Application of OsEBP89 gene in regulation and control of salt tolerance of plants

Abstract

The invention discloses application of an OsEBP89 gene in regulation and control of salt tolerance of plants, and belongs to the technical field of biology. According to the application of the OsEBP89 gene in regulating and controlling the salt tolerance of the plant, the genomic sequence of the OsEBP89 gene is shown as SEQ ID No.1, the coding sequence of the OsEBP89 gene is shown as SEQ ID No.2, and the amino acid sequence coded by the OsEBP89 gene is shown as SEQ ID No.3. The invention further discloses the application of the OsEBP89 gene in regulating and controlling the salt tolerance of the plant. It is clear that overexpression of the OsEBP89 gene can improve the salt tolerance of rice, and a new thought is provided for cultivation of salt-tolerant rice.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving OsEBP89 Application of genes in regulating plant salt tolerance. Background Technology

[0002] Soil salinization is a growing global problem; salt stress damages plant growth and development, impacting crop yields. Rice ( Oryza sativa L. Rice is widely cultivated in over 100 countries and regions, serving as a staple food for approximately half the world's population. Salt stress can lead to reduced yields or even crop failure. Improving the salt tolerance of rice is crucial for ensuring rice production and expanding its cultivation area. Currently, a systematic understanding of rice salt stress tolerance genes and the signaling pathways and molecular networks responding to salt stress is lacking. Therefore, identifying rice salt stress tolerance genes, analyzing related signaling pathways and molecular networks, and breeding new salt-tolerant rice varieties through molecular design have significant theoretical and practical value.

[0003] OsEBP89 The gene encodes a nuclear-localized ERF-type transcription factor with transcriptional activation activity and constitutive expression characteristics; the SnRK1 subfamily protein kinase OSK3 interacts with the OsEBP89 protein, which can enhance... OsEBP89 Transcriptional activity of genes. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides OsEBP89 The application of genes in regulating plant salt tolerance has been clarified. OsEBP89 Gene overexpression can enhance the salt tolerance of rice, providing new ideas for the breeding of salt-tolerant rice.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: to provide OsEBP89 Application of genes in regulating plant salt tolerance OsEBP89 The genome sequence of the gene is shown in SEQ ID No. 1. OsEBP89 The coding sequence of the gene is shown in SEQ ID No. 2.

[0006] Based on the above technical solution, the present invention can be further improved as follows.

[0007] further, OsEBP89 The amino acid sequence encoded by the gene is shown in SEQ ID No. 3.

[0008] Furthermore, by overexpressing in rice OsEBP89 Genes are used to enhance the salt tolerance of rice.

[0009] Furthermore, overexpression in rice OsEBP89The steps of gene generation are as follows: (1) Construction OsEBP89 Gene overexpression vectors; (2) Preparation containing OsEBP89 Recombinant bacteria with gene overexpression vectors; (3) Preparation using recombinant bacteria OsEBP89 Overexpression of genes in strains.

[0010] Furthermore, in step (1), a root-specific gene expression method was used. Os03g01700 Promoter.

[0011] Furthermore, by knocking out rice OsEBP89 Genes are used to reduce the salt tolerance of rice.

[0012] Furthermore, with OsEBP89 The nucleotide sequence shown in SEQ ID No. 6 in the genome is the target for gene knockout.

[0013] Furthermore, knocking out [the virus] in rice OsEBP89 The specific steps of gene generation are as follows: (1) The target sequence was constructed in the pYLCRISPR / Cas9 vector to obtain an expression cassette for targeting the target; (2) Preparation of recombinant bacteria containing expression cassettes; (3) Preparation of knockout bacteria using recombinant bacteria OsEBP89 Mutant strains of the gene.

[0014] Furthermore, the above OsEBP89 Application of genes in rice breeding.

[0015] Furthermore, it can be used to improve the salt tolerance of rice.

[0016] The beneficial effects of this invention are: utilizing CRISPR / Cas9 technology to... OsEBP89 Gene knockout resulted in mutant plants exhibiting significantly different characteristics compared to the wild type. OsEBP89 Gene expression levels decreased, and after salt treatment, the survival rate was significantly lower than that of the wild type; OsEBP89 Genes regulate multiple levels of molecular physiological pathways, including Na+. + / K + Factors such as reactive oxygen species homeostasis affect rice salt tolerance; mutations in this gene result in salt-sensitive plants. Overexpression in rice... OsEBP89 After salt treatment, the average survival rates of the overexpressing lines and the control group were 76.7% and 58.5%, respectively, with a significant difference (t-test p<0.05). This invention provides... OsEBP89 Genes play a crucial role in regulating salt tolerance in rice, laying a solid foundation for improving rice's salt tolerance. Overexpression of these genes in plants is essential. OsEBP89 Genes are of great significance for high-quality rice breeding. OsEBP89 Gene research has laid the foundation for a deeper understanding of the molecular physiological mechanisms and molecular networks of salt tolerance in rice, and provided molecular elements for the design breeding of salt-tolerant rice. Attached Figure Description

[0017] Figure 1 for OsEBP89 The genome structure of the gene and the results of gene editing in three rice mutants; Figure 2 In WT and osebp89 mutant plants OsEBP89 Results of gene expression level detection; Figure 3 The spectrum of unloaded pTFLGHAU; Figure 4 The image shows the recombinant expression vector pTFLGHAU-pro03g01700-1077bp+OsEBP89+linker eGFP (pTFLGHAU modified vector); Figure 5 For WT, OsEBP89 overexpression lines and osebp89 mutants OsEBP89 Results of gene expression level detection; Figure 6 Phenotypic (A) and survival rate (B) of WT and osebp89 mutant plants after salt treatment. Scale bar = 3 cm. Data are expressed as mean ± standard deviation (n = 3 biological replicates, approximately 24 plants in each biological replicate). t-tests were used to generate P values, with ** indicating P < 0.01. Figure 7 Survival statistics (A) and phenotype (B) of WT and OsEBP89 overexpression lines after salt treatment. Each point represents one replicate (24 seedlings) of one line. All five overexpression lines had three replicates. Detailed Implementation

[0018] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0019] Example 1 1. Sequence OsEBP89 The genome sequence is shown in SEQ ID No. 1. OsEBP89 The coding sequence of the gene is shown in SEQ ID No. 2. OsEBP89 The amino acid sequence encoded by the gene is shown in SEQ ID No. 3, rice root-specific expression gene. Os03g01700The nucleotide sequence of the promoter region is shown in SEQ ID No. 4, and the nucleotide sequence of linker-eGFP is shown in SEQ ID No. 5.

[0020] II. Obtaining the rice OsEBP89 mutant using the CRISPR / Cas9 system Using the CRISPR / Cas9 system to analyze rice genome OsEBP89 Gene editing was used to obtain transgenic rice. OsEBP89 Genetic mutation type. The specific steps are as follows: 1. Target sequence selection Target T: AAGTCGACGTCGACTTCGAGG (SEQ ID No. 6), located in the rice shown in SEQ ID No. 1. OSEBP89 Nucleotides 181-202 of the genome sequence (i.e., in the rice shown in SEQ ID No. 2) OSEBP89 Nucleotides 127-148 in the coding sequence of the gene.

[0021] 2. Construction of CRISPR / Cas9 vectors The sequence of target T was constructed into the pYLCRISPR / Cas9 vector using the following method to obtain an expression cassette for targeting target T. The specific construction method is as follows: (1) Adapter primer design: The F chain and R chain of the target site were mixed and denatured and annealed to prepare the T target site adapter.

[0022] Connector primer sequences targeting target T: F chain: 5'-GTCAAAGTCCGACGTCGACTTCG-3' (SEQ ID No. 7); R chain: 5'-AAACCGAAGTCGACGTCGGACTT-3' (SEQ ID No. 8).

[0023] In the F and R strand primers for the target site: the first 4 bases at the 5' end are Bsa The complementary bases of the sticky ends after I enzyme digestion, and the complementary bases of the F chain are used to interact with the enzyme. Bsa The exposed OsU3 ends of the sgRNA-OsU3 obtained after I enzyme digestion are complementaryly linked, and the complementary bases of the R strand are used to bind with the OsU3 ends obtained after I enzyme digestion. Bsa The exposed ends of sgRNA in sgRNA-OsU3 obtained after I enzyme digestion are complementary; the remaining nucleotides in the F and R chains are the target sequence or its reverse complementary sequence.

[0024] (2) Connect the T-target connector to BsaAfter mixing I-HF (NEB, trade name BsaI-HF®v2, catalog number #R3733), pYLsgRNA vector, and T4 ligase, the gRNA gene expression cassette gRNA-U3-OSEBP89-T with T target site driven by OsU3 promoter was obtained.

[0025] The pYLsgRNA vector includes the following elements: sgRNA, a U3 / U6 promoter (any one of OsU3, OsU6a, OsU6b, or OsU6c; in this embodiment, it is the OsU3 promoter), and other necessary elements. The pYLsgRNA vector contains three... Bsa I restriction endonuclease recognition site, via Bsa After I enzyme digestion, sgRNA-promoter elements (i.e., any one of sgRNA-OsU3, sgRNA-OsU6a, sgRNA-OsU6b or sgRNA-OsU6c, sgRNA-OsU3 in this example) can be obtained.

[0026] (3) Two rounds of PCR amplification of gRNA-U3-OSEBP89-T were performed using the gRNA expression cassette amplification primers, which were then used to ligate pYLCRISPR / Cas9.

[0027] The primer information for gRNA expression cassette amplification is as follows: First-round amplification primers: UF: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID No. 9); gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID No. 10).

[0028] The second round of amplification primers, specifically target-specific primers, are as follows: Uctcg-B1':5'-TTCAGAGGTCTCTCTCGACTAGTGGAATCGGCAGCAAAGG-3' (SEQ IDNo. ​​11); gRcggt-BL:5'-AGCGTGGGTCTCGACCGACGCGTCCATCCACTCCAAGCTC-3' (SEQ IDNo. ​​12); B1' is the upstream primer of the promoter (U3), and BL is the downstream primer of the gRNA in the second round of PCR; Uctcg-B1' is complementary to the end of BL on the RB side of the pYLCRISPR / Cas9 vector after BsaI digestion. gRcggt-BL is complementary to the end of BR on the RB side of the pYLCRISPR / Cas9 vector after BsaI digestion.

[0029] (4) The amplified gRNA expression cassette was subjected to... Bsa I enzyme digestion, then with Bsa The plasmid pYLCRISPR / Cas9, after being digested with enzyme I, was ligated, transformed into DH5α, and the recombinant plasmid was obtained and sequenced. The correctly sequenced vector was named pYLCRISPR / Cas9-U3T, which contains a gRNA gene expression cassette (U3T) driven by the U3 promoter with T as the target site.

[0030] In this embodiment, the pYLCRISPR / Cas9 and pYLsgRNA vector are described in the literature " A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants Xingliang Ma, Qunyu Zhang, Qinlong Zhu, Wei Liu, Yan Chen, Rong Qiu, Bin Wang, Zhongfang Yang, Heying Li, Yuru Lin, Yongyao Xie, Rongxin Shen, Shuifu Chen, Zhi Wang, Yuanling Chen, Jingxin Guo, Letian Chen, Xiucai Zhao, Zhicheng Dong and Yao-Guang Liu. Molecular Plant. 2015 August;8:1274-1284. The pYLCRISPR / Cas9 vector used in this embodiment is named "pYLCRISPR / Cas9Pubi-H" in the aforementioned literature.

[0031] 3. Obtaining genetically modified rice The pYLCRISPR / Cas9-U3T vector obtained in step 2 above was transformed into Agrobacterium tumefaciens competent cells EHA105 using a freeze-thaw method to obtain recombinant bacteria EHA105 / pYLCRISPR / Cas9-U3T. This was then transformed into Nipponbare wild-type rice using Agrobacterium-mediated genetic transformation to obtain... OSEBP89 Homozygous mutant rice with gene mutation OsEBP89 The specific conversion method is as follows: (1) Seed disinfection: Soak Nipponbare wild-type rice seeds in ethanol with a volume fraction of about 75% for 1 minute, then soak them in sodium hypochlorite solution with a mass fraction of 2.5% for about 1 hour, and finally wash them 5 times with sterile water.

[0032] (2) Callus induction: Place the sterilized seeds on callus induction medium N6D and culture them in a light incubator at 32℃ for 24 hours for about 6 days. Cut off the callus tissue and transfer it to fresh callus induction medium N6D and continue to culture it in a continuous light incubator at 32℃ for 3 days.

[0033] (3) Infection: Collect and culture to OD 600nm The recombinant bacteria EHA105 / pYLCRISPR / Cas9-U3T with a pH of approximately 0.7 was resuspended in approximately 40 mL of AAM liquid medium to obtain the EHA105 / pYLCRISPR / Cas9-U3T infection solution. Callus tissue was then immersed in the infection solution for approximately 3 minutes.

[0034] (4) Co-culture: The infected callus tissue was transferred to co-culture medium 2N6-AS lined with sterile filter paper and cultured at 25°C in the dark for 3 days.

[0035] (5) Screening: First, wash the callus tissue several times with sterile water until the Agrobacterium is cleaned. Then, add sterile water containing carbenicillin (concentration of 500 mg / L) to wash it and blot it dry on sterile filter paper. Finally, transfer it to the screening medium S1 and incubate it in the dark at 25°C for about 4 days.

[0036] (6) Screening: Transfer to screening medium S2 and screen for about 2 weeks in a 32°C continuous light incubator, then transfer to differentiation medium RE-III.

[0037] (7) Differentiation: Cultured on differentiation medium RE-III for about 7 days, then transferred to fresh differentiation medium and cultured for another 7 days. Under normal circumstances, seedlings will grow on the second differentiation medium. Once the seedlings mature, they are marked as T0 generation rice.

[0038] The culture medium formulations used in the above-mentioned transgenic process are shown in Table 1.

[0039] Table 1. Formulations of various culture media used in the transgenic process Culture medium name Culture medium formulation pH value N6D medium 4g / L Chu (N6) Basal Salt Mixture powder + 2mg / L glycine + 0.5mg / L niacin + 0.5mg / L vitamin B6 + 1mg / L vitamin B1 + 1mg / L 2,4-D + 0.3g / L hydrolyzed casein + 2.878g / L proline + 0.1g / L inositol + 30g / L sucrose + 4g / L plant gel 5.8 Co-culture medium 2N6-AS 4g / L Chu (N6) Basal Salt Mixture powder + 2mg / L glycine + 0.5mg / L niacin + 0.5mg / L vitamin B6 + 1mg / L vitamin B1 + 1mg / L 2,4-D + 0.3g / L hydrolyzed casein + 0.1g / L inositol + 30g / L sucrose + 30g / L glucose + 5g / L plant gel + 10mg / L acetylsylgenone 5.2 AAM medium <![CDATA[AAM in large amounts (0.25 g / L MgSO4·7H2O + 0.15 g / L NaH2PO4·2H2O + 0.15 g / L CaCl2·2H2O + 3 g / L KCl), AAM in trace amounts (10 mg / L MnSO4·4H2O + 2 mg / L MnSO4·4H2O + 0.025 mg / L CuSO4·5H2O + 0.25 mg / L Na2MoO4·2H2O + 0.025 mg / L CoCl2·6H2O + 0.75 mg / L KI + 3 mg / L H3BO3) + iron salts (27.8 mg / L FeSO4·7H2O + 37.3 mg / L Na2-EDTA) + 0.5 g / L hydrolyzed casein + 7.5 mg / L glycine + 0.1767 g / L arginine + 0.9 g / L glutamine + 0.3 g / L aspartic acid + 0.1 g / L inositol + 1 mg / L niacin + 1 mg / L vitamin B6 + 10 mg / L vitamin B1 + 68.5 g / L sucrose + 36 g / L glucose + 10 mg / L acetosyringone]]> 5.2 Screening medium S1 N6D medium + 25 mg / L hygromycin + 400 mg / L carbenicillin 5.8 Screening medium S2 N6D medium + 50 mg / L hygromycin + 400 mg / L carbenicillin 5.8 Differentiation medium RE-III 4.4 g / L MS (Sigma) + 2 g / L hydrolyzed casein + 0.0002 mg / L NAA + 2 mg / L KT + 30 g / L sucrose + 30 g / L sorbitol + 4 g / L plant gel + 50 mg / L hygromycin + 200 mg / L carbenicillin 5.8 4. OsEBP89 Identification of transgenic rice with gene mutations Extract the T0 generation rice genomic DNA obtained in step 3 and perform PCR amplification using the following primer pair (amplified fragment length is 470bp).

[0040] OsEBP89-F: 5'-AGGTTTGCCGTCAGGTGAAAA-3' (SEQ ID No. 13); OsEBP89-R: 5'-ACGGAGACTAGACAGCCCAT-3' (SEQ ID No. 14).

[0041] The PCR amplification products were sent to a biotechnology company for sequencing and analysis to obtain... OsEBP89 Gene mutation information. Compared to wild-type rice. OsEBP89 Gene comparison was performed, and the results were as follows: Figure 1 As shown, the T0 generation rice mutant osebp89-1 in OsEBP89 The first exon of the gene has a 19bp deletion, resulting in a frameshift; osebp89-3 is located in... OsEBP89 The first exon of the gene has a T base deletion, resulting in a frameshift; osebp89-4 is located in... OsEBP89 A T base is inserted in the first exon region of the gene, resulting in a frameshift.

[0042] 5. Genotyping of T1 generation transgenic rice The T0 generation identified in step 4 OsEBP89 The genetically mutated rice plant was harvested as a single seed to obtain T1 generation seeds, which were then propagated to obtain homozygous T2 generation seeds.

[0043] Identification of T1 generation transgenic rice OsEBP89 The genotype of the gene was determined using the following method: Genomic DNA from leaves of T1 generation transgenic rice was used as a template, and PCR amplification was performed using primers OsEBP89-F (SEQ ID No. 13) and OsEBP89-R (SEQ ID No. 14) to obtain PCR amplification products from different lines. Based on the sequencing results, [the following lines were selected]. OSEBP89 The single plant with the homozygous mutant gene was named homozygous mutant OsEBP89 rice for subsequent research.

[0044] Compared with wild-type rice, homozygous mutant OsEBP89 rice has two homologous chromosomes... OSEBP89 The following mutations occurred in all genes: osebp89-1 OsEBP89 A 19 bp deletion occurs in the first exon region of the gene, resulting in a frameshift; osebp89-3 is present in... OsEBP89 The first exon of the gene has a T base deletion, resulting in a frameshift; osebp89-4 is located in... OsEBP89 A T base is inserted in the first exon region of the gene, resulting in a frameshift.

[0045] Using RT-PCR to detect homozygous mutant OsEBP89 rice OsEBP89 Gene expression levels were detected using the following primers: OsEBP89-RT-F: 5'-CTGAGCGCGGCAAAGTG-3' (SEQ ID No. 15); OsEBP89-RT-R: 5'-CGCTCGCCATCTGTTCGT-3' (SEQ ID No. 16); Ubi F: 5'-ACCACTTCGACCGCCACTACT-3' (SEQ ID No. 17); Ubi R: 5'-ACGCCTAAGCCTGCTGGTT-3' (SEQ ID No. 18).

[0046] The results are as follows Figure 2 As shown, compared to the wild-type control, the mutants... OsEBP89 Gene expression levels decreased significantly.

[0047] Example 2 Obtaining OsEBP89 overexpression lines 1. Carrier Construction Genes specifically expressed in rice roots Os03g01700 Using the promoter region as a template, high-fidelity polymerase Q5 was used to amplify the polymerase using primer pairs pro1077-F1 and pro1077-R1. Os03g01700 The 1077bp promoter region was amplified using E89-F2 and E89-R2. OsEBP89 The coding sequence of the gene; the coding sequence of eGFP was amplified using eGFP-F3 and eGFP-R3. Then, the three amplification products and the pTFLGHAU vector (as shown in the diagram) were... Figure 3 (As shown) An overexpression vector was constructed through homologous recombination. After verifying the sequence was correct, it was transformed into E. coli DH5α, and the plasmid was extracted to obtain the recombinant expression vector pTFLGHAU-pro03g01700-1077bp+OsEBP89+linker eGFP (as shown in the figure). Figure 4 As shown in the figure, the vector is named pTFLGHAU. The primer sequences are as follows: pro1077-F1: 5'-ATGACCATGATTACGAATTCAGCTAGCTCGCGATTAACC-3' (SEQ ID No. 19); pro1077-R1: 5'-GGATTGCACCGCCGCACATCTTCTTCTTCTTCGATCGACC-3' (SEQ ID No. 20); E89-F2: 5'-GGTCGATCGAAGAAGAAGAAGATGTGCGGCGGTGCAATCC-3' (SEQ ID No. 21); E89-R2: 5'-GCAGCGGCAGCCGCAGCTGCGTCGAGCAGAAGAGAGGCCTG-3' (SEQ ID No. 22); eGFP-F3: 5'-CAGGCCTCTCTTCTGCTCGACGCAGCTGCGGCTGCCGCTGC-3' (SEQ ID No. 23); eGFP-R3: 5'-CAGTCTAGAGTCGACGGATCCCTTGTACAGCTCGTCCATG-3' (SEQ ID No. 24).

[0048] 2. Preparation of recombinant bacteria containing overexpression vectors The recombinant expression vector pTFLGHAU-pro03g01700-1077bp+OsEBP89+linker eGFP was introduced into Agrobacterium tumefaciens EHA105 via the freeze-thaw method to obtain the recombinant strain EHA105 / pTFLGHAU-pro03g01700-1077bp+OsEBP89+linker eGFP.

[0049] 3. Preparation of overexpression lines The wild-type rice variety Zhonghua 11 was transformed using Agrobacterium-mediated genetic transformation to obtain... OSEBP89 The homozygous overexpression of the gene in rice. The specific transformation method is the same as step 3 in Example 1, obtaining transgenic rice, except that the recombinant bacteria used in the infection process of step 3 here is EHA105 / pTFLGHAU-pro03g01700-1077bp+OsEBP89+linkereGFP. The final result is... OSEBP89 Homozygous overexpression of the gene in T0 generation rice.

[0050] 4. RT-PCR detection of overexpression lines OsEBP89 Gene expression level Using RT-PCR to detect the expression of rice OsEBP89 Gene expression levels were detected using primers OsEBP89-RT-F (SEQ ID No. 15), OsEBP89-RT-R (SEQ ID No. 16), Ubi F (SEQ ID No. 17), and Ubi R (SEQ ID No. 18).

[0051] The results are as follows Figure 5 As shown, qRT-PCR was used to identify the expression in the 14-day-old young roots of five overexpression lines (1077-1, 1077-9, 1077-11, 1077-12, and 1077-19). OsEBP89 Gene expression levels, with controls at two biological replicates of wild-type WT1 and WT2, and OsEBP89 Gene knockout lines (prepared in Example 1); compared to the wild-type control, the expression level of OsEBP89 was increased in all overexpression lines.

[0052] Example 3 Salt solution treatment OsEBP89 Rice with different gene expression levels (1) The WT and osebp89 mutants (prepared in Example 1) and the overexpression line seeds (prepared in Example 2) planted and harvested in the same batch were soaked in distilled water at 37°C for 24 hours to allow the seeds to absorb water. Then the seeds were transferred to filter paper and placed in a 37°C incubator overnight for germination.

[0053] (2) Seeds with relatively consistent germination (wild type, mutant and overexpression) were seeded on 96-well plates (without bottom), and Kimura B rice nutrient solution was replaced and cultured until the three-leaf stage.

[0054] (3) Prepare Kimura B rice nutrient solution containing 200mM NaCl (for osebp89 mutant treatment) or Yoshida rice nutrient solution containing 200mM NaCl (for overexpression treatment). Treat rice seedlings cultured to the three-leaf stage with 200mM NaCl solution for 5 days, and set up an untreated control group.

[0055] (4) Five days after salt stress treatment, the rice seedlings were transferred to normal Kimura B rice nutrient solution (for osebp89 mutant treatment) or Yoshida rice nutrient solution (for overexpression treatment) to recover growth for 15 days. The photos were taken and the survival rate was recorded.

[0056] Results of mutant rice as follows Figure 6 As shown, the survival rates of WT and the osebp89-1, osebp89-3, and osebp89-4 mutants were 72.9%, 31.3%, 12.5%, and 9.38%, respectively. The results for the overexpressing rice mutant are as follows... Figure 7As shown, the average survival rates of the overexpression lines and the control were 76.7% and 58.5%, respectively, with a significant difference (t-test p<0.05). The survival rates of lines 1077-1, 1077-9, 1077-11, 1077-12, and 1077-19 were 61.5%, 77.1%, 76.7%, 85.4%, and 83%, respectively. Statistical results indicate that compared to WT, the survival rate of the osebp89 mutant was significantly lower, while the survival rate of the overexpression lines was higher, suggesting that the osebp89 mutant is salt-sensitive and salt-tolerant after overexpression.

[0057] OSEBP89 The genome sequence of the gene (SEQ ID No. 1):

[0058] OsEBP89 The coding sequence of the gene (SEQ ID No. 2): ATGTGCGGCGGTGCAATCCTCGCCGATTTCACCCCGGCGAGGGTGCCCCGGCGGCTGACCGCCGCCGAGCTCCTGCCGGTGACCCCGACTCCCCCCGCCGCCGAGAGGAGAACCACCCGGAAGCGCAAGTCCGACGTCGACTTCGAGGCGGAGTTCGAGCTTTTCGAGGACGACGACGACGACGATGAGTTCGAGCTTTCCGACGATGGCGACGAGAGTTTGGCCGTGTCATGTGTGTCGTCCCCCAAGTCGAAGGCAGTACCTTCGTTTTCTTTTTCGTCGGATGTCTCCTCGAGCTCCAGGCCGCGGCGGCGCGTGGCGGCGGCGGCGGCCGGTCGTCGGAAGGCGAGCAAGAAGAGCAAGTACAGGGGCGTCCGGCGCCGGCCGTCGGGGAGGTTCGCGGCGGAGATCAGGGACCCCAAGAAGGGGCGGCGCGTGTGGCTCGGCACGTACGGCAGCGCCGAGGAGGCCGCCATGGCCTACGACCGCGAGGCCCGCCGCATCCGCGGCAAGGGCGCGAGGCTCAACTTCCCCCGCGACGGCGATGGCTCCCCTCGCCGGAGTAACGACCGGCCCTGCTGGACCATCGACCTCAACCTCCCCGCGGCGGCCGTCTCCGGTGACGACGACGACGCCATGGCCGTCGACGCCGCAGACGCAGACGCAGGCAGTGCTGGCCGTGCAGCAGCCTATGCAGATCAAGAAGCACTGAGCGCGGCAAAGTGCAAGATCAAGCAGTGTCCTCGCGACGAACAGATGGCGAGCGCCACACCTGAGCTCATGGAGGAGGACGCGAGCAGCAGCAGAAACATGGTGCCCCTGTCCATGGCGCTGCAGCTGCAGTATGCGGCGATGATCGCCGAATGCGACCGCGAGATGGAGGAGATCGCCGCCGTGGAGAGGGACCTCGAGAGGCGCAGGAGGCAGGTGTTCGAGCGCAGAGGCCACCTGGTCAGGCAGGCCTCTCTTCTGCTCGACTGA。

[0059] OsEBP89 The amino acid sequence encoded by the gene (SEQ ID No. 3): MCGGAILADFTPARVPRRLTAAELLPVTPTPPAAERRTTRKSDVDFEAEFELFEDDDDEFELSDDGDESLAVSCVSSPKSKAVPSFSFSSDVSSSSRPRRRVAAAAAGRRKASKKSKYRGVRRRPSGRFAAEIRDPKKGRRVWLGTYGSAEEAAMAYDR EARRIRGKGARLNFPRDGDGSPRRSNDRPCWTIDLNLPAAAVSGDDDDAMAVDAADADAGSAGRAAAYADQEALSAAKCKIKQCPRDEQMASATPELMEEDASSSRNMVPLSMALQLQYAAMIAECDREMEEIAAVERDLERRRRQVFERRGHLVRQASLLLD.

[0060] Rice root-specific expression genes Os03g01700 The nucleotide sequence of the promoter (SEQ ID No. 4):

[0061] The nucleotide sequence of Linker-eGFP (SEQ ID No. 5): .

[0062] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. OsEBP89 The application of genes in regulating plant salt tolerance, the aforementioned OsEBP89 The genomic sequence of the gene is shown in SEQ ID No.

1. OsEBP89 The coding sequence of the gene is shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that: The OsEBP89 The amino acid sequence encoded by the gene is shown in SEQ ID No.

3.

3. The application according to claim 1, characterized in that: By overexpressing the above in rice OsEBP89 Genes are used to enhance the salt tolerance of rice.

4. The application according to claim 3, characterized in that, Overexpression of the above in rice OsEBP89 The steps of gene generation are as follows: (1) Construct the above OsEBP89 Gene overexpression vectors; (2) Preparation of the product containing the above OsEBP89 Recombinant bacteria with gene overexpression vectors; (3) Prepare the recombinant bacteria using the recombinant bacteria OsEBP89 Overexpression of genes in strains.

5. The application according to claim 4, characterized in that: In step (1), a root-specific gene expression method is used. Os03g01700 Promoter.

6. The application according to claim 1, characterized in that: By knocking out the rice OsEBP89 Genes are used to reduce the salt tolerance of rice.

7. The application according to claim 6, characterized in that: As stated OsEBP89 The nucleotide sequence shown in SEQ ID No. 6 of the gene is the target site for gene knockout.

8. The application according to claim 7, characterized in that: Knockout in rice OsEBP89 The specific steps of gene generation are as follows: (1) The sequence of the target point is constructed in the pYLCRISPR / Cas9 vector to obtain an expression cassette for targeting the target point; (2) Prepare recombinant bacteria containing the expression cassette; (3) Prepare the knockout strain using the recombinant bacteria. OsEBP89 Mutant strains of the gene.

9. The claim 1 OsEBP89 Application of genes in rice breeding.

10. The application according to claim 9, characterized in that: The application is to improve the salt tolerance of rice.

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