WO2004058975A1

WO2004058975A1 - Method of enhancing tolerance to environmental stresses of plant

Info

Publication number: WO2004058975A1
Application number: PCT/JP2003/016558
Authority: WO
Inventors: Hideo Takahashi; Kan Tanaka; Harumi Kamachi
Original assignee: Showa Denko K.K.
Priority date: 2002-12-25
Filing date: 2003-12-24
Publication date: 2004-07-15
Also published as: AU2003292749A1; JPWO2004058975A1

Abstract

A method of enhancing the tolerance to environmental stresses (for example, strong light stress, high salt concentration stress, high osmotic pressure stress, low temperature and so on) of a higher plant characterized by promoting the production of a photosynthesis-related protein (PSII active center D2 protein) in the plant; a plant having a high tolerance to environmental stresses which is obtained by this method; and a method of constructing a plant having a high tolerance to environmental stresses by using this method. Thus, damaged photosynthesis function of a plant can be repaired and the plant can made tolerant to environmental stresses caused by strong light, high salt concentration, high osmotic pressure (dryness), low temperature and so on.

Description

How to increase environmental stress tolerance in plants

Technical field

The present invention increases the resistance of plants to environmental stress (high light, high salt concentration, high osmotic pressure, low temperature, etc.), and cultivates useful plants for food, ornamental use, etc. under various conditions.

Technology that enables book

Background art

A variety of environmental stresses, such as drought, high temperature, low temperature, or salt, are the main causes of poor plant growth and reduced crop yields and death. Plants have acquired various stress-tolerant mechanisms during evolution. For example, low-temperature-tolerant plants (Arabidopsis, spinach, lettuce, endo, oats, sugar beet, etc.) are more resistant to unsaturated fatty acids in biological membrane lipids than low-temperature-sensitive plants (corn, rice, kabochia, kiyuri, banana, tomato, etc.). It has a low content of glycerol, so that even when exposed to low temperatures, phase transition of lipids in biological membranes is unlikely to occur, and low-temperature damage is unlikely to occur. Although these are environmental adaptations in the long-term evolution of plants, the growing need to grow crop plants in environments that are unfavorable for plant growth (low-temperature regions, arid regions, etc.) and to prevent desertification by greening are increasing. However, it has been strongly desired to produce an environmentally resistant plant that exceeds the natural environmental stress.

In plant improvement, methods such as line selection and crossing have been used in the class, but the selection method requires a lot of time.On the other hand, the crossing method should be used only among limited species. As a result, it was difficult to produce plants with high environmental stress tolerance. With the recent advances in genetic engineering, techniques for introducing and expressing specific genes into plants, such as drying, low-temperature, Attempts have been made to produce resistant plants.

So far, genes used to create environmental stress-tolerant plants include synthase genes for osmotic pressure regulators (such as mannitol, proline, and glycine betaine) and gene enzymes for modifying cell membrane lipids. Specifically, the mannitol synthase gene is Escherichia coli-derived mannitol 1-phosphate dehydrogenase gene (Science, vol. 259, 1993, pp. 508-510), and the proline synthase gene is bean-derived Δ 1-proline 1 5 One carboxylate synthetase gene (PlantPhysiology, 108, 1995, pp. 1387-1394), and a glycine betaine synthase gene are bacterial choline dehydrogenase genes (The Plant Journal, 12, 1997, 1334-1342. Examples of cell membrane lipid-modifying genes include the Arabidopsis thaliana ω-3 fatty acid desaturase gene (Plant Physiology, Vol. 105, pp. 601-605, 1994) and the cyanobacterial Δ9 desaturase gene (Nature Biotechnology, Vol. 14). , 1996, pp. 1003-1006). However, none of the plants into which these genes have been introduced have been put into practical use due to problems such as unstable stress resistance and low resistance level.

In recent years, it has been reported that several genes act to acquire tolerance to drought, low temperature, or salt stress, resulting in stress resistance in plants (Plant Physiology, 115, 1997, 327). -334). Therefore, a gene encoding a transcription factor capable of simultaneously activating the expression of a plurality of genes involved in obtaining stress resistance has been introduced into plants, and plants having high stress resistance have been produced ( The Plant Cell, Vol. 10, 1998, pp. 1-17). However, when a gene that induces the expression of a plurality of genes is introduced as described above, the plurality of genes are activated at the same time, so that the energy of the host plant is limited to the production of the gene product and the inheritance of the gene. Host plants are often slowed to grow or dwarfed, being directed to intracellular metabolism due to offspring products. In order to solve this problem, a stress-responsive element is placed downstream of a stress-responsive promoter that is expressed in response to a specific stress stimulus. Resistance to environmental stresses (such as drought stress, low temperature stress, and salt stress), including DNA-linked genes that bind to proteins and encode proteins that regulate the transcription of genes downstream of the element. It has also been reported to produce transgenic plants that do not cause dwarfing (JP-A-2000-116259). In addition, a new gene that enhances environmental stress tolerance when introduced into a plant has been discovered (US Patent Application Publication No. 2002/0102695). However, there is no known method for positively improving the damage caused by environmental stress on the photosynthetic system, which is the root of plant survival.

It is known that, among the damages caused by environmental stress to the photosynthetic system, the most damaged by environmental stress is the D2 protein, which is the weakest protein in the photosynthetic system, and is the Ps II active center.

On the other hand, the gene sig 5 (formerly called sig E) is presumed to be responsible for controlling the production of PS II active center, and sig 5 is known to be expressed by blue light ( FEBS Letters, vol. 516, 2002, pp. 225-228), expression under environmental stress (high light, high salt concentration, low temperature, etc.) according to the present invention, and the fact that the PS II activity center D2 protein is exposed to environmental stress. It is not known that it is produced in response, and it is not anticipated that damage to the photosynthetic system due to environmental stress can be solved by promoting the repair function until these two points are discovered That was it. Disclosure of the invention

The present invention is based on a new mechanism that actively restores the plant's resistance to environmental stresses such as strong light, high salt concentration, high osmotic pressure (dryness), and low temperature, and actively recovers the damage to the photosynthetic function of these stresses. One of the tasks is to provide a method for increasing the environmental stress, and another object is to provide a method for obtaining a plant generally having high environmental stress tolerance by the method. The present inventors have found that expression of sig5 is induced by environmental stress such as strong light, high salt concentration, high osmotic pressure (dryness), and low temperature. Furthermore, they found that the P2 protein active center D2 protein, the weakest protein in the photosynthetic system, was promoted by environmental stress such as strong light, high salt concentration, high osmotic pressure (dryness), and low temperature. From this, we found that sig5 is a key factor in photosynthetic response to environmental stress, and to further enhance this function, that is, to recover the photosynthetic system damage caused by environmental stress early, to improve the photosynthetic environment. It has been discovered that stress resistance can be obtained. Based on these findings, the ability of sig5 to be highly expressed even when environmental stress is not applied, and whether or not sig5 is more highly expressed in response to a stimulus, can enhance the environmental stress tolerance of plants, and can enhance the environmental stress tolerance. The present inventors have found that a plant having a high yield can be obtained, and completed the present invention.

That is, the present invention includes the following items.

1. A method for enhancing the resistance of plants to environmental stress, which promotes the production of photosynthesis-related proteins in higher plants.

2. The method for enhancing tolerance to environmental stress of a plant according to the above item 1, wherein the photosynthesis-related protein of a higher plant is a PS II active center D2 protein.

3. The method for increasing the resistance of a plant to environmental stress as described in 1 or 2 above, wherein the sig5 component gene is expressed during growth.

4. The sig5 gene is linked downstream of the transcriptional regulatory region that strongly expresses the sig5 component gene by the gene conversion method, and is incorporated into plants to express the sig5 component gene during growth as described in 1 or 2 above. A method for increasing the resistance of the described plant to environmental stress.

5. By the gene conversion method, the sig5 gene is ligated downstream of the promoter, which is strongly induced by environmental stress, and transformed with Agrobacterium via T-DNA. Sig 5 genetic by the method 3. The method for increasing the resistance of a plant to environmental stress according to the above item 1 or 2, wherein the offspring are introduced into a plant body.

6. The method for increasing the resistance of a plant to environmental stress according to any one of the above items 1 to 5, wherein the environmental stress is high light stress.

7. The method for increasing the resistance of a plant to environmental stress according to any one of the above items 1 to 5, wherein the environmental stress is high salt concentration stress.

8. The method for increasing the tolerance of a plant to environmental stress according to any one of the above items 1 to 5, wherein the environmental stress is hyperosmotic stress.

9. The method according to any one of the preceding items 1 to 5, wherein the environmental stress is low-temperature stress, and the plant has an increased resistance to environmental stress.

10. A plant having high resistance to environmental stress obtained by using the method for increasing resistance to environmental stress of a plant according to any one of 1 to 9 above.

11. A method for producing a plant having high resistance to environmental stress, which comprises using the method for increasing resistance of a plant to environmental stress according to any one of 1 to 9 above.

Hereinafter, the present invention will be described in detail.

The sigma factor is a factor that binds to the enzyme part (core enzyme) responsible for R Ν Α synthesis of DNA-dependent RNA polymerase to recognize the promoter on the genome and initiate transcription. Only when the sigma factor is present, the first stage of protein production is transferred to DNA → R Ν D. And factors are a group of proteins that control the expression of various genes at the transcriptional stage.

Chloroplasts are known as organelles that perform photosynthesis, and have their own genome and gene expression system. The chloroplast genome encodes an intrinsic bacterial RNA polymerase (PEP) and is mainly involved in the expression of photosynthesis-related genes. Confer promoter recognition specificity to the core enzyme subunit of this PEP The sigma factor gene is located on the nuclear chromosome.

It has been clarified that there are six types of sigma factors (sig1 to sig6, formerly called sigA to sigF) that bind to the chloroplast PEP. The sigma factor is highly conserved among organisms, and it is presumed that the six species found in plants (Arabidopsis) are almost the same in other higher plants, and their functions will be the same.

Although the function of the sigma factor is being elucidated in part, the function of sig 5, which is the subject of the present application, was hardly known.

sig5 is one of the six sigma factors found in plants (Arabidopsis thaliana), but we have identified that this gene can be used in plants such as strong light, high salt concentration (high osmotic pressure), and low temperature. Was found to be highly expressed by environmental stimuli that would be stressful for the children. Similarly, they found that gene expression of a protein (PS II active center) that is susceptible to environmental stress is also promoted by environmental stress.

As a result of high expression of sig5, it was proved that among the photosynthesis-related genes, gene expression in the center of PS II active I ¹ was promoted. By this action, they discovered that they function to acquire environmental resistance by restoring the photosynthetic pathway damaged by stress.

In other words, by performing a modification such that sig5 is always highly expressed irrespective of the presence or absence of environmental stimulus, it is possible to produce a plant which is essentially resistant to environmental stress.

Since the function of the σ factor is common to higher plants, the principle of producing a stress-tolerant plant according to the present invention can be applied to higher plants in general.

A general sig5 expression method, gene conversion method, and the like will be described. In the present invention, the environmental stress means a stress that causes some damage to a plant body, and includes those present in a normal plant growth environment such as light and oxygen. In the present invention, intense light refers to light that exists in the natural environment and impairs a plant, and varies depending on the type of the plant. For example, the amount of light that is twice or more the normal growth environment of a certain plant, That is, for a plant that normally grows sufficiently with a light quantity of 25 μE / m ² · s, the value is 50 μE Zm ² · s or more (microinsteinine square meters.sec).

In the present invention, the high salt concentration means a salt concentration that damages a plant, and varies depending on the type of a plant.

In the present invention, the low temperature means a low temperature that damages a plant, and is, for example, a temperature lower than the environment where a certain plant normally grows by 1 ° C. or more.

The sig5 gene used in the present invention is registered in the gene number as accession number AB0211120. The sig5 gene can be obtained by chemical synthesis, by PCR using the cDNA or genomic DNA of the present gene as type III, or by hybridization using a DNA fragment having the base sequence as a probe. it can.

The sig5, which is similar to the sig5 gene under conditions of stringent hybridization with the sig5 gene, can also be used as long as the protein encoded by the DNA has a function of activating RNA polymerase by binding to PEP. Can be used. The stringent condition is, for example, a condition at a formamide concentration of 30 to 50%, preferably 50%, and a temperature of 37 to 50 ° C, preferably 42 ° C. Say.

By introducing the sig5 gene into plant hosts using genetic engineering techniques, it is possible to develop resistance to environmental stress, especially strong light, light salt concentration, high osmotic pressure (dryness), and low temperature stress. A transgenic plant having the same can be produced. Methods for introducing a gene into a plant host include indirect methods such as the agrobacterium infection method, the particle gun method, the polyethylene glycol method, and the liposome method. Examples include direct introduction methods such as a sosome method and a microinjection method. When the agrobacterium infection method is used, a transgenic plant can be prepared as follows.

(1) Preparation of Plant Introducing Recombinant Vector and Transformation of Agrobacterium Transplantation The plant introducing recombinant vector is obtained by cutting the DNA containing sig5 with an appropriate restriction enzyme, if necessary. It can be obtained by ligating an appropriate linker and inserting it into a cloning vector for plant cells. As a cloning vector, a binary vector plasmid such as pBI2113Not, pBI2113, pBI101, pBI121, pGA482, pGAH, pBIG, or an intermediate vector plasmid such as pLGV23Neo, pNCAT, or pMON200 can be used.

When a binary vector-based plasmid is used, a target gene is inserted between the boundary sequences (LB, RB) of the binary vector, and the recombinant vector is amplified in Escherichia coli. Next, the amplified recombinant vector is introduced into Agrobacterium tumefaciens C58, LBA4404, EHA101, C58ClRifR, EHA105, or the like by a freeze-thaw method, an electoral poration method, or the like, and the agrobacterium is transduced into a plant. Used for

In addition to the above method, in the present invention, agrobacterium for plant infection containing the sig5 gene can be prepared by a three-way conjugation method (Nucleic Acids Research, 12: 8711 (1984)). That is, Escherichia coli having a plasmid containing a target gene, Escherichia coli having a helper plasmid (for example, pRK2013), and agrobacterium are mixed cultured, and cultured on a medium containing rifampicin and kanamycin. It is possible to obtain a conjugate aglopacterium for plant infection.

The sig5 gene activates the chloroplast PEP and increases the production rate of the PSII active center D2 protein, the weakest protein in the photosynthetic system. sig 5 is the expression of specific genes such as PS II active center D 2 protein in chloroplast Because it is a controlling factor, an unspecified number of genes are activated, and the resulting increase in energy consumption and metabolic activation do not suppress the growth of the plant itself. However, it is thought that it is expressed only when it receives a stimulus that causes environmental stress, and connects the sig5 gene downstream of the stress-responsive promoter. Examples of such promoters include the rd29A gene promoter (The Plant Cell, 6, 251-264 (1994)) and the rd29B gene promoter (The Plant Cell, 6, 251-264 (1994)). ), Rd17 gene promoter (Plant Physiol., 115, 1287 (1997)), rd22 gene promoter (Mol. Gen. Genet., 247, 391-398 (1995)), DREB1A gene promoter (Biochem Biophys. Res. Com., 250, 161-170 (1998)), cor6.6 gene promoter (Plant Mol. Biol "28, 619-634 (1995)), cor15a gene promoter (Plant Mol. Biol., 24, 701-713 (1994)), erd 1 gene promoter (Plant J., 12, 851-861 (1997)), kin 1 gene promoter (Plant Mol. Biol., 28, 605-617 (1995) These promoters are obtained by PCR using a primer designed based on the nucleotide sequence of the DNA containing the promoter and using the genomic DNA as a 錶 type. Can be obtained.

In addition, a terminator that directs the termination of transcription can be connected downstream of the DREB gene, if necessary. Examples of the terminator include a cauliflower mosaic virus-derived ゃ nopaline synthase gene terminator. However, the terminator is not limited to this, and may be any terminator known to function in a plant.

If necessary, an intron sequence between the promoter sequence and the sig5 gene, which enhances gene expression, such as the intron of maize alcohol-nordehydrogenase (Adh1) (Genes & Developmentl, 1183-1200) (1987)) can be introduced.

Furthermore, effective selection markers are used to efficiently select the desired transformed cells. Preferably, the Kerr gene is used in combination with the sig5 gene. The selectable markers used in this case include the kanamycin resistance gene (Ν Ρ ΤΠ), the hygromycin phosphotransferase (htp) gene that confers resistance to the antibiotic hygromycin to plants, and the resistance to bialaphos. One or more genes selected from phosphinothricin acetyltransferase (bar,) genes and the like to be conferred can be used. The sig5 gene and the selectable marker gene may be integrated together into a single vector, or two types of recombinant DNAs each integrated into separate vectors may be used.

(2) Introduction of sig5 gene into plant host

In the present invention, a plant host refers to a plant cultured cell, the whole plant of a cultivated plant, a plant organ (eg, leaf, petal, stem, root, rhizome, seed, etc.), or a plant tissue (eg, epidermis, phloem) , Parenchyma, xylem, vascular bundle, etc.). Plants that can be used as plant hosts include Arabidopsis, tobacco, rice, corn and the like. For the sig5 gene, a vector containing the sig5 gene in a collected plant section can be introduced into the above-mentioned plant host by an agrobacterium infection method, a particle gun method, a polyethylene dalicol method, or the like. Alternatively, a vector containing the sig5 gene can be introduced into protoplasts by the electroporation method.

When a gene is introduced by the Agrobacterium infection method, it is necessary to infect a plant host with the Agrobacterium containing a plasmid containing the target gene. This step can be performed by a vacuum infiltration method (CR Acad. Sci. Paris, Life Science, 316, 1194 (1993)). That is, a plant host is directly immersed in a culture medium of agrobacterium containing a plasmid containing the sig5 gene, put in a desiccator, and aspirated with a vacuum pump until the pressure reaches 65 to 70 mmHg. Leave at room temperature for minutes. Transfer the pot to a tray and cover with plastic wrap to keep humidity. Take the wrap the next day and let the plants grow Harvest the seeds.

Next, seeds from various strains are sown on an MS agar medium supplemented with an appropriate antibiotic in order to select a target gene-bearing individual. Transgenic plants into which the gene used in the present invention has been introduced can be obtained by transferring the plants grown in this medium to pots and growing them.

Generally, a gene introduced into a plant is integrated into the genome of a host plant. In this case, a phenomenon called a position effect in which the expression of the introduced gene differs due to a difference in the position on the genome to be introduced is observed. Transformants in which the transgene is more strongly expressed can be selected by detecting the level of mRNA expressed in the host plant by the Northern method using the DNA fragment of the transgene as a probe. .

To confirm that the target gene has been incorporated into the transgenic plant into which the gene used in the present invention has been introduced and its next generation, DNA can be extracted from these cells and tissues in accordance with a conventional method, and a known PCR method can be used. It can be performed by detecting the introduced gene using the method or Southern analysis.

(3) Analysis of expression level and expression site of sig5 gene in plant tissue

Analysis of the expression level and expression site of the sig5 gene in a transgenic plant into which the sig5 gene has been introduced is performed by extracting RNA from these cells and tissues according to a conventional method, and using a known RT-PCR method or Northern blot method. The analysis can be performed by detecting the mRNA of the sig5 gene.

(4) Changes in mRNA levels of various genes in transgenic plants into which sig5 gene has been introduced

In the transgenic plant into which the sig5 gene has been introduced, a gene whose expression level is considered to have changed due to the action of sig5 can be identified by Northern analysis. In Northern analysis, transgenic plants with the sig5 gene introduced and plants without the sig5 gene were used, The assay can be performed by comparing the mRNA levels of genes considered as target genes according to a conventional method.

For example, a plant grown on a GM agar medium or the like is subjected to environmental stress for a predetermined period (for example, 1 to 2 weeks). At this time, the strong light stress can be given by irradiating light having an intensity of 50 μEZm ² · s (microin stubine / square meter's), preferably 500 E / m ² · s. High salt stress can be imparted to normal MS media by adding 25 OmM sodium chloride. Hyperosmotic stress can be imparted to normal MS media by adding 25 OmM mannitol. On the other hand, the load of low-temperature stress can be provided by maintaining the temperature at 15 ° C to 14 ° C, preferably at 4 ° C for 10 minutes to 24 hours. Prepare total RNA from unstressed control plants and stressed plants, perform electrophoresis, and test the expressed genes by Northern analysis or RT-PCR.

(5) Evaluation of tolerance of transgenic plants to environmental stress

The resistance of transgenic plants transfected with the sig5 gene to environmental stress is as follows: Transgenic plants are planted in flower pots containing soil containing bamikiuraito, perlite, etc., resulting in high light, high salt concentration, and high osmotic pressure. It can be evaluated by examining the survival and leaf color when various stresses such as low temperature are applied. When stress causes abnormalities in the photosynthetic system, the green color of the leaves changes clearly and slightly. For example, the resistance to high light stress can be determined by examining its survival under high light for 2-4 weeks, and the resistance to low temperature stress can be determined by placing it at 4 ° C for 5-10 days, then 5-10 days. It can be evaluated by growing at 20 to 25 ° C. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the sig in plants stressed with salt stress according to the method of Example 2. The results of analysis of the expression of 5 and ps bD are shown together with the results of the control not subjected to salt stress (“one NaClj” in the figure).

FIG. 2 shows the results of analyzing the expression of sig5 and psbD in low-temperature stressed plants according to the method of Example 2.

FIG. 3 shows the results of analyzing the expression of sig5 in plants subjected to hyperosmotic stress according to the method of Example 2.

FIG. 4 shows the results of angular analysis of the expression of sig5 and psbD in plants subjected to high light stress according to the method of Example 2.

FIG. 5 shows a sig expression analysis plasmid (pE-GUS) obtained by introducing a 1.8 kb Hindlll-BamHI fragment derived from pEp into the Hindlll-BamHI site of ρΒΙΙΟΙ according to the method of Example 4.

FIG. 6 shows the results of expression of the sig5 promoter by GUS staining of the Arabidopsis thaliana in Example 4 which had been subjected to the salt stress treatment, together with the results of the control not loaded with salt stress (“one Na C 1” in the figure).

FIG. 7 shows a sig expression analysis plasmid (pE-OX) according to Example 5. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples. Example 1: Culture of Arabidopsis thaliana (L.) Heynth ecotype Columbia (Col)

One of the following two methods was selected according to the purpose.

A: Solid culture

Basically according to (Plant Cell Physiol., 41, 1119-1128 (2000)). Arabidopsis The required amount of na seeds was collected, sterilized with 70% (v / v) ethanol and 3% (v / v) sodium hypochlorite, and then 0.4% (w / v) gel light (Wako ) Containing 1% Sucrose-MS plate (Physiol. Plant, 15, 473-497 (1962)) or Jiffy-7 (AS Jiffy Products). After 24 hours of low-temperature treatment at 4 ° C in the dark, the cells were grown at 23 ° C under constant light conditions (50 µmo 1 photons / m ² · s).

B: Liquid culture

The cells were shake-cultured in a 1% Sucrose_MS liquid medium at 23 ° C under continuous white light (50 μπιο1 photons / m ² -s) irradiation conditions. Example 2: Environmental stress load

According to the method of Example 1, the plants were grown on day 10 grown under continuous white light (50 mo 1 photons / m ² · s). Except for the described conditions, the conditions were the same before and after the stress treatment.

A: Salt stress treatment

To the plants cultured in the MS liquid medium, NaCl was added to a final concentration of 250 mM, the growth was observed, and sampling was performed over time.

B: High light stress treatment

Plants grown under 50 mo 1 hotons / m ² · s white light were transferred under 500〃 mo 1 photons / m ² -s white light and sampled over time.

C: Low temperature stress treatment

Plants grown on a solid medium at 23 ° C were transferred to 4 ° C and then sampled over time.

D: Osmotic stress treatment

For plants cultured in liquid medium, mannitol was added to a final concentration of 250 mM, and samples were taken over time. Example 3: Gene expression analysis in response to environmental stress

It was performed by Northern Hydride.

For preparation of RNA from plants, the plants were frozen and crushed in liquid nitrogen, and then all RNA was extracted using TRIzol reagent (Invitrogen). The method followed the protocol of the kit. Spectrophotometer DU640 (BECKMAN) was used for quantification of the extracted total RNA.

The operation of Northern hybridization was according to the literature (Plant Cell Physiol., 40, 832-842 (1999)). Probes for detecting each gene are as follows: sig5 is the corresponding cDNA type D, psbD is the Arabidopsis total DNA type 铸, and amplified by PCR using the primer sets shown in SEQ ID NOs: 1-4. It was prepared by DIG labeling. PCR was performed using ExTaq (Takara) for 25 cycles of 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute. The size of the fragment to be amplified is 500 bp for sig5 and 549 bp for psbD.

Sequence number l: 5 'primer for sig5 sig5—F

Sequence number 2: 5 'primer for sig5 sig5—R

SEQ ID NO: 3 for ps bD 5, primer ps bD-F

SEQ ID NO: 4: Primer p s bD—R for p s bD

According to the method of Example 2, expression of sig5 and psbD in plants subjected to environmental stress was analyzed. The results are shown in Figs. 1 to 4 (Fig. 1: salt stress, Fig. 2: low temperature stress, Fig. 3: hyperosmotic stress, Fig. 4: high light stress). The results clearly show that sig5 and psbD are specifically highly expressed in response to various environmental stresses. Example 4: Expression of sig5 in plants in response to environmental stress

Furthermore, sig 5—promoter region: construction of uid A fusion gene, transformation Preparation and evaluation by SUG staining.

PCR was performed on Arabidopsis chromosome 5 P1 clone MLE8 (provided by Kazusa DNA Research Institute) using the primers of SEQ ID NOS: 5 to 6 to obtain an amplified fragment of 2.5 kb. The 1.8 kb fragment obtained by digesting it with PmaCI and BamHI was introduced into the SmaI-BamHI site of pBluescriptll KS + to obtain pE. The 1.8 kb Hindlll-BamHI fragment derived from pEp was introduced into Hindlll-BamHI 眘 position 15 of ρΒΙΙΟΙ to obtain pE—GUS (FIG. 5). After introducing this plasmid into Agrobacterium tumefaciens strain C58, Arabidopsis plants were transformed by invasion. Kanamycin-resistant strains were selected from the obtained seeds, and 20 lines were obtained and used for analysis. GUS staining was according to the literature (Plant Cell Physiol., 40, 832-842 (1999)). Rooster column number 5: sig5 promoter (promoter) primer D5—B amH

I

SEQ ID NO: 6: sig5 promoter (promoter) primer D5—BamHI2

The plants of the above 20 lines were subjected to salt stress treatment according to Example 2, and the treated plants were GUS-stained to observe expression. Filter paper was placed on an MS plate, seeded on it, and cultured for 14 days under normal conditions. The plant was transferred together with the filter paper onto an MS plate containing 25 OmM NaC1, and the culture was continued for 12 hours. Staining was performed according to the literature (Plant Cell Physiol., 40, 832-842 (1999)). In plants that had been treated with salt, it was observed that the sig5 promoter showed high expression (Fig. 6). Example 5: Production of sig5 overexpressing plant and stress tolerance test

The sig5 cDNA clone was designated as type III, and PCR amplification was performed with the primers shown in SEQ ID NOS: 7 to 8 under the same conditions as in Example 3 to obtain a fragment containing the entire sig5 ORF. SEQ ID NO: 7: Promoter for sig5 ORF sig5 ORF—F SEQ ID NO: 8: Promoter for sig5 ORF sig5 ORF—R After cloning this into pCR2.1-TOPO (Invitrogen) plasmid, BamHI—E It was excised as a coRV fragment. This was cloned into the BamHI-SmaI site of pBI121 to obtain pE-OX (FIG. 7). This plasmid was introduced into Agrobacterium tumefaciens strain C58 and transformed into Arabidopsis plants by invasion. A kanamycin-resistant strain was selected from the obtained seeds, and a line overexpressing sig5 was selected and used for analysis. As a control, a plant transformed with pBI121, a vector introduced into a plant, was used.

In the test for salt stress, the cells were seeded on an MS plate to which 250 mM NaCl was added, and the state of germination under normal growth conditions was observed. As a result, most of the cotyledons were observed to be whitened in the control strain, whereas more than half of the sig5-expressing strains formed green cotyledons.

Test for strong light stress, after seeded on MS plate, 200 / im o 1 photons / m 2 - the culture was performed in white light illumination under s. The control strain became yellow-green from the beginning of the culture and died in about one month, whereas the sig5 high expression strain was darker in green and did not die.

The test for drought was performed by sown on Jiffy-7, cultivated under normal growth conditions for about 3 weeks, then stopped supplying water and compared the time until death. While the control strain died in about one week, the sug5 high-expressing strain exhibited a survival advantage of several days. Industrial applicability

ADVANTAGE OF THE INVENTION According to the method of this invention, the damage caused by the environmental stress to the photosynthetic system which is the root of plant survival can be positively improved, It is possible to avoid the death of houseplants due to the decrease in photosynthetic function caused by the loess and the decrease in crops and yield. In addition, plants that are resistant to environmental stress can be produced by this method.

Claims

The scope of the claims

2. The method according to claim 1, wherein the photosynthesis-related protein of a higher plant is a PS II active center D2 protein.

3. The method for enhancing tolerance to environmental stress of a plant according to claim 1 or 2, wherein the sig5 component gene is expressed during growth.

4. The sig5 gene is ligated downstream of the transcriptional regulatory region that strongly expresses the sig5 component gene by the gene conversion method, and the sig5 component gene is expressed during growth by integration into a plant. Or the method for increasing the resistance of a plant to environmental stress according to 2 above.

5. By the gene conversion method, the sig5 gene is ligated downstream of the promoter, which is strongly induced by environmental stress, and is transformed by Agrobacterium through T-DNA. 3. The method according to claim 1 or 2, wherein the sig5 gene is introduced into the plant body to increase resistance of the plant to environmental stress.

6. The method for increasing the resistance of a plant to environmental stress according to any one of claims 1 to 5, wherein the environmental stress is high light stress.

7. Any of claims 1 to 5, wherein the environmental stress is high salt concentration stress A method for increasing the resistance of plants to environmental stress according to

8. The method according to any one of claims 1 to 5, wherein the environmental stress is high osmotic stress.

9. The method for increasing tolerance of a plant to environmental stress according to any one of claims 1 to 5, wherein the environmental stress is low-temperature stress.

10. A plant having high resistance to environmental stress obtained by using the method for increasing resistance to environmental stress of a plant according to any one of claims 1 to 9.

11. A method for producing a plant having high resistance to environmental stress, comprising using the method for increasing resistance to environmental stress of a plant according to any one of claims 1 to 9.