OsRIPK gene and its coded protein in improving rice salt tolerance

Abstract

The application discloses OsRIPK The application discloses a gene and application of the gene and a coded protein in improving salt tolerance of rice, and belongs to the technical field of plant genetic engineering and molecular breeding. OsRIPK The application first discovers and proves that the gene is a key gene for negatively regulating salt tolerance. OsRIPK The application obtains a mutant with a function loss by targeting and knocking out the gene through CRISPR / Cas9 gene editing technology. OsRIPK Compared with a wild type, the mutant shows a significant phenotype of enhanced salt tolerance under salt stress treatment at a seedling stage. Experiments show that the gene is a key gene for negatively regulating salt tolerance of rice. The gene can effectively improve the salt tolerance of rice by being knocked out through gene editing technology. The application provides a new gene target and valuable germplasm resource for rice salt tolerance molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to rice gene function research and molecular breeding. More specifically, this invention relates to rice. OsRIPK The function and application of genes in regulating salt tolerance, particularly involving the use of gene editing technology to knock out the gene to obtain rice mutants with improved salt tolerance and its applications. Background Technology

[0002] Salinization is one of the major abiotic stresses restricting global agricultural production. Excessive salt content in the soil (mainly sodium) is a contributing factor. + and Cl - Salt tolerance causes dual damage to plants: initially, it triggers osmotic stress, hindering water absorption, leading to stomatal closure, reduced photosynthesis, and stunted growth; later, it causes ion toxicity, disrupting intracellular ion homeostasis, interfering with enzyme activity, and causing oxidative damage, ultimately affecting crop yield and quality. As a major food crop, improving the salt tolerance of rice is of great significance for ensuring food security.

[0003] Currently, the main approaches to improving salt tolerance in rice include traditional breeding and molecular breeding. Traditional breeding methods (such as hybridization and systematic selection) rely on phenotypic screening, which is a lengthy and inefficient process, and is difficult to cope with complex saline-alkali environments and salt tolerance traits controlled by multiple genes. With the development of molecular biology, marker-assisted selection (MAS) can accelerate the aggregation of multiple salt tolerance-related quantitative trait loci (QTLs), but it still relies on the mining of existing natural variations, and its ability to create new genetic resources is limited.

[0004] In recent years, gene editing technology, especially the CRISPR / Cas9 system, has become a powerful tool for creating new germplasm due to its high efficiency and precision. This technology can directly knock out, knock in, or modify specific genes, thereby rapidly obtaining mutant material with the target trait. However, the prerequisite for successful application of this technology is the identification of key genes that regulate the target trait (such as salt tolerance).

[0005] The OsRIPK gene has previously been reported as a receptor-like cytoplasmic kinase that participates in the production of reactive oxygen species (ROS) by activating NADP-ME2 in plant immune responses. However, its function in responses to abiotic stresses, especially salt stress, remains unclear, and there are no reports of its application in rice salt-tolerant breeding. Therefore, discovering and validating new salt-tolerant regulatory genes and creating corresponding new salt-tolerant germplasm using gene editing technology are urgent technical problems to be solved in current rice salt-tolerant breeding. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies and provide a novel gene that negatively regulates rice salt tolerance and its application. This invention has experimentally identified and confirmed... OsRIPK Deletion or inactivation of genes can significantly enhance the salt tolerance of rice.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for improving the salt tolerance of rice, the method comprising: using gene editing technology to target specific components in rice... OsRIPK Genes are knocked out or mutated to reduce the aforementioned OsRIPK Gene expression levels; wherein, the OsRIPK The CDS sequence of the gene is shown in SEQ ID NO.1.

[0008] Furthermore, the aforementioned OsRIPK The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0009] Furthermore, the gene editing technology is the CRISPR / Cas9 system.

[0010] Furthermore, the nucleotide sequence of the sgRNA used by the CRISPR / Cas9 system is shown in SEQ ID NO.3.

[0011] The present invention also provides OsRIPK Application of genes in improving salt tolerance in rice, the application including reducing the salt content of the rice shown in SEQ ID NO.1 OsRIPK The expression level of genes can be increased, thereby improving the salt tolerance of rice.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses and verifies OsRIPK This gene is a key gene that negatively regulates salt tolerance in rice. Loss-of-function mutations can significantly enhance salt tolerance in rice seedlings, providing a novel gene target for genetic improvement of rice salt tolerance. The obtained... OsRIPK Loss-of-function mutants are unaffected by growth under normal conditions, but exhibit significantly stronger tolerance (such as higher survival rate) under salt stress. They are considered "green" salt-tolerant traits and can be directly used as excellent germplasm resources for molecular design breeding of salt-tolerant rice varieties, thus having high application value. Attached Figure Description

[0013] Figure 1 yes OsRIPK Gene structure diagram and OsRIPK Sequencing peak alignment results of gene knockout lines.

[0014] Figure 2 It shows a phenotypic comparison of wild-type and gene knockout lines under normal conditions and salt stress, as well as a statistical chart of survival rates. Detailed Implementation

[0015] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0016] Example 1: OsRIPK Construction of gene knockout vector 1. Design sgRNA target sequences According to the rice variety "Huazhan" OsRIPK

[0017] 2. Construction of CRISPR / Cas9 expression vector Using the pYLCRISPR / Cas9Pubi-H multi-target vector system developed by Liu Yaoguang's laboratory at South China Agricultural University (reference: Ma X, Zhang Q, Zhu Q, et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants. Mol Plant, 2015, 8(8): 1274~1284), the intermediate vector pYLgRNA-U3 was first linearized using BsaⅠ restriction endonuclease, and then ligated to the U3 promoter. Subsequently, using a "cut-and-ligate" strategy, the ligation product was integrated into the plant genome site-directed editing vector pYLCRISPR / Cas9-MT. After transformation in E. coli, expansion culture of positive clones, plasmid extraction, and sequencing verification, a recombinant vector targeting the OsRIPK gene was successfully obtained and named pYLgRNA-OsRIPK-ko. The correct assembly of this vector was verified by agarose gel electrophoresis and DNA sequencing.

[0018] 3. Amplification and extraction of recombinant plasmids The verified recombinant plasmid pYL-OsRIPK-ko was transformed into E. coli using the heat shock method. Escherichia coli DH5α competent cells were plated on LB agar containing kanamycin and incubated overnight at 37°C. Single colonies were picked for expansion culture, and high-purity plasmid DNA was extracted using a commercial plasmid extraction kit (such as the PlasmidMini Kit from Tiangen Biotech). The plasmid concentration and purity were determined using a NanoDrop 2000 micro-spectrophotometer, and final confirmation was performed by restriction endonuclease digestion and agarose gel electrophoresis.

[0019] Example 2: Agrobacterium-mediated genetic transformation of rice and acquisition of transgenic plants The extracted high-purity recombinant plasmid pYL-OsRIPK-ko was introduced into competent cells of Agrobacterium tumefaciens strain EHA105 using a freeze-thaw method.

[0020] The specific genetic transformation process is as follows: (1) Remove the husk from the rice seeds, disinfect them with alcohol for 2 minutes, disinfect them with 3% sodium hypochlorite for 15 minutes, wash them with sterile water 4-5 times, drain the water, spread them flat on sterilized filter paper and dry them. Transfer the dried seeds to the induction medium and incubate them in the dark at 30°C for 2 weeks to induce the production of callus tissue.

[0021] (2) Streak the Agrobacterium tumefaciens strain GV3101 containing the target gene on an LB agar plate containing the corresponding antibiotic, and incubate at 28°C for 2 days. Inoculate about one loopful of Agrobacterium into 100 ml of suspension medium, and incubate at 28°C and 200 rpm for 30 min on a constant temperature shaker. The concentration of Agrobacterium suspension should be about OD600 = 0.3.

[0022] (3) Collect the callus tissue into a 250 ml sterile Erlenmeyer flask, pour the prepared Agrobacterium suspension into the Erlenmeyer flask containing the callus tissue until all the callus tissue is submerged, and let stand for 10 min.

[0023] (4) Drain the bacterial solution, spread the callus on the filter paper of a sterile dish, and let it air dry for 1-2 hours.

[0024] (5) Use tweezers (or a spoon) to transfer the dried callus particles to the co-culture medium (do not move the callus after it has been transferred to the co-culture medium to reduce the contact between the medium and the callus surface and prevent the overgrowth of Agrobacterium), and seal with sealing glue.

[0025] (6) Co-cultured at 28 °C in the dark for 3 days.

[0026] (7) Transfer the co-cultured callus to a water washing cup, pour in sterile distilled water until the callus is completely submerged, cover and shake for 20-30 seconds, then discard the sterile distilled water. Repeat this water washing process 3-4 times.

[0027] (8) Drain the water from the callus, transfer it to the screening medium and culture it in the dark for 20 days. Observe whether fresh and tender yellow resistant callus grows. If there is still no resistant callus, continue to transfer to a plate for a second screening culture.

[0028] (9) Select small pieces of light yellow, dense, dry, and vigorous resistant callus and place them in differentiation medium. Culture them under light (28°C, 14 h light / 10 h dark) for 30-40 days. Once the differentiated seedlings are 3-5 cm tall, they can be cultured for rooting.

[0029] (10) Root culture in a light culture room for 15-20 days. After the new roots have grown sufficiently, transplant them.

[0030] The regenerated T0 generation seedlings were transplanted to a greenhouse, and the genomic DNA of the gene knockout line was extracted. The target region was amplified and sequenced to identify the mutation type.

[0031] Sequencing results showed ( Figure 1 The ripk-1# strain has an adenine (A) deletion at the target site, resulting in a frameshift mutation; the ripk-2# strain has a two-base deletion near the target site, also resulting in a frameshift mutation. These mutations are expected to cause loss of RIPK protein function.

[0032] Example 3: Evaluation of salt tolerance in transgenic rice materials Experimental materials: Wild-type Huazhan (HZ), two OsRIPK Gene-edited mutant lines (ripk-1#, ripk-2#).

[0033] Select plump T1 generation seeds, disinfect and germinate them, then hydroponically culture them in 1 / 2 Hoagland nutrient solution at 28℃ / 25℃ (day / night) with 14 hours of light until they reach the two-leaf-one-heart stage (approximately 14 days). Select seedlings with uniform growth and randomly divide them into two groups: the control group continues to be cultured in normal nutrient solution; the salt stress group is treated with 1 / 2 Hoagland nutrient solution containing 8‰ NaCl. The nutrient solution is changed every 3 days.

[0034] After 7 days of salt stress treatment, all seedlings were transferred to normal nutrient solution for 7 days of rehydration. After rehydration, wild-type and... OsRIPK Differences in salt tolerance phenotypes among gene-edited mutant lines were investigated, and survival rates were statistically analyzed.

[0035] like Figure 2 As shown, after treatment with 8‰ NaCl, wild-type Hua Zhan exhibited significant wilting and growth inhibition, while OsRIPK The gene-edited mutant lines (ripk-1#, ripk-2#) maintained a good green appearance and had a significantly higher survival rate than the wild type.

[0036] Experimental results show that knockout OsRIPK Genes can significantly improve the salt tolerance of rice, proving that... OsRIPK The gene is a gene that negatively regulates the salt tolerance of rice.

Claims

1. A method for improving the salt tolerance of rice, characterized in that, The method includes: using gene editing technology to manipulate rice... OsRIPK Genes are knocked out or mutated to reduce the aforementioned OsRIPK Gene expression levels; wherein, the OsRIPK The CDS sequence of the gene is shown in SEQ ID NO.

1.

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

2.

3. The method according to claim 1, characterized in that, The gene editing technology used is the CRISPR / Cas9 system.

4. The method according to claim 3, characterized in that, The nucleotide sequence of the sgRNA used by the CRISPR / Cas9 system is shown in SEQ ID NO.

3.

5. OsRIPK The application of genes in improving salt tolerance in rice is characterized by, The application includes reducing the concentration of the substance shown in SEQ ID NO.1 in rice. OsRIPK This increases the expression level of genes, thereby improving the salt tolerance of rice.

6. A salt-tolerant rice variety, characterized in that, It is prepared by the method described in claims 1-4.

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