Application of OsFLZ14 gene in salt stress resistance of rice

Abstract

The invention discloses application of an OsFLZ14 gene in rice salt stress resistance, the nucleotide sequence of the OsFLZ14 gene is shown as SEQ ID NO.1, the OsFLZ14 gene negatively regulates the rice salt tolerance, and specifically, the OsFLZ14 gene is knocked out from rice to improve the tolerance of rice to salt stress. The invention also provides a rice salt-stress-resistant gene mutant which shows a salt-resistant phenotype when basic groups are knocked out or inserted into the nucleotide sequence as shown in SEQ ID NO.1. The invention also provides a rice salt-stress-resistant gene mutant. According to the invention, the function of the FLZ protein OsFLZ14 in adversity stress of rice is enriched, and a new gene resource is provided for cultivation of salt-resistant rice.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering and relates to the application of OsFLZ14 gene in rice salt stress tolerance. Background Art

[0002] In recent years, abiotic stresses such as salt and drought have occurred frequently, severely restricting crop growth and development, resulting in reduced crop yield and quality. According to statistics, about 1 / 5 of the world's arable land and nearly half of the irrigated land are polluted to varying degrees by salinization. Studies have found that high salt stress affects the root system, leaf development, osmotic system, hormone levels, and physiological and biochemical traits of crops, resulting in a significant reduction in crop yield and quality, endangering food security worldwide. Soil salinization has become an important factor restricting rice production. Therefore, the use of molecular breeding technology to cultivate salt-tolerant rice varieties is of great significance to improving the utilization rate of salinized soil, expanding the rice planting area, and ensuring food security.

[0003] OsFLZ14 belongs to the zinc finger protein FLZ (FCS-Like Zinc Finger) gene family. The FLZ gene family is plant-specific and plays an important role in plant growth and development and stress response. Current reports show that this family of genes has an important regulatory effect on plant growth and development, and can regulate flowering period, promote seed germination, root and leaf development, and plant tolerance to salt, low temperature, high temperature, drought and other stresses. Proteins containing the FLZ domain are generally referred to as FLZ proteins. In rice, a total of 29 FLZ proteins have been found (Ma et al., 2021), and the functions of some FLZ proteins have been clarified. For example, OsFLZ2 interacts with OsMADS51 under short-day conditions to reduce the stability of OsMADS51, thereby weakening the transcriptional activation of the rice heading core gene Ehd1, thereby delaying the flowering period of rice (Ma et al., 2022), and OsFLZ18 interacts with OsMADS51, an important factor in regulating the heading period of rice, and inhibits the activity of OsMADS51 to promote the expression of the rice heading core regulatory gene Ehd1, thereby regulating the heading period of rice (Ma Yamei et al., 2022). However, the functions of some FLZ proteins are still unknown, such as the sequence described in SEQ ID NO.1 given by the gene accession number LOC_Os04g49660; in addition, the research on this gene family in rice salt stress has not been reported yet, and it is urgent to study. Summary of the invention

[0004] The first object of the present invention is to provide an application of the OsFLZ14 gene in rice salt stress tolerance in view of the deficiencies in the prior art.

[0005] In order to solve the above technical problems, the present invention provides an application of an OsFLZ14 gene in rice salt tolerance. The nucleotide sequence of the OsFLZ14 gene is shown in SEQ ID NO.1. The OsFLZ14 gene negatively regulates rice salt tolerance.

[0006] Furthermore, knocking out the OsFLZ14 gene in rice will improve rice's tolerance to salt stress.

[0007] The second object of the present invention is to provide a method for improving the salt stress tolerance of rice, specifically: knocking out the OsFLZ14 gene in the target rice germplasm genome, the nucleotide sequence of the OsFLZ14 gene is shown in SEQ ID NO.1.

[0008] The third object of the present invention is to provide a rice salt stress tolerance gene mutant, wherein the rice salt stress tolerance gene mutant is a mutant of the OsFLZ14 gene, which is any one of the following: (a) a mutant cr-osflz14-1, in which a single base T is inserted at positions 116-117 in the first exon of the nucleotide sequence shown in SEQ ID NO.1; the nucleotide sequence of the mutant cr-osflz14-1 is shown in SEQ ID NO.3; (b) The mutant cr-osflz14-2, in which the twenty-three bases GCCCGGCGGCCGCCTCTTCTGCG are deleted at positions 99-121 in the first exon of the nucleotide sequence shown in SEQ ID NO.1; the nucleotide sequence of the mutant cr-osflz14-3 is shown in SEQ ID NO.4.

[0009] The fourth object of the present invention is to provide a method for obtaining the above-mentioned rice salt stress tolerance gene mutant, specifically: CRISPR / Cas9 technology was used to perform site-directed gene editing on the exons of the rice OsFLZ14 gene, and two mutants of different editing types of the gene were obtained. Both mutants showed a salt-tolerant phenotype. The designed target is: CTTCTGCGACCCGTGCGACG.

[0010] The fifth object of the present invention is to provide a recombinant vector containing the above rice salt stress tolerance gene mutant.

[0011] The sixth object of the present invention is to provide the application of the above rice salt stress tolerance gene mutant in the breeding of salt stress tolerance rice.

[0012] The seventh object of the present invention is to provide a rice breeding method, specifically: knocking out the OsFLZ14 gene in rice, thereby improving the salt stress tolerance of rice.

[0013] The eighth object of the present invention is to provide a method for preparing transgenic plants, comprising the following steps: knocking out the OsFLZ14 gene in a starting plant to obtain a transgenic plant with increased salt stress tolerance compared with the starting plant; the nucleotide sequence of the OsFLZ14 gene is shown in SEQ ID NO.1.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses CRISPR / Cas9 technology to perform site-directed gene editing on the exons of the rice OsFLZ14 gene, and obtains two mutants of different editing types of the gene, both of which show a salt-tolerant phenotype. It is speculated that the OsFLZ14 gene can negatively regulate the salt tolerance of rice. The results not only enrich the function of the FLZ protein OsFLZ14 in rice adverse stress, but also provide new gene resources for the cultivation of salt-resistant rice.

[0015] Based on this, the present invention identified a new OsFLZ14 gene related to rice salt stress, and used the most advanced and widely used CRISPR / CAS9 technology in gene editing to create mutants of the rice OsFLZ14 gene and evaluate their ability to tolerate salt stress, providing new germplasm resources for the cultivation of salt-tolerant rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0017] Figure 1 :Schematic diagram of OsFLZ14 gene structure and CRISPR target sites.

[0018] Figure 2 :The phenotypes and related indexes of two OsFLZ14 knockout mutants and their parent Nipponbare rice were different after 8 days of salt stress (140 mM NaCl) and 2 days of rehydration; Among them: Figure A shows the phenotypes of two OsFLZ14 gene knockout mutants and their parent Nipponbare rice after being treated with salt stress (140 mM NaCl) for 8 days and rehydrated for 2 days; the left figure is the control group, and the right figure is the NaCl treatment group.

[0019] Figure BE shows the results of the determination of the survival rate of each rice line (B), the total chlorophyll content (C), plant height and root length (D), and the fresh weight of the aboveground part and the fresh weight of the root (E) after 8 days of salt stress treatment and 2 days of rehydration. The survival rate and chlorophyll were replicated three times each, and the plant height, root length and fresh weight were replicated five times each. The differences between the treatment groups and the control group were compared using the student-t test, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0021] The purpose of the present invention is to study the function of OsFLZ14 gene in rice salt stress tolerance and provide new germplasm resources for the cultivation of salt-tolerant rice. The specific technical scheme is as follows:

[0022] 1) Sequence analysis of the OsFLZ14 gene of the rice variety to be improved, the nucleotide sequence encoded by the OsFLZ14 gene is shown in SEQ ID NO. 1. The amino acid sequence of the protein encoded by the OsFLZ14 gene is shown in SEQ ID NO. 2. Select appropriate sites for gene editing target design.

[0023] 2) Construct a gene editing vector and use Agrobacterium-mediated method to introduce the gene editing vector into the callus tissue of the rice variety to be improved.

[0024] 3) Screen homozygous mutant plants and treat them with salt stress to examine changes in their salt tolerance.

[0025] The present invention is further described below.

[0026] Example 1: Sequence analysis of rice OsFLZ14 and design of gene editing targets The analysis of the rice OsFLZ14 gene structure showed that the gene contained two exons. A target site was designed in the first exon to obtain two types of rice with OsFLZ14 gene mutations. The target site sequences are as follows:

[0027] Target: CTTCTGCGACCCGTGCGACG Example 2: Construction of gene editing vector and acquisition of gene knockout rice system According to the target design of Example 1, the corresponding sgRNA was synthesized, and first annealed to form a dimer (forward primer SEQ ID NO.5 of synthetic target 1: CAGCGTCGCACGGGTCGCAGAAG, reverse primer SEQ ID NO.6: AACCTTCTGCGACCCGTGCGACG; 95°C for 3 min, slowly cooled from 95°C to 25°C, for example -1°C / 20 S or placing the sample tube in a 95°C water bath for 5 min, then taking it out and naturally cooling it to room temperature, and then cloning the sgRNA into the VK005 vector.

[0028] The above recombinant vector was transformed into the callus tissue of the recipient rice Nipponbare by conventional Agrobacterium-mediated method, and the OsFLZ14 gene knockout mutant was created through screening and differentiation of resistant callus. The specific steps are as follows: (1) Using Nipponbare rice seeds as materials, remove the husks, disinfect with 75% ethanol and 1.5% sodium hypochlorite, place in N6D induction medium, and culture in a light incubator at 28℃ / 24℃, 12h light / 12h dark. After 3-4 weeks of induction culture, naturally divided embryonic callus tissues were picked and placed in N6D induction medium. Culture was continued for 7 days. After three subcultures, naturally dispersed, bright yellow, and about 3-5 mm in diameter granular callus tissues were used for Agrobacterium transformation; (2) The VK005 vector containing sgRNA was transferred into the competent Agrobacterium EHA105 by electroporation to obtain an Agrobacterium suspension; (3) Soak the callus obtained in step (1) in the Agrobacterium suspension (to which AAM infection solution has been added) in step (2), let it stand for 30-40 min, then dry the callus on sterile filter paper, inoculate it on the co-culture medium, and culture it in the dark at 25°C for 3 days; (4) Collect the callus obtained in step (3), wash it thoroughly with sterile water, soak it in sterile water containing 300 mg / L carbenicillin sodium for 30 min, and then wash it twice. Finally, place it on sterile filter paper to drain; transfer the dried callus to the screening medium containing 300 mg / L carbenicillin sodium for the first round of screening, and culture it at 28°C under light for 14 days. Transfer the initial callus with resistant callus to the screening medium containing 300 mg / L carbenicillin sodium for the second round of screening until granular resistant callus grows;

[0029] (5) Transfer 3-6 callus tissues obtained in step (4) to a differentiation tank containing differentiation medium, and place it in a constant temperature culture room at 25°C. When the seedlings grow to the top of the lid of the differentiation tank, place them in a rooting medium to root and strengthen the seedlings. After 7 days, open the sealing film and add an appropriate amount of sterile water to prevent bacterial growth in the culture. Harden the seedlings for about 3 days until the seedlings stand upright, then wash off the agar and culture in the solution for 7-14 days before transplanting.

[0030] (6) After the rice plants transplanted in step (5) have formed ears, the leaves of each plant are taken and genomic DNA is extracted using the CTAB method, and PCR amplification is performed using the CTAB method (forward primer SEQ ID NO.7: CGAAGCAAACCAAGCCAAGA, reverse primer SEQ ID NO.8: AGGAGGAAGGAACCGAACAA). The amplified bands are purified and recovered by gel purification and then sequenced to detect whether there are mutations in the sequence near the target site. Finally, three types of homozygous mutations are obtained, namely cr-osflz14-1 and cr-osflz14-2.

[0031] cr-osflz14-1, the nucleotide sequence of which is shown in SEQ ID NO.3; cr-osflz14-2, the nucleotide sequence of which is shown in SEQ ID NO.4; Schematic diagram of OsFLZ14 gene structure and CRISPR target sites Figure 1 As shown; cr-osflz14-1 is a single base T insertion, and cr-osflz14-2 is a twenty-three base GCCCGGCGGCCGCCTCTTCTGCG deletion.

[0032] (7) The two homozygous mutant rice plants obtained in step (6) are harvested to obtain homozygous T1 generation mutants. In order to obtain homozygous plants with Cas9-Free gene knockout, the present invention performs antibiotic screening on the T2 generation seeds of the homozygous T1 generation plants (select 15 seeds of each homozygous T1 generation plant for about 3 days after germination, add 50 mg / L hygromycin solution to the culture medium, if all seedlings die, the T1 generation strain is Cas9-Free homozygous), and the seeds of the obtained homozygous strain are T2 generation homozygous mutant rice, which is used for the subsequent Example 3.

[0033] Example 3: Analysis of Salt Stress Phenotype of OsFLZ14 Gene Edited Mutant Rice (1) The homozygous T2 mutant rice (cr-osflz14-1, cr-osflz14-2) and wild-type rice (Nipponbare) obtained in Example 2 were placed in a 42°C oven for one week to break dormancy, soaked at 28°C for 48 h, and germinated at 30°C until germination, and planted together in a hydroponic box. After culturing in pure water for one week, the water solution was changed to Yoshida nutrient solution, which was changed every 3 days. When the seedlings grew to two leaves and one heart, salt stress treatment was carried out, and the stress solution (140 mM NaCl was added to the Yoshida nutrient solution) was changed every 3 days.

[0034] (2) After 8 days of treatment, observe the phenotypic differences between the mutant and wild-type rice. Take leaves at the same position, remove the main veins, and cut them into small pieces with a side length of 2-3 mm with scissors. Soak 0.15-0.2 g of the sample to be tested in 25 mL of a mixture of acetone and anhydrous ethanol (95% acetone: anhydrous ethanol = 2:1) and incubate in the dark at 26°C for 24 hours. Use a spectrophotometer to measure the absorbance values ​​of chlorophyll A (maximum absorption peak 470 nm), chlorophyll B (maximum absorption peak 645 nm) and carotenoids (maximum absorption peak 663 nm), respectively. Referring to the method of Arnon (1949), calculate the chlorophyll content in each rice, and replicate three times for each treatment. At the same time, measure the plant height, root length and fresh weight of each rice. The results are as follows: Figure 2 shown.

[0035] (3) The results showed that after 8 days of salt treatment, the degree of leaf wilting, curling and yellowing of the two OsFLZ14 gene-edited mutant rice was significantly lower than that of the wild-type rice Nipponbare, indicating that the OsFLZ14 gene-edited mutant rice is more tolerant to salt.

[0036] In summary, knocking out the OsFLZ14 gene in rice makes rice insensitive to salt stress and increases its salt tolerance.

[0037] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

[0038] The sequence table involved in the present invention is as follows: SEQ ID NO.1: OsFLZ14 gene nucleotide sequence ATGGCGCGCTACCACCCCACCTCCATCCACGACGACATCGAGGCCGGCTTCTCCGGCCACTCCGCCTCCCCCGTGAAGCCCGCGGCGTCGCCTCGGCGGCCCGGCGGCCGCCTCTTCTGCGACCCGTGCGACGACGCCGACGACCTCCTCGGCCACCACCACTACCTCGACATCTGCTTCCGCTGCCGGAGACCCCTCGGAGGAAACAGAGACATCTTCATGTACAGAGGTGACATGCCCTTCTGCAGCGAGGAGTGCAGGCAGGAGCAGATCGAGATCGACGAGGCGAGAGAGCAGAGATCGAAGCAGACGGGGAGGGCAGAGCAGGAGAGGCAGAGGCAGCAGAAGGCGAGCCACCCGAGGATCCCGGTCTGGGCATGGTAA SEQ ID NO.2: Amino acid sequence of the protein encoded by the OsFLZ14 gene MARYHPTSIHDDIEAGFSGHSASPVKPAASPRRPGGRLFCDPCDDADDLLGHHHYLDICFRCRRPLGGNRDIFMYRGDMPFCSEECRQEQIEIDEAREQRSKQTGRAEQERQRQQKASHPRIPVWAW* SEQ ID NO.3: Amino acid sequence of the cr-osflz14-1 gene ATGGCGCGCTACCACCCCACCTCCATCCACGACGACATCGAGGCCGGCTTCTCCGGCCACTCCGCCTCCCCCGTGAAGCCCGCGGCGTCGCCTCGGCGGCCCGGCGGCCGCCTCTTTCTGCGACCCGTGCGACGACGCCGACGACCTCCTCGGCCACCACCACTACCTCGACATCTGCTTCCGCTGCCGGAGACCCCTCGGAGGAAACAGAGACATCTTCATGTACAGAGGTGACATGCCCTTCTGCAGCGAGGAGTGCAGGCAGGAGCAGATCGAGATCGACGAGGCGAGAGAGCAGAGATCGAAGCAGACGGGGAGGGCAGAGCAGGAGAGGCAGAGGCAGCAGAAGGCGAGCCACCCGAGGATCCCGGTCTGGGCATGGTAA SEQ ID NO.4: Amino acid sequence of the cr-osflz14-2 gene ATGGCGCGCTACCACCCCACCTCCATCCACGACGACATCGAGGCCGGCTTCTCCGGCCACTCCGCCTCCCCCGTGAAGCCCGCGGCGTCGCCTCGGCGACCCGTGCGACGACGCCGACGACCTCCTCGGCCACCACCACTACCTCGACATCTGCTTCCGCTGCCGGAGACCCCTCGGAGGAAACAGAGACATCTTCATGTACAGAGGTGACATGCCCTTCTGCAGCGAGGAGTGCAGGCAGGAGCAGATCGAGATCGACGAGGCGAGAGAGCAGAGATCGAAGCAGACGGGGAGGGCAGAGCAGGAGAGGCAGAGGCAGCAGAAGGCGAGCCACCCGAGGATCCCGGTCTGGGCATGGTA。

Claims

1. Application of OsFLZ14 gene in rice salt stress tolerance, characterized in that: The nucleotide sequence of the OsFLZ14 gene is shown in SEQ ID NO.

1. The OsFLZ14 gene negatively regulates the salt tolerance of rice.

2. The use according to claim 1, characterized in that: Knocking out the OsFLZ14 gene in rice improves rice's tolerance to salt stress.

3. A method for improving rice's tolerance to salt stress, characterized in that: The method specifically comprises: knocking out the OsFLZ14 gene in the target rice germplasm genome, wherein the nucleotide sequence of the OsFLZ14 gene is shown in SEQ ID NO.

1.

4. A rice salt stress tolerance gene mutant, characterized in that: The rice salt stress tolerance gene mutant is any one of the following: (a) a mutant cr-osflz14-1, in which a single base T is inserted at positions 116-117 in the first exon of the nucleotide sequence shown in SEQ ID NO.1; the nucleotide sequence of the mutant cr-osflz14-1 is shown in SEQ ID NO.3; (b) The mutant cr-osflz14-2 has the bases GCCCGGCGGCCGCCTCTTCTGCG deleted at positions 99-121 in the first exon of the nucleotide sequence shown in SEQ ID NO.1; the nucleotide sequence of the mutant cr-osflz14-2 is shown in SEQ ID NO.

4.

5. A method for obtaining the rice salt stress tolerance gene mutant according to claim 4, characterized in that: The method is specifically as follows: CRISPR / Cas9 technology was used to perform site-directed gene editing on the exons of the rice OsFLZ14 gene, and two mutants of different editing types of the OsFLZ14 gene were obtained. Both mutants showed a salt-tolerant phenotype. The designed target is: CTTCTGCGACCCGTGCGACG.

6. A recombinant vector containing the rice salt stress tolerance gene mutant according to claim 4.

7. Use of the rice salt stress tolerance gene mutant according to claim 4 in breeding salt stress tolerance rice.

8. A rice breeding method, characterized in that: The method specifically comprises: knocking out the OsFLZ14 gene in rice, thereby improving the salt stress tolerance of rice.

9. A method for preparing a transgenic plant, characterized in that: The method comprises the following steps: knocking out the OsFLZ14 gene in a starting plant to obtain a transgenic plant with increased salt stress tolerance compared with the starting plant; the nucleotide sequence of the OsFLZ14 gene is shown in SEQ ID NO.1.

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