Application of OsMT9 protein and coding gene thereof in regulating and controlling salt tolerance of rice

Abstract

The invention relates to the field of rice gene engineering, and particularly provides an application of OsMT9 protein and a coding gene thereof in regulating and controlling the salt tolerance of rice. According to the invention, sgRNA (target sequences are as shown in SEQ ID NO.4 and SEQ ID NO.5) of a specific targeting OsMT9 gene coding region is designed, and a gene editing vector is constructed. The rice Zhonghua 11 is transformed through an agrobacterium-mediated method, and mutants with OsMT9 gene function deletion, such as mutants 22-1 and 23-15, are obtained. Salt stress experiments show that the survival rate (about 43%) of the mutant is remarkably higher than that (about 6%) of a wild type. The invention provides a new gene target and a valuable germplasm resource for salt-tolerant breeding of rice, and has important significance on utilization of saline-alkali soil.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the application of OsMT9 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 effective approach. Because rice grows in an aquatic environment, it can leach soluble salts and alkalis in the soil, thus being 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, rice salt-tolerant breeding mainly relies on quantitative trait loci (QTLs), such as those on chromosome 1. qSKC-1 and Saltol With the development of molecular biology, the discovery of salt-tolerant genes through gene editing technology and their application in genetic improvement have become an effective way to enhance the salt tolerance of rice. Metallothionein (MT) is a non-enzymatic small molecule protein rich in thiol groups or thiol clusters. Plant metallothioneins have multiple biological functions, including chelating heavy metals and detoxifying them; transporting trace essential metals, maintaining essential metal homeostasis, and participating in plant growth and development; and acting as highly efficient scavengers of reactive oxygen species (ROS), reducing free radicals and ROS in cells after stress (Qiu et al., 2021). Under abiotic stress, plant metallothioneins play an important role in improving plant stress resistance. Previous studies have extensively investigated the functions of metallothioneins in plants (Saeed-ur-Rahman et al., 2020). The rice MT family contains 13 genes. OsMT-3a It can increase the Cd content of the variety 2+ Resistance; OsMT2c Cu-induced increased expression OsMT2c Overexpression increases copper tolerance (Liu et al., 2015). OsMT It can improve the resistance of rice to drought stress. OsMT1e-P and OsMT4 It participates in the defense mechanisms against salt stress. OsMT9 There are currently no reports of individuals participating in adversity-related coercion.

[0004] With the development of molecular biology technology, using mutants to isolate and mine salt stress genes of rice and for rice genetic engineering to assist breeding and improve salt stress resistance have extremely important significance for effectively controlling the harm of salt stress to rice, improving rice yield and improving rice quality. However, its specific implementation also faces great challenges. When using CRISPR / Cas9 system for gene editing, the design of single guide RNA (sgRNA) is the core key and main technical bottleneck. The editing efficiency and specificity of sgRNA are highly uncertain, and its design needs to comprehensively consider dozens of complex factors such as sequence length, PAM adjacent sequence, GC content, polyT structure, seed sequence specificity, off-target effect and optimal action site on target gene. Although there are various sgRNA design software, the algorithms of these tools are different, the evaluation standards for off-target effect are different, and most of the prediction results lack reliable experimental verification support (Xie Shengsong, 2015). From thousands of possible sequences, successfully screening and obtaining an effective sgRNA that can efficiently and accurately edit the target gene and ultimately produce the expected agronomic traits is a process that requires creative labor and repeated practice verification.

[0005] Therefore, it is necessary to first disclose OsMT9 the direct application value of the gene in regulating the overall salt tolerance of rice, and it is also necessary to overcome the technical implementation obstacles and provide a verified and efficient and specific gene editing path, which is of great significance for quickly creating new salt-tolerant rice germplasm through molecular breeding technology and effectively responding to salt stress hazards. SUMMARY

[0006] The purpose of the present application is to provide OsMT9 the application of the gene in regulating the salt tolerance of rice.

[0007] To achieve the above purpose, the present application provides the following technical solutions: In a first aspect, the present application provides a knockout OsMT9 application of the gene, which is any one of the following: A1) application in improving the salt tolerance of rice; A2) application in preparing a product for improving the salt tolerance of rice; A3) application in breeding salt-tolerant rice; A4) application in preparing a product for breeding salt-tolerant rice; A5) application in salt breeding of rice or improvement of salt-tolerant germplasm resources of rice; The knockout OsMT9 The nucleotide sequence of the mutant OsMT9 gene obtained after the knockout The OsMT9 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 3.

[0008] The second aspect of the present application provides a method for improving the salt tolerance of rice plants, which introduces a single base insertion and base deletion mutation or a large fragment deletion mutation in the endogenous OsMT9 The single base insertion mutation makes the 359th base from the start codon ATG of the OsMT9 gene be a T base, and 5 bases ATCTG from 564th to 568th base be deleted, so as to obtain a mutant osmt9-ko-22-1 gene with a nucleotide sequence shown as SEQ ID NO. 6, and further obtain a rice plant with improved salt tolerance; the large fragment deletion mutation makes the 359th to 568th base from the start codon ATG of the OsMT9 gene be deleted by 210 bases, so as to obtain a mutant osmt9-ko-23-15 gene with a nucleotide sequence shown as SEQ ID NO. 9. osmt9 The mutant gene is constructed by a CRISPR / Cas9 system, which uses sgRNA targeting sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5.

[0009] The method introduces a single base insertion and base deletion mutation or a large fragment deletion mutation in the endogenous OsMT9 gene of the genome of the rice plant, including the following steps: Step one, constructing a CRISPR / Cas9 gene editing vector, which targets the nucleotide sequence from the 356th base to the 375th base from the start codon ATG of the OsMT9 gene and the nucleotide sequence from the 552nd base to the 571st base from the start codon ATG of the OsMT9 gene shown as SEQ ID NO. 5; the OsMT9 nucleotide sequence of the gene is shown as SEQ ID NO. 1; Step two, constructing an agrobacterium genetic engineering bacterium containing the CRISPR / Cas9 gene editing vector of step one; Step three, transforming the agrobacterium genetic engineering bacterium of step two into a rice plant to obtain a rice plant carrying the single base insertion mutation or the large fragment deletion mutation.

[0010] The third aspect of the present application provides a breeding method of a salt-tolerant rice strain, comprising: using the salt-tolerant rice obtained by the above method as a parent to cross with a target material, backcrossing the obtained F1 generation with the target material, and obtaining a backcross progeny with salt tolerance.

[0011] Further, the backcross progeny with salt tolerance and the parent have the same mutant type OsMT9 gene; the nucleotide sequence of the mutant type OsMT9 gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the mutant type OsMT9 gene is shown as SEQ ID NO. 6 or SEQ ID NO. 9.

[0012] The present application has the following beneficial effects: (1) The new function of the gene is disclosed for the first time, and a new salt-tolerant breeding target is provided: the gene is found and verified for the first time to negatively regulate the salt tolerance of rice. By knocking out the gene, the salt tolerance of rice can be significantly improved, which provides a new theoretical basis for understanding the molecular mechanism of plant salt tolerance and provides a new target gene for salt-tolerant breeding. OsMT9

[0013] (2) A new breeding material with significantly enhanced salt tolerance is created: using the CRISPR / Cas9 gene editing technology, a functional loss mutant of the gene (such as OsMT9 and osmt9-ko-22-1 ) is successfully obtained. Under salt stress, the survival rate of the mutant (about 43%) is significantly higher than that of the wild type (about 6%), and the phenotype is outstanding, providing valuable germplasm resources that can be directly used for breeding. osmt9-ko-23-15

[0014] (3) An efficient and precise salt tolerance improvement technical solution is established: The sgRNA (target sequence: SEQ ID NO. 4 and SEQ ID NO. 5) and the corresponding gene editing vector specifically targeting the gene are designed. This method is precise, efficient, and stable in mutation, and can realize the site-directed knockout of the gene, which is suitable for the directional improvement of salt tolerance of different rice varieties, and has strong technical repeatability. OsMT9 OsMT9

[0015] (4) It has great potential for production and application: the technical solution and mutant material provided by the present application can be directly used to cultivate new salt-tolerant rice varieties. This has important and far-reaching significance for the development and utilization of large areas of saline-alkali land and the expansion of rice planting area. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Schematic diagram of the transcriptome of the metal-sulfur transporter in the aboveground and underground parts of rice after salt stress. ​​​​

[0017] Figure 2 For OsMT9 Gene structure and CRISPR / Cas-OsMT9 vector target sequence element schematic diagram and sequencing result peak chart; wherein the upper graph is the wild type ZH11 reference sequence, and the lower graph is the mutant osmt9-ko-22-1 and osmt9-ko-23-15 Target point sequencing peak chart.

[0018] Figure 3 For osmt9 Mutant 13-day seedlings, 10‰ salt treatment for 8 days, and 7-day rehydration culture salt tolerance phenotype identification chart, bar = 7 cm.

[0019] Figure 4 For ZH11 and osmt9 Survival rate statistics before and after salt treatment. The values shown are the mean ± standard deviation, n = 3. *, significant difference, P < 0.05; **, extremely significant difference, P < 0.01, and the statistical analysis method is one-way ANOVA.

[0020] Figure 5 For backcross breeding route schematic diagram. DETAILED DESCRIPTION

[0021] The specific embodiments of the present application are described below to facilitate those skilled in the art to understand the present application, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all applications utilizing the concept of the present application are within the scope of protection. Example 1

[0022] This embodiment provides a rice OsMT9 gene function and application, specifically including the following: 1. Rice OsMT9 Gene sequence and expression pattern analysis In the Ensembl Plants library (http: / / plants.ensembl.org / index.html), the rice OsMT9 gene was found, and the nucleotide sequence in japonica rice Zhonghua 11 is shown in SEQ ID NO. 1, and the CDS sequence is shown in SEQ ID NO. 2. The encoded protein contains 78 amino acids, and the sequence is shown in SEQ ID NO. 3. In order to study the function of the gene, the present application first analyzes the expression pattern of the gene in different tissue parts of rice by using gene expression database, and the expression analysis result shows that OsMT9 The gene is specifically expressed in the root of rice.

[0023] 2. RiceOsMT9 Transcriptome analysis of genes WT and ko-1 aerial leaf samples under salt stress for 6, 30 and 102 h were sampled, 3 biological replicates for each sample, 3 plants randomly selected for each replicate, frozen with liquid nitrogen and stored at -80℃ for standby. Total plant RNA was extracted using the TRIzol method, mRNA with polyA tail was enriched by Oligo(dT) magnetic beads, and mRNA was broken into 250-300 bp fragments by ion breaking, to construct the mRNA library. Double-end sequencing was performed based on the Illumina NovaSeq 6000 platform. Total RNA extraction and quality detection, cDNA library construction and sequencing were completed by Beijing Baire and Kang Biotechnology Co., Ltd. After filtering the raw data, removing the adapter and low-quality reads, the HISAT2 software was used to align the obtained high-quality reads to the reference genome of rice 'Nipponbare' (IRGSP-1.0) (Mortazavi et al. 2008). According to the alignment results, the number of reads covered in the range from the start to the end of each gene was counted, and the FPKM method (Trapnell et al. 2010) was used to calculate the expression of each gene and the Pearson correlation coefficient between WT and ko-1 replicate samples. The DESeq2 (Love et al. 2014) software was used to analyze the difference in gene expression level between WT and ko-1, and the standard for screening differentially expressed genes (DEGs) was: expression difference fold |log2 (Fold Change)|≥1, P≤0.05. In order to further analyze the biological function and metabolic pathway of DEGs, GO enrichment analysis was performed based on the hypergeometric distribution principle. By analyzing the transcriptome data before and after salt stress, it was found that the members of the metallothionein family responded to salt stress Figure 1 ) 3. Rice OsMT9 Verification of the function of the gene In order to clarify OsMT9 the function of the gene in rice, the present application uses CRISPR / Cas9 gene editing method to site-specifically mutate the gene sequence and knock out the function of the gene in rice.

[0024] The present application selects rice conventional rice Zhonghua 11 (hereinafter referred to as ZH11) as the receptor material for gene editing. The present application selects the target sequence 1 of the nucleotide sequence from the 356th base to the 375th base from the start codon ATG, as shown in SEQ ID NO. 4; and the target sequence 2 of the nucleotide sequence from the 356th base to the 375th base from the start codon ATG, as shown in SEQ ID NO. 5, as the target sequence of the gene. OsMT9The target sequence 2 is the nucleotide sequence from base 552 to base 571 of the gene coding region, starting from the start codon ATG, as shown in SEQ ID NO. 5 (see SEQ ID NO. 5). Figure 2 ).

[0025] (1) OsMT9 Construction of CRISPR / Cas9 gene editing vector The gene editing vector of this invention is pEGCas9Pubi-B-OsMT9, 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: i) Design of target gRNA. [The following is likely a separate, unrelated sentence:] OsMT9 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. 4.

[0026] 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-OsMT9. The recombinant vector pEGCas9Pubi-OsMT9 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 multiple genome editing in plants. BMC plant biology 14:327."

[0027] iii) Sequencing verification.

[0028] The positive clone with correct sequencing result is the successfully constructed pEGCas9Pubi-OsMT9 gene editing vector.

[0029] (2) Agrobacterium-mediated genetic transformation of rice The successfully constructed pEGCas9Pubi-OsMT9 gene editing vector was transformed into Agrobacterium EHA105 by heat shock method, and the bacterial solution was stored at -80°C after PCR identification and addition of glycerol.

[0030] Freshly peeled 1.5 mm or so of hybrid rice Zhonghua 11 embryo was used as the recipient material, and the peeled rice embryo was placed in a 2 mL plastic centrifuge tube containing 1.8 mL of suspension, and the placement time was not more than 1 hour, about 100 embryos were placed in each centrifuge tube; the suspension was sucked off, and the embryos were washed twice with new suspension, a small amount of suspension was reserved at the bottom of the tube to cover the embryos, then 43°C heat shock for 2 minutes, followed by ice bath for 1 minute, the residual washing solution was sucked off with a pipette, and 1.0 mL of Agrobacterium infection solution was added, shaken for 30 seconds, then placed in the dark for 8 minutes.

[0031] Next, the embryos in the centrifuge tube and the infection solution were poured onto the co-culture medium, shaken evenly, and the excess infection solution was sucked out with a pipette, all the embryos were placed with the scutes upwards, and co-cultured at 23°C in the dark for 3 days.

[0032] After co-culture, the embryos were transferred to recovery medium with sterile forceps, and cultured at 28°C for 7-14 days, and the growing sprouts on the embryos were removed in time during the process.

[0033] After recovery culture, the embryos were placed on 1.5 mg / L Bialaphos selection medium for 3 rounds of selection, each round for 2 weeks, and then transferred to 2 mg / L Bialaphos selection medium for 2 rounds of selection, each round for 2 weeks.

[0034] The resistant callus was transferred to the expansion medium and cultured at 28°C in the dark for 2 weeks. Then the expanded resistant callus was transferred to the induction medium and cultured at 28°C in the dark for 2 weeks. Then it was transferred to the differentiation medium and cultured at 25°C under 5000 lx light for 2 weeks.

[0035] After culture, the differentiated seedlings were separated into single seedlings and placed in rooting medium, and cultured at 25°C under 5000 lx light until rooting; the seedlings were transferred to small pots for growth, and after growth and survival, they were transplanted to a greenhouse, and the offspring seeds were harvested after 3-4 months.

[0036] (3) T0 generation plant CRISPR / Cas9 mutation result detection To determine the T0 generation plant CRISPR / Cas9 mutation result, the following steps are taken for detection: The present application first extracts rice leaf DNA by CTAB method, and the specific method is as follows: the DNA extraction method refers to the traditional CTAB method (Rogers and Bendich, 1985). Put 3 cm rice leaves into a sterilized 2 mL centrifuge tube, add 6 mm steel balls, use a cell crusher to break the tissue, then extract by CTAB method, finally add 200 μL of sterilized water (ddH2O) to the extracted sample to dissolve the air-dried sample DNA. After the DNA is completely dissolved, 2 μL of sample is taken for nucleic acid OD value (A260 / A280) and nucleic acid concentration determination using ultraviolet spectrophotometer (Nanodrop 2000). Dilute the DNA sample to 50 ng / μL for standby.

[0037] PCR uses 5 μL of 2x PCR premix (containing Mg2+; Taq DNA Polymerase; 2.5 mM dNTPs; 10x PCR Buffer) of Biomiga, 1 μL of primer (containing 0.5 μL of forward and reverse primer), 1 μL of template DNA, and ddH2O to make up 10 μL. The PCR amplification program is the conventional 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 of amplification, and finally 72℃ extension for 5 min). The amplified product is electrophoresed on an 8% non-denaturing polyacrylamide gel, stained with 0.1% AgNO3, and developed with formaldehyde and NaOH for photography.

[0038] Mutant osmt9 Nucleotide sequence alignment analysis found that Figure 2 Compared with the unedited wild type (WT), the mutation of OsMT9 Gene mutant osmt9-ko-22-1 It inserts a T base at the 359th position from the start codon ATG, and 5 bases ATCTG are deleted from the 564th to 568th position. The nucleotide sequence is shown in SEQ ID NO. 6, the CDS sequence is shown in SEQ ID NO. 7, and the amino acid sequence is shown in SEQ ID NO. 8. The OsMT9 Gene mutant osmt9-ko-23- 15It has 210 large fragment base deletions from the start codon ATG to the 359th to the 568th, the nucleotide sequence is shown as SEQ ID NO. 9, the CDS sequence is shown as SEQ ID NO. 10, and the amino acid sequence is shown as SEQ ID NO. 11. The nucleotide deletion encoded by the mutant causes a frameshift and causes premature termination of amino acid translation. Example 2

[0039] In this embodiment, the mutant obtained in Example 1 osmt9 was subjected to phenotype analysis, specifically as follows: 1. Identification of salt tolerance of the mutant osmt9 The parameters of light and dark alternating culture are as follows: light intensity is 120 μmol·m -2 ·s -1 , temperature is 28℃ / 25℃ (day / night), light period is 10h light / 14h dark.

[0040] The rice seeds to be tested are OsMT9 - L1 The mutant T1 generation homozygous seeds, ZH11 and empty control. The experiment was repeated 3 times to take the average value, and the steps of each repetition were as follows: (1) For each material, 24 rice seeds to be tested were taken and packed in kraft paper bags, and then soaked in water at 28-30℃ for 48h.

[0041] (2) After completing step 1, the seeds were germinated at 28-30℃ for 24h (the seeds need to be kept moist during the germination process), and the germinated seeds were obtained.

[0042] (3) After completing step 2, 96-well plates were taken, the lower edge of each well was cut, and then 1 germinated seed was placed in each well (embryo bud upward, embryo root downward).

[0043] (4) After completing step 3, the 96-well plates (with germinated seeds on them) were placed on a plastic box containing Yoshida rice culture solution and the germinated seeds were immersed in the culture solution, and light and dark alternating culture was carried out for 3 weeks to obtain rice seedlings grown to the three-leaf stage. During the light and dark alternating culture period, the Yoshida rice culture solution needs to be replaced every 7d.

[0044] (5) After completing step 4, the 96-well plates (with rice seedlings grown to the three-leaf stage on them) were placed in a plastic box containing 10% NaCl Yoshida rice culture solution and the roots were completely immersed in the culture solution, and light and dark alternating culture was carried out under high salt stress for 8d (during the high salt stress period, the Yoshida rice culture solution was replaced every 2d).

[0045] ​(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 recover for 7 days under alternating light and dark culture.

[0046] Observe the growth status of rice seedlings and calculate the survival rate. Survival rate = (Number of surviving rice seedlings / 12) × 100%.

[0047] See the growth status of rice seedlings before treatment. Figure 3 Survival rate statistics can be found in Figure 4 , .

[0048] The results showed that before salt treatment, osmt9 The growth pattern was basically the same as that of ZH11; freshwater control ZH11 and mutant osmt9 The survival rate was 100%. After salt solution treatment, the survival rate of ZH11 was about 6%, while the mutant... osmt9 The survival rate was approximately 43%, according to statistical analysis results. osmt9 The survival rate of [the strain] was significantly higher than that of ZH11, indicating that... osmt9 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

[0049] This embodiment performs osmt9 Transgenic experiments using mutant genes, using mutants osmt9 The R3261 variety was hybridized, backcrossed, and self-crossed with recipients exhibiting excellent agronomic traits, with molecular markers used throughout the process. OsMT9 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 5 As shown.

[0050] The above example uses R3261 as a breeding example, but it is not limited to R3261 and can be any rice material.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not 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; and these 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. Knockout OsMT9 The application of genes in improving salt tolerance in rice is characterized by, The OsMT9 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the knockout... OsMT9 mutants obtained after gene generation OsMT9 The nucleotide sequence of the gene is shown in SEQ ID NO.6 or SEQ ID NO.

9.

2. The application according to claim 1, characterized in that, The OsMT9 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

3.

3. A method for improving the salt tolerance of rice plants, characterized in that, In the endogenous nature of the rice plant genome OsMT9 Introducing single-base insertion and deletion mutations or large-fragment deletion mutations into a gene: The single-base insertion and deletion mutations cause the gene to... OsMT9 The gene inserts a T base at position 359, starting from the start codon ATG, and simultaneously deletes 5 bases (ATCTG) from positions 564 to 568, resulting in the mutant nucleotide sequence shown in SEQ ID NO.

6. osmt9-ko-22-1 Genes, thereby obtaining rice plants with improved salt tolerance; the large segment deletion mutation makes the... OsMT9 The gene had 210 bases deleted from position 359 to 568, starting from the start codon ATG, resulting in the mutant nucleotide sequence shown in SEQ ID NO.

9. osmt9-ko-23-15 Gene.

4. The method according to claim 3, characterized in that, The mutant OsMT9 The gene was constructed using a CRISPR / Cas9 system, the sgRNA used in which the CRISPR / Cas9 system targets sequences as shown in SEQ ID NO.4 and SEQ ID NO.

5.

5. The method according to claim 3, characterized in that, The endogenous nature of the rice plant genome OsMT9 Introducing single-base insertion and deletion mutations or large-fragment deletion mutations into a gene includes the following steps: Step 1: Construct a CRISPR / Cas9 gene editing vector, wherein the gene editing vector targets the target shown in SEQ ID NO.

4. OsMT9 The nucleotide sequence of the gene from base 356 to base 375, starting from the start codon ATG, and as shown in SEQ ID NO.

5. OsMT9 The nucleotide sequence of the gene from base 552 to base 571, starting from the start codon ATG; OsMT9 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 plants carrying the single-base insertion and base deletion mutations or large-fragment deletion mutations.

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