Application of OsCBL10 gene in regulating salt tolerance of rice

Abstract

The application discloses a kind of OsCBL10 The application discloses application of a gene in regulating salt resistance of rice OsCBL10 The application discloses application of a gene in regulating salt resistance of rice OsCBL10 The application discloses application of a gene in regulating salt resistance of rice OsCBL10 The application discloses application of a gene in regulating salt resistance of rice The application enriches the molecular regulation mechanism of rice response to salt stress, and provides a new candidate gene and improvement strategy for salt-tolerant rice breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and specifically relates to a... OsCBL10 Application of genes in regulating salt tolerance in rice. Background Technology

[0002] Soil salinization and secondary salinization damage arable land resources and cause huge losses to agricultural production. Improving the salt tolerance of crops is one of the most effective ways to solve this agricultural problem and reduce agricultural losses. Rice is my country's most important food crop and also the first choice for improving saline-alkali land. Analyzing the salt tolerance mechanism of rice and cultivating salt-tolerant rice varieties is of great significance for increasing rice yield in high-salinity areas and further expanding the rice planting area.

[0003] Salt stress damages plants primarily through osmotic stress, ion toxicity, and the resulting secondary stresses such as nutrient imbalance and oxidative stress. Rice salt tolerance is a comprehensive manifestation of various salt-tolerant physiological and biochemical responses, a quantitative trait controlled by multiple genes, and has a complex genetic basis. Improving rice salt tolerance using traditional breeding methods is difficult and progresses slowly. However, using molecular marker-assisted selection and genetic engineering techniques can accelerate the breeding process of new salt-tolerant rice varieties.

[0004] Therefore, identifying key salt-tolerant genes and elucidating their molecular regulatory networks and mechanisms of action is crucial for improving salt tolerance in rice. Summary of the Invention

[0005] The purpose of this invention is to provide a OsCBL10 Application of the gene (calcineurin B-like protein 10) in regulating salt tolerance in rice. OsCBL10 Loss-of-function mutations significantly reduce salt tolerance in rice, and overexpression... OsCBL10 Genes can significantly improve the salt tolerance of rice.

[0006] The technical solution adopted in this invention is:

[0007] In a first aspect, the present invention provides the application of a calcineurin B protein (OsCBL10) encoding gene in regulating salt tolerance in rice. OsCBL10 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence encoding the protein is shown in SEQ ID NO.2. The regulation includes reducing or increasing the salt tolerance of rice.

[0008] Due to the specific nature of nucleotide sequences, any variant of the nucleotide sequence shown in SEQ ID NO.1, provided it shares more than 90% homology with the polynucleotide, falls within the scope of protection of this invention. Variants of the polynucleotide include substitution variants, deletion variants, and insertion variants. These variants do not substantially alter the function of the encoded amino acid.

[0009] Due to the specificity of amino acid sequences, any fragment or variant thereof containing the amino acid sequence shown in SEQ ID NO.2, such as its conserved variants, bioactive fragments or derivatives, as long as the fragment or variant of the polypeptide has more than 95% homology with the aforementioned amino acid sequence, is within the scope of protection of this invention.

[0010] Secondly, the present invention provides OsCBL10 Application of genes in reducing salt tolerance in rice.

[0011] Furthermore, the application involves using the CRISPR / Cas9 system to alter the rice genome. OsCBL10 The gene sequence may be altered, or the expression of that gene may be reduced, disrupting its function. OsCBL10 Gene function, acquisition OsCBL10 Rice with missing genes has reduced salt tolerance.

[0012] Furthermore, the alteration of the rice genome OsCBL10 The gene sequence includes substitutions and / or insertions and / or deletions of one or more bases of the nucleotides shown in SEQ ID NO.1.

[0013] Furthermore, the reduction of gene expression includes substituting and / or deleting and / or adding one or more amino acid residues to the protein shown in SEQ ID NO.2.

[0014] Furthermore, the application method is as follows: using the CRISPR / Cas9 system, in OsCBL10 Gene editing target sequences were designed in the genome, amplified, and gene editing vectors were constructed. These vectors were then transferred into the rice genome using Agrobacterium-mediated transformation, followed by screening. OsCBL10 Plants with altered gene sequences are mutant rice plants.

[0015] The primers for amplifying the target sequence are as follows:

[0016] The upstream primer is: GGCAGCCTTGTTTAGAACACCGGC;

[0017] The downstream primer is: AAACGCCGGTGTTCTAAACAAGGC.

[0018] Thirdly, the present invention also provides OsCBL10 Application of genes in improving the salt resistance of rice.

[0019] Furthermore, the application involves overexpression in the rice genome. OsCBL10 Genes or proteins can increase the salt tolerance of rice.

[0020] Furthermore, the method of application is as follows: OsCBL10 The gene was ligated into the pCAMBIA1300-35S-GFP vector, and then Agrobacterium-mediated transgenesis was used to... OsCBL10 Genes were transferred into rice callus tissue for screening. OsCBL10 Rice plants with overexpressed genes and increased salt tolerance.

[0021] Compared with the prior art, the beneficial effects of this invention are mainly reflected in the following: This invention is the first to discover that the calcineurin B-like protein OsCBL10 participates in regulating rice salt tolerance and reduces [the salt content of rice] within the plant. OsCBL10 The expression or disruption of gene function can reduce salt tolerance in rice; while increasing it can decrease salt tolerance. OsCBL10 Gene expression can enhance the salt tolerance of rice. This invention enriches the molecular regulatory mechanisms of rice response to salt stress, providing new candidate genes and improvement strategies for salt-tolerant rice breeding. Attached Figure Description

[0022] Figure 1 for OsCBL10 Overexpression transgenic lines and OsCBL10 The results of identifying mutant lines of the gene. A represents the RT-qPCR identification results of the overexpressing transgenic lines, and NIP indicates wild type. CBL10-OE-1 and CBL10-OE-2 Indicates two OsCBL10 Overexpression transgenic lines; B is a mutant line. cbl10-1 and cbl10-2 The sequencing results.

[0023] Figure 2 Wild type (NIP) OsCBL10 mutant strains ( cbl10-1 and cbl10-2 )and OsCBL10 Overexpression transgenic lines ( CBL10-OE-1 and CBL10-OE-2 Salt-tolerant phenotype of ) . A is the one-week wild-type and OsCBL10 The mutant lines were treated with 0 and 120 mM NaCl nutrient solutions for 7 days, followed by 5 days of culture in normal nutrient solution (Bar = 5 cm). Figure B shows the survival rate of treated plants after treatment in Figure A. Figure C shows the survival rate of one-week-old wild-type and... OsCBL10The phenotype of the overexpression transgenic lines treated with 0 and 120 mM NaCl nutrient solutions for 9 days, followed by 5 days of culture in normal nutrient solution, is shown in Figure C. Bar = 5 cm. D represents the survival rate of the treated plants.

[0024] Figure 3 For a week, wild type, OsCBL10 mutant lines cbl10-1 and OsCBL10 overexpression transgenic lines CBL10-OE-1 Fresh weight (row A) and dry weight (row B) of aboveground parts and roots after treatment with 120 mM NaCl for 0 hours to 9 days.

[0025] Figure 4 For a week, wild type, OsCBL10 mutant lines cbl10-1 and OsCBL10 overexpression transgenic lines CBL10-OE-1 The sodium and potassium ion concentrations and sodium to potassium ion ratios in the aboveground parts (column A) and roots (column B) after treatment with 120 mM NaCl for 0 hours to 9 days were determined.

[0026] Figure 5 for OsCBL10 Overexpression transgenic seedlings (i.e.) CBL10-OE-1 Subcellular localization results of OsCBL10-GFP extracted from protoplasts. FM4-64 is a cell membrane fluorescent dye used to indicate the cell membrane; Bar = 5 μm.

[0027] Figure 6 for OsCBL10 GUS staining results (indicators) of various tissues of promoter-fused GUS transgenic lines OsCBL10 (The tissue expression sites). A shows the staining results of different parts of rice seedlings at 1 week old, Bar = 1 cm; B shows the staining results of root tips of 1-month-old rice, Bar = 500 μm; C shows the staining results of the root maturity zone of 1-month-old rice, Bar = 500 μm; D shows a cross section of the root tip of 1-month-old rice, Bar = 50 μm; E shows a cross section of the root maturity zone of 1-month-old rice, Bar = 50 μm; F shows a cross section of the leaf of 1-month-old rice, Bar = 50 μm; G shows a cross section of the stem of 1-month-old rice, Bar = 50 μm. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: the experimental operation and conditions for Agrobacterium-mediated transfer of exogenous genes into the rice genome in the embodiments of the present invention refer to (Li et al., 2025, Nat. Commun.).

[0029] Example 1 OsCBL10 Construction of gene overexpression transgenic lines

[0030] 1. OsCBL10 Construction of gene overexpression transgenic vectors and Agrobacterium-mediated transformation

[0031] Searching for the gene ID LOC_Os01g5142 from the Rice Genome Annotation Project yielded... OsCBL10 Genomic information of genes. Using wild-type rice Nipponbare (… Oryza sativa Japonica Group (taxid:39947) cDNA was used as a template. OsCBL10 Full-length amplification primers (with stop codons removed), using the PCR amplification system in Table 1, were amplified by PCR with KODFX enzyme (purchased from TOYOBO). OsCBL10 The full-length coding sequence (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) was obtained, and the target fragment size was 798 bp. The fragment was recovered directly using a Gel and PCR Clean-up Kit (MACHEREY-NAGEL) and then ligated using a Clone Express® II One Step Cloning Kit (Novizan). Company , BamHI The pCAMBIA1300-35s-GFP vector (purchased from Thermo Fisher Scientific) was digested with enzymes and transformed into E. coli DH5α (purchased from Vidi). Positive clones were extracted, and after successful sequencing, plasmids were extracted using a plasmid extraction kit (purchased from MACHEY-NAGEL) to obtain pCAMBIA1300-35s. - OsCBL10-GFP plasmid.

[0032] OsCBL10 Full-length amplification primers:

[0033] OsCBL10-CDS-F: GAGCTGGTACCATGGACTCCTCCCGC (Bold text represents...) Company Enzyme cleavage site (SEQ ID NO. 4);

[0034] OsCBL10-CDS-R: GACTCTAGAGGATCCGTCTTCAACTTGTGT (Bold text represents...) BamHI Enzyme cleavage site (SEQ ID NO.5).

[0035] SEQ ID NO.1:

[0036] ATGGACTCCTCCCGCTCCTCCAACTCTCTCGATTCGGGGAGCTCTCTGACGCTGGGGGAGCTCGCGTGCGCGGCGCTGATTCCGGTGCTCGCCCTGGTGGACGCGGTCGTGTTCGCGGCGGCGCAATGCTTCCAGAAGCGCCCGCCGGGGCTGCTGCCCGCTACACTCGCCGCCCGCGCGCGCCGCCGCGCCGGTGGCCGCCTCACCTTCCGTGAGCTCGCCGACCTCGCCGACGAGTCCCGCTGCTTCTCGGTGAACGAGGTGGAGGCACTGTACGAGCTCTACAAGAAGATCAGCTGCTCCATCGTCGATGACGGCCTGATCCATAAGGAAGAGCTGCAATTAGCCTTGTTTAGAACACCGGCTGGAAAGAATCTTTTTCTAGATAGAGTTTTTGATCTGTTTGATGAAAAGAAAAATTCTGTTATTGAATTTGAAGAGTTTATTCACGCAATAAGTGTATTTCATCCTAATACTCCTCTTGAAGACAAAATTGATTTTTCATTCAGATTATATGATTTGAGGCAAACCGGTTTCATTGAACGTGAAGAGGTAAAACAAATGGTTGTTGCTACCTTATTGGAGTCAGAGGTGCAGCTGTCTGATGATCTTGTGGAAGCCATACTAGACAAGACATTTGAGGATGCTGACACTGACAAGGATAACAGGATTAGCAAAGAGGAGTGGAAAGCTTTTGTACTGAAGCACCCGTCTGTTATAAAGAAGATGACCCTGCCCACCCTGAAGGACACTACAGCGGCGTTCCCCAGCTTCATTTTCAATACACAAGTTGAAGACTAG

[0037] SEQ ID NO.2:

[0038] MDSSRSSNSLDSGSSLTLGELACAALIPVLALVDAVVFAAAQCFQKRPPGLLPATLAARARRRAGGRLTFRELADLADESRCFSVNEVEALYELYKKISCSIVDDGLIHKEELQLALFRTPAGKNLFLDRVFD LFDEKKNSVIEFEEFIHAISVFHPNTPLEDKIDFSFRLYDLRQTGFIEREEVKQMVVATLLESEVQLSDDLVEAILDKTFEDADTDKDNRISKEEWKAFVLKHPSVIKKMTLPTLKDTTAAFPSFIFNTQVED

[0039] Table 1. PCR amplification system

[0040]

[0041] PCR amplification program: pre-denaturation 94℃ 2 min; denaturation 98℃ 10 sec, annealing 58℃ 30 sec, extension 68℃ 1 min, 29 cycles; extension 68℃ 5 min.

[0042] pCAMBIA1300-35s - The OsCBL10-GFP plasmid was transformed into Agrobacterium EHA105 (purchased from Vidi), and the 35S::OsCBL10-GFP was integrated into the genome of wild-type rice Nipponbare via Agrobacterium-mediated integration. Hygromycin resistance selection was then performed to obtain... OsCBL10 Transgenic positive lines with overexpression of genes are denoted as CBL10-OE (Right now 35S::OsCBL10- GFP ).

[0043] 2. OsCBL10 RT-qPCR identification of overexpression transgenic lines:

[0044] For the obtained OsCBL10 RNA was extracted from overexpression transgenic lines and wild-type Nipponbare (NIP), reverse transcribed, and detected using RT-qPCR. OsCBL10 The relative expression level, specifically, is as follows:

[0045] (1) The obtained T0 generation OsCBL10-OE The roots of the overexpression transgenic lines and wild-type Nipponbare plants were immersed in the nutrient solution shown in Table 2 and cultured in a culture chamber with a photoperiod of 14 h light culture / 10 h dark culture; day and night temperatures were 30℃ and 22℃, respectively; the light source was a bulb with a light intensity of 200 μmol / L. m -2 s -1 The humidity was 60%. Unless otherwise specified, the following examples illustrate rice cultivation under these conditions.

[0046] Table 2. Rice nutrient solution formula (10 L) (the remainder should be made up with water).

[0047]

[0048] (2) After culturing under the conditions of step (1) for 7 days, take 50-100 mg of rice leaves, wrap them in aluminum foil, and place them in liquid nitrogen. Extract total RNA using TRIzol (Thermo Fisher Scientific) as follows:

[0049] 1) Grind the sample in liquid nitrogen, then place the sample in a 2 mL centrifuge tube, immediately add 1 mL TRIzol, vortex and place on ice for 15 min.

[0050] 2) Add 250 μL of chloroform, shake vigorously, and let stand on ice for 10 min to separate the layers. Centrifuge (13,000 rpm, 4℃, 10 min) and collect 200 μL of the supernatant.

[0051] 3) Add 200 μL of isopropanol to the supernatant, mix gently, and place on ice for 10 min.

[0052] 4) After centrifugation (13,000 rpm, 4℃, 10 min), discard the supernatant. Add 75% ethanol (prepared with DEPC water) to wash the RNA precipitate, centrifuge (13,000 rpm, 4℃, 1 min), and discard the washing solution. Repeat the washing once. Aspirate the residual ethanol solution with a pipette tip and evaporate the ethanol on a clean bench for 20 min.

[0053] 5) Dissolve RNA in 50 μL of DEPC water. Assess RNA quality and total RNA concentration by electrophoresis on a 1% agarose gel. Store samples at -80℃.

[0054] (3) Reverse transcription:

[0055] cDNA synthesis was performed using the Invitrogen SuperScript II RT kit, with a total RNA content of 1 μg in the reverse transcription system.

[0056] (4) RT-qPCR:

[0057] Quantitative PCR was performed using the FastStart Universal SYBR Green Master kit (Roche). Quantitative analysis was performed using a LightCycler 480 Real-Time PCR instrument (Roche). OsACTIN As an internal reference gene.

[0058] Quantitative PCR reaction system (5 μL): cDNA 0.2 μL, PCR Forward Primer (10 μM) 0.1 μL, PCR Reverse Primer (10 μM) 0.1 μL, SYBR Green I (2×) 2.5 μL, ddH2O to make up to 5 μL.

[0059] The PCR reaction conditions were: 95℃ for 10 min; 95℃ for 5 sec, 58℃ for 10 sec, 72℃ for 20 sec, for 45 cycles; 72℃ for 10 min.

[0060] Primers required for quantification

[0061] ACTIN-qRT-F: CAACACCCCTGCTATGTACG (SEQ ID NO. 6);

[0062] ACTIN-qRT-R: CATCACCAGAGTCCAACACAA (SEQ ID NO. 7).

[0063] CBL10-qRT-F: CCGGTTTCATTGAACGTGAAGAGG (SEQ ID NO. 8);

[0064] CBL10-qRT-R: CCTGTTATCCTTGTCAGTGTCAGC (SEQ ID NO. 9).

[0065] OsCBL10 RT-qPCR identification results of overexpression transgenic lines ( Figure 1 A) indicates that the sample obtained in Example 1 OsCBL10 Overexpression transgenic lines CBL10-OE-1 , CBL10-OE-2 In OsCBL10 The relative expression level of the gene was significantly higher than that of the wild type (NIP), with relative expression levels being 100-fold and 160-fold higher, respectively. OsCBL10 The overexpression lines of the gene did indeed achieve enhanced expression. OsCBL10 The effect of the gene indicates that we have successfully obtained OsCBL10 Transgenic materials with enhanced gene expression can be used for further experiments.

[0066] Example 2 OsCBL10 Construction of mutant lines

[0067] 1. OsCBL10 Construction of gene-targeted editing vectors

[0068] CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is a technique that uses RNA-guided Cas9 nucleases to target and edit genes. The pYLCRISPR / Cas9-MH(B) plasmid used in this example was provided by Academician Yaoguang Liu's team, and its construction method referenced "A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants" (Ma et al., 2015, Mol Plant).

[0069] The specific steps for constructing the OsCBL10 CRISPR / Cas9 vector are as follows:

[0070] (1) Design OsCBL10 Target sites for gene mutation:

[0071] Search OsCBL10 The 20 bp sequence upstream of NGG in the genome that is G or A is preferentially selected as the target sequence; if it is not G or A, the upstream 20 bases are selected as the target sequence.

[0072] (2) Synthetic target site adapter primers

[0073] Based on the target site in step (1), the adapter primers were designed and synthesized, and dissolved in water to prepare a 10 μM stock solution. 10 μL of each primer was mixed with 80 μL of water to dilute the adapter primers to 1 μM. After denaturation at 95℃ for 1 min, the primers were cooled to room temperature to complete the annealing.

[0074] Target site adapter primers:

[0075] OsCBL10-Cri-F: GGCAGCCTTGTTTAGAACACCGGC (SEQ ID NO. 10);

[0076] OsCBL10-Cri-R: AAACGCCGGTGTTCTAAACAAGGC (SEQ ID NO. 11).

[0077] (3) Enzyme digestion of gRNA vector

[0078] Take 1 μg pYLgRNA-OsU3 plasmid (Zhu et al., 2024, New Phytol), add 2.5 μL of 10x FastDigest Buffer and 1 μL EcoR31I (Thermo Scientific) to construct a 25 μL reaction system (add ddH2O), digest at 37℃ for 30 min, and then heat at 70℃ for 5 min to inactivate the enzyme.

[0079] (4) Connecting gRNA expression cassette

[0080] The target site adapter primers annealed in step (2) were ligated with the pYLgRNA-OsU3 vector recovered by enzyme digestion in step (3). The reaction system is shown in Table 3. The ligation was carried out at room temperature for 15 min.

[0081] Table 3 gRNA expression cassette ligation reaction system

[0082]

[0083] (5) Amplification of gRNA expression cassette

[0084] First round of amplification: Take 1 μL of the ligation product from step (4) as the PCR reaction template, use UF as the reverse primer for the adapter and gRNA-R as the forward primer for the adapter, and use KOD-FX high-fidelity enzyme for the first round of amplification. The amplification program is as follows: 95℃ for 2 min; 95℃ for 15 s, 60℃ for 15 s, 68℃ for 20 s, 28 cycles; and finally 68℃ for 5 min.

[0085] Second round of amplification: Take 1 μL of the product from the first round of PCR reaction, dilute it 10 times with ddH2O, and then take 1 μL as the PCR template. Perform the second round of PCR reaction using the gRNA expression cassette position-specific primer B1'+BL. The amplification program is as follows: 95℃ for 2 min; 95℃ for 15 s, 58℃ for 15 s, 68℃ for 20 s, 20 cycles; and finally 68℃ for 5 min.

[0086] UF: CTCCGTTTTACCTGTGGAATCG (SEQ ID NO. 12).

[0087] gRNA-R: CGGAGGAAAATTCCATCCAC (SEQ ID NO. 13).

[0088] B1': TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG (SEQ ID NO. 14).

[0089] BL: AGCGTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC (SEQ ID NO. 15).

[0090] (6) Ligating the gRNA expression cassette to the pYLCRISPR / Cas9-MH (B) plasmid:

[0091] First, the gel was cut and the second-round amplification product (i.e., the gRNA expression cassette purified product) was recovered. Then, the product was ligated with the pYLCRISPR / Cas9-MH (B) plasmid using a digest-ligation-liquidation method. The digestion and ligation system is shown in Table 4. After mixing the reaction system, it was incubated at 37℃ for 10 min. Then, 1.5 μL of 10 x Takara T4 DNA ligase buffer and 0.5 μL of T4 DNA ligase were added for temperature-controlled cyclic digestion and ligation. The reaction program was: 37℃ for 2 min, 10℃ for 3 min, 20℃ for 5 min, 15 cycles; and finally 37℃ for 5 min.

[0092] Table 4 pYLCRISPR / Cas9-MH (B) linkage reaction system

[0093]

[0094] (7) The ligation product of step (6) was transformed into Escherichia coli DH5α by chemical heat shock method. Positive clones were selected by bacterial PCR, and plasmids were extracted and sent for sequencing after verification. After verification, the plasmids were stored at -20℃.

[0095] 2. OsCBL10 Construction of mutant lines:

[0096] After the constructed vector was verified to be correct by sequencing, it was transformed into Nipponbare callus using an Agrobacterium-mediated transgenic method. OsCBL10 Mutant lines with altered gene sequences. The steps for identifying mutant lines are as follows:

[0097] (1) Rapid extraction method for rice DNA:

[0098] For the obtained T0 generation OsCBL10The mutant lines and wild-type Nipponbare (NIP) were cultured for 7 days according to the method described in Example 1 above. 2-5 mm long rice leaves were directly placed in 2 mL centrifuge tubes, and 200 μL of rice DNA extraction solution and a grinding bead were added. The samples were ground completely using a Retsch MM400 grinder, incubated in a 65℃ oven for about 15 min, and then briefly centrifuged at 8000 rpm. The supernatant (the extracted rice genomic DNA) was collected and used for subsequent PCR experiments.

[0099] Rice DNA extraction solution formulation (1 L): 1 M Tris-HCl 100 mL; 0.5 M EDTA 40 mL; 5 M NaCl 100 mL; 10% SDS 150 mL; ddH2O 610 mL.

[0100] (2) PCR amplification:

[0101] Using the DNA obtained in step (1) as a template, the DNA containing the target site was amplified using ABclonal DNA polymerase Powerpol 2X PCR mixwith Dye. OsCBL10 The genome sequence was obtained, with a fragment size of 892 bp. The amplification products were detected by electrophoresis on a 1% agarose gel, and the experimental results were recorded using a Gel Doc™ XR+ (BIO-RAD, USA) imaging system. The target band was recovered using a gel recovery kit from MN (Germany), and the recovered products were sent to Urogene for sequencing.

[0102] The amplification primers are:

[0103] CBL10-sequence-F: AGTCTTTTGAACCAATTAGGACGG (SEQ ID NO. 16);

[0104] CBL10-sequence-R:GGAGAAGAAAAGCTAACTCAAATGG (SEQ ID NO. 17).

[0105] PCR reaction system (15 μL): cDNA template 1.5 μL; forward / reverse primers 0.3 μL; 2xMix enzyme 7.5 μL; ddH2O 5.4 μL.

[0106] The PCR reaction conditions were: 95℃ for 5 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 50 sec, for 32 cycles; 72℃ for 8 min.

[0107] (3) OsCBL10 Identification of mutant lines:

[0108] Using Snapgene software, with wild-type sequencing results as a template, the sequencing data was analyzed. OsCBL10 The sequencing results of the mutant lines were compared and identified, resulting in two homozygous lines. OsCBL10 Mutant strains with gene mutations cbl10-1 and cbl10-2 ( Figure 1 (Middle B). cbl10-1 The OsCBL10 coding sequence is missing 37 bases, causing premature termination of transcription and resulting in a protein with only 139 amino acids. cbl10-2 An additional base was added to the OsCBL10 coding sequence, causing premature termination of transcription and resulting in a protein with 129 amino acids, which is shorter than the wild-type protein with 266 amino acids.

[0109] (4) OsCBL10 Obtaining mutant lines:

[0110] In step (3) cbl10-1 and cbl10-2 The mutant line was backcrossed with wild-type Nipponbare to obtain F1 generation heterozygous mutant materials. After one generation (F2 generation), DNA extraction, PCR amplification, and identification were performed according to the above steps to screen for homozygous mutants free of hygromycin. cbl10-1 and cbl10-2 Mutant strains.

[0111] Hygromycin amplification primers are:

[0112] HYG-F: TTTCTTTGCCCTCGGACGAGT (SEQ ID NO. 18);

[0113] HYG-R: ATGAAAAAGCCTGAACTCACC (SEQ ID NO. 19).

[0114] Example 3: Phenotypic Analysis, Sodium and Potassium Ion Content Analysis of Transgenic Rice Seedlings

[0115] 1. Salt stress treatment of rice:

[0116] Wild-type rice Nipponbare (NIP) and the rice obtained in Example 2 OsCBL10 mutant strains ( cbl10-1 and cbl10- 2Rice seeds were soaked in 20 mL of 1% (v / v) dilute nitric acid solution and left at room temperature for 20 h. The nitric acid was then replaced with tap water, and the seeds were placed in a 37°C oven to germinate until they showed white sprouts. Twenty-four sprouted seeds were then sown in 96-well black troughs and cultured for 7 days according to the methods and conditions in Example 1 (the nutrient solution was changed every 3 days, as shown in Table 2). The nutrient solution in the black troughs was then replaced with a nutrient solution containing 0 or 120 mM sodium chloride. After 7 days of normal or salt stress treatment, the solution was replaced with fresh nutrient solution, and the culture continued for another 5 days. Phenotypic results are shown in […]. Figure 2 For example, in the case of Chinese A, the survival rate statistics can be found in the chart below. Figure 2 B.

[0117] Wild-type rice Nipponbare (NIP) and the rice obtained in Example 2 OsCBL10 Overexpression transgenic lines ( CBL10-OE- 1 and CBL10-OE-2 Rice seeds were culturing for 7 days after dormancy breaking and sowing as described above, followed by 9 days of treatment with nutrient solution containing 0 or 120 mM sodium chloride. The nutrient solution was then replaced with the solution shown in Table 2, and culturing continued for 5 days. Phenotypic results are shown below. Figure 2 For C, see the survival rate statistics chart. Figure 2 D.

[0118] The results showed that, compared with the wild type, both OsCBL10 mutant strains ( cbl10-1 and cbl10-2 The survival rate was low after salt stress treatment and subsequent recovery to normal culture, while the two CBL10 Overexpression transgenic lines ( CBL10-OE-1 and CBL10-OE-2 Salt tolerance is enhanced. Figure 2 ).

[0119] 2. Determination of fresh and dry weight of aboveground parts and roots

[0120] Wild-type rice Nipponbare and the rice obtained in Example 2 OsCBL10 mutant strains ( cbl10-1 ), obtained in Example 1 OsCBL10 Overexpression transgenic lines ( CBL10-OE-1 After being cultured normally for 7 days according to the method and conditions of Example 1, the samples were transferred to a nutrient solution containing 120 mM sodium chloride and subjected to salt stress for 0-9 days. After being rinsed with deionized water, the aboveground parts and roots were taken and weighed as fresh weight. They were then dried in an oven at 65℃ for 7 days and weighed as dry weight.

[0121] The results showed that before salt treatment (0 h). OsCBL10 mutant strains ( cbl10-1 ), OsCBL10 Overexpression transgenic lines ( CBL10-OE-1 The fresh and dry weights of the [type] were not significantly different from those of the wild type. After 3 days of salt treatment, cbl10-1 The fresh weight of the above-ground parts and roots was significantly lower than that of the wild type; after 6 days of salt treatment, cbl10-1 The dry weight of the aboveground parts and roots was significantly lower than that of the wild type. Figure 3 Salt treatment for 0-9 days. OsCBL10 Overexpression transgenic lines ( CBL10-OE-1 The fresh weight and dry weight of the ) were not significantly different from those of the wild type. Figure 3 ).

[0122] 3. Na from the above-ground parts and roots + and K + Content determination:

[0123] (1) Cleaning the tubes. Clean the digestion tubes (specific rectangular tubes for the digester) in advance, wash twice with deionized water and once with ultrapure water. Dry them for later use.

[0124] (2) Weighing the sample. Take the wild-type (NIP) sample dried in step 2 above. OsCBL10 mutant strains ( cbl10-1 ), OsCBL10 Overexpression transgenic lines ( CBL10-OE-1 All above-ground parts or roots (not more than 0.4 g) were added to digestion tubes.

[0125] (3) Add acid. 5 mL concentrated HNO3 + 1 mL H2O2. Digest (CEM MARS6 high-throughput closed microwave digester) for 40 minutes.

[0126] (4) Acid removal. Turn on the Heating Block and set the temperature to 160℃. After about 1 hour, when about 1 mL of sample remains, turn off the Heating Block and allow it to cool before making up to volume.

[0127] (5) Volume adjustment. Rinse the digestion tube three times with ultrapure water and then adjust the volume to 30 mL.

[0128] (6) Measurement. Na was measured on an ICP (Inductively Coupled Plasma Atomic Emission Spectrometer). + and K + Content. Results are shown in Figure 4 The results showed that after 6 hours of salt stress cbl10-1 Na root + Initially, it decreased significantly compared to NIP, and after 12 hours, the root K... + The content was significantly increased compared to NIP; CBL10-OE-1 12 hours later, root Na + It started to rise significantly higher than NIP, K + The content was significantly lower than that of NIP.

[0129] Example 4: Subcellular localization analysis of the OsCBL10 gene

[0130] The method obtained in Example 1 OsCBL10 Overexpression transgenic lines ( CBL10-OE-1 T2 generation seedlings were cultured for 20 days according to the methods and conditions in Example 1. Stems were cut into 0.5 cm lengths and immersed in culture dishes containing the enzymatic hydrolysate shown in Table 5. The dishes were incubated in the dark at 40 rpm for 3 h at 28°C. After enzymatic hydrolysis, the mixture was gently pipetted several times to pass through a 0.45 μm microporous nylon membrane (purchased from Millipore). The protoplasts were filtered into a new 50 mL centrifuge tube. The culture dish and nylon membrane were washed with four times the volume of pre-cooled W5 solution shown in Table 6. The filtrate was filtered into a centrifuge tube containing the protoplasts, and the centrifuge tube was placed on ice for 30 minutes. The centrifuge tube was centrifuged at 100-120 g for 10 min at room temperature (with the deceleration brake set to 1). After removing the supernatant, the protoplast precipitate was resuspended in 1 mL of W5 solution and transferred to a 2 mL centrifuge tube. The tube was centrifuged at 0.1 g for 2 minutes at room temperature. After min, remove the supernatant; resuspend the precipitate in 100 μL of W5 solution, add 1 μL of FM4-64 staining solution (MedChemExpress) to the precipitate; mix well, take 10 μL to a glass slide and observe and photograph the fluorescence under a fluorescence confocal microscope, with an excitation wavelength of 488 nm and an emission wavelength of 507 nm.

[0131] Table 5. Composition of each 10 mL enzymatic hydrolysate (pH adjusted to 5.6 with 1 M KOH, solvent is deionized water, filtered through a 0.22 μm filter membrane for later use).

[0132]

[0133] Table 6. Composition of W5 solution per liter (solvent is deionized water, pH adjusted to 5.8 with 1M KOH)

[0134]

[0135] The measurement results showed that OsCBL10 was located in the vacuolar membrane ( Figure 5 ).

[0136] Example 5: GUS staining analysis OsCBL10 Tissue expression sites of genes

[0137] 1. PBI101.3- OsCBL10 PRO - GUS plus vector construction: Gene ID LOC_Os01g51420 was searched from the Rice Genome Annotation Project to obtain... OsCBL10 Genomic information of the gene was obtained by selecting the promoter of the OsCBL10 gene, which is located 2500 bp above the open reading frame. Primers were designed as follows, using the genome of wild-type rice Nipponbare (NIP) (…). Oryza sativa Japonica Group Using (taxid:39947) as a template, PCR amplification was performed according to the method in Example 1 to obtain... OsCBL10 The promoter (nucleotide sequence shown in SEQ ID NO.3) was recovered directly using a Gel and PCR Clean-up kit (MACHEREY-NAGEL) and ligated into a PBI101.3-GUS plus vector digested with BamHI and SalI (Thermo Fisher Scientific) using a Clone Express® II One Step Cloning Kit (Novizan) (Zhu et al., 2024, New Phytol). This vector was then transformed into *E. coli* DH5α (TAKARA). Positive clones were extracted, and after successful sequencing, plasmids were extracted using a plasmid extraction kit (MACHEREY-NAGEL) to obtain plasmid PBI101.3-. OsCBL10 PRO -GUS plus. After sequencing verification, the gene containing the GUS (β-glucuronidase, β-D-glucuronidase) will be inserted... OsCBL10 PBI101.3- promoter expression OsCBL10 PRO The GUS plus plasmid was transformed into Agrobacterium EHA105 (purchased from Takara), and integrated into the genome of wild-type rice Nipponbare (Zhu et al., 2024, NewPhytol) through infection. T0 generation GUS transgenic lines were obtained by hygromycin screening.

[0138] OsCBL10-Pro-F: TGCCTGCAGGTCGACAACATGTACTTGCTTGCT (SEQ ID NO.20)

[0139] OsCBL10-Pro-R: TGGTACCGTGGATCCCGGCCGTGCCGCCGGCGG (SEQ ID NO. 21)

[0140] SEQ ID NO.3:

[0141]

[0142] 2. GUS staining and microscopic observation

[0143] The T0 generation GUS transgenic line seedlings obtained in step 1 were propagated to obtain the T1 generation seedlings.

[0144] After culturing T1 generation GUS transgenic seedlings in the nutrient solution (Table 2) under the methods and conditions of Example 1 for 7 days, the entire seedling was immersed in the GUS staining solution (Table 7). The mixture was then vacuum-treated at room temperature (25-30℃) for 20 min, followed by a 37℃ incubation. Once the sample turned blue, it was transferred to FAA fixative (formaldehyde: glacial acetic acid: 70% ethanol aqueous solution = 1:1:18, v:v:v), vacuum-treated for 1 min, and the reaction was terminated. The sample was then photographed (Nikon D70s). Figure 6 As shown in Figure A.

[0145] After culturing GUS transgenic seedlings in the nutrient solution shown in Table 2 for one month, the roots, stems, and leaves were immersed in the GUS staining solution shown in Table 7. Following the steps described above, staining, fixation, and vacuuming were performed. Observation was then conducted under a LEICA MZ95 stereomicroscope, and photographs were taken using a LEICA DC100 camera. Results for root tips and root maturation zones are shown below. Figure 6 B and C. The leaves and stems were then transferred to a 70% ethanol aqueous solution for decolorization 2-3 times until they turned white. After embedding the roots, stems, and leaves in agarose, they were transversely sectioned using a vibrating microtome and observed under a Nikon ECLIPSE 90i microscope. Images were taken using a Nikon DS-Fi1 camera. The results are shown in [Figure 1]. Figure 6 Use a 20x scope for images D and E, a 10x scope for image F, and a 4x scope for image G. Figure 6 The blue sites B, C, D, E, F, and G are GUS expression sites.

[0146] Table 7. Composition of GUS dye solution

[0147]

[0148] GUS staining results indicate that the seedling stage OsCBL10 Genes are mainly expressed in the roots, during the one-month-old seedling stage. OsCBL10 It is expressed in the roots, leaves, and stems. OsCBL10 Genes are primarily expressed in thin-walled cells.

Claims

1. A kind OsCBL10 The application of genes in improving the salt tolerance of rice is characterized by, The application involves overexpression in the rice genome. OsCBL10 Genes or proteins can increase the salt tolerance of rice.

2. The application as described in claim 1, characterized in that, The OsCBL10 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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