Rice salt-tolerant ccx2 mutant gene and application thereof

Abstract

This invention discloses a salt-tolerant CCX2 mutant gene in rice and its applications, relating to the field of biotechnology. The CDS sequence of the CCX2 mutant gene is shown in SEQ ID NO.3. The proposed CCX2 mutant gene and its salt-sensitive mutant ccx2 can lead to salt sensitivity and reduced survival rate in rice, while overexpression of this gene significantly enhances rice salt tolerance. Simultaneously, physiological and biochemical analyses revealed that under salt stress, the wild-type (WT) rice exhibited significantly higher SOD and CAT activities than the salt-sensitive mutant ccx2, while its POD activity, MDA content, and H2O2 content were significantly lower. Therefore, this CCX2 mutant gene and its salt-sensitive mutant ccx2 can be applied to research on rice growth and salt stress response, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of rice salt-sensitive mutation technology, specifically to a rice salt-tolerant CCX2 mutant gene and its application. Background Technology

[0002] Soil salinization is one of the most serious abiotic stresses threatening modern agriculture and global food security. Due to its semi-aquatic growth habits and high sensitivity to sodium accumulation, salt stress can lead to reduced germination rate, growth stagnation, and decreased grain filling rate in rice, ultimately resulting in a significant reduction in yield.

[0003] Rice primarily responds to salt stress through three major physiological pathways: ion balance regulation, osmotic regulation, and antioxidant defense. Among these, cation transporters play a central role in maintaining intracellular ion homeostasis and mitigating salt ion toxicity. Calcium-sodium exchangers (CCXs) are important members of this family, involved in ion balance and stress adaptation. Their functional conservation has been confirmed in various plants; for example, Arabidopsis thaliana's AtCCX1 regulates Na+... + / Ca 2+ Ion transport is involved in salt stress signal transduction. In rice, some members of the CCX family have been reported to participate in ion transport, but the function of CCX2 under salt stress remains unclear, hindering research on molecular design breeding based on the CCX2 gene and the development of salt-tolerant rice varieties. However, there are no reports on proposing a CCX2 mutant gene and constructing salt-sensitive mutant ccx2 rice plants to elucidate the functional mechanism of CCX2 under salt stress. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to propose a salt-tolerant CCX2 mutant gene and construct a salt-sensitive mutant ccx2 rice plant, and then study and reveal the response pathway and genetic analysis of the rice salt-tolerant gene CCX2 under salt stress.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] This invention proposes a salt-tolerant CCX2 mutant gene, the CDS sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also proposes a protein encoded by the salt-tolerant CCX2 mutant gene, the amino acid sequence of which is shown in SEQ ID NO.4.

[0008] The CCX2 mutant gene LOC_Os03g45370 has a 2 bp deletion in its coding region (the 53rd and 54th bases in the coding region are deleted), which leads to premature termination of translation.

[0009] This invention also proposes the application of the above-mentioned salt-tolerant CCX2 mutant gene in the construction of salt-sensitive mutant ccx2 rice plants.

[0010] This invention also proposes a method for constructing a salt-sensitive mutant ccx2 rice plant, comprising the following steps: The CCX2 mutant gene was introduced into the target rice variety using CRISPR / Cas9 gene editing technology.

[0011] The present invention also proposes the application of the above-mentioned salt-tolerant CCX2 mutant gene or protein in the growth and / or salt stress response of rice.

[0012] This invention also proposes the application of the above-mentioned salt-tolerant CCX2 mutant gene or protein in studying the mechanism of changes in physiological and biochemical indicators related to antioxidant defense under salt stress.

[0013] Preferably, the physiological and biochemical indicators related to antioxidant defense include one or more of SOD activity, MDA content, POD activity, CAT activity, and H2O2 content.

[0014] Preferably, by upregulating the activities of superoxide dismutase (SOD) and catalase (CAT), the content of malondialdehyde (MDA), peroxidase (POD) activity, and hydrogen peroxide (H2O2) content are reduced, thereby enhancing the antioxidant capacity of cells and improving the salt tolerance of rice.

[0015] This invention also proposes the application of the above-mentioned salt-tolerant CCX2 mutant gene in testing whether the ccx2 salt-tolerant phenotype is controlled by a single gene.

[0016] This invention also proposes the application of the above-mentioned salt-tolerant CCX2 mutant gene in testing whether the ccx2 salt-tolerant phenotype in Wuyunjing 7 is affected by the genetic background.

[0017] This invention also proposes a method for constructing a CCX2 expression vector based on the gateway method, comprising the following steps: (1) Using cDNA from leaves of Wuyunjing 7 as a template, PCR products with att1 / att2 sites were designed for BP recombination reaction. The CCX2 gene with attB sites at both ends was amplified by high-fidelity enzyme-mediated PCR. (2) The PCR product was subjected to BP recombination reaction, and the reaction product was transformed into Escherichia coli. After screening and sequencing verification, the correct entry vector was obtained. (3) The initiation clone and the target vector were mixed and subjected to LR recombination reaction. After transformation into Escherichia coli, the plant expression vector was obtained by screening with appropriate antibiotics.

[0018] Preferably, the method for identifying the salt tolerance of CCX2 overexpression is to germinate the overexpression rice material identified as homozygous and wild-type rice, cultivate them to a seedling age of 14 days, treat them in a high concentration of 0.9% NaCl solution for 7 days, rehydrate them for 7 days, and then count the seedling survival rate.

[0019] The present invention also proposes a CCX2 expression vector constructed by the above method.

[0020] This invention also proposes the application of the above-mentioned CCX2 expression vector in the cultivation of salt-tolerant rice plants.

[0021] LOC_Os03g45370 is the salt tolerance gene calcium-sodium exchange protein gene CCX2, whose CDS sequence is shown in SEQ ID NO.1 and amino acid sequence is shown in SEQ ID NO.2.

[0022] This invention utilizes heavy ion irradiation to obtain the salt-sensitive mutant ccx2 from the Wuyunjing 7 rice variety. Through genetic population construction and statistical analysis of salt tolerance samples in the F2 segregating population, it was found that the salt-tolerant gene CCX2 is controlled by a single dominant gene. Map-based cloning revealed a 2 bp (GC) deletion at positions 331-332 in the coding region of the salt-sensitive mutant ccx2, leading to an altered amino acid sequence and premature termination. Gene knockout of CCX2 and overexpression of the CCX2 gene demonstrated that this gene can improve the survival rate of rice under salt stress, exhibiting salt tolerance characteristics.

[0023] Preferably, the method for identifying the salt tolerance of the salt-sensitive mutant ccx2 is as follows: after disinfecting the seeds of Wuyunjing 7 and the salt-sensitive mutant ccx2, the seeds are germinated and hydroponically cultured until the seedlings are 14 days old. The seedlings are then treated in a 0.6% NaCl solution for 7 days and then rehydrated for 7 days. The survival rate of the seedlings is then calculated.

[0024] The beneficial effects of this invention are as follows: 1. The salt-tolerant CCX2 mutant gene and its salt-sensitive mutant ccx2 proposed in this invention can lead to salt sensitivity and reduced survival rate in rice, while overexpression of this gene significantly enhances rice salt tolerance. Simultaneously, physiological and biochemical analyses revealed that under salt stress, the wild-type (WT) rice exhibited significantly higher SOD and CAT activities than the salt-sensitive mutant ccx2, while its POD activity, MDA content, and H2O2 content were significantly lower. Therefore, this salt-tolerant CCX2 mutant gene and its salt-sensitive mutant ccx2 can be applied to research on rice growth and salt stress response, showing promising application prospects.

[0025] 2. The salt-sensitive mutant rice identified by this invention was cloned into LOC_Os03g45370. Through phenotypic, physiological and genetic analysis, the relationship and mechanism of action between the physiological and biochemical indicators related to antioxidant defense and rice salt tolerance were clarified, the molecular regulatory network of rice response to high salt stress was improved, and molecular design breeding with resistance as the target was carried out in a targeted manner.

[0026] This invention primarily targets the inhibitory effect of salt stress on rice growth. A new salt-tolerant gene was obtained through the creation of a salt-sensitive mutant of the CCX2 gene and the functional verification of the CCX2 salt-tolerant gene. Mutations in this gene impair the antioxidant defense capabilities of rice under salt stress, affecting seedling survival rates. CCX2 can be introduced into rice as the target gene to improve its salt tolerance.

[0027] 3. Overexpression of the CCX2 gene demonstrated a significantly improved survival rate under salt stress, indicating salt tolerance. Furthermore, this invention confirms that the salt-tolerant gene CCX2 participates in the rice salt stress response, maintaining antioxidant defenses and contributing to the rice's salt stress response. Therefore, this invention has significant advantages in the development of saline-alkali land agriculture.

[0028] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0029] Figure 1 This is a comparison diagram of wild-type WT and mutant ccx2 before (a) and after (b) salt treatment in Example 1 of the present invention; Figure 2 The survival rates of wild-type WT and mutant ccx2 after salt treatment and rehydration in Example 1 of this invention; Figure 3 The H2O2 content (a), MDA content (b), POD activity (c), SOD activity (d), and CAT activity (e) of wild-type WT and mutant ccx2 before and after salt treatment in Example 1 of this invention are shown. Figure 4 The images show map-based cloning diagram (a) and gene alignment diagram (b) of the salt tolerance gene CCX2 in Example 2 of this invention. Figure 5 Design of the salt tolerance gene CCX2 editing site in Example 2 of the present invention (a) and gene alignment (b); Figure 6 This is a verification of the salt tolerance gene CCX2 gene knockout in Example 2 of the present invention, where (a) is before salt treatment, (b) is after salt treatment, and (c) is a comparison of survival rates. Figure 7The images show a comparison of the CCX2 overexpression homozygous line and wild-type WT before salt treatment (a), after salt treatment (b), and the survival rate comparison (c) in Example 2 of this invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0031] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0032] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0033] Example 1: Obtaining the salt-sensitive mutant ccx2 and its application in rice growth and / or salt stress response. (1) Phenotypic analysis of the salt tolerance gene CCX2 Heavy ion mutagenesis was used to mutate the seeds of Wuyunjing 7 japonica rice, which were then planted in the field until the phenotypic stable mutant ccx2 was obtained. Wuyunjing 7 and ccx2 mutant seeds were placed in petri dishes, disinfected with 5% sodium hypochlorite for 20 min, washed, and germinated at 33℃ for 3 days. After hydroponics until 14 days of seedling age, they were treated with 0.6% NaCl for 7 days and then rehydrated for 7 days. Compared with ccx2, wild-type (WT) Wuyunjing 7 showed continued growth, while most of the ccx2 mutants died. Figure 1 As shown, WT has a high survival rate of approximately 80%, while ccx2 has a survival rate of only 11%, a decrease of about 70% compared to WT. Figure 2 As shown.

[0034] (2) Salt stress response pathway of the salt tolerance gene CCX2 under salt stress After 14 days of hydroponic cultivation, WT and the mutant ccx2 were used to test fresh leaves for physiological and biochemical indicators related to antioxidant defense (SOD activity, MDA content, POD activity, CAT activity, and H2O2 content). No significant differences were found in these indicators among the leaves. After treatment with 0.6% NaCl, leaves were taken for further testing of these physiological and biochemical indicators related to antioxidant defense. The results showed that WT had significantly higher SOD and CAT activities than ccx2, while its POD activity, MDA content, and H2O2 content were significantly lower. (The results are as follows...) Figure 3 As shown.

[0035] Example 2: Map-based cloning of salt tolerance genes (1) Genetic analysis of CCX2 A genetic segregating population was constructed by crossing the ccx2 mutant with Wuyunjing 7. F2 seeds were dried and placed in petri dishes, sterilized with 5% sodium hypochlorite for 20 min, washed, and germinated at 33℃ for 3 days. After 14 days of hydroponics, 435 seedlings were obtained. These were treated with 0.6% NaCl for 7 days and rehydrated for 7 days. 327 salt-tolerant plants and 108 salt-sensitive plants were then identified. The chi-square test showed that the salt-tolerant phenotype of ccx2 was controlled by a single dominant gene pair. Statistical analysis of agronomic traits revealed no significant differences between ccx2 and Wuyunjing 7 in plant height, panicle length, number of tillers, number of grains per panicle, seed setting rate, and thousand-grain weight, indicating that the salt-tolerant phenotype was not influenced by genetic background.

[0036] (2) Gene location of CCX2 After expanding the salt tolerance population, 856 salt-sensitive individual plants were obtained. Gene mapping using map-based cloning revealed significant linkage on chromosome 3. Sequencing analysis of five genes within this region showed a 2 bp deletion in the coding region of LOC_Os03g45370, leading to premature translation termination. Figure 4 As shown.

[0037] (3) Validation of CCX2 gene knockout The CCX2 gene in rice was knocked out at a specific site using CRISPR / Cas9 technology, and the resulting gene-edited materials were molecularly identified and subjected to salt tolerance phenotype analysis. The knockout target site was designed in the start region of the CCX2 gene coding region, near the downstream of the start codon (ATG). Sequencing analysis of the target site revealed that, compared with the wild type, the ccx2-crispr material exhibited a one-base insertion at the target site, resulting in a change in the coding sequence (e.g., ...). Figure 5 As shown in the figure, this leads to premature termination of translation of the protein, and the function of the CCX2 protein has been severely affected.

[0038] CCX2-CRISPR seedlings and wild-type seeds were placed in petri dishes, sterilized with 5% sodium hypochlorite for 20 min, washed, and germinated at 33℃ for 3 days. After hydroponics for 14 days, they were cultured with 6% NaCl for 7 days and then rehydrated for 7 days. The results showed that under normal growth conditions, there were no significant differences in growth status and overall morphology between 2-week-old CCX2-CRISPR seedlings and wild-type seedlings (e.g., ...). Figure 6 (As shown in Figure a). After treatment with 100 mM NaCl for 7 days and rehydration for 7 days, ccx2-crispr plants exhibited a significant salt-sensitive phenotype, with significantly higher levels of leaf yellowing and wilting compared to the wild type (e.g., ...). Figure 6 (As shown in b). Survival statistics (e.g.) Figure 6 As shown in Figure c), under salt stress, the survival rate of WT plants was approximately 70%, while that of ccx2-crispr plants was only approximately 12%, a decrease of about 58% compared to WT, and the difference was highly significant.

[0039] (4) Validation of overexpression of salt tolerance gene CCX2 To verify that LOC_Os03g45370 is the salt-tolerant CCX2 gene, RNA was extracted from the leaves of Wuyunjing 7 seedlings and reverse transcribed into cDNA. An overexpression vector was constructed using the Gateway method, and the pActin::CCX2-Flag overexpression vector was transformed into Zhonghua 11 rice using Agrobacterium-mediated transformation. Transformed plants were identified, and the homozygous transgenic line CCX2-OE, with higher expression levels and protein accumulation than Zhonghua 11 rice, was obtained. After treating 14-day-old Zhonghua 11 and three CCX2 overexpression lines with 0.9% NaCl for 7 days and rehydrating for 7 days, it was found that all three CCX2 overexpression lines were more salt-tolerant and had higher survival rates than Zhonghua 11 rice. Figure 7 As shown.

[0040] SEQ ID NO.1: SEQ ID NO.2: MALLRRRGYSAAVPSACCFLLLLLVLSASHLLPTRRGHGGVLEGLALRGSASRSRSGSSSSSSAGEEQGSCQELQSIEGGEARCLYLRTHPPCAPAGYVDYLRLFYCGFAHAPAAGYAAAVLWLAVLFYLLGDTASEYFCASLEGLSAELRLPPAIAGVTLLSLGNGAPDVFASVVSFAAGDGGGVGLNSALGGALFVSTVVAGVVALAAASRAGRGGVVVELRGFVRDICFLLLALCSLLAILVTGTVTVWVSASFVSLYVAYVLLVWTSHCCSEPGKPPQADLAAPLLLDDDGGVTPLPSYSKNSAPSKKRAYLHCLLSAILIPLYLPRRLTIPDIAGHRWSRPCAVASLALAPVLLAATWASSCRHALAVLLGGALLGLLLAALAAATTEAASPPRGRWRRVPWLAAGFLMSVLWAYTLARELVALLVAIGYMVGVRASVLGVTVLAWGDSLGDLVSNVAMALHGGAGGAQTAVSGCYAGPLFNTVVGLGLSLTLAAGSQYPAPFAIPAGGAVYEAVGFLGAGLAWALLVVPARGMRLDRVYGMGLIAIYLAFVTIRVFDSLGLWTHSWWPA SEQ ID NO.3: SEQ ID NO.4: MALLRRRGYSAAVPSACFPPPPAARPLRLPPPAHSPRPWRRPRRVGP The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. 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 salt-tolerant CCX2 mutant gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

3.

2. The application of the salt-tolerant CCX2 mutant gene as described in claim 1 in the construction of salt-sensitive mutant ccx2 rice plants.

3. The application of the salt-tolerant CCX2 mutant gene as described in claim 1 in the growth and / or salt stress response of rice.

4. The application of the salt-tolerant CCX2 mutant gene as described in claim 1 in studying the mechanisms of changes in physiological and biochemical indicators related to antioxidant defense under salt stress, characterized in that... The physiological and biochemical indicators related to antioxidant defense include one or more of the following: SOD activity, MDA content, POD activity, CAT activity, and H2O2 content.

5. The application according to claim 3, characterized in that, By upregulating the activities of superoxide dismutase (SOD) and catalase (CAT), and reducing the content of malondialdehyde (MDA), peroxidase (POD) activity, and hydrogen peroxide (H2O2), the antioxidant capacity of cells is enhanced, thereby improving the salt tolerance of rice.

6. The application of the salt-tolerant CCX2 mutant gene as described in claim 1 in testing whether the ccx2 salt-tolerant phenotype is controlled by a single gene.

7. The application of the salt-tolerant CCX2 mutant gene as described in claim 1 in testing whether the ccx2 salt-tolerant phenotype in Wuyunjing 7 is affected by the genetic background.

8. A method for constructing a CCX2 expression vector based on the gateway method, characterized in that, Includes the following steps: (1) Using cDNA from leaves of Wuyunjing 7 as a template, PCR products with att1 / att2 sites were designed for BP recombination reaction. The CCX2 gene with attB sites at both ends was amplified by high-fidelity enzyme-mediated PCR. (2) The PCR product was subjected to BP recombination reaction, and the reaction product was transformed into Escherichia coli. After screening and sequencing verification, the correct entry vector was obtained. (3) The initiation clone and the target vector were mixed and subjected to LR recombination reaction. After transformation into Escherichia coli, the plant expression vector was obtained by screening with appropriate antibiotics.

9. The salt-tolerant CCX2 expression vector constructed by the method of claim 8.

10. The application of the salt-tolerant CCX2 expression vector according to claim 9 in the cultivation of salt-tolerant rice plants.

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