Application of osSGR1 protein and its encoding gene in regulating rice salt tolerance

Abstract

This invention relates to the field of rice genetic engineering, disclosing the application of the OsSGR1 protein and its encoding gene in regulating salt tolerance in rice. This invention uses CRISPR / Cas9 technology to edit the OsSGR1 protein gene, introduces it into the japonica rice variety Zhonghua 11 using Agrobacterium-mediated transformation, and screens for knockout mutants; experimental results demonstrate the effectiveness of the knockout mutant. OsSGR1 The rice mutant of the gene, after being treated with 150 mM NaCl and 200 mM NaCl for 11 days at the 2-leaf-1-heart stage and then rehydrated for 7 days, showed a significantly higher survival rate than the wild-type Zhonghua 11 plants. This provides a simple and effective technical means for rapidly creating new salt-tolerant rice lines. Therefore, the OsSGR1 protein and its encoding gene of this invention can regulate the salt tolerance of rice, which is of great significance for breeding salt-tolerant transgenic rice.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically the application of the OsSGR1 protein and its encoding gene in regulating salt tolerance in rice. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops, feeding more than half the world's population, and its production is of great significance. However, rice is frequently subjected to various abiotic stresses throughout its growth process, among which salt stress is one of the main threats to rice production. Faced with the contradiction between population growth and limited arable land, developing and utilizing coastal tidal flats and inland saline-alkali land resources is one of the effective ways to ensure food security. Because rice grows in an aquatic environment, it can leach soluble salts and alkalis in the soil, and is therefore considered the preferred food crop for developing saline-alkali land. Improving rice's salt tolerance through genetic modification is a core strategy for expanding its planting area and increasing yield.

[0003] Currently, research on rice salt tolerance mainly focuses on ion homeostasis (such as regulating Na+ through HKT and HAK family genes). + / K + Direct pathways such as balance and osmotic regulation are involved. In recent years, the association between salt stress and leaf senescence and yellowing has also received increasing attention. Salt stress is known to induce leaf senescence and yellowing, characterized by chlorophyll degradation and photosynthetic decline. Receptor-activated C kinase 1B (OsRACK1B), as a scaffold protein, has been reported to participate in the salt stress response. Recent studies have shown that OsRACK1B delays salt stress-induced leaf senescence and yellowing by directly interacting with the STAY-GREEN protein (OsSGR) and negatively regulating its transcription and translation levels. This study confirms that... OsSGR It is a key regulator of chlorophyll degradation pathways, and its expression level is positively correlated with the leaf senescence and yellowing process. These findings provide a new perspective for understanding the molecular mechanisms of leaf senescence and yellowing under salt stress.

[0004] However, existing research mainly focuses on elucidating OsRACK1B The signal transduction function of [the substance] and its regulation of the leaf senescence and yellowing phenotype, the technical implication of which lies in how to utilize [this substance] OsRACK1B This influences the yellowing process of leaves. As for direct manipulation... OsSGRWhether the genes themselves can be transformed for genetic improvement of rice salt tolerance, especially whether they can significantly improve the overall survival rate and agronomic traits of rice under long-term salt stress, remains unclear and lacks any reported or clear technical implications. Currently, the salt tolerance QTLs used in breeding are mainly located at the qSKC-1 and Saltol loci on chromosome 1, and the gene resources are relatively limited.

[0005] With the development of molecular biotechnology, the use of mutants to isolate and discover salt stress-tolerant genes in rice, and their application in rice genetic engineering for assisted breeding and alkali stress improvement, is of paramount importance for effectively controlling the damage of salt stress to rice, increasing rice yield, and improving rice quality. However, its implementation also faces significant challenges. When using the CRISPR / Cas9 system for gene editing, the design of single-guide RNA (sgRNA) is the core key and major technical bottleneck determining success or failure. The editing efficiency and specificity of sgRNA are highly uncertain, and its design requires a comprehensive consideration of dozens of complex factors, including sequence length, PAM neighboring sequences, GC content, polyT structure, seed sequence specificity, off-target effects, and the optimal site of action on the target gene. Although various sgRNA design software exist, the algorithms underlying these tools vary greatly, the evaluation criteria for off-target effects differ, and most prediction results lack reliable experimental verification support (Xie Shengsong, 2015). Successfully screening and obtaining an effective sgRNA from thousands of possible sequences that can efficiently and accurately edit the target gene and ultimately produce the desired agronomic traits is a process that requires creative labor and repeated practical verification.

[0006] Therefore, it is not only necessary to reveal it for the first time. OsSGR To realize the direct application value of genes in regulating the overall salt tolerance of rice, it is necessary to overcome technical obstacles and provide a validated, efficient and specific gene editing pathway. This is of vital importance for rapidly creating new salt-tolerant rice germplasm through molecular breeding technology and effectively addressing the harm of salt stress. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the OsSGR1 protein and its encoding gene in regulating rice salt tolerance and in creating salt-tolerant rice varieties.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] The first aspect of this invention provides the application of genes that knock out the OsSGR1 protein, said application being any of the following:

[0010] A1) Application in improving salt tolerance in rice;

[0011] A2) Application in the preparation of products that improve the salt tolerance of rice;

[0012] A3) Application in the cultivation of salt-tolerant rice;

[0013] A4) Application in the preparation of products for cultivating salt-tolerant rice;

[0014] A5) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources;

[0015] The amino acid sequence of the OsSGR1 protein is shown in SEQ ID NO. 3.

[0016] A second aspect of the invention provides the application of biomaterials related to the OsSGR1 protein, wherein the application is any of the following:

[0017] B1) Application in improving salt tolerance in rice;

[0018] B2) Application in the preparation of products that improve the salt tolerance of rice;

[0019] B3) Application in the cultivation of salt-tolerant rice;

[0020] B4) Application in the preparation of products for cultivating salt-tolerant rice;

[0021] B5) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources;

[0022] The biomaterial is any one of the following C1) to C3):

[0023] C1) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the OsSGR1 protein;

[0024] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0025] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0026] C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3), wherein the microorganism is Agrobacterium.

[0027] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the OsSGR1 protein is shown in SEQ ID NO. 2.

[0028] A third aspect of the present invention provides a method for improving the salt tolerance of rice, the method comprising knocking out the gene for the OsSGR1 protein in rice to obtain rice with improved salt tolerance, wherein the amino acid sequence of the OsSGR1 protein is shown in SEQ ID NO.3.

[0029] In the above method, knocking out the OsSGR1 protein gene in rice involves using gene knockout technology to reduce the expression level of the gene encoding the OsSGR1 protein.

[0030] In the above method, the expression level of the gene encoding the OsSGR1 protein is reduced by using gene knockout technology. This is achieved by using a CRISPR / Cas9 system containing the target site shown in SEQ ID NO.5 to perform targeted knockout of the OsSGR1 protein gene.

[0031] The fourth aspect of the present invention provides a method for breeding salt-tolerant rice varieties, comprising: using the salt-tolerant rice obtained by the above method as a parent, hybridizing it with a target material, and backcrossing the obtained F1 generation with the target material so that the backcross offspring acquire the same salt tolerance trait as the parent.

[0032] Furthermore, the salt-tolerant backcross offspring and the parents possess the same mutated gene; OsSGR1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, with a single-base insertion mutation as follows: OsSGR1 The gene inserts a "T" base at position 1363, starting from the start codon ATG. The nucleotide sequence of the mutated gene is shown in SEQ ID NO. 4.

[0033] The beneficial effects of this invention are:

[0034] (1) This invention is the first to discover and confirm that OsSGR1 This gene negatively regulates salt tolerance in rice. Inhibiting or knocking out this gene actually enhances the salt tolerance of rice. This invention provides a new perspective and target for the molecular mechanism of rice's response to salt stress, enriching the theoretical basis of plant salt tolerance.

[0035] (2) Specifically, this invention provides a method for precise knockout using CRISPR / Cas9 gene editing technology. OsSTA230 The gene-based approach includes specific target sites (SEQ ID NO.5) and corresponding targeting vectors. This method is targeted, efficient, and quick, overcoming the bottlenecks of traditional breeding where selecting for salt tolerance traits is difficult and time-consuming.

[0036] (3) Through the above methods, we successfully obtained OsSGR1 Gene knockout mutants. Rigorous physiological experiments demonstrated that after treatment with 150mM and 200mM NaCl salt stress, the survival rate of the mutants (75%) was significantly higher than that of the wild type (8.33%). This provides genetic material and gene resources with direct application value for salt-tolerant rice breeding.

[0037] (4) This invention not only provides a method for creating core salt-tolerant germplasm, but also clarifies a breeding strategy for rapidly introducing this salt-tolerant trait into other superior rice varieties through hybridization and backcrossing. This means that the technological achievement can be quickly applied to actual production to cultivate new rice varieties adapted to saline-alkali land. Attached Figure Description

[0038] Figure 1 for OsSGR1 Schematic diagram of gene structure and target sequence elements of CRISPR / Cas-OsSGR1 vector.

[0039] Figure 2 These are peak diagrams of sequencing results; the top diagram shows the sequencing peaks of the wild-type ZH11 target, and the bottom diagram shows the peaks of the mutant. ossgr1 Target sequencing peak diagram.

[0040] Figure 3 for ossgr1 Salt tolerance test of mutants. A represents ZH11 and... ossgr1 Plant growth status before and after salt treatment, bar = 5 cm; B represents the survival rate statistics of ZH11 and ossgr1 before and after salt treatment. Values ​​shown are mean ± standard deviation, n = 3. * indicates significant difference, P < 0.05; ** indicates extremely significant difference, P < 0.01. Statistical analysis method was one-way ANOVA; C represents the growth status of ZH11 and... ossgr1 Comparison of plant survival rates.

[0041] Figure 4 This is a schematic diagram of the backcrossing and breeding route. Detailed Implementation

[0042] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0043] CRISPR / Cas vector BGK03: Hangzhou Baige Biotechnology Co., Ltd., product catalog number BGK03. Example 1

[0044] This embodiment provides rice. OsSGR1 The functions and applications of genes include the following:

[0045] 1. Rice OsSGR1 Gene sequence and expression pattern analysis

[0046] The Ensembl Plants database (http: / / plants.ensembl.org / index.html) contained information on rice. OsSGR1 The nucleotide sequence of the gene in *Zhonghua 11* of japonica rice is shown in SEQ ID NO. 1, and the CDS sequence is shown in SEQ ID NO. 2. Its encoded protein contains 274 amino acids, and its sequence is shown in SEQ ID NO. 3. To study the function of this gene, this invention first used a gene expression database to analyze the expression pattern of this gene in different tissues of rice. The expression analysis results showed... OsSGR1 The gene is predominantly expressed in the rice roots, suggesting that it may be involved in the rice root development process.

[0047] 2. Rice OsSGR1 Functional verification of genes

[0048] To clarify OsSGR1 This invention addresses the function of genes in rice by using CRISPR / Cas9 gene editing to site-directedly mutate gene sequences, resulting in mutant genes. OsSGR1 This invention involves knocking out the function of a gene in rice. ZH11 (hereinafter referred to as ZH11), a common rice variety, was selected as the recipient material for gene editing. The nucleotide sequence from base 1346 to base 1365 of the gene coding region, starting from the start codon ATG, was selected as the target region for CRISPR / Cas9 gene editing (see [link to CRISPR / Cas9 gene editing instructions]). Figure 1 The mutant OsSGR1 The gene was constructed using sgRNA, the sequence of which is shown in SEQ ID NO.5.

[0049] (1) OsSGR1 Construction of CRISPR / Cas9 gene editing vector

[0050] The gene editing vector of this invention is pEGCas9Pubi-B-OsSGR1, and the base vector of this vector is pEGCas9Pubi-B. This invention involves designing target sites on primers, obtaining MT-sgRNA via PCR, and then ligating it into the base vector using a one-step cloning method. The specific construction process is as follows:

[0051] i) Design of target gRNA. [The following is likely a separate, unrelated sentence:] OsSGR1 The gene sequence was input into https: / / zlab.bio / guide-design-resources for target design, and the PAM sequence was set to NGG. The DNA sequence of the target region selected in this invention is shown in SEQ ID NO. 5.

[0052] ii) Amplify the sgRNA expression cassette by overlap PCR and nested PCR. Primer pairs containing the above-mentioned sgRNA target sequences were synthesized and annealed. Then, the primer pairs were ligated with the binary vector pEGCas9Pubi-B (see Ma X, Zhang Q, Zhu Q. et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants, Mol Plant. 2015, 8(8):1274-1284, vector pEGCas9Pubi-B was kindly provided by Professor Long Tuan of Hainan University) to obtain the recombinant vector pEGCas9Pubi-OsSGR1. The recombinant vector pEGCas9Pubi-OsSGR1 was transformed into E. coli DH5α, and positive clones were selected for sequencing. The specific steps were as described in the reference "Xing, H.L., Dong, L., Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., and Chen, Q.J. (2014). A CRISPR / Cas9toolkit for multiplex genome editing in plants. BMC plant biology 14:327."

[0053] iii) Sequencing verification.

[0054] The successful construction of pEGCas9Pubi-OsSGR1 was verified by sequencing.

[0055] (2) Agrobacterium-mediated genetic transformation of rice

[0056] The constructed pEGCas9Pubi-OsSGR1 vector was transformed into Agrobacterium EHA105 via heat shock. After PCR identification, the bacterial culture was stored at -80 °C with glycerol. Freshly peeled embryos of rice hybrid Zhonghua 11 (approximately 1.5 mm in diameter) were used as recipient materials. The peeled rice embryos were placed in 2 mL plastic centrifuge tubes containing 1.8 mL of suspension and left for no more than 1 hour. Approximately 100 embryos were placed in each centrifuge tube. The suspension was removed, and the embryos were washed twice with fresh suspension, leaving a small amount of suspension at the bottom of the tube to submerge the embryos. The tubes were then heat-shocked at 43 °C for 2 minutes, followed by an ice bath for 1 minute. The remaining wash solution at the bottom of the tube was aspirated with a pipette, and 1.0 mL of Agrobacterium infection solution was added. The tubes were gently shaken for 30 seconds and then incubated in the dark for 8 minutes. Next, pour the embryos and infection solution from the centrifuge tubes onto the co-culture medium, shake well, and then use a pipette to remove excess infection solution. Ensure all embryos have their scutellaria facing upwards and co-culture at 23°C in the dark for 3 days. After co-culture, use sterile forceps to transfer the embryos to recovery medium and culture at 28°C for 7-14 days. During this process, carefully remove any sprouts that appear on the embryos. After recovery culture, place the embryos on 1.5 mg / L Bialaphos selection medium for 3 rounds of selection, each round lasting 2 weeks. Then transfer them to 2 mg / L Bialaphos selection medium for 2 rounds of selection, each round lasting 2 weeks. Transfer the resistant callus to propagation medium and culture at 28°C in the dark for 2 weeks. Then transfer the propagated resistant callus to induction medium and culture at 28°C in the dark for 2 weeks. Finally, transfer it to differentiation medium and culture at 25°C, 5000 lx under light for 2 weeks. After the culture is completed, the differentiated seedlings are separated into individual seedlings and placed in a rooting medium. They are cultured at 25 ℃, 5000 lx, and under light until they root. The seedlings are then transferred to small nutrient pots for growth. After they have survived, they are transplanted into a greenhouse. The offspring seeds are harvested 3-4 months later.

[0057] (3) Detection of CRISPR / Cas9 mutation results in T0 generation plants

[0058] To determine the CRISPR / Cas9 mutation results in T0 generation plants, the following steps were taken for detection:

[0059] This invention first employs the CTAB method to extract DNA from rice leaves. The specific method is as follows: DNA extraction is performed according to the traditional CTAB method (Rogers and Bendich, 1985). A 3 cm rice leaf is placed in a sterilized 2 mL centrifuge tube, a 6 mm steel ball is added, and the tissue is disrupted using a cell disruptor. Then, CTAB extraction is performed. Finally, 200 μL of sterile water (ddH2O) is added to dissolve the air-dried DNA sample, which is then set aside. After the DNA is completely dissolved, 2 μL of the sample is taken and the nucleic acid OD value (A260 / A280) and nucleic acid concentration are determined using a UV spectrophotometer (Nanodrop 2000). The DNA sample is then diluted to 50 ng / μL for later use.

[0060] PCR was performed using Biomiga's 2×PCR premix (containing Mg). 2+ The following reagents were used: 5 μL of Taq DNA Polymerase, 2.5 mM dNTPs, and 10×PCR Buffer; 1 μL of primers (containing 0.5 μL each of forward and reverse primers); 1 μL of template DNA; and ddH2O to a final volume of 10 μL. The PCR amplification program was a standard SSR program (94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and a final extension at 72℃ for 5 min). The amplified products were subjected to 8% non-denaturing polyacrylamide gel electrophoresis, stained with 0.1% AgNO3, and photographed after formaldehyde and NaOH staining.

[0061] For mutants ossgr1 Nucleotide sequence alignment analysis revealed that ( Figure 2 Compared to the unedited wild type (WT), the mutated ossgr1 A T base is inserted at position 1363 starting from the start codon ATG, as shown in SEQ ID NO.4. The deletion of the nucleotide encoded by the mutant causes a frameshift of the amino acid and leads to premature termination of amino acid translation. Example 2

[0062] This embodiment describes the mutant obtained in Example 1. ossgr1 Phenotypic analysis was performed, as follows:

[0063] 1. Mutant ossgr1 Salt tolerance assessment

[0064] The parameters for alternating light and dark culture are as follows: light intensity is 120 μmol·m⁻¹. -2 ·s -1 The temperature is 28℃ / 25℃ (day / dark), and the photoperiod is 10h light / 14h darkness.

[0065] The rice seeds to be tested areossgr1 - L1 Homozygous seeds of mutant T1 generation, along with its background material ZH11 and empty vector control. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows:

[0066] 1. For each material, take 12 rice seeds to be tested, put them into kraft paper bags, and soak them in water at 28℃~30℃ for 48 hours.

[0067] 2. After completing step 1, germinate the seeds at 28℃~30℃ for 24 hours (keep the seeds moist during germination) to obtain germinated seeds.

[0068] 3. After completing step 2, take a 96-well plate, cut off part of the lower edge of each well, and then put one germinated seed into each well (embryo facing up, radicle facing down).

[0069] 4. After completing step 3, place the 96-well plate (containing the germinated seeds) on a plastic box containing Yoshida rice culture medium, immersing the germinated seeds in the medium. Culture in alternating light and dark conditions for 3 weeks to obtain rice seedlings that have reached the three-leaf stage. During the alternating light and dark culture period, the Yoshida rice culture medium should be replaced every 7 days.

[0070] 5. After completing step 4, place the 96-well plate (containing rice seedlings that have grown to the three-leaf stage) on a plastic box containing 150 mM NaCl Yoshida rice culture solution and ensure that the roots are completely immersed in the culture solution. After 3 days of treatment, replace the Yoshida rice culture solution with 200 mM NaCl and treat again. After 3 days, replace the Yoshida rice culture solution with 150 mM NaCl and treat again. Perform high salt stress for 11 days under alternating light and dark conditions (during the high salt stress period, replace the Yoshida rice culture solution every 2 days).

[0071] 6. After completing step 5, place the 96-well plate (with rice seedlings on it) on a plastic box containing Yoshida rice culture medium and allow it to recover for 7 days under alternating light and dark conditions.

[0072] Observe the growth status of rice seedlings and calculate the survival rate. Survival rate = (Number of surviving rice seedlings / 12) × 100%.

[0073] See the growth status of rice seedlings before treatment. Figure 3 The growth status after treatment A is shown in the figure. Figure 3 The survival rate statistics for B are shown in the table below. Figure 3 C.

[0074] The results showed that before salt treatment, ossgr1 Slightly shorter than ZH11; freshwater control ZH11 and mutant ossgr1The survival rate was 100%. After salt solution treatment, the survival rate of ZH11 was 8.33%, while that of the mutant... ossgr1 The survival rate was 75%, and statistical analysis results showed that... ossgr,1 The survival rate of [the strain] was significantly higher than that of ZH11, indicating that... ossgr1 The salt tolerance of the mutant was significantly improved. The phenotype and survival rate of the empty vector control were basically consistent with those of the background material ZH11, with no statistical difference. Example 3

[0075] This embodiment performs OsSGR1 Transgenic experiments using mutant genes, using mutants ossgr1 The R3261 variety was hybridized, backcrossed, and self-crossed with recipients exhibiting excellent agronomic traits, with molecular markers used throughout the process. OsSGR1 Genetic and genetic prospect selection ultimately yields a restorer line with a homozygous mutant gene in the R3261 background. The technical route for hybridization and conversion is as follows: Figure 4 As shown.

[0076] The above example uses R3261 as a breeding example, but it is not limited to R3261 and can be any rice material.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the salt tolerance of rice plants, characterized in that, In the endogenous nature of the rice plant genome OsSGR1 Introducing a single-base insertion mutation into the gene makes the... OsSGR1 The gene inserts a T base at position 1363, starting from the start codon ATG, resulting in a mutant nucleotide sequence as shown in SEQ ID NO.

4. OsSGR1 Genes were used to obtain rice plants with improved salt tolerance; the mutant strain OsSGR1 The gene was constructed using sgRNA; the sequence of the sgRNA is shown in SEQ ID NO.

5.

2. The method according to claim 1, characterized in that, The endogenous nature of the rice plant genome OsSGR1 Introducing a single-base insertion mutation into a gene includes the following steps: Step 1: Construct a CRISPR / Cas9 gene editing vector, wherein the vector targets the gene editing vector as shown in SEQ ID NO.

5. OsSGR1 The nucleotide sequence of the gene from base 1346 to base 1365, starting from the start codon ATG; OsSGR1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; Step 2: Construct Agrobacterium genetically engineered bacteria containing the CRISPR / Cas9 gene editing vector described in Step 1; Step 3: Transform rice plants with the Agrobacterium tumefaciens genetically engineered bacteria described in Step 2 to obtain rice carrying the single-base insertion mutation.

3. The method according to claim 2, characterized in that, The OsSGR1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

3.

4. A method for breeding salt-tolerant rice varieties, characterized in that, include: Using the salt-tolerant rice obtained by the method of any one of claims 1-3 as the parent, hybridize it with the target material, and then backcross the F1 generation obtained with the target material to obtain backcross offspring with salt tolerance.

5. The breeding method according to claim 4, characterized in that, The salt-tolerant backcross offspring and the parent have the same mutant type. OsSGR1 Genes; the stated OsSGR1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the mutant... OsSGR1 The nucleotide sequence of the gene is shown in SEQ ID NO.4.

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