Application of rice OsNAC113 gene in regulation and control of rice salt tolerance

Abstract

The invention provides an application of a rice OsNAC113 gene in regulation and control of rice salt tolerance, a CRISPR / Cas9 technology is utilized to design a specific sgRNA for the OsNAC113 gene, and a CRISPR / Cas9 gene editing mutant in which the rice OsNAC113 gene is knocked out is constructed. And performing genome editing on the adversity stress response factor OsNAC113 to obtain a new rice germplasm with higher stress resistance. According to the invention, the OsNAC113 knockout mutant material with stronger salt tolerance is obtained. The method can be directly applied to agricultural production.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to the application of rice OsNAC113 gene in regulating rice salt tolerance. Background Art

[0002] Globally, more than 6% of the land area is increasingly affected by salt accumulation. High salt harms plant growth, crop production and ecosystem balance. Soil salinization has become one of the main abiotic stresses facing agriculture around the world. Plants are affected by high osmotic potential in high-salt environments, which is essentially an osmotic stress that seriously affects plant growth. Under high-salt conditions, plants may wither and die even if there is sufficient water and various nutrients. The imbalance of salt content may interfere with the absorption of basic nutrients, leading to competition between elements (i.e. antagonism). Sodium ions affect the structure and properties of the soil, indirectly causing damage to plant roots. Under high-salt stress, plant cell walls undergo compositional and structural changes and enhance polysaccharide synthesis and the expression of related modification genes to maintain cell integrity and improve adaptability to salt stress. At present, salt stress has been widely studied in many plants such as Arabidopsis thaliana, Sesuvium portulacastrum, and soybean (Glycine max). These plants have evolved various mechanisms to resist salt stress, including osmotic regulation, reactive oxygen species (ROS) scavenging, ion homeostasis, and regulation of plant hormones. At the same time, excessive salt may also disrupt amino acid synthesis, resulting in a decrease in the synthesis of some proteins related to adverse stress.

[0003] The essence of transcription factors is a type of regulatory gene that regulates gene expression. It can bind to the cis-acting elements in the promoter region of the target gene to activate or inhibit the transcription of the target gene, ultimately ensuring that the regulated gene is accurately expressed in time and space. Transcription factors play an important role in the regulation of plant cell cycles, the balance of metabolic pathways, and the response to stress. There are a large number of stress-responsive transcription factors in plants. In rice, 56 families with a total of 2,408 transcription factors have been identified. Among them, there are many family members such as AP2 / ERF (APETALA2) / (Ethylene responsive element binding), bZIP (basic region / leucine zipper motif), DREB (dehydration-responsive element binding protein), NAC, MYB (v-myb avianmyeloblastosis viral oncogene homolog) and WRKY, which have been found to play an important role in stress response.

[0004] NAC transcription factors are one of the largest transcriptional regulatory factor families in plants, and members of the gene family play an important role in participating in the process of adverse stress. NAC transcription factors are involved in regulating plant resistance to drought, high salt, low temperature, viruses and other stress damage, and are involved in regulating plant morphology, root growth and extension, and leaf senescence. According to current literature, although the NAC transcription factor family has an important regulatory role, its family members are large, the research content is not in-depth, and the biological functions of most members are still unclear, which causes great trouble for application in production practice.

[0005] The NAC transcription factors that the research focuses on are a family of transcription factors unique to plants. In different plants, their family members play an important regulatory role in the response to adverse stress. At present, a total of 158 NAC transcription factor family members have been annotated in the rice genome, but the number of members with clear gene functions has been reported to be very limited. Due to the lack of mutant materials, the functions of most NAC family members are difficult to analyze, and the number of natural rice mutants is very small and the background is unclear. The creation of mutants by T-DNA insertion and physical and chemical mutagenesis has a long cycle and high cost, and it is difficult to accurately create mutants for the target gene. Therefore, traditional mutant creation methods are no longer sufficient to meet the requirements of the experiment. The cutting-edge technology CRISPR-Cas9 genome editing technology studied in recent years can effectively make up for this defect. Its advantages are that it can not only quickly and accurately edit the target sequence, but also can obtain plants without exogenous vectors through separation and screening during the offspring propagation process, preventing exogenous genes from affecting subsequent research on mutants.

[0006] Gene editing technology uses site-specific nucleases to cut a specific DNA fragment in the genome, causing a double-strand break (DSB). Then, the organism's own repair system is used to delete, insert, replace and mutate specific DNA fragments, thereby achieving sequence changes in the target gene and affecting gene expression and function. In the absence of a specific protein, detecting the operation and abnormality of the signal pathway can provide a deep understanding of the role played by the protein. It can play a direct functional role or be part of a feedback regulation process. At present, the gene editing technology CRISPR / Cas system has been widely used in research. Different from the first and second generation gene editing technologies, which are both complex and time-consuming, CRISPR / Cas can easily, accurately and efficiently change various genomes. Genome editing has completely changed the DNA manipulation of eukaryotes, achieving precise mutation, insertion or deletion of single base pairs, DNA fragment replacement and nucleotide base conversion. The specificity of gene editing has been greatly improved. This technology further improves the potential of DNA editing. Summary of the invention

[0007] In view of this, the present invention aims to overcome the defects in the prior art and proposes the application of rice OsNAC113 gene in regulating rice salt tolerance.

[0008] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0009] The first aspect of the present invention: The present invention provides the use of rice OsNAC113 gene in regulating rice salt tolerance, the nucleotide sequence of the rice OsNAC113 gene is shown in SEQ ID NO.1; the amino acid sequence is shown in SEQ ID NO.3.

[0010] Preferably, the application is to reduce the expression level of the rice OsNAC113 gene or weaken the function of the rice OsNAC113 gene, thereby improving the salt tolerance of rice.

[0011] Preferably, the application is to use CRISPR / Cas technology to target and knock out the OsNAC113 gene in rice.

[0012] The second aspect of the present invention: The present invention provides the application of the CRISPR / Cas9 gene editing system for targeted mutation of rice OsNAC113 gene in constructing OsNAC113 gene mutants.

[0013] The third aspect of the present invention: The present invention provides a sgRNA for targeting the mutation of the rice OsNAC113 gene, the nucleotide sequence of the sgRNA is shown in SEQ ID NO.2, and the sgRNA is designed at the first exon of the rice OsNAC113 gene.

[0014] The fourth aspect of the present invention: The present invention also provides a method for improving the salt tolerance of rice, by reducing the expression level of the above-mentioned rice OsNAC113 gene or weakening the function of the rice OsNAC113 gene to improve the salt tolerance of rice.

[0015] The fifth aspect of the present invention: The present invention also provides a knockout vector, which is a CRISPR / Cas expression vector constructed by goldengate cloning technology and contains the targeted knockout rice OsNAC113 gene.

[0016] The sixth aspect of the present invention: The present invention also provides a method for creating a rice mutant with improved salt tolerance, comprising the following characteristic steps:

[0017] The OsNAC113 gene was used as the editing target gene. The CRISPR / Cas9 gene editing system was used to knock out the OsNAC113 gene and then the homozygous mutant plants were obtained through propagation, which were rice mutants with improved salt tolerance.

[0018] Among them, the nucleotide sequence of the sgRNA of the CRISPR / Cas9 gene editing system is shown in SEQ ID NO.2, and the nucleotide sequence of the OsNAC113 gene is shown in SEQ ID NO.1.

[0019] There are 158 NAC transcription factors identified in rice, but few have been studied and their functions have been proven. The present invention analyzes the tissue specificity and stress-induced expression characteristics of OsNAC113, combines CRISPR / Cas9 technology, performs site-directed mutagenesis on the target gene, and obtains OsNAC113 functional loss mutants that respond to rice stress. The growth of mutant OsNAC113 under salt stress conditions in the germination and seedling stages is significantly better than that of the wild type. This shows that after knocking out OsNAC113, the salt tolerance of the plant is enhanced. Physiological indexes of the plants after stress treatment were tested, and it was found that the relative water content, chlorophyll content, and oxidoreductase-related activity mutants were all higher than the wild type, and the accumulation of MDA and other harmful substances was higher than that of the wild type. This result also shows that after knocking out OsNAC113, the functional loss mutant can enhance the plant's tolerance to high salt stress. Therefore, the present invention establishes and discloses a new method for improving rice salt tolerance. The method is to improve rice salt tolerance by blocking or weakening the expression of the OsNAC113 gene in rice. CRISPR / Cas gene editing technology is used to create a rice directed editing mutant with knockout of the OsNAC113 gene, and a mutant plant with significantly improved salt tolerance is obtained.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The present invention uses CRISPR / Cas9 technology to design specific sgRNA for the OsNAC113 gene, and constructs a CRISPR / Cas9 gene-edited mutant of the rice OsNAC113 gene. The adversity stress response factor OsNAC113 is subjected to genome editing to obtain a new rice germplasm with stronger stress resistance. The present invention obtains an OsNAC113 knockout mutant material with stronger salt tolerance. It can be directly applied to agricultural production. Therefore, the present invention has a relatively good prospect for the promotion of results both in theory and in the creation of new germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the design diagram of the sgRNA site in the OsNAC113 exon;

[0023] Figure 2 This is the electrophoresis result of the recombinant vector PCR identification product;

[0024] Figure 3 This is the result of OsNAC113 genotype identification;

[0025] Figure 4 The OsNAC113 mutant exhibits a salt-resistant phenotype;

[0026] Figure 5 is the relative water content of the mutant under salt stress;

[0027] Figure 6 is the chlorophyll content of the mutant under salt stress;

[0028] Figure 7 is the soluble sugar content of the mutant under salt stress;

[0029] Figure 8 is the SOD activity of the mutant under salt stress;

[0030] Fig. 9 is the POD activity of the mutant under salt stress;

[0031] Fig.10 is the MDA content of the mutant under salt stress;

[0032] Fig.11 is the CAT activity of the mutant under salt stress;

[0033] Fig.12 This is the positive identification result of transgenic regenerated plants. DETAILED DESCRIPTION

[0034] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0035] The present invention will be described in detail below in conjunction with embodiments.

[0036] Example 1

[0037] 1. Construction of CRISPR-Cas9 editing expression vector

[0038] 1.1sgRNA primer design

[0039] (1) The OsNAC113 gene sequence was downloaded from the database website NCBI; sgRNA was designed and screened using the online website Crispr-P, and sgRNA targets with low predicted off-target efficiency and GC content less than 50% were screened for primer synthesis, which was then used for vector construction.

[0040] The resulting primer sequence is (5'-3'):

[0041] OsNAC113-sgRNA1-F: GTGTGCGGGGAGGAGGGGTGTACGGOsNAC113-sgRNA1-R: AAACCCGTACACCCCTCCTCCCCGC

[0042] 1.2 Knockout vector construction

[0043] (1) Annealing sgRNA primers

[0044] 10ul of primers carrying upstream and downstream fragments of sgRNA were mixed, denatured at 98℃ for 5min and cooled at room temperature. According to the pZHY988 backbone vector, restriction endonuclease BsaI digestion and ligation of sgRNA were completed at the same time. The specific enzyme digestion and ligation reaction system (20ul) is shown in Table 1:

[0045] Table 1 Enzyme digestion and ligation reaction system

[0046]

[0047]

[0048] The following program was set for the reaction: 37°C, 5 min → 16°C, 10 min) for 10 cycles → 37°C, 15 min → 80°C, 10 min.

[0049] (3) Escherichia coli competent cell transformation

[0050] 2 μL of ligation product was added to 30 μL of competent medium in a thawed state on ice, mixed and placed on ice for 30 min; heat shock at 42°C for 30 s, and placed on ice for 3-5 min; resuscitation was performed in non-resistant LB medium, incubated at 37°C, 200 rpm for 1 h; part of the bacterial solution was spread on LB solid medium (containing 100 mg / L Kan) and cultured overnight at 38°C for 12 h.

[0051] 1.3 PCR identification and sequencing of bacterial culture

[0052] In the clean bench, 10 single colonies were selected for simultaneous inoculation of 1.5 ml EP tubes (adding 500 μL of liquid LB medium containing Kan, incubating at 37°C, 200 rpm for 3-4 hours) and PCR product identification. 1 μL of bacterial solution was taken for PCR detection (Table 2), the reaction system is shown in Table 3, and the reaction procedure is shown in Table 4. 1-3 positive bands were selected for Sanger sequencing.

[0053] Table 2 Primer sequences used

[0054]

[0055] Table 3 PCR system

[0056]

[0057]

[0058] Table 4 PCR reaction program

[0059]

[0060] 1.4 Identification of recombinant plasmid

[0061] The knockout vector plasmid was extracted using the Zhuangmeng plasmid extraction kit, and the pZHY988-F / R at the left and right ends were used as primers for PCR amplification and identification. The PCR system and reaction procedures are shown in Tables 2 and 3. Two samples with bands were selected and sent to Genewise for sequencing. The plasmid with correct sequencing was used for the following experiments. The specific steps for plasmid extraction are as follows:

[0062] (1) 8 mL of bacterial solution cultured overnight on a shaker was added in batches to 1.5 mL centrifuge tubes at 12000 rpm for 1 min to obtain bacterial cells;

[0063] (2) Add 250 μL of solution 1 into the centrifuge tube and vortex to resuspend the cells;

[0064] (3) Add 250 μL of solution 2 and gently invert the tube 5-6 times;

[0065] (4) Add another 350 μL of solution 3, gently invert 5-6 times, and centrifuge at 12000 rpm for 10 min;

[0066] (5) The supernatant obtained after centrifugation was added to the adsorption column, centrifuged at 12000 rpm for 1 min, and then the waste liquid was discarded;

[0067] (6) Pipette 700 μL of rinse solution W2 and add it to the adsorption column. Centrifuge for 20 seconds and then discard the waste liquid.

[0068] (7) Pipette 500 μL of rinse solution W2 and add it to the adsorption column. Centrifuge for 20 seconds and then discard the waste liquid.

[0069] (8) After placing the adsorption column, centrifuge at the same speed for 2 min to remove the residual waste liquid;

[0070] (9) Place the centrifuged column in a new 1.5 mL centrifuge tube, add 80 μL of elution buffer TE to the center of the adsorption membrane, and centrifuge at the same speed for 1 min to obtain the plasmid and measure the concentration for subsequent experiments.

[0071] 1.5 Transformation of recombinant plasmid into Agrobacterium competent cells

[0072] Take 1 μL of the successfully constructed knockout vector plasmid and add it to EHA105 competent cells, and place it in an ice bath for 30 minutes; put it in liquid nitrogen treatment for 5 minutes and then place it in a 37°C water bath for 5 minutes. After the end, place it on ice again for 3 minutes; add 600 μL LB non-resistant liquid culture medium and culture it at 28°C shaker at 200rpm for 4-5 hours; centrifuge at 8000rpm for 2 minutes to collect the bacteria, discard the supernatant, and evenly apply LB+50mg / LKan+50mg / L Rif after resuspending. After inverted culture in a 28°C incubator for 2-3 days, select a single colony for PCR verification and save it for subsequent rice genetic transformation.

[0073] 2. Steps for stable transformation of rice

[0074] Rice seeds were shelled and washed with 20mL 75% ethanol for 90s; washed with sterile water for 30s, repeated twice; sterilized with 15% NaClO (with one drop of Tween) for 15min, washed once; sterilized with 15% NaClO solution for 15min; rinsed with distilled water for 90s after sterilization, and dried with filter paper after 6 times; inoculated with seeds on N6D medium, cultured in a 32℃ light incubator for 7d, and callus was obtained for transformation. Agrobacterium was expanded (28℃, 200rpm) to about OD600 = 0.1. The bacterial liquid was collected in a sterile centrifuge tube, and the bacteria were collected by centrifugation at 6000rpm for 5min at room temperature. The callus tissue cultured for 7 days was infected in the above-mentioned AAM Agrobacterium resuspension for about 90s, and shaken until uniformly infected. After infection, the callus tissue was sterilized to remove excess bacterial liquid; co-cultured on the medium 2N6D-AS, cultured in the dark at 25℃ for 3-3.5d. After infection, the callus was washed 5 times with appropriate amount of sterile water, each time for 90 seconds; washed with sterile water containing 400 mg / L carbenicillin (Carb) for 30 minutes; dried with filter paper and placed in a screening medium with corresponding resistance, cultured at 32°C under continuous light for 2 weeks, and the medium was changed until the plant was regenerated. Cultured on differentiation and regeneration medium REIII for 4 weeks. The plant was transferred to rooting medium HF to induce rooting. After identification, the transgenic positive was transplanted into nutrient soil and cultured in the greenhouse.

[0075] 3. Identification of rice CRISPR-Cas9 positively transformed regenerated plants

[0076] First, DNA from some leaves of the regenerated plants was extracted for transgenic positive identification, and the primers used were partial specific sequences of CAS9 protein. The transgenic positive plants were subjected to target gene fragment amplification and Sanger sequencing, and the specific steps were as follows.

[0077] 3.1 Extraction of rice genomic DNA

[0078] Take more than 200g of leaves from the regenerated plants and freeze them in liquid nitrogen before extracting rice DNA. The detailed steps for extracting DNA from plants are as follows:

[0079] (1) Take about 100 mg of fresh rice leaves and put them into a 1.5 mL centrifuge tube. After grinding thoroughly with liquid nitrogen, add 400 μL Buffer LP1 and 6 μL RNase A (10 mg / ml).

[0080] (2) Vortex to mix thoroughly and let stand at room temperature for 10 min;

[0081] (3) Add 130 μL Bufer LP2, shake and mix, centrifuge at 12000 rpm for 300 s, and transfer the supernatant to a new centrifuge tube;

[0082] (4) Add 195 μL of Buffer LP3 and mix evenly;

[0083] (5) Add the liquid and precipitate in the above centrifuge tube to the column placed in the collection tube, centrifuge at the same speed for 60 seconds, and then discard the waste liquid;

[0084] (6) Add 500 μL of Buffer GW2 to the column, centrifuge at the same speed for 60 s, and discard the waste liquid;

[0085] (7) Repeat step 6.

[0086] (8) After centrifugation at the same speed for 120 seconds, discard the waste liquid and dry it at room temperature for 300 seconds;

[0087] (9) Place the centrifuged column in a new 1.5 mL centrifuge tube and add 80 μL of Buffer GE to the center of the adsorption membrane. Place the column at room temperature for 300 s and then centrifuge it at the same speed for 60 s to obtain DNA and measure its concentration for subsequent experiments.

[0088] 3.2 PCR transgenic positive detection of regenerated plants

[0089] The detection primer sequences are shown in Table 2.

[0090] The PCR system (20 μL) for detecting whether rice is transgenic positive is as follows: PCRMix 12.5 μL; template 2 μL; DCAS9-F 1 μL; DCAS9-R 1 μL; ddH2O 8.5 μL.

[0091] The PCR reaction procedure is shown in Table 4. The PCR products were detected by electrophoresis on 1% agarose gel at 130V for about 0.5 h, and the PCR products with the correct bands were selected and sent to Sanger sequencing.

[0092] 3.3 Mutant genotype detection

[0093] Primers were designed upstream and downstream of the knockout site, and the target fragment was amplified and sent to the company for sequencing. The OsNAC113 detection primers were:

[0094] 113-F:AGGAGGAGATGAGTTTCATAGGC

[0095] 113-R:AAGCCACTCTGCTTCACTTTG

[0096] The PCR amplification system (25 μL) was as follows: Template 2 μL; 1 μL of 113-F; 1 μL of 113-R; 12.5 μL of 2×MasterMix; and ddH20 to make up to 25 μL.

[0097] The PCR amplification program is shown in Table 3.

[0098] In this experiment, the osnac113 mutant was detected by Sanger sequencing.

[0099] 3.4 Germination observation

[0100] After the seeds were sterilized, they were operated under sterile conditions and placed in MS solid culture medium for culture observation. The culture temperature of the light incubator was 28°C, and 16h / 8h (light / dark) was the culture condition for rice seeds. The differences in germination conditions were observed from 0 to 7 days.

[0101] 3.5 Phenotypic analysis after stress treatment

[0102] The wild-type and mutant plants were cultured in the field, and 8-week-old rice plants of the wild-type (WT) and osnac113 mutant were selected and subjected to 200 mM high salt stress for phenotypic observation.

[0103] 4. Results Analysis

[0104] 4.1 Creation of OsNAC113 Targeted Editing Mutants

[0105] OsNAC113-sgRNA01 was designed in the first exon sequence, and a targeted knockout expression vector was constructed through golden gate ( Figure 1 ), perform preliminary PCR identification on the expression vector, and after the product band size is correct, perform Sanger sequencing ( Figure 2 ). The vector plasmid carrying the target sgRNA was transferred into Agrobacterium EHA105 for standby use, and the regenerated plants were obtained through rice genetic transformation resistance screening. The Cas9-related sequence primers were used to preliminarily identify whether the vector was transferred into the regenerated plants, and the transgenic positive plants were determined to be the next mutant genotype detection ( Figure 3 ).

[0106] 4.2 Identification of transgenic rice regenerated plants

[0107] After transformation, a total of 7 regenerated plants were obtained. After agarose gel electrophoresis and PCR identification of transgenic positive results, it was found that all regenerated plants were transgenic positive ( Fig.12 ), so all 7 transgenic plants can be used for mutant genotype analysis.

[0108] 4.3 Genotype identification of OsNAC113 regenerated plants

[0109] DNA was extracted from leaves of transgenic positive plants, and the corresponding fragments were amplified and knocked out for Sanger sequencing. The sequencing results were analyzed to further analyze the gene type of the mutant and whether mutation cutting occurred. After testing, 5 mutants were found among the 7 transgenic positive plants, with a mutation rate of 71.4%. Among them, OsNAC113-sgRNA04 and 05 were wild-type genotypes and no mutation occurred ( Figure 3 A. Figure 3 B). OsNAC113-sgRNA01-01, 02, and 03 are mutants with a base deletion ( Figure 3 A. Figure 3 C). OsNAC113-sgRNA01-06 and 07 are mutants with a single base A inserted, and are biallelic gene mutations, laying a good foundation for subsequent experimental material screening ( Figure 3 A. Figure 3D). The translated protein sequence of the mutant obtained by knocking out the OsNAC113 gene using gene editing technology changed greatly. In the mutant OsNAC113 (-1bp / -1bp), the amino acid sequence is: MSFIGMVEARMPPGFRFHPRDDELVLDYLLHKLAAGGRGGGVSAAAAASPSSTSTSTSASHGTFOTRRAWEARSGTSSASATASTPPATAPTAPPAPATGRPPARTAPSPAAPPSPPASPPPPPPPPPSACARRSSSTAAAPPRAARPSGSCTSSGSNRSLSTSRRTGCYAGCSTRPDKQFQAHHLKRPSHCPMS*TCLPRLRSRPSSMPTSPSIAHRRRRHRWSGATSKCP ASPACRHCO*RAPSALGTCWPWTPLORRRPSGYCTTATLOSWSSP OIGDRRVACHRCGTPN; it can be seen that the translation of amino acids terminates prematurely. The amino acid sequence translated from the mutant OsNAC113 (+A / +A) is: MSFIGMVEARMPPGFRFHPRDDELVLDYLLHKLAAGGRGGGV*RRRRRRRHRRRRPQQVRAMGPSRRGVRGRQGVVLLQPPRPQVRHRPPHQPRHPLRLLEGHROGPLHHPPLLHLLRRALLLRRRRRRRHAODARLLPRPRPOGPODRVGHARVPARTAASPPOGGLGAMOGVLQDOTNNSKPII*RGRHTAQ*ARPACHAFAPAPHRCLHRLR*RTDDDAIDGRELRASVLLLRPAGIANEGLHOLWGPAGHGHLCREEGHOGTA QQQHCKAGALPRLGTGEWPVTDVEPPI. It can be seen that the amino acids are also very different from the wild type.

[0110] In summary, single-base deletion mutants (-1 / -1) and single-base insertion mutants (A / A) were screened for subsequent experiments. Since the gene editing knockout system used in this experiment is highly efficient, double-allelic mutants were obtained in the T0 generation, providing a more reliable material basis for subsequent phenotypic analysis.

[0111] 4.4 Phenotypic analysis of OsNAC113 under salt stress

[0112] The rice seedlings with normal growth and those treated with salt stress were photographed and observed. Figure 4 A. Figure 4B. Under normal growth conditions, the mutant grew better; after treating the OsNAC113 mutant and wild-type rice with 200mmol / L sodium chloride for one week, the mutant plants showed higher resistance. After treatment with 200mmol / L sodium chloride, the rice seedlings were photographed and observed. Figure 4 C. Under salt stress, the mutant plants showed higher resistance. Leaves of the wild type and mutant plants at maturity were treated with salt (200mmol / L, 3d) and photographed. Figure 4 D, The leaves of the mutant are relatively greener. In summary, OsNAC113 has enhanced salt resistance during the germination and maturity stages.

[0113] In summary, the present invention constructed an OsNAC113 gene editing knockout vector, and used rice Nipponbare as the genetic transformation receptor. The gene was transferred into rice callus through a rice stable transformation system to obtain regenerated plants. After identification and screening, a total of 7 transgenic positive strains were obtained, and the transgenic positive conversion rate was 100%. The sequencing results showed that 5 double-allelic homozygous mutant strains were obtained, and the knockout efficiency was 71.4%. After screening and identification, stable genetic plants were obtained for subsequent functional verification experiments. The growth state and survival rate of the mutant under high salt stress were significantly higher than those of the wild type. It was found that the relative water content, chlorophyll content and soluble sugar content of the mutant under salt stress were higher than those of the wild type ( Figure 5 , Figure 6 , Figure 7 ).

[0114] In addition, after the stress treatment, the plants were damaged. At this time, the ROS scavenging mechanism in the cell will play a role, and the enzymes related to scavenging ROS include CAT, POD and SOD. The degree of plant damage can be directly reflected by the MDA content and the activity of various enzymes in the redox pathway. The experimental results are as follows Figure 8-Figure 11 As shown, it can be seen that the MDA content in the OsNAC113 mutant is 6% lower than that in the wild type, and the activities of SOD, POD and CAT related enzymes in OsNAC113 are 18.7%, 24.5% and 22.5% higher than those in the wild type, respectively. This indicates that under salt stress, the oxidative damage of the OsNAC113 mutant is smaller and its tolerance to salt treatment is significantly enhanced.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of rice OsNAC113 gene in regulating rice salt tolerance, the nucleotide sequence of the rice OsNAC113 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The application is to reduce the expression level of the rice OsNAC113 gene or weaken the function of the rice OsNAC113 gene, thereby improving the salt tolerance of rice.

3. The use according to claim 1, characterized in that: The application is to use CRISPR / Cas technology to target and knock out the OsNAC113 gene in rice.

4. Application of the CRISPR / Cas9 gene editing system for targeted mutation of rice OsNAC113 gene in the construction of OsNAC113 gene mutants.

5. An sgRNA for targeting and mutating the rice OsNAC113 gene, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO.2, and the sgRNA is designed at the first exon of the rice OsNAC113 gene.

6. A method for improving salt tolerance of rice, characterized in that: The salt tolerance of rice is improved by reducing the expression level of the rice OsNAC113 gene or weakening the function of the rice OsNAC113 gene.

7. A knockout vector, characterized in that: The knockout vector is a CRISPR / Cas expression vector constructed by golden gate cloning technology and contains the targeted knockout rice OsNAC113 gene.

8. A method for creating a rice mutant with improved salt tolerance, characterized in that: The following characteristic steps are included: The OsNAC113 gene was used as the editing target gene. The CRISPR / Cas9 gene editing system was used to knock out the OsNAC113 gene and then the homozygous mutant plants were obtained through propagation, which were rice mutants with improved salt tolerance. Among them, the nucleotide sequence of the sgRNA of the CRISPR / Cas9 gene editing system is shown in SEQ ID NO.2, and the nucleotide sequence of the OsNAC113 gene is shown in SEQ ID NO.1.

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