Application of knocking out os sid1 gene and method for improving salt tolerance of rice

Abstract

This application relates to the field of plant genetic engineering technology and provides a knockout method. OsSID1 Application of genes and methods for improving salt tolerance in rice. The application includes any one of the following: (1) application in improving salt tolerance in rice; (2) application in cultivating salt-tolerant rice; (3) application in salt-tolerant rice breeding or improvement of salt-tolerant rice germplasm resources. The methods for improving salt tolerance in rice include: […]. OsSID1 Introduced into the gene OsSID1 Mutations leading to loss of gene function. This invention is the first to discover and verify... OsSID1 This gene negatively regulates the salt tolerance of rice; knocking out this gene can significantly improve the salt tolerance of rice.

Description

Technical Field

[0001] This application relates to the field of plant genetic engineering technology, specifically to a knockout method. UsSID1 The application of genes and methods to improve the salt tolerance of rice. Background Technology

[0002] Rice ( Rice Rice (L.) is susceptible to various biotic and abiotic stresses during its growth and development. Salt stress is one of the main abiotic stresses restricting its growth, development, and yield, and the degree of harm varies with the growth stage, with seedlings at the 2-3 leaf stage and the greening stage being particularly sensitive. During the 2-3 leaf stage, as salt concentration increases, rice leaves become shorter, lighter in color, and the central leaf curls. Currently, the salt tolerance of rice seedlings is typically assessed using survival rate, plant height, root length, water content, dry and fresh weight, and sodium (Na+). + / K + A comprehensive evaluation was conducted using indicators such as ratios, chlorophyll content, and malondialdehyde (MDA) accumulation. Cultivating salt-tolerant food crops is one of the most economical, effective, and sustainable methods for developing and utilizing saline-alkali land resources.

[0003] Currently, most rice salt-tolerant breeding methods improve salt tolerance through conventional breeding techniques. These methods suffer from low efficiency and a lack of precise and effective molecular targets, significantly limiting the breeding and promotion of salt-tolerant rice varieties. With the development of molecular biology, discovering new salt-tolerant genes through gene editing and genetically modifying them has become an effective way to improve rice salt tolerance. Indeterminate domain (IDD) proteins are a plant-specific C2H2-type zinc finger transcription factor subfamily that widely participates in plant growth and development processes and regulates biotic and abiotic stress responses. Studies have confirmed that IDD transcription factors play a crucial role in the regulation of plant abiotic stress resistance and are important components of plant stress resistance mechanisms. The rice IDD family has 15 members, among which… OsIDD2 It negatively regulates secondary cell wall formation and mediates rice cell proliferation; OsIDD3 Positively regulating cold tolerance in rice, it is a key gene in rice's response to low-temperature stress; OsIDD6 It mediates the reproductive development process of rice and participates in flowering regulation; OsIDD12 and OsIDD4 Mediates auxin transport and regulates the morphogenesis and development of rice leaf veins; however, current understanding of... OsIDD4 ( UsSID1 Functional studies on this gene are limited to its regulatory role in the flowering process of rice. No studies have yet confirmed that this gene participates in the regulation of rice's response to abiotic stress. Its salt tolerance function and molecular regulatory mechanism still need to be further explored and elucidated.

[0004] With the continuous development and advancement of molecular biology techniques, the analysis and discovery of novel rice salt-tolerance genes using mutants, and their application in gene-assisted breeding and salt-tolerance improvement, are of great significance for improving rice salt tolerance, increasing yield, and improving rice quality. However, achieving this goal also faces many challenges. While CRISPR / Cas9 gene editing offers numerous advantages such as high efficiency, short cycle time, broad spectrum, multiplexing capability, and rich functionality, it also has limitations, including off-target effects, uneven editing efficiency, PAM sequence limitations, and delivery system bottlenecks. Off-target effects are the most critical issue, and improving the specificity of single-guide RNA (sgRNA) and separating it from the DNA strand when mismatches occur is key to solving this problem. sgRNA design requires comprehensive consideration of numerous factors, including PAM sequence requirements, target sequence length and structure, GC content, seed sequence specificity, off-target effects, optimal target gene action location, transcription efficiency, and backbone compatibility, making the design process more complex. Although various sgRNA design software programs have been developed, due to significant differences in algorithms, inconsistent off-target evaluation criteria, and the lack of sufficient experimental verification for most prediction results, it often requires creative strategies and repeated experimental verification to screen effective sgRNAs from a massive number of candidate sequences that can efficiently and accurately edit target genes and stably produce the expected agronomic traits.

[0005] Therefore, it was clarified for the first time UsSID The direct function and application value of gene 1 in regulating the overall salt tolerance of rice is not only a breakthrough in scientific discovery, but also an important step in overcoming technical obstacles. Summary of the Invention

[0006] This invention aims to solve the problems in the prior art and provide a knockout method that can lay a solid foundation for the rapid creation of new salt-tolerant rice germplasm through molecular breeding and effectively cope with salt stress. UsSID1 The application of genes and methods to improve the salt tolerance of rice.

[0007] To achieve the above objectives, the first aspect of this application provides a knockout method. UsSID1 The application of genes, the UsSID1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the application includes any of the following:

[0008] (1) Application in improving the salt tolerance of rice; (2) Application in the cultivation of salt-tolerant rice; (3) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources.

[0009] In the above application, preferably, the knockout UsSID1 Genes are created through gene editing; the gene editing enables... UsSID1A frameshift mutation occurs in the gene, causing premature termination of the coding process.

[0010] In the above-described applications, preferably, the gene editing employs the CRISPR / Cas9 system, and the sgRNA targeting sequence used by the CRISPR / Cas9 system is shown in SEQ ID NO.4 and EQ ID NO.5.

[0011] In the above application, preferably, the knockout UsSID1 mutants obtained after gene generation UsSID1 The nucleotide sequence of the gene is shown in SEQ ID NO.12 or SEQ ID NO.15.

[0012] The above-mentioned applications, preferably, are as follows: UsSID1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3.

[0013] A second aspect of this application provides a method for improving salt tolerance in rice, comprising: [the following is a description of an endogenous process in the rice plant genome]. UsSID1 Introduced into the gene UsSID1 Loss-of-gene mutations, the aforementioned UsSID1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0014] In the above method, preferably, the mutation includes a base deletion or a single base insertion mutation; the base deletion causes the... UsSID1 The gene undergoes a 3-base GAC deletion from position 58 to 60, starting from the start codon ATG, and a 1-base G deletion at position 139; this single-base insertion mutation results in... UsSID1 The gene inserts a C base at position 62 starting from the start codon ATG, and a T base at position 139.

[0015] The above method, preferably, involves the introduction of... UsSID1 Loss-of-gene mutations are achieved using the CRISPR / Cas9 gene editing system, involving the following steps: (1) Construct a CRISPR / Cas9 gene editing vector, wherein the vector contains a target UsSID1 sgRNA of a gene; (2) Transform the carrier into rice cells or tissues; (3) Screen rice plants that have undergone the mutation.

[0016] In the above method, preferably, the target sequence of the sgRNA is shown in SEQ ID NO.4 and SEQ ID NO.5.

[0017] In the above method, preferably, the vector is pYLCRISPR / Cas9Pubi; the transformation is performed using Agrobacterium-mediated transformation.

[0018] Compared with the prior art, this application has the following beneficial effects: (1) First time revealing a new gene function, providing a new target for salt-tolerant breeding: This invention is the first to discover and verify UsSID1 This gene negatively regulates the salt tolerance of rice. Knocking out this gene can significantly improve the salt tolerance of rice. This provides a new theoretical basis for understanding the molecular mechanism of salt tolerance in plants and provides a new target gene for salt-tolerant breeding.

[0019] (2) Creating new breeding materials with significantly enhanced salt tolerance: This invention utilizes CRISPR / Cas9 gene editing technology to successfully obtain UsSID1 Loss-of-function mutants (e.g.) ossid1-1 and ossid1-2 Under salt stress, the survival rate of the mutants (approximately 33% and 43%) was significantly higher than that of the wild type (approximately 15%), exhibiting a prominent phenotype and providing valuable germplasm resources that can be directly utilized for breeding.

[0020] (3) An efficient and precise salt tolerance improvement technology solution was established: This invention designed a specific targeting UsSID1 The method involves the sgRNA of the gene (target sequences are SEQ ID NO.4 and SEQ ID NO.5) and the corresponding gene editing vector. This method is precise, efficient, and mutationally stable, and can achieve… UsSID1 Targeted gene knockout is applicable to the targeted improvement of salt tolerance in different rice varieties, and the technology is highly reproducible.

[0021] (4) It has great potential for production and application: The technical solution provided by this invention can be directly used to cultivate new salt-tolerant rice varieties. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 for UsSID1 Gene structure and sequencing results peak diagram; where A is the wild-type NIP reference sequence and B is the mutant sequence. ossid1-1 , ossid1-2 Sequence and sequencing peak diagram.

[0024] Figure 2 For NIP, ossid1 Photo A of the mutant before salt stress and photo B of the mutant after 11 days of treatment with 8‰ NaCl and 7 days of rehydration culture.

[0025] Figure 3 For NIP and ossid1 Survival statistics after salt stress. Values ​​shown are mean ± standard deviation, n = 5. The difference was highly significant (P < 0.001), and the statistical analysis method was one-way ANOVA.

[0026] Figure 4 For NIP and ossid1 Chlorophyll content statistics before and after salt stress treatment. Values ​​shown are mean ± standard deviation, n = 3. The difference was highly significant (P < 0.001), and the statistical analysis method was one-way ANOVA.

[0027] Figure 5 For NIP and ossid1 Statistical analysis of MDA (malondialdehyde) content before and after salt stress treatment. Values ​​shown are mean ± standard deviation, n = 3. The difference was highly significant (P < 0.001), and the statistical analysis method was one-way ANOVA. Detailed Implementation

[0028] To facilitate understanding of this application, the following description will be more comprehensive and detailed in conjunction with the accompanying drawings and preferred embodiments, but the scope of protection of this application is not limited to the following specific embodiments.

[0029] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of this application.

[0030] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0031] Example 1: Rice UsSID1 CRISPR / Cas9 gene knockout and mutant acquisition This embodiment provides rice. UsSID1 The CRISPR / Cas9 gene editing knockout method and mutant acquisition, specifically including the following: 1. Rice UsSID1 Gene sequence analysis The Ensembl Plants database (http: / / plants.ensembl.org / index.html) contained information on rice. UsSID1 The gene, with its nucleotide sequence in Nipponbare japonica rice as shown in SEQ ID NO.1 and its CDS sequence as shown in SEQ ID NO.2, encodes a protein containing 616 amino acids, the sequence of which is shown in SEQ ID NO.3.

[0032] 2. Rice UsSID1 Functional verification of genes To clarify UsSID1 To investigate the function of a gene in rice, this invention employs the CRISPR / Cas9 gene editing method to site-directedly mutate the gene sequence and knock out its function in rice.

[0033] This invention selects conventional rice, Nipponbare (hereinafter referred to as NIP), as the recipient material for gene editing. This invention selects... UsSID1 The nucleotide sequence from base 45 to base 63 of the gene coding region starting from the start codon ATG is designated as target sequence 1 (as shown in SEQ ID NO. 4), and the nucleotide sequence from base 123 to base 142 of the start codon ATG is designated as target sequence 2 (as shown in SEQ ID NO. 5) (see...). Figure 1 ).

[0034] (1) UsSID1 Construction of CRISPR / Cas9 gene editing vector The gene editing vector of this invention is pYLCRISPR / Cas9Pubi, and the specific construction process is as follows: i) Design of target gRNA. [The following is likely a separate, unrelated sentence:] UsSID1 The gene sequence was input into https: / / zlab.bio / guide-design-resources for target design, and the PAM sequence was set to NGG. The DNA sequences of the target regions selected in this invention are shown in SEQ ID NO. 4 and SEQ ID NO. 5.

[0035] ii) Amplification of the sgRNA expression cassette. Design and synthesize primers containing the target sequence described above: Forward primer for target 1: 5'-TATGGCGCCATCCAACGCCGGTTTTAGAGCTAGAAAT-3' (as shown in SEQ ID NO. 6); Target 1 reverse primer: 5'-CGGCGTTGGATGGCGCCATACGGCAGCCAAGCCAGCA-3' (as shown in SEQ ID NO. 7); Forward primer for target 2: 5'-AATTAGGGATGGCGACCATGGTTTTAGAGCTAGAAAT-3' (as shown in SEQ ID NO. 8); Target 2 reverse primer: 5'-CATGGTCGCCATCCCTAATTCAACACAAGCGGCAGC-3' (as shown in SEQ ID NO. 9).

[0036] The above primer pairs were annealed to form double-stranded gRNA.

[0037] Subsequently, the gRNA sequence was combined with the Bsa I-digested binary vector pYLCRISPR / Cas9Pubi (see Ma X, Zhang Q, Zhu Q, et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants). Molecular Plant (2015, 8(8):1274-1284) were linked to obtain the recombinant vector pYLCRISPR / Cas9Pubi-OsSID1.

[0038] iii) Transformation of E. coli and Sequencing Verification. The recombinant vector pYLCRISPR / Cas9Pubi-OsSID1 was transformed into E. coli DH5α, and positive clones were selected for sequencing. The specific transformation steps were as follows: DH5α competent cells were removed from -80℃ and placed on ice. After 5 minutes, the target DNA was added, and the mixture was gently stirred by tapping the bottom of the EP tube. The cells were then incubated on ice for 20 minutes. A heat shock was performed at 42℃ for 45 seconds, followed by immediate return to ice and incubation for 2 minutes. 700 μL of antibiotic-free LB solution was added, and the cells were incubated at 37℃ and 200 rpm for 30 minutes. The cells were then plated. The plates were inverted and incubated overnight at 37℃. Positive clones were screened and sequenced for verification. Positive clones with correct sequencing results were considered successfully constructed pYLCRISPR / Cas9Pubi-OsSID1 gene editing vectors.

[0039] 3. Agrobacterium-mediated genetic transformation of rice The successfully constructed pYLCRISPR / Cas9Pubi-OsSID1 gene-editing binary vector was transformed into *Agrobacterium tumefaciens* competent cells GV3101 (pSoup-p19) using a heat shock method. Single clones were selected and confirmed to be correct by colony PCR and sequencing. Then, sterile glycerol at a final concentration of 20-30% was added, and the cells were stored at -80°C for later use.

[0040] Genetic transformation of the japonica rice variety "Nipponbare" ( Oryza sativaL . ssp. japonicacv. Nipponbare mature seed-induced immature embryos as recipient material, Agrobacterium-mediated transformation and subsequent screening and regeneration. Reference: "Cui Ying, Cai Zhaoxia, Lin Yongjun, Chen Hao. (2018). Agrobacterium-mediated rapid transformation of rice." Bio-101e1010176. The method described in DOI:10.21769 / BioProtoc.1010176 is as follows: The specific steps are as follows: (1) Callus induction.

[0041] Select mature and plump rice seeds, remove the husks; disinfect with 75% alcohol for 2 min, then discard the alcohol; rinse twice with sterile distilled water; soak in 0.15% mercuric chloride (containing 0.1% Tween 20) for 15 min, shaking several times during this period; discard the mercuric chloride, and rinse five times with sterile distilled water. Inoculate the sterilized seeds into callus induction medium and culture at 32 °C under light for 7 days.

[0042] (2) Agrobacterium streak activation.

[0043] Two days before infection, Agrobacterium was streaked onto LB medium containing 50 mg / L kanamycin and incubated at 28 °C.

[0044] (3) Suspension, infection and co-culture of Agrobacterium.

[0045] Before infection, activated Agrobacterium was scraped into a suspension medium and cultured at 28 °C with shaking at 180 rpm for 3–3.5 h. The bacterial concentration was then adjusted to OD600 = 0.1–0.2 using the suspension medium. Callus tissue induced for 7 days was placed in the Agrobacterium suspension and infected for 1.5 min. The bacterial suspension was discarded, and the surface of the callus was blotted dry with sterile filter paper. The callus surface was covered with sterile filter paper and dried in a laminar flow hood for 30 min. After drying, the callus was transferred to a co-culture medium covered with a layer of sterile filter paper, incubated overnight in the dark at 20 °C, and then transferred to a 25 °C incubator for another 2 days in the dark.

[0046] (4) Clean bacteria.

[0047] After co-culturing, the callus tissue was transferred to an empty sterile container using forceps. The callus was repeatedly washed 7 times with sterile distilled water, with the first 3 washes being quick and the subsequent 4 washes involving soaking for 5 minutes each. Finally, the callus was soaked in sterile distilled water containing 500 mg / L Cn for 30 minutes. The Cn solution was discarded, and the surface moisture of the callus was blotted dry as much as possible with sterile filter paper. The callus surface was then covered with another layer of sterile filter paper and dried in a laminar flow hood for 1 hour.

[0048] (5) Screening.

[0049] After sterilization, the callus was placed on a selection medium and cultured at 32 °C under light for 14 days.

[0050] (6) Differentiation.

[0051] After 14 days of selection, the resistant callus was transferred to differentiation medium and cultured at 28 °C (photocycle of 14 h light / 10 h dark).

[0052] (7) Rooting.

[0053] Once the resistant callus has formed a 3-4 cm tall regenerated seedling on the differentiation medium, it is transferred to the rooting medium for further culture until a complete plant is formed.

[0054] After the plants have grown and survived, they are transplanted into a greenhouse. The offspring seeds were harvested 4 months later.

[0055] 4. Detection of CRISPR / Cas9 mutation results in T0 generation plants To determine the CRISPR / Cas9 mutation results in T0 generation plants, the following steps were taken for detection: (1) Extraction of rice genomic DNA.

[0056] Rice genomic DNA was extracted using the CTAB method (Rogers and Bendich, 1985). The specific method is as follows: Fresh rice leaves (2-3 cm in diameter) were placed in sterile 2 mL centrifuge tubes containing 6 mm steel balls. After flash freezing in liquid nitrogen, the leaves were thoroughly homogenized using a tissue homogenizer. Rice DNA was then extracted, followed by chloroform-isoamyl alcohol extraction, isopropanol precipitation, and ethanol washing. The DNA precipitate was air-dried at room temperature, dissolved in 200 μL (adjusted according to the precipitate volume) of nuclease-free ultrapure water (ddH2O), and stored at -20℃ for later use. 2 μL of DNA sample was taken, and the A260 / A280 ratio and concentration were determined using a NanoDrop 2000 UV spectrophotometer. Qualified samples were uniformly diluted to 50 ng / μL and used as templates for target site PCR amplification and subsequent CRISPR / Cas9 mutation type identification.

[0057] (2) Target site PCR amplification.

[0058] PCR was performed using vazyme's 2×PCR premix (containing Mg). 2+The following reagents were added: Taq DNA Polymerase; 2.5 mM dNTPs; 10×PCR Buffer (5 μL), forward and reverse primers (0.5 μL each), template DNA (1 μL), and ddH2O to a final volume of 10 μL. The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 30 s, for 35 cycles, followed by a final extension at 72℃ for 5 min.

[0059] The PCR primer sequences used to amplify target sites for mutation detection are as follows: Detection primer-F: 5'-CTGCTACTAGCCCAAGCCAA-3' (as shown in SEQ ID NO.10); Detection primer-R: 5'-ACCTGGTAAGGTTCTCTTCTTC-3' (as shown in SEQ ID NO.11).

[0060] The amplified products were subjected to agarose gel electrophoresis at a concentration of approximately 1%, and sequencing was performed to verify the correct band size.

[0061] (3) Sequencing results analysis.

[0062] For mutants ossid1 Nucleotide sequence alignment analysis revealed that ( Figure 1 Compared to the unedited wild type (WT), the mutated UsSID1 Gene mutants ossid1-1 The OsSID1 gene mutant has a 3-base GAC deletion from position 58 to 60 of the start codon ATG, and a 1-base G deletion at position 139. The nucleotide sequence is shown in SEQ ID NO. 12, the CDS sequence in SEQ ID NO. 13, and the amino acid sequence in SEQ ID NO. 14. ossid1-2 The mutant inserts a C base at position 62 of the start codon ATG and a T base at position 139. The nucleotide sequence is shown in SEQ ID NO. 15, the CDS sequence is shown in SEQ ID NO. 16, and the amino acid sequence is shown in SEQ ID NO. 17. The deletion of the nucleotide encoded by the mutant causes a frameshift in the amino acids, leading to premature termination of amino acid translation.

[0063] Example 2: Rice UsSID1 Salt tolerance phenotype identification of gene mutants This embodiment describes the mutant obtained in Example 1. ossid1 Phenotypic analysis was performed, as follows: 1. Mutant ossid1 Salt tolerance assessment.

[0064] The cultivation parameters are as follows: light intensity is 120 μmol·m -2 ·s -1 The temperature is 30℃ / 28℃ (day / dark), and the photoperiod is 10h light / 14h darkness.

[0065] The rice seeds to be tested are ossid1 The T1 generation homozygous seeds of the mutant (obtained by self-pollination of the T0 generation) and their background material NIP. The experiment was repeated 5 times, and the average survival rate was taken. The steps for each repetition are as follows: (1) Take the rice seeds of the material to be tested, put them into mesh bags, and soak them in water at 28-30℃ for 48 hours.

[0066] (2) Germinate the seeds at 28-30℃ for 24 hours (keep the seeds moist during the germination process) to obtain germinated seeds.

[0067] (3) Take a 96-well plate without holes and put one germinated seed in each well (embryo facing up, radicle facing down).

[0068] (4) Place the above-mentioned 96-well plate (with pre-germinated seeds) in a blue turnover box filled with pure water and culture it under alternating light and dark conditions for 5 days. Then transfer it to a black hydroponic box filled with Yoshida rice culture medium and continue to culture for 14 days to obtain rice seedlings that have grown to the three-leaf stage. During the alternating light and dark culture period, change the Yoshida rice culture medium every 4 days.

[0069] (5) Transfer rice seedlings at the three-leaf stage to a black hydroponic box containing Yoshida rice culture solution with 8‰ NaCl, so that the roots are completely immersed in the culture solution, and culture under high salt stress for 11 days under alternating light and dark conditions (during the high salt stress period, the Yoshida rice culture solution containing 8‰ NaCl is replaced every 4 days).

[0070] (6) Transfer the rice seedlings to a black hydroponic box containing Yoshida rice culture solution and allow them to recover for 7 days under alternating light and dark conditions. Observe the growth status of the rice seedlings and calculate the survival rate. Survival rate = number of surviving rice seedlings / total number of rice seedlings × 100%, with the survival criterion being whether new leaves have emerged.

[0071] 2. Salt tolerance test results.

[0072] See the growth status of rice seedlings before treatment. Figure 2 Survival rate statistics can be found in Figure 3 .

[0073] The results showed that before salt stress treatment, the mutant ossid1 The growth was basically the same as that of NIP; after salt stress treatment, the survival rate of NIP was about 15%, while that of the mutants... ossid1-1 and ossid1-1 The survival rates were approximately 33% and 43%, respectively. Statistical analysis results indicate that the mutants... ossid1 The survival rate of the mutant was significantly higher than that of NIP, indicating that the mutant... ossid1 Its salt tolerance has been significantly improved.

[0074] Example 3: Rice UsSID1 Chlorophyll content determination of gene mutants This embodiment describes the mutant obtained in Example 1. ossid1 Chlorophyll content analysis was performed. Chlorophyll is the main pigment in photosynthesis, and its content directly reflects the photosynthetic potential of leaves. The specific measurement steps are as follows: 1. The culture conditions in Example 2 were followed. ossid1 The T1 generation of homozygous mutant seeds and their background material NIP were cultured. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows: (1) Before high salt stress (salt stress treatment method is the same as in Example 2) (treatment 0 days) and after treatment (treatment 11 days), 0.2g of each material rice leaf sample was placed in a mortar, liquid nitrogen was added for quick freezing and then ground into powder, and transferred to a 10mL centrifuge.

[0075] (2) Add 3 mL of 80% acetone to the sample and extract chlorophyll overnight at 4°C in the dark.

[0076] (3) After overnight centrifugation, centrifuge the sample at 5000 rpm for 5 min at 4°C, and transfer the supernatant to a new centrifuge tube using a pipette.

[0077] (4) Measure the absorbance of the sample at 645 nm and 663 nm. Use 80% acetone as a blank control to zero the sample during the measurement. Calculate the chlorophyll content according to the following formula: C a+b (mg / g)=[8.026×A663+20.206×A645]×V / 1000×W; In the formula, V is the volume of the extract (mL), W is the fresh weight of the leaf (g), and A663 and A645 are the absorbance values ​​of the sample at 663 nm and 645 nm, respectively.

[0078] 2. Chlorophyll content measurement results.

[0079] See the statistical results of chlorophyll content. Figure 4 The results showed that before salt stress, the mutant... ossid1(ossid1-1) and ossid1-2) The chlorophyll content of NIP was basically the same as that of the mutant, with no significant difference; after salt stress treatment, the chlorophyll content of NIP was 0.62 mg / g, while that of the mutant was... ossid1-1 and ossid1-2 The chlorophyll contents were 1.23 mg / g and 1.39 mg / g, respectively. Statistical analysis results showed that the mutant... ossid1 The chlorophyll content of the mutant was significantly higher than that of NIP, indicating that the mutant... ossid1 Its salt tolerance has been significantly improved.

[0080] Example 4: Rice UsSID1 Determination of MDA (malondialdehyde) content in gene mutants This embodiment describes the mutant obtained in Example 1. ossid1 MDA content analysis was performed. MDA is the final product of cell membrane lipid peroxidation, and its content reflects the degree of cell membrane damage under salt stress; higher content indicates more severe membrane lipid peroxidation damage. The specific measurement steps are as follows: 1. The culture conditions in Example 2 were followed. ossid1 The T1 generation of homozygous mutant seeds and their background material NIP were cultured. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows: (1) Before (day 0 of treatment) and after (day 11 of treatment) high salt stress treatment, 0.2g of rice leaf samples of each material were placed in a mortar, liquid nitrogen was added for quick freezing and then ground into powder, and transferred into 10mL centrifuge tubes.

[0081] (2) Add 10% trichloroacetic acid to the sample and mix well. Let it stand at 4°C for 1 hour, then centrifuge at 5000 rpm for 10 minutes. Use a pipette to collect the supernatant and transfer it to a new centrifuge tube.

[0082] (3) Using 10% trichloroacetic acid as a control, add an equal volume of 0.6% thiobarbituric acid (TBA) to the supernatant of the sample after centrifugation, and boil in a water bath for 15 min.

[0083] (4) After boiling in the water bath, the sample was rapidly cooled and centrifuged at 4℃, 12000 rpm, and for 10 min. The absorbance of the sample was measured at wavelengths of 532 nm, 600 nm, and 450 nm. The MDA content was calculated using the following formula: C(μmol / L)=6.45×(A532-A600)-0.56×A450; MDA(μmol / g FW)=[C×V×0.001] / W; Where C represents the concentration of MDA in the sample (μmol / L), V is the total volume of the extract (mL), W is the fresh weight of the sample (g), and A450, A532, and A600 are the absorbance values ​​of the sample at 450 nm, 532 nm, and 600 nm, respectively.

[0084] 2. MDA content determination results.

[0085] The statistical results of MDA content are shown in [the table]. Figure 5 The results showed that before salt stress, the mutant... ossid1(ossid1-1) and ossid1- 2） The MDA content of NIP was basically the same as that of NIP, with no significant difference; after salt stress treatment, the MDA content of NIP was 0.82 μmol / g, while that of the mutant was... ossid1-1 and ossid1-2 The MDA contents were respectively Statistical analysis results showed that the mutants were 0.37 μmol / g and 0.35 μmol / g. ossid1 The MDA content was significantly lower than that of NIP, indicating that ossid1 The mutant exhibited significantly improved salt tolerance.

[0086] The above are merely preferred embodiments of this application. It should be noted that this application is not limited to the above embodiments. For those skilled in the art, several improvements and modifications can be made without departing from the principles of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should also be considered within the scope of protection of this application.

[0087] Sequence List: SEQ ID NO.1:

[0088] SEQ ID NO.2:

[0089] SEQ ID NO.3: MASNSSAAAVAALFGIRDGDHEDQIKPLFAQQQQHHHHQPPMAPSNAAAAASAAGSAAGQAAVAAPPAKKKRTLPDPDAEVIALSPKTLLATNRFVCEVCNKGFQREQNLQLHRRGHNLPWKLKQKNPLQAQRRRVYLCPEPTCVHHDPSRALGDLTGIKKHFCRKHGEKKWKCDKCSKRYAVQSDWKAHSKICGTREYRCDCGTLFSRRDSFITHRAFCDALAQESARLPPAAAGHLYGSAGAANMALSLSQVGSHLASTLQDHGHHHHHHGASPDLLRFGGSGGGAMAARLEHLLSSSSASAFRPLPPPQQQPPAPFLLGAAPQGFGDGGDGSGPHGFLQGKPFHGLMQLPDLQGNGTGGPSPSGPGLYNLGYIANSANSSGTSSHGHASQGQMTNTDQFSEGGGGGGGGGGSETSAAALFGAGGNFSGGDHHQVSPAGMYANDQAMMLPQMSATALLQKAAQMGSSTSSANGAGASVFGGGFAGSSAPSSIPHGRGTTMVDQGQMHLQSLMNSLAGGGNADHQGMFGSGSMIDPRLYDMDQHEVKFSLQRGGGGGGDGDVTRDFLGVGGGGFMRGMSMARGEHHGGGGSDMHGTLEAEMKSASSSFNGGRMQ。

[0090] SEQ ID NO.4: AATTAGGGATGGCGACCAT。

[0091] SEQ ID NO.5: TATGGCGCCATCCAACGCCG。

[0092] SEQ ID NO.6: TATGGCGCCATCCAACGCCGGTTTTAGAGCTAGAAAT。

[0093] SEQ ID NO.7: CGGCGTTGGATGGCGCCATACGGCAGCCAAGCCAGCA。

[0094] SEQ ID NO.8: AATTAGGGATGGCGACCATGGTTTTAGAGCTAGAAAT。

[0095] SEQ ID NO.9: CATGGTCGCCATCCCTAATTCAACACAAGCGGCAGC。

[0096] SEQ ID NO.10: CTGCTACTAGCCCAAGCCAA。

[0097] SEQ ID NO.11: ACCTGGTAAGGTTCTCTTCTTC。

[0098] SEQ ID NO.12:

[0099] SEQ ID NO.13:

[0100] SEQ ID NO.14: MASNSSAAAVAALFGIRDGHEDQIKPLFAQQQQHHHHQPPMAPSNPRRRLLRQGRRPVKRPWRRHQRRRREPYQTRTRR。

[0101] SEQ ID NO.15:

[0102] SEQ ID NO.16:

[0103] SEQ ID NO.17: WHPTHQRQLWRRCLELGMATHEDQIKPLFAQQQQHHHHQPPMAPSNVRGGGFCGRVGGRSSGRGGATSEEEENLTRPGRGGDSAVAEDAAGDEPVRVRGVQQGVPAGAEPAAAPARAQPAVEAEAEEPAAGAAPPGVPVPGADVRPPRPLPRPRRPHRHQEALLPQARREEVEVRQVLQALRRPVRLEGPLQDLRHPRVPLRLRHPLLPEGQLHHPPRLLRRPRPGERAAATRRRRPPLRLRRRRQHGAQPLPGRLPPRLHPPGPRPPPPPSRRLPGPPPLRRQRRWRHGCTPRAPPVVVQRLRVPAPAAAAAAASGAVPPRRGAAGVRRRRRRQWSARILAG。

Claims

1. Knockout OsSID1 The application of genes is characterized by, The OsSID1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the application includes any of the following: (1) Application in improving the salt tolerance of rice; (2) Application in the cultivation of salt-tolerant rice; (3) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources.

2. The application according to claim 1, characterized in that, The knockout OsSID1 Genes are created through gene editing; the gene editing enables... OsSID1 A frameshift mutation occurs in the gene, causing premature termination of the coding process.

3. The application according to claim 2, characterized in that, The gene editing was performed using the CRISPR / Cas9 system, and the sgRNA targeting sequence used by the CRISPR / Cas9 system is shown in SEQ ID NO.4 and EQ ID NO.

5.

4. The application according to claim 2, characterized in that, The knockout OsSID1 mutants obtained after gene generation OsSID1 The nucleotide sequence of the gene is shown in SEQ ID NO.12 or SEQ ID NO.

15.

5. The application according to claim 1, characterized in that, The OsSID1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

3.

6. A method for improving the salt tolerance of rice, characterized in that, include: In the endogenous nature of the rice plant genome OsSID1 Introduced into the gene OsSID1 Loss-of-gene mutations, the OsSID1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The method according to claim 6, characterized in that, The mutation includes a base deletion or a single base insertion mutation; the base deletion causes the... OsSID1 The gene undergoes a 3-base GAC deletion from position 58 to 60, starting from the start codon ATG, and a 1-base G deletion at position 139; this single-base insertion mutation results in... OsSID1 The gene inserts a C base at position 62 starting from the start codon ATG, and a T base at position 139.

8. The method according to claim 6 or 7, characterized in that, The introduction makes OsSID1 Loss-of-gene mutations are achieved using the CRISPR / Cas9 gene editing system, involving the following steps: (1) Construct a CRISPR / Cas9 gene editing vector, wherein the vector contains a target OsSID1 sgRNA of a gene; (2) Transform the carrier into rice cells or tissues; (3) Screen rice plants that have undergone the mutation.

9. The method according to claim 8, characterized in that, The target sequence of the sgRNA is shown in SEQ ID NO.4 and SEQ ID NO.

5.

10. The method according to claim 8, characterized in that, The vector was pYLCRISPR / Cas9Pubi; the transformation was performed using Agrobacterium-mediated transformation.

Images