Salt stress regulation gene osnca1b of rice and application thereof

Abstract

This invention relates to the field of molecular biology, and more particularly to rice salt stress regulatory genes. OsNCA1b Its applications. The CDS sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2. This invention creates... OsNCA1b Genetic materials from the rice variety Kitaake were overexpressed and knocked out using CRISPR-CAS9 editing. The salt-sensitive phenotype of these materials under 100 mM NaCl salt stress treatment and 0.5% saline-alkali soil planting conditions was examined. The results demonstrated that: OsNCA1b CRISPR gene-edited lines showed salt tolerance compared to wild-type rice. OsNCA1b The overexpression lines exhibited high salt sensitivity, indicating that this gene is a negative regulator of salt stress in rice. This invention provides genetic resources and technical methods for breeding salt-tolerant rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, and specifically relates to a rice salt stress regulatory gene. OsNCA1b And its applications. Background Technology

[0002] Soil salinization has become one of the major abiotic factors affecting crop yield. Years ago, scientists proposed two main approaches to address this problem: saline soil remediation and the biological approach of planting halophytes or salt-tolerant crops. Planting salt-tolerant crops is undoubtedly a fundamental and cost-effective method. Cultivating salt-tolerant crops can not only improve saline soil but also increase crop yield. This urgently requires us to discover key genes for crop salt stress adaptation, analyze their mechanisms of action, and apply them to genetic engineering breeding to cultivate salt-adapted crop varieties with stable yield potential. With the expansion of saline-alkali land, especially affected by climate change and the reduction of land resources, rice is facing increasingly severe salt stress challenges. Saline-alkali land significantly affects the growth, yield, and quality of rice.

[0003] In recent years, researchers have used functional genomics and transcriptomics techniques to identify several genes associated with salt stress. A collaborative study by Professor Wu Dezhi's team at Hunan Agricultural University and Professor Zhang Guoping's team at Zhejiang University discovered that knocking out… OsCaM1 The -1 gene significantly reduces salt tolerance in rice, while its overexpression enhances salt tolerance. This study clarifies... OsCaM1 -1 positively regulates the molecular mechanism of rice salt tolerance by controlling the sodium-potassium ion balance. Furthermore, research by Wang Juan et al. indicates that: OsDSK2a Gene-edited mutants osdsk2aS2G It is characterized by reduced plant height and increased salt tolerance. SnRK1A exist OsDSK2a The upstream plays a regulatory role. SnRK1A Regulation OsDSK2a S2 phosphorylation. Under salt stress conditions. SnRK1A Decreased activity, leading to OsDSK2a Phosphorylation and reduced abundance of [a substance] limit plant growth. Haplotype analysis of 3K-RG data revealed [the following]. OsDSK2a -S2 exhibits natural variation, and OsDSK2a The -G allele can reduce plant height while increasing grain yield under salt stress. The study revealed... OsDSK2a The novel mechanism of stability provides a valuable target for salt-tolerant crop breeding, helping to address the impact of salt stress on yield. Furthermore, the study found that rice exhibits a positive response to salt stress through pathways such as sucrose and starch metabolism, flavonoid synthesis, and glutathione metabolism. Despite significant progress in salt stress signaling and detoxification responses, further research is needed to uncover new genes or regulatory elements that may be used to produce salt-tolerant crops.

[0004] NCA1 Genes are primarily found in higher plants, and were first identified in Arabidopsis thaliana, where the genome contains only one copy. In contrast, two copies have been identified in the rice genome. OsNCA1a ( Os01g0104100 )and OsNCA1b ( Os02g0795300 Researchers Liu Jianzhe, Cui Lili, Xie Zongwang, and others discovered that both genes can interact with and activate catalase (CAT), and possess functional redundancy. Their expression patterns are similar, with the highest expression levels observed in leaves. Compared to the wild type, the single mutant... nca1a or nca1b There were no significant differences in phenotype and CAT activity, while the double mutant showed no significant differences. nca1anca1b The CAT activity of the double mutant was significantly reduced (to only about 5%), and it exhibited a distinct lesion-like phenotype on the leaves, even showing whitening. When the double mutant was grown under high CO2 conditions of 3500 ppm, photorespiration was inhibited, H2O2 production decreased, and the lesion-like phenotype was eliminated, although CAT activity was still reduced by more than 95%. This indicates that the lesion phenotype of the double mutant may be caused by H2O2 accumulation induced by photorespiration. This invention cloned rice using homologous cloning technology. OsNCA1b Genes, through creation OsNCA1b Gene overexpression and CRISPR-CAS9 editing knockout of the rice variety Kitaake were used to detect the salt-sensitive phenotype of these genetic materials under 100 mM NaCl salt stress treatment and 0.5% saline-alkali soil planting conditions, demonstrating that it plays a negative regulatory role in the rice salt stress response. This invention provides target genes and technical methods for breeding salt-tolerant rice varieties. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the purpose of this invention is to provide a rice salt stress regulatory gene. OsNCA1b and its applications, the OsNCA1b Genes play a negative regulatory role in rice salt tolerance, providing genetic resources and technical methods for breeding salt-tolerant rice varieties.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for regulating the salt tolerance of rice. OsNCA1b Genes, the ones mentioned OsNCA1b A gene is any of the following: A1) The encoded sequence is: SEQ ID NO:1 in the sequence list. OsNCA1b Gene CDS sequence; A2) The nucleotide is: SEQ ID NO:1 in the sequence listing. OsNCA1b Gene CDS sequence.

[0007] The present invention also provides the above. OsNCA1b The OsNCA1b protein encoded by a gene, wherein the OsNCA1b protein is any one of the following: B1): The amino acid sequence is that of the protein listed as SEQ ID NO:2 in the sequence listing; B2): A protein that is more than 90% identical to the protein shown in B1) and is associated with plant salt tolerance, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2 in the sequence listing. B3): A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0008] The present invention also provides a biomaterial relating to at least one of the following; C1) OsNCA1b Nucleic acid molecules of genes; OsNCA1b protein; It contains at least one of the following: C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) A transgenic plant cell line containing the nucleic acid molecule described in C1), or a transgenic plant cell line containing the expression cassette described in C2); C6) Transgenic plant tissue containing the nucleic acid molecules described in C1), or transgenic plant tissue containing the expression cassette described in C2); C7) A transgenic plant organ containing the nucleic acid molecule described in C1), or a transgenic plant organ containing the expression cassette described in C2); C8) Reduce the expression of the OsNCA1b protein in nucleic acid molecules; C9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in C8).

[0009] The present invention also provides a rice OsNCA1b Gene knockout mutant, wherein the rice mutant is a mutant that knocks out the nucleotide sequence shown in SEQ ID NO: 1.

[0010] The present invention also provides a method for regulating the salt stress response of plants, wherein the expression of the encoding gene of salt stress response-related protein in the target plant is regulated, wherein the salt stress response-related protein is any one of the OsNCA1b proteins mentioned above.

[0011] The present invention also provides a plant salt-tolerant substance, wherein the plant salt-tolerant substance contains the aforementioned OsNCA1b The gene and / or the OsNCA1b protein, and / or the biological material; the plant is a monocotyledonous or dicotyledonous plant.

[0012] This invention also provides rice salt stress regulatory genes. OsNCA1b The application is implemented based on at least one of the following: The aforementioned OsNCA1b Any of the genes or any of the OsNCA1b proteins or any of the biological materials described; The applications include one or more of the following: D1) Application in regulating plant salt tolerance; D2) Application in cultivating salt-tolerant / salt-intolerant plants; Application of D3 in the preparation of products that improve / reduce plant salt tolerance; Application of D4 in plant breeding.

[0013] Preferably, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0014] The present invention also provides a method for cultivating transgenic plants with improved salt tolerance, comprising reducing the expression of the gene encoding the OsNCA1b protein in the target plant to obtain a transgenic plant with higher salt tolerance than the target plant; The reduction of the expression of the gene encoding the OsNCA1b protein in the target plant is achieved by using the CRISPR / Cas9 gene editing system to inhibit the content and / or activity of the OsNCA1b protein in the target plant.

[0015] Preferably, the target plant is a monocotyledonous plant or a dicotyledonous plant.

[0016] The present invention has the following technical effects and advantages: This invention successfully constructed the pCAMBIA1305APFH-OsNCA1b overexpression vector and the pYLCRISPRCas9Pubi9-H-OsNCA1b editing vector, genetically transformed the rice variety Kitaake, and finally obtained 19 T2 generation plants. OsNCA1b Overexpression homologous lines, 26 T2 generation strains OsNCA1bEdit pure line; under 100mM NaCl salt stress treatment and 0.5% saline-alkali soil conditions, OsNCA1b The edited strains showed some salt tolerance. OsNCA1b The overexpression lines exhibited high salt sensitivity; agronomical traits such as effective tillering, ear grain weight, and thousand-grain weight were analyzed and found to be similar to the wild type in both the overexpression and edited lines. Results showed: OsNCA1b Genes play a negative regulatory role in rice salt tolerance, providing genetic resources and technical methods for breeding salt-tolerant rice varieties. Attached Figure Description

[0017] Figure 1 OsNCA1b RNA expression of genes after salt stress treatment at different times.

[0018] Figure 2 OsNCA1b RNA expression was observed in the T2 generation overexpression transgenic pure lines. CK was the Kitaake wild-type control. OENCA1b for OsNCA1b Overexpression of Kitaake transgenic pure lines.

[0019] Figure 3 pYCRIPR / Cas9Pubi-H- OsNCA1b Identification of editing targets in transgenic Kitaake rice lines. A represents the amplified DNA sequence at the target site, and B represents the sequence obtained after sequencing the amplified band. OsNCA1b Forms of gene editing and their impact on protein structure.

[0020] Figure 4 Phenotypic images of aboveground rice seedlings after 100 mM NaCl treatment. A and D represent the aboveground phenotype of rice seedlings treated with 0 mM NaCl; B and E represent the aboveground salt-sensitive phenotype of seedlings treated with 100 mM NaCl for 9 days; C and F represent the aboveground salt-sensitive phenotype of seedlings treated with 100 mM NaCl for 9 days and rehydrated for 5 days. CK is the Kitaake wild-type control. nca1b for OsNCA1b Editing pure system, OENCA1b for OsNCA1b Overexpression transgenic pure lines.

[0021] Figure 5 Survival rate of aboveground parts of seedlings after treatment with 100 mM NaCl.

[0022] Figure 6 Salt sensitivity phenotypes of various combinations in 0.5% saline-alkali soil. Among them, A represents the salt sensitivity phenotypes of the control and three edited lines in 0.5% saline-alkali soil, and B represents the salt sensitivity phenotypes of the control and two overexpression lines in 0.5% saline-alkali soil.

[0023] Figure 7 OsNCA1b The agronomic traits of pure lines and overexpression pure lines were analyzed. A represents the number of effective panicles; B represents the number of effective grains per panicle; and C represents the thousand-grain weight. Detailed Implementation

[0024] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0026] Example 1 Harvested Kitaake rice seeds were treated in a 45℃ oven for 36 hours to break dormancy. After treatment, the seeds were individually packaged and clearly labeled, then disinfected with a 5% sodium hypochlorite solution for 50 minutes, and repeatedly rinsed with deionized water until the sodium hypochlorite was completely removed. Next, the seeds were soaked and germinated in a 37℃ constant temperature incubator for 48-72 hours. After germination, seeds with uniform growth were selected and transplanted into black hydroponic boxes containing rice nutrient solution. The black hydroponic boxes were disinfected with a 5% sodium hypochlorite solution for 24 hours beforehand. After transplanting, the boxes were placed in a light incubator for cultivation. The nutrient solution was changed every 3 days. The conditions in the light incubator were: 16000 lx light, 28℃, 12 hours; darkness stage, 24℃, 12 hours. Kitaake material was cultured in rice nutrient solution (product code NSP1040-250g, purchased from Beijing Cooler Master Technology Co., Ltd.) until it reached the three-leaf-one-heart stage, then treated with 100mM NaCl, and leaves were collected at different time points for quantitative fluorescence analysis. The results are as follows: Figure 1 As shown, OsNCA1b The expression level increased significantly, with the highest expression levels at 12h and 72h.

[0027] Example 2: OsNCA1b Creation of overexpression materials By visiting the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), for OsNCA1b The gene sequence was analyzed. OsNCA1b The coding region of a gene contains 1098 nucleotides, encoding 365 amino acids.

[0028] OsNCA1b The CDS sequence of the gene is shown in SEQ ID NO:1: >LOC_Os02g55200 (1098bp) The amino acid sequence of the OsNCA1b protein is shown in SEQ ID NO:2: >LOC_Os02g55200 MSSLCPFAKLASAGATCPVKSDNKTTSCPVTANNHTDDDDNEKTGNANTDPRVVPAKCPFGYDSNNTFKLGPLSCVVCHALLHQSSKCTPCSHKFCKACILRFKDCPLCGADIQGIEPDDELQGLVDRFIDGHARIKRSHAAGDGEAASDKTKVIYEHVSMERGAFLVQQAMRAFRAQNIESA KSRLSMCAEDIREELKSKEDNQELCSQLGAVLGMLGDCCRTLGDAPSAITYYEESAEFLSKLPKKDLELVHTLSVSLNKIGDLCYYDGDLHSARSYYARSLDVRRSAVKEHSAVASQVIDVATSLAKVADVDRNLGNESMAVEGFEEAIKCLENLKLESGEASLEQRRLSVLDFLQKQLDDK*. Using the cDNA of wild-type rice Kitaake as a template, specific primer pairs were used... OsNCA1b -MluI-IF-F1 / OsNCA1b -MluI-IF-R1 amplification OsNCA1b The full-length CDS sequence of the gene was obtained. The PCR reaction system consisted of: 30 μl of 2×PCR Buffer and 1.2 μl of 40 mM dNTPs. OsNCA1b -MluI-IF-F1 0.9μl, OsNCA1b MluI-IF-R1 0.9 μl, cDNA 1.2 μl, KOD Fx Neo 0.6 μL, ddH2O 25.2 μl; PCR reaction parameters: 94℃ 4 min; 94℃ 30 s, 55℃ 30 s, 72℃ 1 min, for a total of 36 cycles; 72℃ 7 min. KOD Fx Neo, catalog number KFX-201, was purchased from Beijing Dongge Boye Biotechnology Co., Ltd. The amplified fragment was recovered using a microcolumn DNA concentration gel extraction kit (microcolumn DNA concentration gel extraction kit, catalog number ZPV202, Beijing Zhuangmeng International Biotechnology Co., Ltd.).

[0029] The recovered target fragment was constructed into the pCAMBIA1305APFH vector via homologous recombination to obtain... OsNCA1b Gene overexpression vector pCAMBIA1305APFH- OsNCA1b The cells were then transformed into *E. coli* DH10B competent cells (Beijing Bomei Biotechnology Co., Ltd., catalog number BC106), cultured overnight at 37°C, and clones were picked and cultured to extract plasmids (using the plasmid mini-prep kit from Tiangen Biotech (Beijing) Co., Ltd., catalog number DP103). Restricted enzyme digestion and PCR identification were then performed. The correctly identified positive single clones were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Sequencing results were compared with the original sequences using SEContral software, and the peak diagram of the sequencing results was analyzed using Chromas software to ensure accuracy. OsNCA1b The sequence is correct. Plasmids with correct sequence alignment and peak plot analysis results were selected and transformed into Agrobacterium competent cells EHA105 to obtain pCAMBIA1305APFH-. OsNCA1b Agrobacterium strain was used to genetically transform the Kitaake rice variety. Hefei Jinjie Ruihao Biotechnology Co., Ltd. was commissioned to complete the pCAMBIA1305APFH- OsNCA1b Rice genetic transformation. Using pC1305-APFH-F1 / OsNCA1b The -Mlu-IF-R1 primer pair was used for transgenic positive identification via PCR. After two generations of planting and screening, the final result was... OsNCA1b Three transgenic lines that were overexpressed were randomly selected for further processing. OsNCA1b Overexpression detection using primer pairs OsNCA1b qPCR-F1 / OsNCA1b qPCR-R1 was used for detection. Figure 2 The three transgenic pure lines OENCAb1#22-1, OENCAb1#24-1, and OENCAb1#26-2 were shown. OsNCA1b The expression status of [the substance / organism]. Real-time quantitative PCR results showed: OsNCA1b The gene expression level was significantly higher than that of the wild type, indicating that OsNCA1b Gene overexpression. The nucleotide sequences of the primers used in the creation of the overexpression material are shown in Table 1.

[0030] Table 1 Primers used in the creation of overexpression materials

[0031] Example 3: OsNCA1b Editing materials creation 1. First, construct LacZ-U6a-sgRNA- OsNCA1b Expression Box Using the U6a-LacZ-1 plasmid as a template (Mol. Plant. 2015, 8, 1274-284), primer pair Cas9U6-F / U6a was used. OsNCA1baT1 and gR OsNCA1b PCR was performed using T1 / gR-Rcas9 to obtain PCR product 1 and PCR product 2, respectively.

[0032] PCR reaction system for PCR product 1: U6a-LacZ-1 2 µL, KOD Fx Neo 0.5 µL, Cas9U6-F 0.75 µL, U6a OsNCA1b The reaction mixture consisted of 0.75 µL of T1, 1 µL of 40 mM dNTP, 25 µL of 2×Buffer, and 20 µL of ddH2O, for a total of 50 µL. The PCR reaction parameters were as follows: 94℃ for 4 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 40 s, 36 cycles, and 72℃ for 7 min.

[0033] The reaction system for PCR product 2 consisted of: 2 µL U6a-LacZ-1, 25 µL 2×Buffer, 0.5 µL KOD Fx Neo, 0.75 µL gR-Rcas, 0.75 µL gROsNCA1bT1, 1 µL 40 mM dNTP, and 20 µL ddH2O, for a total of 50 µL. The PCR reaction parameters were as follows: 94℃ for 4 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 20 s, 36 cycles, followed by 72℃ for 7 min.

[0034] PCR product 1 and PCR product 2 were diluted separately, and the products from both rounds were mixed and used as a template. Overlapping PCR was performed using Cas9U6-F and gR-Rcas9 primers, and the amplified product was 862 bp LacZ-U6a-sgRNA. OsNCA1b Expression cassette. The PCR conditions for the amplification reaction were as follows: 2 µL of a mixture of PCR product 1 and PCR product 2, 25 µL of 2×Buffer, 0.5 µL of KOD Fx Neo, 0.75 µL of gR-Rcas9, 0.75 µL of Cas9U6-F, 1 µL of 40 mM dNTP, and 20 µL of ddH2O. The PCR reaction parameters were as follows: 94℃ for 4 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 36 cycles, and 72℃ for 7 min.

[0035] 2. pYCRIPR / Cas9Pubi-H - OsNCA1b Editing carrier construction The constructed LacZ-U6a-sgRNA- OsNCA1bThe expression cassette was recombined into the pYCRISPRCas9PUbi-H vector (Mol. Plant. 2015, 8, 1274–1284). First, the pYCRISPRCas9PUbi-H vector was digested with enzymes (10 µL pYCRISPRCas9PUbi-H vector, 6 µL 10 × Cutsmart Buffier, 1.2 µL BsaI-HPv2, 42.8 µL ddH2O), and reacted at 37°C for 30 min. The large fragment of the digested vector was then recovered using a microcolumn concentrated DNA gel extraction kit (Beijing Zhuangmeng International Biotechnology Co., Ltd., catalog number ZPV202-2). The linearized pYCRISPRCas9PUbi-H vector fragment, after digestion and gel extraction, was then combined with LacZ-U6a-sgRNA- OsNCA1b Homologous recombination was performed using the expression cassette (pYCRISPRCas9PUbi-H 4 μL, LacZ-U6a-sgRNA expression cassette 1 μL, 2×MultiF SeamLessAssembly Mix 10 μL, ddH2O 5 μL). The recombinant plasmid was transformed into competent *E. coli* cells, and positive single clones were identified by colony PCR (colony, 2×Buffer 10 µL, KOD Fx Neo 0.2 µL, Cas9gR-R 0.3 µL, Cas9U6-F 0.3 µL, 40 mM dNTP 0.4 µL, ddH2O 8.8 µL). The PCR reaction parameters were as follows: 94℃, 4 min; 94℃, 30 s; 55℃, 30 s; 72℃, 30 s; 36 cycles; 72℃, 7 min. Plasmids were extracted using the plasmid mini-prep kit (catalog number: DP103) from Tiangen Biotech (Beijing) Co., Ltd. Enzyme digestion (pYLCRISPR-Cas9Pubi-H-) was then performed. OsNCA1b 2 µL, 10X Cutsmart Buffier 2 µL, BsaI-HPv2 0.4 µL, ddH2O 15.2 µL) and sequencing identification to ensure pYCRIPR / Cas9Pubi-H - OsNCA1b Successfully constructed. The correctly identified positive monoclonal bacterial cultures, confirmed by PCR and enzyme digestion, were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the original sequences using SECentral software, and the peak diagram of the sequencing results was analyzed using Chromas software. Plasmids with correct sequence alignment and peak diagram analysis were selected for transformation into Agrobacterium competent cells, pending genetic transformation in rice.

[0036] 3. pYCRIPR / Cas9Pubi-H - OsNCA1bRice genetic transformation and editing pure line screening and identification Hefei Jinjie Ruihao Biotechnology Co., Ltd. was commissioned to complete the pYLCRISPR / Cas9Pubi9-H- OsNCA1b The rice genetic transformation experiment was conducted, transforming the rice variety Kitaake. Results were obtained through PCR amplification and sequencing analysis. OsNCA1b Edit pure rice lines.

[0037] Hitom OsNCA1b PCR positive identification was performed using the F1 / R1 primer pair, and the results were as follows: Figure 3 As shown, the target band size is 335 bp. OsNCA1b The target sequence was located at positions 62-81 on the sequence. PCR detection and sequence alignment using SEcentral software confirmed that the target sequence in the positive strain had been edited. Sequencing peak diagrams were viewed using Chromas software to confirm the edited pure lines. After statistical analysis of the sequencing results and editing status, the following results were obtained: Figure 3 The data shows 26 edited pure lines with four editing forms. The four editing forms are: 76-77 with 5 bases inserted, 74-81 with 8 bases deleted, 69-81 with 13 bases deleted, and 77-78 with 1 base inserted. OsNCA1b The nucleotide sequences of the primers used in the creation of the edited material are shown in Table 2.

[0038] Table 2 OsNCA1b Primers used in the creation of editing materials

[0039] Example 4: OsNCA1b Phenotypic identification of pure and overexpression lines under salt stress treatment Harvested rice seeds were treated in a 45℃ oven for 36 hours to break dormancy. After treatment, the seeds were separated and clearly labeled, then disinfected with a 5% sodium hypochlorite solution for 50 minutes, and repeatedly rinsed with deionized water until the sodium hypochlorite was completely removed. Next, the seeds were soaked and germinated in a 37℃ constant temperature incubator for 48-72 hours. After germination, seeds with uniform growth were selected and transplanted into black hydroponic boxes containing rice nutrient solution. The black hydroponic boxes were disinfected with a 5% sodium hypochlorite solution for 24 hours before transplanting. After transplanting, the boxes were placed in a light incubator for cultivation. The nutrient solution was changed every 3 days. The conditions in the light incubator were: 16000 lx light, 28℃, 12 hours; and 24℃, 12 hours in darkness. At the three-leaf stage, salt stress treatments were applied, with 0 mM NaCl and 100 mM NaCl concentrations used for each salt concentration. Uniformly germinated rice seeds were placed in the wells of a 96-well plate (8×12). From left to right, the CK (Kitaake) and three other seedlings were transplanted into the 96-well plate. OsNCA1b Edited strains and CK (Kitaake), three OsNCA1b Overexpression lines were treated for 9 days, and photographs were taken. Then, the salt solution was replaced with rice nutrient solution for rehydration treatment for 7 days, and photographs were taken again. Survival rate indicators were statistically analyzed.

[0040] Phenotypic results of 100 mM NaCl treatment (see) Figure 4 Compared to CK, OsNCA1b The edited lines showed milder leaf yellowing or wilting and leaf curling both before and after rehydration, with a significantly reduced number of leaves exhibiting curved or yellowing edges. A small portion of the weaker seedlings also showed softening stems, indicating a clear salt-tolerant phenotype. OsNCA1b The overexpression transgenic pure lines exhibited a significant phenotype of sensitivity to salt stress, indicating that under salt stress, rice seedlings... OsNCA1b The gene has a negative regulatory function on salt tolerance in rice. Survival rate statistical analysis was performed, and the results are as follows: Figure 5 As shown in the figure. These genetic materials were planted in 0.5% saline soil at the Caofeidian Crop Salt Tolerance Identification and Evaluation Base in Hebei Province to observe their salt sensitivity. The planting was conducted using the same arrangement as the control, edited lines, and control / overexpression lines. The results are as follows. Figure 6 As shown, the phenotype is consistent with that treated with 100 mM NaCl: the edited strains exhibited a certain degree of salt tolerance, while OsNCA1b The overexpression transgenic pure lines showed significant sensitivity to salt stress.

[0041] Example 5: OsNCA1b A survey of agronomic traits of pure lines and overexpression pure lines. According to the standards for investigating agronomic traits of rice, statistical analysis was performed on three important agronomic traits: effective tiller number, number of grains per panicle, and thousand-grain weight. The results are as follows: Figure 7 As shown, OsNCA1b There were no significant differences in the number of effective spikes / tillers, effective grains per spike, and thousand-grain weight between the edited and overexpression lines and the control Kitaake.

[0042] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for cultivating transgenic plants with enhanced salt tolerance, characterized in that, This includes reducing the expression of the gene encoding the OsNCA1b protein in the target plant to obtain a transgenic plant with higher salt tolerance than the target plant; The reduction of the expression of the gene encoding the OsNCA1b protein in the target plant is achieved by using the CRISPR / Cas9 gene editing system to inhibit the content and / or activity of the OsNCA1b protein in the target plant. The amino acid sequence of the OsNCA1b protein is shown in SEQ ID NO:2; The OsNCA1b protein is encoded by a gene whose nucleotide sequence is shown in SEQ ID NO:

1. OsNCA1b a gene whose nucleotide sequence is shown in SEQ ID NO:

1. The target plant is rice.

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