent years, many cases have been reported in which drought response and dehydration tolerance are enhanced by modification of various transcription factor genes involved in drought stress response and drought stress tolerance of plants (Non-Patent Documents 3 to 6). Many of these cases have a strong response to short-term relatively drought stress and dehydration tolerance, and are often exposed to various timings, periods and degrees of intermittent drying, and at the same time exposed to other stresses. There is a need for further technical development for application to the growth of practical crops in the cultivation environment (Non-Patent Documents 7 and 8). Moreover, such enhancement of traits such as drought response and dehydration tolerance often has adverse effects on crop productivity such as poor growth (Non-Patent Documents 3 and 4), and production. Since it is necessary to select useful traits according to the environmental characteristics of the ground, genetic resources related to various types of dry adaptability are required (Non-Patent Document 9). Therefore, the search for further useful genes and various factors related to productivity under dry conditions such as vegetative growth / flowering, water absorption / transpiration / photosynthesis / water use efficiency, or response to complex stress such as high temperature and high salt. Identification of genes involved in complex traits and development of breeding techniques for improving complex traits in an integrated manner are being promoted (Non-Patent Documents 4, 8, and 10-12). For example, transcription factor genes that are promising for improving productivity under dry conditions have been reported (Non-Patent Documents 13 and 14).

From the viewpoint of breeding techniques such as improvement of biomass productivity in plants, control of the timing of flower bud formation, that is, the vegetative long term is also an important issue (Non-patent Document 15). Plant biomass production occurs primarily during the vegetative growth process, resulting in growth. In addition, the biomass produced during the vegetative growth period is converted into a stored material for accumulation in seeds, fruits and the like. Therefore, in many cultivated plants, controlling the vegetative long-term period and the timing of flower bud formation is an important technical issue for improving biomass productivity as a plant body and improving the quality and productivity of seeds and fruits. Yes (Non-Patent Document 15).
In several plant species, genes involved in flower bud formation have been clarified. Especially in Arabidopsis thaliana, many genes involved in the control of flower bud formation have been clarified (Non-patent Documents 15 and 16). Attempts have been made to improve the expression of such genes to promote or delay flower bud formation (Non-patent Document 15). For example, a flower bud formation regulator comprising an unsaturated fatty acid derivative having a flower bud formation inhibitory action as an active ingredient (Patent Document 1), a method for controlling flower bud formation using a flower bud formation inhibitory protein protein or an antisense DNA thereof (Patent Document 2) In addition, a method for producing a flower bud formation delayed plant using a fusion protein of a flower bud formation promoting transcription factor and a functional peptide of the ERF transcription factor family (Patent Document 3) is known.

Under these circumstances, the present inventor has been proceeding with functional genome analysis of transcription factors for the development of plant production control technology in plants, and in the process, high expression of BZF1 gene, In addition, it was found that the biomass amount can be increased and the viability under drought conditions can be improved (Patent Document 4). However, no other transcription factor having such a function has been known so far.
The chloroplasts of higher plants contain chlorophyll and carotenoids as photosynthetic pigments. Chlorophyll plays a major role in the mechanism that collects light and converts light energy into chemical energy. On the other hand, carotenoids have a protective function against photodamage as well as a function as an auxiliary pigment in photosynthesis. Carotenoids are also important as biosynthetic precursors such as vitamin A and abscisic acid, a type of plant hormone.
Until now, several methods for modifying the content and composition of carotenoids using genetic recombination techniques have been reported (Non-Patent Document 19), but none of them is a technique using a transcription factor. Regarding the chlorophyll content, the only technique utilizing a transcription factor has been reported to increase the chlorophyll content by highly expressing the GLK gene belonging to Group 1 of the GARP type MYB family (Non-patent Document 17). 18), however, there are no reports regarding improved drought tolerance and increased vegetative growth.

The present inventors have been proceeding with functional analysis of a MYBL transcription factor having an MYB domain of Arabidopsis thaliana for the purpose of searching for a transcription factor gene useful for efficient production of useful substances and biomass in plants.
A transcription factor with MYB domain was first discovered as an avian myeloblastosis virus oncogene (Non-patent Document 20), but transcription factors with 1 to 3 MYB domains also exist in plants. And was known to form a gene family (Non-patent Documents 21 and 22). The MYB protein contains a large group of transcriptional regulators of eukaryotes and is involved in various biological functions. A common feature of the MYB protein is the presence of a functional DNA binding domain (MYB domain) that is conserved among animals, plants, and yeast. Generally, MYB domains are called R1, R2, and R3, and Consists of 3 incomplete (not identical) repeats. Each repeat is about 50 amino acids long and contains three helices in which the second and third helices form a helix-turn-helix (HTH) DNA binding structure. MYB proteins are classified into subfamilies based on the number of repeats in the domain and the characteristics of the amino acid sequence of each repeat. The MYB family in plants is known to contain R1R2R3-MYB (or 3R-MYB) containing R1, R2 and R3 (or 3R-MYB) and R2R3-MYB containing R2R3-MYB, which contains typical MYB domain repeats. (Non-patent document 29).
Among MYB transcription factors, it is known that MYB8 (HOS10) belonging to the R2R3-MYB subfamily can enhance plant stress response to low temperature, drought, osmotic stress, abscisic acid and salt ( Patent Document 5), an attempt to control cell cycle and cell division by targeting R3 type tobacco-derived MYB transcription factor (Ntmyb protein) gene was also known (Patent Document 6).
In recent years, it has been known that the MYB family having a MYB domain in plants has a type that is completely different from the type containing typical repetitive sequences of R1, R2, and R3 (non-non-type). Patent Document 30).
The present inventors called MYBL (MYB-like) a type having a MYB domain that does not contain typical repetitive sequences such as R1, R2, and R3. A homology search for the MYBL protein sequence of Arabidopsis thaliana in the database Many were identified. So far, 147 MYBL (MYB-like) transcription factors have been found, and molecular phylogenetic analysis and comparative analysis of conserved domain motifs have been performed.
By comparative analysis of amino acid sequences, MYBL transcription factors are classified into several subfamilies. Among them, the type having the repeat containing the SHL / AQKY motif in the MYB domain was the most common, and 85 were found from the Arabidopsis genome database. These are further classified into a type called GARP having a SHLQKY motif and a type having a SHAQKY motif including the transcription factor CCA1, which is supposed to function centrally in the circadian rhythm control system. The MYB family with SHL / AQKY was divided into 10 groups by molecular phylogenetic analysis (FIG. 1). Groups 1-9 have one repeat containing the SHL / AQKY motif. Group 10 has 2 repeats and contains a SHAQKY motif in the second repeat. In the GARP type, 54 members were found and divided into groups 1-6. The GLK gene and B-type ARR gene for which the above-mentioned chlorophyll content increasing action was found belong to group 1, KANADI gene belongs to group 6, NSR1 gene and APL gene belong to group 4, and PHR1 gene belongs to group 5. ing. As described above, regarding the MYBL transcription factor, detailed studies on the expression pattern and function have been gradually advanced (Non-Patent Documents 23 to 28). However, there was no report on the function of the MYB gene group belonging to Group 3 including the RGM1 gene.

JP 2003-73209 A JP 2004-16201 A JP 2005-295878 A JP 2009-72063 A JP 2006-522608 A JP 2004-290193 A

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  It is an object of the present invention to provide a method for improving drought tolerance and / or a method for increasing vegetative growth of plants and / or a method for increasing the chlorophyll and carotenoid content of plants using transcription factor genes. And

While the present inventors are focusing on the RGM1 transcription factor gene belonging to the GARP family of the "MYBL transcription factor" of Arabidopsis thaliana, and examining its function, the RGM1 gene was introduced into Arabidopsis thaliana and multiple T1 generations were introduced. Transformed plants were prepared, and T3 and T4 generation plants were obtained by passage through self-pollination for each T1 plant, and it was confirmed that the RGM1 gene was expressed at a high level. By comparing the traits of T4 generation plants with wild-type plants in detail, the possibility that the RGM1 gene confers drought tolerance on plants, the possibility of increasing vegetative growth, and the possibility of improving chlorophyll content We noticed that there were some traits such as tolerance to drought stress, vegetative growth period and amount, chlorophyll and carotenoid content. As a result, it was confirmed that the transformed plant showed remarkable drought tolerance, increased vegetative growth, and significantly higher chlorophyll and carotenoid content per fresh weight than the wild type. Since the above traits have been confirmed in the T4 generation of multiple transformants, the traits of improved drought tolerance, increased vegetative growth, and increased chlorophyll and carotenoid content are traits that are stably inherited by offspring. It is thought that.
The present inventors have found that the RGM1 gene is introduced into a plant and expressed, thereby improving the drought tolerance of the plant, increasing vegetative growth, and increasing the content of chlorophyll and carotenoid. It came to complete.

The present invention relates to the use of the RGM1 transcription factor gene and provides the invention described below.
[1] An action for improving drought tolerance of a plant, characterized in that it is a DNA encoding RGM1 and contains a recombinant DNA containing at least one of the following (a) to (d) as an active ingredient: An agent having one or more of the following actions: increasing the vegetative growth of plants, increasing the chlorophyll or carotenoid content of plants;
(a) DNA comprising the base sequence represented by SEQ ID NO: 1,
(b) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1,
(c) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(d) DNA encoding a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2.
[2] The drug according to [1], wherein the recombinant DNA is incorporated into a vector that can be expressed in plant cells.
[3] A transformed plant cell or tissue transformed with the agent according to [1] or [2], and generating or redifferentiating a plant organ or an individual from the cell or tissue A transformed plant cell or tissue capable of obtaining a transformed plant with improved drought tolerance and / or increased vegetative growth and / or increased plant chlorophyll and carotenoid content.
[4] Improved drought tolerance and / or increased vegetative growth and / or plant chlorophyll and / or transformed with the agent according to [1] or [2] A transformed plant or a part thereof having an increased carotenoid content.
[5] The transformed plant or part thereof according to [4], wherein the transformed plant or part thereof is a plant individual, seed, plant organ, plant tissue, or plant cell.
[6] Drought tolerance characterized by including a step of transforming a plant with a recombinant DNA containing RGM1 and containing at least one of the following DNAs (a) to (d): Improved transgenic plants and / or increased vegetative growth and / or increased plant chlorophyll and carotenoid content;
(a) DNA comprising the base sequence represented by SEQ ID NO: 1,
(b) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1,
(c) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(d) DNA encoding a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2.
[7] The production method of [6], wherein the recombinant DNA is incorporated into a vector that can be expressed in plant cells.
[8] The transformed plant produced by the production method according to [6] or [7] is further obtained by self-pollination, mating method, or cell fusion method, or the transformed plant A method for producing a plant with improved drought tolerance and / or increased vegetative growth and / or increased plant chlorophyll and carotenoid content, characterized in that the clone is obtained using a part.
[9] A progeny of a transformed plant obtained by the production method according to [8] or a part thereof.
[10] A step of transforming a plant using a recombinant DNA containing DNA encoding RGM1 and containing at least one of the following DNAs (a) to (d): Improving the drought tolerance of plants and / or increasing vegetative growth and / or increasing the chlorophyll and carotenoid content of plants;
(a) DNA comprising the base sequence represented by SEQ ID NO: 1,
(b) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1,
(c) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(d) DNA encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2,
[11] The method according to [10], wherein the recombinant DNA is incorporated into a vector that can be expressed in plant cells.

  The present invention provides a transformed plant having improved drought tolerance. In addition, the present invention provides a transformed plant having increased vegetative growth. Also provided are transformed plants having increased chlorophyll and carotenoid content. Furthermore, the present invention provides a transformed plant to which these traits are imparted in combination. Increasing vegetative growth enables increased production of plant biomass resources. Increased drought tolerance or increased drought tolerance and increased vegetative growth, enabling more efficient plant production under drought or low irrigation conditions, saving labor and saving water, and expanding cultivation areas In addition, it is possible to improve the yield of a plant individual of a cultivated plant or a useful part thereof, increase the yield, reduce the production cost, and the like. Further, the improvement of plant productivity is effective in improving the productivity of various useful substances synthesized and accumulated in the plant body. Furthermore, by increasing the content of chlorophyll and carotenoids, it can be applied to the development of plants that are beneficial to human health and plants that are excellent for greening, as well as to the conversion of plant light energy or photodamage modification technology. Be expected.

FIG. 1 is a diagram showing a molecular phylogenetic tree of the SHL / AQKY type MYB family of Arabidopsis thaliana. It was generated using repetitive amino acid sequences containing SHLQKY or SHAQKY motifs. In this case, the groups 9 and 10 are not distinguished from each other, but the type having two repetitions is group 10 and is classified into 10 groups. RGM1 belongs to 3 in the group. FIG. 2 is a schematic diagram of a vector for constitutively expressing the RGM1 gene of SEQ ID NO: 1 in plants. FIG. 3 shows the results of RT-PCR showing the overexpression of the RGM1 gene of SEQ ID NO: 1 in the transformed plant. RGM1 gene expression is shown for each of two individuals (# 1 and # 2) in a wild-type plant and four RGM1-transformed plants (T3 or T4 generation). PCR was performed using forward and reverse primers. The RGM1 gene is overexpressed in three RGM1-transformed plants. (A) is a photograph showing that the amount of chlorophyll in the transformed plant is increased by high expression of the RGM1 gene. The plant after cultivating the plant for 21 days is shown. Compared to wild-type plants, green color increases in RGM1-transformed plants due to an increase in chlorophyll content. (B) is a graph showing that the chlorophyll content and carotenoid content of the transformed plant are increased by high expression of the RGM1 gene. FIG. 5 is a photograph showing that drought tolerance of transformed plants has been improved by high expression of RGM1 gene. Fig. 4 shows a plant 4 days after cultivating the plant for 25 days, after stopping irrigation for 14 days and restarting irrigation. The wild type did not recover but died as it was, but the RGM1-transformed plant recovered and showed normal growth again. (A) is a photograph showing that the vegetative growth of the transformed plant was increased by the expression of the RGM1 gene. When wild-type plants and RGM1-transformed plants are cultivated under long-day conditions, which are the conditions for promoting flower bud formation, on the 35th day after sowing, in the wild-type, flower stems are extracted, whereas RGM1-transformed plants Then, flower bud formation is not seen. (B) shows the number of real leaves (average of 17 to 18 individuals) until the flower stems in the wild type plant and the RGM1 transformed plant are extracted. Comparing the number of true leaves when flower bud formation began to be observed, the number of RGM1-transformed plants was higher than that of wild-type plants.

1. In the present invention, “to improve drought tolerance of a plant” means to improve the tolerance of a target plant to drought compared to a wild type plant. As shown in Example 3, the drought tolerance of a plant was determined after cultivating a plant transformed with the gene of the present invention together with a wild type plant, stopping irrigation for a certain period, and starting irrigation again. It can be evaluated by examining the survival rate.

2. In the present invention, “vegetative growth is increased” means that the vegetative growth period and / or the amount of vegetative growth of the target plant is increased as compared to the wild type plant. As shown in Example 4, vegetative growth of the present invention can be evaluated by examining the number of days required for flower stems to be extracted or the number of true leaves when flower stem extraction is observed. In addition, the raw weight or dry weight at the time when flower buds are formed may be used as an index.

3. In the present invention, “the content of chlorophyll and carotenoid in the plant is increased” means that the content of chlorophyll and carotenoid in the green leaf portion of the target plant is increased as compared with the wild type plant. As shown in Example 2, the increase in the chlorophyll and carotenoid content of the plant was achieved by cultivating the plant transformed with the gene of the present invention together with the wild-type plant, and then increasing the chlorophyll and carotenoid per fresh leaf weight. It can be evaluated by measuring the amount of.

4). In the present invention, the RGM1 gene can be exemplified by DNA of SEQ ID NO: 1 encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 typically derived from Arabidopsis thaliana.
In addition, DNA having a function of improving drought tolerance, an effect of increasing vegetative growth, and an action of increasing the chlorophyll content can be expressed as “DNA and a string comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1. Also included is DNA that hybridizes under gentle conditions. Such DNA can be obtained by preparation and hybridization of a gene library well known to those skilled in the art, and the setting of “stringent conditions” is well known to those skilled in the art (for example, Maniatis et al. (1989) Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Typically, an appropriate primer pair is designed using the nucleotide sequence information represented by SEQ ID NO: 1 encoding RGM1 of SEQ ID NO: 2, and PCR is performed using the mRNA prepared from the plant as a template using the primer pair. And a plant-derived cDNA library prepared by conventional means using the obtained amplified DNA fragment as a probe can be obtained.
Further, it is a DNA that imparts an effect of improving drought tolerance, an effect of increasing vegetative growth, and an effect of increasing the chlorophyll content to a transformed plant, in the amino acid sequence represented by SEQ ID NO: 2, one or several A DNA encoding a protein consisting of an amino acid sequence in which amino acids are deleted, substituted or added is also used in the present invention.
Therefore, the “DNA encoding RGM1” of the present invention typically includes the following DNA.
(a) DNA comprising the base sequence represented by SEQ ID NO: 1,
(b) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1,
(c) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(d) DNA encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2,

5). Method for producing transformed plant The plant to be transformed with the gene of the present invention may be either a monocotyledonous plant or a dicotyledonous plant. Particularly preferred plants include rice, corn, soybean, poplar, Chinese cabbage, rapeseed and grape.
The vector for inserting the DNA is not particularly limited as long as the inserted gene can be expressed in plant cells. For example, using a vector having a promoter (eg, cauliflower mosaic virus 35S promoter) for constitutive gene expression in plant cells or a vector having a promoter that is inducibly activated by external stimuli Is also possible. The vector into which the DNA is inserted may be introduced into plant cells by methods known to those skilled in the art, such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, particle gun method, etc. Can do. In the examples of the present invention, a typical Agrobacterium method was used.
The present invention provides a plant having improved drought tolerance, grown or regenerated from a transformed plant cell into which the above DNA or vector has been introduced. The “plant having improved drought tolerance” in the present invention refers to a plant having a drought-resistant drought resistance compared to a wild-type plant.

The present invention also provides a plant with delayed flower bud formation. “Plant with delayed flower bud formation” refers to a plant with artificially delayed flower bud formation compared to a wild-type plant.
Furthermore, the present invention provides a plant with improved drought tolerance and delayed flower bud formation.
The present invention provides not only a plant produced from a cell into which the DNA has been introduced, but also its progeny or clone. Once a transformed plant into which the above DNA or vector has been introduced into the genome is obtained, progeny or clones can be obtained from the plant by sexual reproduction, asexual reproduction, tissue culture, cell culture, cell fusion, etc. Is possible. Further, a propagation material (for example, seed, fruit, cut ear, tuber, tuberous root, strain, adventitious bud, adventitious embryo, callus, protoplast, etc.) is obtained from the plant or its progeny or clone, and the plant is based on them. It is also possible to mass-produce the body.
The present invention also relates to a transformation method for improving the drought tolerance of a plant, for delaying flower bud formation of a plant, and for improving the drought tolerance of a plant and delaying the flower bud formation of a plant. I will provide a. The method includes a step of introducing the DNA or vector into a plant cell and a step of growing or regenerating a plant from the cell into which the DNA or vector has been introduced.
The vector having the DNA inserted therein can be introduced into plant cells by methods known to those skilled in the art. The step of growing or regenerating a plant body from the transformed plant cell can be performed by a method known to those skilled in the art depending on the type of plant. For example, for Arabidopsis thaliana, the method (Clough et al., 1998, Plant J. 16: 735-743) or the method (Akama et al., 1992, Plant Cell Reports 12: 7-11) may be mentioned. For rice, the method (Hiei et al., 1994, Plant J.6: 271-281) or (Fujimura et al., 1985, Plant Tissue Culture Lett. 2: 74-75) can be used. .
And in the present invention, when referred to as a “transformed plant”, not only a transformed plant prepared by introducing the above DNA, but also a progeny plant in which seeds obtained by sexual reproduction of the plant are grown, asexual reproduction, It is a concept including all of progeny plants or clones obtained by tissue culture, cell culture, cell fusion, etc., and further progeny plants obtained by subculture them. In the examples of the present invention, the T3 generation or the T4 generation in which seeds obtained by self-pollination of the transformed plant introduced with the DNA of SEQ ID NO: 1 are grown is used.

EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
Regarding the experimental method of the present invention, unless otherwise stated, “Maniatis et al. (1989) Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York”, “Gelvin and Schilperoort edition (1994) Plant Molecular Biology Manual, second edition, Kluwer Academic Publishers "," Shimmoto et al. (2005) Model plant experiment protocol, Shujunsha "or other experimental documents, or instructions for commercial kits used in the experiment Was followed.

Preparation of (Example 1) Isolation of a transgene into a gene of a plant, recombinant vectors and transformed plant for gene transfer can be carried out by known methods. In this example, RGM1 cDNA was isolated from an Arabidopsis plant, a plant expression vector was prepared, and transformed into Arabidopsis using Agrobacterium to obtain an RGM1-overexpressing transformed plant.
(1-1) Cloning of RGM1 gene Total RNA was extracted from Arabidopsis thaliana plant roots, and using this as a template, oligo (dT) and reverse transcriptase were allowed to act to synthesize single-stranded cDNA. Using this single-stranded cDNA as a template, PCR was carried out using forward primer: 5'-CACCATGATCAAGAACTTAAGTAATATG-3 '(SEQ ID NO: 3), reverse primer: 5'- TTATGATATTATTTTTGCATCTTTCGT-3' (SEQ ID NO: 4), and cDNA was single-ended. Released. PCR conditions were 98 ° C. for 30 seconds (98 ° C. for 10 seconds, 58.6, 55.5 ° C., 30 seconds, 72 ° C. for 1 minute) × 30 cycles, 72 ° C. for 10 minutes, and then kept at 4 ° C. The amplified fragment was about 1100 bp (1078 accurately including cacc) bp, and was the desired length.

(1-2) Preparation of RGM1 gene plant expression vector
A plant expression vector for constitutively expressing the RGM1 gene in a plant was prepared as follows. An entry clone containing the full-length cDNA of the RGM1 gene obtained in (1) above and an expression vector (destination vector pK2GW7, (Karimi et al., 2002, Trends in Plant Science; 7 (5): 193-195 ), And an expression clone containing the full-length cDNA of RGM1 gene was obtained by LR reaction according to the Invitrogen Gateway PCR cloning system manual.The constructed RGM1 gene high expression vector (pK2GW7-P35S :: RGM1) 2, it contains a CaMV 35S promoter region, a polynucleotide encoding the RGM1 cDNA of the present invention, and a CaMV 35S terminator region, which were used for subsequent gene introduction into Arabidopsis thaliana.

(1-3) Introduction of RGM1 gene into Arabidopsis thaliana The expression vector constructed in (2) above was introduced into Agrobacterium tumefaciens LBA4404 by the freeze-thaw method. The introduction of the gene was confirmed by colony PCR. The Arabidopsis thaliana for transformation was cultivated at 23 ° C. for long days, and the number of buds was increased by pinching. On the day of infection, the pods and flowered flowers were removed, leaving the buds.
On the other hand, transformed Agrobacterium was pre-cultured at 28 ° C for about 24 hours using YEB medium containing 100 mg / L spectinomycin and 200 mg / L streptomycin, and then cultured at 27 ° C for about 24 hours. Went. The obtained culture broth was collected and suspended in a transformation medium.
According to the Floral-dip method (Clough et al., 1998, Plant J. 16: 735-743), the cocoons were immersed in the suspension for about 1 minute. The treated plants were prevented from drying with wraps, and after being left overnight at 23 ° C. for long days, the suspension was diluted by passing water through the soil. Growing as it was under the same conditions, T1 seeds were obtained. T1 seeds were selected by germination and growth under long-day conditions at 23 ° C. using MS medium containing 50 mg / L kanamycin and 100 mg / L carbenicillin. T2 seeds were obtained from this T1 plant. T2 seeds were selected by germination and growth under conditions of long days at 23 ° C. using MS medium containing 50 mg / L kanamycin. T3 seeds were obtained from this T2 plant. T3 seeds were subjected to kanamycin selection in the same manner as T2 seed selection described above. T4 seeds were obtained from this T3 plant.

(1-4) Expression of RGM1 gene in transformed plants
Total RNA was extracted from four transgenic plants and wild-type plants into which RGM1 gene was introduced, and 0.5 ug was used as a template, and oligo (dT), reverse transcriptase was allowed to act to synthesize single-stranded cDNA. Results of PCR using this single-stranded cDNA as a template and forward primer: 5'-CTCTTTGCGTAGAGCTCGTGA-3 '(SEQ ID NO: 5), reverse primer: 5'-CACTGCCACCATCGCTAGTA-3' (SEQ ID NO: 6) 3 shows. From this result, it was shown that three lines out of four lines of transformed plants expressed the transgene at a high level.

(Example 2) Content of chlorophyll and carotenoid in transformed Arabidopsis plants
Chlorophyll content was examined using T4 generation plants of transgenic plants expressing high levels of RGM1 gene.
After planting RGM1-transformed plants and wild-type plants expressing the introduced RGM1 gene at high levels on artificial soil for 21 days, extract and measure chlorophyll and carotenoids from plant leaves, The amount of chlorophyll was calculated. As a result, as shown in FIG. 4, it was shown that the RGM1-transformed plant had a higher content of chlorophyll and carotenoid in any leaf as compared with the wild type plant. From these results, it was shown that, by expressing the RGM1 gene at a high level, the content of chlorophyll and carotenoid in the transformed plant increased per fresh weight.

(Example 3) Drought tolerance of transformed Arabidopsis thaliana
RGM1 transformed plant (33-2-1 #) and wild type plant (Col-0) were cultivated for 21 days after sowing in artificial soil under long day conditions, and then irrigation was stopped for 14 days Drought. Thereafter, irrigation was resumed and the survival rate after 4 days was examined. As a result, as shown in FIG. 5, in the wild type plant, the above-ground part of the plant body was almost withered 12 to 14 days after irrigation was stopped, and it died without being recovered even after resumption of irrigation. The transformant did not wither on day 13 after cessation of irrigation, and finally the ground part began to wither after 14 days. After that, when irrigation was resumed, it recovered and showed normal growth again. From these results, it was shown that the transgenic plant acquires drought tolerance by expressing the RGM1 gene at a high level. In addition, since no significant abnormality in growth or morphology was observed in the transformed plant, it was considered that overexpression of the RGM1 gene does not have an extreme adverse effect on the growth of the plant.
That is, when irrigation is stopped and drought is performed, RGM1-transformed plants have a slower wilt of the plant body than wild-type plants and are resistant to drought. In addition, when irrigation was resumed in a drought condition where the majority of wild-type plants were wilt, the wild-type plants did not recover but died as they were, but most of the RGM1 transformants recovered and resumed normal growth again. .

(Example 4) Increase in vegetative growth of transformed Arabidopsis plants
RGM1 transformed plants were cultivated under long day conditions with wild type plants (Col-0). FIG. 6A shows the state of the wild-type plant and the RGM1-transformed plant after 35 days. RGM1-transformed plants have delayed flower bud formation compared to wild-type plants (Col-0), while the main leaves that develop are thicker and more in number than wild-type plants and control-transformed plants. became.
(B) of FIG. 6 shows the result of examining the number of true leaves in each plant until the flower stalk is extracted from 17 to 18 individuals. RGM1 transformed plants (33-2-1 #) have an average of 19.0, whereas wild-type plants have an average of 10.5, confirming a 1.8-fold increase in the number of true leaves in RGM1 transformed plants did.

  The transformed plant with enhanced expression of the RGM1 gene of the present invention can be used for increasing the production of agricultural crops and various resource plants because vegetative growth is increased. Furthermore, since drought tolerance is further improved, it is possible to improve the production efficiency of crops and resource plants under drought / low irrigation conditions, save labor and save water, and expand the cultivation area. Not only plant production in traditional agriculture and forestry, but also greening in arid areas, increasing carbon dioxide absorption, increasing production efficiency under artificial weather conditions such as building rooftops and plant factories, saving labor and resources Available. As a form of utilization of plant biomass to be produced, utilization as biomass fuel and raw material is conceivable in addition to the use of traditional agricultural and forestry products. It is also effective in improving the productivity of various useful substances synthesized and accumulated in plants. Furthermore, by extending the life span of a harvested plant or part of the plant and improving the maintenance of freshness, it can be used for reducing the reduction in commercial value and reducing transportation and storage costs. Furthermore, the increased content of chlorophyll and carotenoids can be used to make plants with enhanced human health benefits.

SEQ ID NO: 1: Base sequence of RGM1 gene SEQ ID NO: 2: Amino acid sequence of RGM1 protein
SEQ ID NO: 3: Forward primer for amplification of RGM1 gene (1)
SEQ ID NO: 4: Reverse primer for amplification of RGM1 gene (1)
Sequence number 5: Forward primer for RGM1 gene amplification (2)
SEQ ID NO: 6: Reverse primer for amplification of RGM1 gene (2)

Claims (11)

An action for improving drought tolerance of a plant, characterized by comprising a recombinant DNA comprising RGM1-encoding DNA and at least one of the following DNAs (a) to (d) as an active ingredient: A drug having one or more of the effects of increasing vegetative growth and increasing the content of chlorophyll or carotenoid in plants;
(a) DNA comprising the base sequence represented by SEQ ID NO: 1,
(b) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1,
(c) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(d) DNA encoding a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2.   The drug according to claim 1, wherein the recombinant DNA is incorporated into a vector that can be expressed in a plant cell.   Drought tolerance is improved by generating or redifferentiating a plant organ or individual from a transformed plant cell or tissue transformed with the agent according to claim 1 or 2. And / or transformed plant cells or tissues capable of obtaining transformed plants with increased vegetative growth and / or increased plant chlorophyll and carotenoid content.   Improved drought tolerance and / or increased vegetative growth and / or increased plant chlorophyll and carotenoid content, characterized by being transformed with the agent according to claim 1 or 2 A transformed plant or part thereof.   The transformed plant or a part thereof according to claim 4, wherein the transformed plant or a part thereof is a plant individual, a seed, a plant organ, a plant tissue, or a plant cell. The method comprises a step of transforming a plant with a recombinant DNA containing RGM1 and containing at least one of the following DNAs (a) to (d): And / or a method of producing a transformed plant with increased vegetative growth and / or increased plant chlorophyll and carotenoid content;
(a) DNA comprising the base sequence represented by SEQ ID NO: 1,
(b) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1,
(c) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(d) DNA encoding a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2.   The production method according to claim 6, wherein the recombinant DNA is incorporated into a vector that can be expressed in a plant cell.   The transformed plant produced by the production method according to claim 6 or 7, further obtaining its progeny by self-pollination, mating method, or cell fusion method, or clone thereof using a part of the transformed plant A process for producing a plant with improved drought tolerance and / or increased vegetative growth and / or increased chlorophyll and carotenoid content of the plant, characterized in that The offspring of the transformed plant obtained by the manufacturing method of Claim 8, or its part. Comprising a step of transforming a plant with a recombinant DNA containing DNA encoding RGM1 and containing at least one of the following DNAs (a) to (d): Methods for improving drought tolerance of plants and / or increasing vegetative growth and / or increasing the chlorophyll and carotenoid content of plants;
(a) DNA comprising the base sequence represented by SEQ ID NO: 1,
(b) DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1,
(c) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 2,
(d) DNA encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, The method according to claim 10, wherein the recombinant DNA is incorporated into a vector that can be expressed in a plant cell.