OsMT9 protein and its coding gene in regulating rice salt tolerance

Abstract

This invention relates to the field of rice genetic engineering, specifically providing the application of the OsMT9 protein and its encoding gene in regulating rice salt tolerance. This invention designs a specific targeting... OsMT9 The sgRNA in the coding region of the gene (target sequences shown in SEQ ID NO.4 and SEQ ID NO.5) was extracted, and a gene editing vector was constructed. The gene was then transformed into rice Zhonghua 11 using Agrobacterium-mediated transformation, yielding... OsMT9 Mutants with loss of gene function, such as mutants 22-1 and 23-15. Salt stress experiments showed that the survival rate of these mutants (approximately 43%) was significantly higher than that of the wild type (approximately 6%). This invention provides new gene targets and valuable germplasm resources for salt-tolerant rice breeding, and is of great significance for the utilization of saline-alkali land.

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 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.

[0005] Therefore, it is not only necessary to reveal it for the first time. OsMT9 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

[0006] The purpose of this invention is to provide OsMT9 Application of genes in regulating salt tolerance in rice.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides knockout OsMT9 Applications of genes, wherein the application is any of the following:

[0009] A1) Application in improving salt tolerance in rice;

[0010] A2) Application in the preparation of products that improve the salt tolerance of rice;

[0011] A3) Application in the cultivation of salt-tolerant rice;

[0012] A4) Application in the preparation of products for cultivating salt-tolerant rice;

[0013] A5) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources;

[0014] 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;

[0015] The OsMT9 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 3.

[0016] Secondly, this invention provides a method for improving the salt tolerance of rice plants, by utilizing the endogenous components 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 mutation causes 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; 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.

[0017] In the method described, 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:

[0018] Step 1: Construct a CRISPR / Cas9 gene editing vector, the vector targeting the gene as 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;

[0019] Step 2: Construct Agrobacterium genetically engineered bacteria containing the CRISPR / Cas9 gene editing vector described in Step 1;

[0020] 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 mutation or large-fragment deletion mutation.

[0021] In a third aspect, 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 to obtain backcross offspring with salt tolerance.

[0022] Furthermore, the salt-tolerant backcross offspring and the parent have the same mutant type. OsMT9 Genes; the stated OsMT9 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the mutant... OsMT9 The nucleotide sequence of the gene is shown in SEQ ID NO.6 or SEQ ID NO.9.

[0023] The beneficial effects of this invention are:

[0024] (1) First time revealing a new gene function, providing a new target for salt-tolerant breeding: First time discovering and verifying OsMT9 This gene negatively regulates salt tolerance in rice. Knocking out this gene significantly improves rice salt tolerance, providing a novel theoretical basis for understanding the molecular mechanisms of plant salt tolerance and offering a new target gene for salt-tolerant breeding.

[0025] (2) Creating new breeding materials with significantly enhanced salt tolerance: Using CRISPR / Cas9 gene editing technology, new materials with significantly enhanced salt tolerance were successfully obtained. OsMT9 Loss-of-function mutants (e.g.) osmt9-ko-22-1 and osmt9-ko-23-15 Under salt stress, the survival rate of the mutant (approximately 43%) was significantly higher than that of the wild type (approximately 6%), exhibiting a prominent phenotype and providing valuable germplasm resources that can be directly utilized for breeding.

[0026] (3) An efficient and precise salt tolerance improvement technology solution was established:

[0027] Designed specific targets OsMT9 The method involves the sgRNA of the gene (target sequences are SEQ ID NO.4 and SEQ ID NO.5) and the corresponding gene editing vector. This method is precise, efficient, and mutationally stable, and can achieve… OsMT9 Targeted gene knockout is applicable to the targeted improvement of salt tolerance in different rice varieties, and the technology is highly reproducible.

[0028] (4) Significant potential for production and application: The technical solution and mutant materials provided by this invention can be directly used to cultivate new salt-tolerant rice varieties. This is of great and far-reaching significance for developing and utilizing large areas of saline-alkali land and expanding the rice planting area. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the transcriptome of metal sulfur transporter protein expression in the aboveground and underground parts of rice after salt stress.

[0030] Figure 2 for OsMT9 Schematic diagram of gene structure and CRISPR / Cas-OsMT9 vector target sequence elements, along with sequencing result peaks; the top image shows the wild-type ZH11 reference sequence, and the bottom image shows the mutant. osmt9-ko-22-1 and osmt9-ko-23-15 Target sequencing peak diagram.

[0031] Figure 3 for osmt9 Salt tolerance phenotype identification diagram of 13-day-old mutant seedlings treated with 10‰ salt for 8 days and rehydrated for 7 days, bar = 7cm.

[0032] Figure 4 For ZH11 and osmt9 Survival rates before and after salt treatment were statistically analyzed. Values ​​shown are mean ± standard deviation, n = 3. * indicates significant difference (P < 0.05); ** indicates highly significant difference (P < 0.01). One-way ANOVA was used for statistical analysis.

[0033] Figure 5 This is a schematic diagram of the backcrossing and breeding route. Detailed Implementation

[0034] 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. Example 1

[0035] This embodiment provides rice. OsMT9 The functions and applications of genes include the following:

[0036] 1. Rice OsMT9 Gene sequence and expression pattern analysis

[0037] The Ensembl Plants database (http: / / plants.ensembl.org / index.html) contained information on rice. OsMT9The 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 78 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... OsMT9 The gene is specifically expressed in the roots of rice.

[0038] 2. Rice OsMT9 Transcriptome analysis of genes

[0039] Aboveground leaves of WT and ko-1 rice plants subjected to salt stress for 6, 30, and 102 h were sampled, with three biological replicates per sample. Three plants were randomly selected from each replicate, flash-frozen in liquid nitrogen, and stored at −80℃ for later use. Total RNA was extracted from the plants using the TRIzol method. mRNA with polyA tails was enriched using Oligo(dT) magnetic beads, and the mRNA was fragmented into 250–300 bp fragments using ion fragmentation to construct mRNA libraries. Paired-end sequencing was performed using the Illumina NovaSeq 6000 platform. Total RNA extraction and quality control, cDNA library construction, and sequencing were all performed by Beijing Berry Genomics Co., Ltd. After filtering the raw data to remove adapters and low-quality reads, the high-quality reads obtained were aligned to the reference genome (IRGSP-1.0) of rice 'Nipponbare' (Mortazavi et al., 2008) using HISAT2 software. Based on the comparison results, the number of reads covered by each gene from start to end was counted. The expression level of each gene and the Pearson correlation coefficient between WT and ko-1 replicates were calculated using the FPKM method (Trapnell et al. 2010). Differential expression levels between WT and ko-1 were analyzed using DESeq2 software (Love et al. 2014). The criteria for screening differentially expressed genes (DEGs) were: fold change |log2 (Fold Change)| ≥ 1, P ≤ 0.05. To further analyze the biological functions and metabolic pathways of DEGs, GO enrichment analysis was performed based on the hypergeometric distribution principle. Analysis of transcriptome data before and after salt stress revealed that all members of the metallothionein family responded to salt stress (…). Figure 1 )

[0040] 3. Rice OsMT9 Functional verification of genes

[0041] To clarify OsMT9To investigate the function of a gene in rice, this invention employs the CRISPR / Cas9 gene editing method to site-directedly mutate the gene sequence and knock out its function in rice.

[0042] This invention selects the conventional rice variety ZH11 as the recipient material for gene editing. The invention selects the target sequence 1, as shown in SEQ ID NO.4, a nucleotide sequence from base 356 to base 375 starting from the start codon ATG; and the target sequence... OsMT9 The 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 ).

[0043] (1) OsMT9 Construction of CRISPR / Cas9 gene editing vector

[0044] 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:

[0045] 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.

[0046] 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."

[0047] iii) Sequencing verification.

[0048] A positive clone with correct sequencing results is a successfully constructed pEGCas9Pubi-OsMT9 gene editing vector.

[0049] (2) Agrobacterium-mediated genetic transformation of rice

[0050] The pEGCas9Pubi-OsMT9 gene editing vector successfully constructed above was transformed into Agrobacterium EHA105 via heat shock. After identification by PCR, the bacterial culture was stored at -80 °C with glycerol.

[0051] Freshly peeled embryos of the hybrid rice variety Zhonghua 11, approximately 1.5 mm in size, 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 washings at the bottom of the tubes were aspirated with a pipette, and 1.0 mL of Agrobacterium infection solution was added. The tubes were gently shaken for 30 seconds and then left to stand in the dark for 8 minutes.

[0052] 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 any excess infection solution. Place all embryos with their scutes facing upwards and co-culture at 23°C in the dark for 3 days.

[0053] 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, be careful to remove any sprouts that grow on the embryos.

[0054] After the recovery culture was completed, the immature embryos were placed on a selection medium containing 1.5 mg / L Bialaphos for 3 rounds of selection culture, with each round lasting 2 weeks. Then, they were transferred to a selection medium containing 2 mg / L Bialaphos for 2 rounds of selection culture, with each round lasting 2 weeks.

[0055] The resistant callus was transferred to propagation medium and cultured in the dark at 28°C for 2 weeks. The propagated resistant callus was then transferred to induction medium and cultured in the dark at 28°C for 2 weeks. Finally, it was transferred to differentiation medium and cultured under light at 25°C and 5000 lx for 2 weeks.

[0056] 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 after 3-4 months.

[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 5 μL of Biomiga 2×PCR premix (containing Mg2+, 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 analyzed by 8% non-denaturing polyacrylamide gel electrophoresis, stained with 0.1% AgNO3, and photographed after color development with formaldehyde and NaOH.

[0061] For mutants osmt9 Nucleotide sequence alignment analysis revealed that ( Figure 2 Compared to the unedited wild type (WT), the mutated OsMT9 Gene mutants osmt9-ko-22-1 It inserts a T base at position 359 starting from the start codon ATG, and simultaneously deletes 5 bases (ATCTG) from positions 564 to 568. 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. OsMT9 Gene mutants osmt9-ko-23- 15 The mutant exhibits a large deletion of 210 bases from position 359 to 568, starting from the start codon ATG. The nucleotide sequence is shown in SEQ ID NO. 9, the CDS sequence in SEQ ID NO. 10, and the amino acid sequence in SEQ ID NO. 11. This nucleotide deletion causes a frameshift in the amino acids, leading to premature termination of translation. Example 2

[0062] This embodiment describes the mutant obtained in Example 1.osmt9 Phenotypic analysis was performed, as follows:

[0063] 1. Mutant osmt9 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 are OsMT9 - 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 24 rice seeds to be tested, put them into kraft paper bags, and soak them in water at 28℃~30℃ for 48h.

[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 (with germinated seeds on it) on a plastic box containing Yoshida rice culture solution and immerse the germinated seeds in the culture solution. Culture in alternating light and dark for 3 weeks to obtain rice seedlings that have grown to the three-leaf stage. During the alternating light and dark culture period, the Yoshida rice culture solution needs to be replaced every 7 days.

[0070] (5) After completing step 4, place the 96-well plate (on which rice seedlings that have grown to the three-leaf stage) on a plastic box containing 10% NaCl Yoshida rice culture solution and immerse the roots completely in the culture solution. Under high salt stress for 8 days under alternating light and dark conditions (during the high salt stress period, the Yoshida rice culture solution is replaced 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 recover for 7 days under alternating light and dark culture.

[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 Survival rate statistics can be found in Figure 4 , .

[0074] 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

[0075] 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.

[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. 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 Genes; the stated OsMT9 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. 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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