Application of OsSTA230 protein and coding gene thereof in regulating and controlling salt tolerance of rice

Abstract

The invention relates to the field of rice gene engineering, and discloses an application of OsSTA230 protein and a coding gene thereof in regulating and controlling the salt tolerance of rice. The gene of the OsSTA230 protein is edited on the basis of a CRISPR / Cas9 technology, an agrobacterium-mediated method is utilized to introduce a japonica rice variety Zhonghua 11, and a knockout mutant is obtained through screening; tests prove that the survival rate of the rice mutant with the OsSTA230 gene knocked out is remarkably higher than that of a wild type Zhonghua 11 plant after the rice mutant is subjected to stress treatment for 11 days by 150 mM NaCl and 200 mM NaCl salt and then is rehydrated for 7 days after the rice mutant is subjected to two-leaf and one-core stage. And a simple and effective technical means is provided for rapidly creating a new salt-tolerant rice strain. Therefore, the OsSTA230 protein and the coding gene thereof disclosed by the invention can regulate and control the salt tolerance of the rice, and are of great significance to the cultivation of salt-tolerant transgenic rice.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically the application of the OsSTA230 protein and its encoding gene in regulating salt tolerance in rice. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops, feeding more than half of the world's population. However, rice is frequently subjected to various abiotic stresses throughout its growth process, such as salinity, drought, flooding, and extreme temperatures. Among these, salinity stress is one of the most significant abiotic stresses threatening rice production.

[0003] Soil salinization has led to a reduction in arable land, contributing to the food crisis. Faced with a growing population, limited available arable land, and increasingly severe secondary soil salinization due to inefficient irrigation, making it difficult to significantly increase rice yields, developing and utilizing coastal and inland saline-alkali land resources is an effective way to ensure arable land availability. Rice is a moderately salt-sensitive crop, growing in aquatic environments; rice cultivation can leach soluble salts and alkalis from the soil. Therefore, rice is the preferred food crop for developing coastal and saline-alkali land.

[0004] Improving rice's salt tolerance through genetic modification is one of the effective ways to increase rice planting area and yield. Currently, the salt-tolerant QTLs used in breeding are mainly located at two loci on rice chromosome 1: qSKC-1 and Saltol. With the development of molecular biotechnology, using mutants to isolate and discover salt-stress-tolerant genes in rice, and then using them for rice genetic engineering for assisted breeding and alkali-stress improvement, is of paramount importance for effectively controlling the damage of salt stress to rice, increasing rice yield, and improving rice quality. OsSTA230 Encoding a monovalent cation transporter: belonging to the proton antiporter-2 family, currently no information is available regarding... OsSTA230 Reports on improving rice's resistance to salt stress. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the OsSTA230 protein and its encoding gene in regulating rice salt tolerance and in creating salt-tolerant rice lines.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention provides the application of genes that knock out the OsSTA230 protein, said application being any of the following: A1) Application in improving salt tolerance in rice; A2) Application in the preparation of products that improve the salt tolerance of rice; A3) Application in the cultivation of salt-tolerant rice; A4) Application in the preparation of products for cultivating salt-tolerant rice; A5) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources; The amino acid sequence of the OsSTA230 protein is shown in SEQ ID NO. 3.

[0007] A second aspect of the present invention provides the application of biomaterials related to the OsSTA230 protein, wherein the application is any of the following: B1) Application in improving salt tolerance in rice; B2) Application in the preparation of products that improve the salt tolerance of rice; B3) Application in the cultivation of salt-tolerant rice; B4) Application in the preparation of products for cultivating salt-tolerant rice; B5) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources; The biomaterial is any one of the following C1) to C3): C1) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the OsSTA230 protein; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3), wherein the microorganism is Agrobacterium.

[0008] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the OsSTA230 protein is shown in SEQ ID NO. 2.

[0009] A third aspect of the present invention provides a method for improving the salt tolerance of rice, the method comprising knocking out the gene for the OsSTA230 protein in rice to obtain rice with improved salt tolerance, wherein the amino acid sequence of the OsSTA230 protein is shown in SEQ ID NO. 3.

[0010] In the above method, knocking out the OsSTA230 protein gene in rice involves using gene knockout technology to reduce the expression level of the gene encoding the OsSTA230 protein.

[0011] In the above method, the expression level of the gene encoding the OsSTA230 protein is reduced by using gene knockout technology to perform targeted knockout of the OsSTA230 protein gene using a CRISPR / Cas9 system containing the target site shown in SEQ ID NO.5.

[0012] The fourth aspect of the present invention provides a method for breeding salt-tolerant rice varieties, comprising: using the salt-tolerant rice obtained by the above method as a parent, hybridizing it with a target material, and backcrossing the obtained F1 generation with the target material so that the backcross offspring acquire the same salt tolerance trait as the parent.

[0013] Furthermore, the salt-tolerant backcross offspring and the parents possess the same mutated gene; OsSTA230 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the mutated gene is... OsSTA230 The gene inserts an "A" base at position 3750, starting from the start codon ATG, and its nucleotide sequence is shown in SEQ ID NO. 4.

[0014] The beneficial effects of this invention are: (1) This invention is the first to discover and confirm that OsSTA230 This gene negatively regulates salt tolerance in rice. Inhibiting or knocking out this gene actually enhances the salt tolerance of rice. This invention provides a new perspective and target for the molecular mechanism of rice's response to salt stress, enriching the theoretical basis of plant salt tolerance.

[0015] (2) Specifically, this invention provides a method for precise knockout using CRISPR / Cas9 gene editing technology. OsSTA230 The gene-based approach includes specific target sites (SEQ ID NO.5) and corresponding targeting vectors. This method is targeted, efficient, and quick, overcoming the bottlenecks of traditional breeding where selecting for salt tolerance traits is difficult and time-consuming.

[0016] (3) Through the above methods, we successfully obtained OsSTA230 Gene knockout mutants. Rigorous physiological experiments demonstrated that after treatment with 150 mM and 200 mM NaCl salt stress, the survival rate of the mutants (58.33%) was significantly higher than that of the wild type (8.33%). This provides genetic material and gene resources with direct application value for salt-tolerant rice breeding.

[0017] (4) This invention not only provides a method for creating core salt-tolerant germplasm, but also clarifies a breeding strategy for rapidly introducing this salt-tolerant trait into other superior rice varieties through hybridization and backcrossing. This means that the technological achievement can be quickly applied to actual production to cultivate new rice varieties adapted to saline-alkali land. Attached Figure Description

[0018] Figure 1 for OsSTA230 Gene expression pattern analysis diagram Figure 2 for OsSTA230 Gene structure and CRISPR / Cas- OsSTA230 Schematic diagram of carrier target sequence elements Figure 3 These are peak diagrams of sequencing results; the top diagram shows the sequencing peaks of the wild-type ZH11 target, and the bottom diagram shows the peaks of the mutant. ossta230 Target sequencing peak diagram.

[0019] Figure 4 for ossta230 Salt tolerance test of mutants. A represents ZH11 and... ossta230 Plant growth status before and after salt treatment, bar = 5cm; B represents ZH11 and ossta230 Survival rates before and after salt treatment were statistically analyzed. Values ​​shown are mean ± standard deviation, n = 3. * indicates significant difference (P < 0.05); ** indicates highly significant difference (P < 0.01). One-way ANOVA was used for statistical analysis. Detailed Implementation To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0020] Unless otherwise specified, the experimental methods described below can be performed using conventional methods in this field. Unless otherwise specified, commercially available materials can be used for the experiments described below.

[0021] CRISPR / Cas vector BGK03: Hangzhou Baige Biotechnology Co., Ltd., product catalog number BGK03. Example 1

[0022] This embodiment provides rice. OsSTA230 The functions and applications of genes include the following: 1. Rice OsSTA230 Gene sequence and expression pattern analysis The Ensembl Plants database (http: / / plants.ensembl.org / index.html) contained information on rice. OsSTA230The nucleotide sequence of the gene in *Zhonghua 11* of japonica rice is shown in SEQ ID NO.1, and the CDS sequence is shown in SEQ ID NO.2. Its encoded protein contains 279 amino acids, and its sequence is shown in SEQ ID NO.3. To study the function of this gene, this invention first analyzed the expression pattern of this gene in different tissues of rice using a gene expression database. Figure 1 The expression analysis results show OsSTA230 The gene is specifically expressed in the rice panicle, suggesting that it may be involved in the development of the rice panicle.

[0023] 2. Rice OsSTA230 Functional verification of genes To clarify OsSTA230 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. This invention selects the conventional rice variety ZH11 as the recipient material for gene editing. This invention selects the nucleotide sequence from base 3735 to base 3757 of the gene coding region, starting from the start codon ATG (as shown in SEQ ID NO. 5), as the target region for CRISPR / Cas9 gene editing (see [link to target region]). Figure 2 ).

[0024] (1) OsSTA230 Construction of CRISPR / Cas9 gene editing vector The gene editing vector of this invention is pEGCas9Pubi-B- OsSTA230 The base vector for this vector is pEGCas9Pubi-B. This invention involves designing target sites on primers, obtaining MT-sgRNA via PCR, and then ligating it into the base vector using a one-step cloning method. The specific construction process is as follows: i) Design of target gRNA. [The following is likely a separate, unrelated sentence:] OsSTA230 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 sequence of the target region selected in this invention is shown in SEQ ID NO.5.

[0025] ii) Amplification of the sgRNA expression cassette by overlap PCR and nested PCR. Primer pairs containing the above-mentioned sgRNA target sequences were synthesized and annealed. Then, the primer pairs were ligated with the binary vector pEGCas9Pubi-B (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, Mol Plant. 2015, 8(8):1274-1284, vector pEGCas9Pubi-B was kindly provided by Professor Long Tuan of Hainan University) to obtain the recombinant vector pEGCas9Pubi- OsSTA230 The recombinant vector pEGCas9Pubi- OsSTA230 Transformed E. coli DH5α, selected positive clones for sequencing, and followed the specific steps in the reference “Xing, H.L., Dong, L., Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., and Chen, Q.J. (2014). A CRISPR / Cas9toolkit for multiplex genome editing in plants. BMC plant biology 14:327.”

[0026] iii) Sequencing verification.

[0027] pEGCas9Pubi- was validated by sequencing. OsSTA230 Successfully built.

[0028] (2) Agrobacterium-mediated genetic transformation of rice The pEGCas9Pubi- constructed above OsSTA230The vector was transferred into Agrobacterium EHA105 via heat shock. After PCR identification, the bacterial culture was stored at -80 °C with glycerol. Freshly peeled embryos (approximately 1.5 mm in diameter) of the hybrid rice variety Zhonghua 11 were used as recipient material. The peeled embryos were placed in 2 mL plastic centrifuge tubes containing 1.8 mL of suspension for no more than one hour, with approximately 100 embryos per tube. The suspension was removed, and the embryos were washed twice with fresh suspension, leaving a small amount at the bottom of the tube to submerge the embryos. The tubes were then heat-shocked at 43 °C for 2 minutes, followed by an ice bath for 1 minute. The remaining wash solution at the bottom of the tube was aspirated, and 1.0 mL of Agrobacterium infection solution was added. The tubes were gently shaken for 30 seconds and then incubated in the dark for 8 minutes. Next, the embryos and infection solution were poured onto a co-culture medium, mixed well, and excess infection solution was aspirated with a pipette. All embryos were cultured with their scutes facing upwards at 23 °C in the dark for 3 days. After co-culture, the immature embryos were transferred to recovery medium using sterile forceps and cultured at 28°C for 7-14 days, during which time any emerging shoots should be removed promptly. After recovery culture, the immature embryos were placed on a selection medium containing 1.5 mg / L Bialaphos for three rounds of selection, each round lasting two weeks, and then transferred to a selection medium containing 2 mg / L Bialaphos for two more rounds of selection, each round lasting two weeks. The resistant callus was transferred to propagation medium and cultured in the dark at 28°C for two weeks. Subsequently, the propagated resistant callus was transferred to induction medium and cultured in the dark at 28°C for two weeks. Then, it was transferred to differentiation medium and cultured under light at 25°C and 5000 lx for two weeks. After culture, the differentiated seedlings were separated into individual seedlings and placed in rooting medium at 25°C and 5000 lx under light until rooting. The seedlings were then transferred to small nutrient pots for further growth. Once established, they were transplanted into a greenhouse, and the offspring seeds were harvested after 3-4 months.

[0029] (3) 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: This invention first employs the CTAB method to extract DNA from rice leaves. The specific method is as follows: DNA extraction is performed according to the traditional CTAB method (Rogers and Bendich, 1985). A 3 cm rice leaf is placed in a sterilized 2 mL centrifuge tube, a 6 mm steel ball is added, and the tissue is disrupted using a cell disruptor. Then, CTAB extraction is performed. Finally, 200 μL of sterile water (ddH2O) is added to dissolve the air-dried DNA sample, which is then set aside. After the DNA is completely dissolved, 2 μL of the sample is taken and the nucleic acid OD value (A260 / A280) and nucleic acid concentration are determined using a UV spectrophotometer (Nanodrop 2000). The DNA sample is then diluted to 50 ng / μL for later use.

[0030] PCR was performed using Biomiga's 2×PCR premix (containing Mg). 2+ The following reagents were used: 5 μL of Taq DNA Polymerase, 2.5 mM dNTPs, and 10×PCR Buffer; 1 μL of primers (containing 0.5 μL each of forward and reverse primers); 1 μL of template DNA; and ddH2O to a final volume of 10 μL. The PCR amplification program was a standard SSR program (94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and a final extension at 72℃ for 5 min). The amplified products were subjected to 8% non-denaturing polyacrylamide gel electrophoresis, stained with 0.1% AgNO3, and photographed after formaldehyde and NaOH staining.

[0031] For mutants ossta230 Nucleotide sequence alignment analysis revealed that ( Figure 2 Compared to the unedited wild type (WT), the mutated ossta230 An A base is inserted at position 3750 of the start codon ATG, as shown in SEQ ID NO.4. The deletion of the nucleotide encoded by the mutant causes a frameshift of the amino acid and leads to premature termination of amino acid translation. Example 2

[0032] This embodiment describes the mutant obtained in Example 1. ossta230 Phenotypic analysis was performed, as follows: 1. Mutant ossta230 Salt tolerance assessment The parameters for alternating light and dark culture are as follows: light intensity is 120 μmol·m⁻¹. -2 ·s -1 The temperature is 28℃ / 25℃ (day / dark), and the photoperiod is 10h light / 14h darkness.

[0033] The rice seeds to be tested are ossta230 - L1 Homozygous seeds of mutant T1 generation, along with its background material ZH11 and empty vector control. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows: 1. For each material, take 12 rice seeds to be tested, put them into kraft paper bags, and soak them in water at 28℃~30℃ for 48 hours.

[0034] 2. After completing step 1, germinate the seeds at 28℃~30℃ for 24 hours (keep the seeds moist during germination) to obtain germinated seeds.

[0035] 3. After completing step 2, take a 96-well plate, cut off part of the lower edge of each well, and then put one germinated seed into each well (embryo facing up, radicle facing down).

[0036] 4. After completing step 3, place the 96-well plate (containing the germinated seeds) on a plastic box containing Yoshida rice culture medium, immersing the germinated seeds in the medium. Culture in alternating light and dark conditions for 3 weeks to obtain rice seedlings that have reached the three-leaf stage. During the alternating light and dark culture period, the Yoshida rice culture medium should be replaced every 7 days.

[0037] 5. After completing step 4, place the 96-well plate (containing rice seedlings that have grown to the three-leaf stage) on a plastic box containing 150 mM NaCl Yoshida rice culture solution and ensure that the roots are completely immersed in the culture solution. After 3 days of treatment, replace the Yoshida rice culture solution with 200 mM NaCl and treat again. After 3 days, replace the Yoshida rice culture solution with 150 mM NaCl and treat again. Perform high salt stress for 11 days under alternating light and dark conditions (during the high salt stress period, replace the Yoshida rice culture solution every 2 days).

[0038] 6. After completing step 5, place the 96-well plate (with rice seedlings on it) on a plastic box containing Yoshida rice culture medium and allow it to recover for 7 days under alternating light and dark conditions.

[0039] Observe the growth status of rice seedlings and calculate the survival rate. Survival rate = (Number of surviving rice seedlings / 12) × 100%.

[0040] See the growth status of rice seedlings before treatment. Figure 4 The growth status after treatment A is shown in the figure. Figure 4 The survival rate statistics for B are shown in the table below. Figure 4 C.

[0041] The results showed that before salt treatment, ossta230 Slightly shorter than ZH11; freshwater control ZH11 and mutant ossta230 The survival rate was 100%. After salt solution treatment, the survival rate of ZH11 was 8.33%, while that of the mutant... ossta230 The survival rate was 58.33%, and statistical analysis results showed that... ossta230 The survival rate of [the strain] was significantly higher than that of ZH11, indicating that... ossta230 The salt tolerance of the mutant was significantly improved. The phenotype and survival rate of the empty vector control were basically consistent with those of the background material ZH11, with no statistical difference.

Claims

1. The application of the gene for knocking out the OsSTA230 protein, characterized in that, The application is any one of the following: A1) Application in improving salt tolerance in rice; A2) Application in the preparation of products that improve the salt tolerance of rice; A3) Application in the cultivation of salt-tolerant rice; A4) Application in the preparation of products for cultivating salt-tolerant rice; A5) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources; The amino acid sequence of the OsSTA230 protein is shown in SEQ ID NO.

3.

2. The application of biomaterials related to the OsSTA230 protein described in claim 1, characterized in that, The application is any one of the following: B1) Application in improving salt tolerance in rice; B2) Application in the preparation of products that improve the salt tolerance of rice; B3) Application in the cultivation of salt-tolerant rice; B4) Application in the preparation of products for cultivating salt-tolerant rice; B5) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources; The biomaterial is any one of the following C1) to C3): C1) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the OsSTA230 protein as described in claim 1; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3), wherein the microorganism is Agrobacterium.

3. The application according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the OsSTA230 protein is shown in SEQ ID NO.

2.

4. A method for improving the salt tolerance of rice, characterized in that, The method involves knocking out the gene for the OsSTA230 protein in rice to obtain rice with improved salt tolerance, the amino acid sequence of which is shown in SEQ ID NO.

3.

5. The method according to claim 4, characterized in that, The gene that knocks out the OsSTA230 protein in rice is obtained by using gene knockout technology to reduce the expression level of the gene encoding the OsSTA230 protein.

6. The method according to claim 5, characterized in that, The reduction in the expression level of the gene encoding the OsSTA230 protein using gene knockout technology is achieved by using a CRISPR / Cas9 system containing the target site shown in SEQ ID NO.5 to perform targeted knockout of the OsSTA230 protein gene.

7. A method for breeding salt-tolerant rice varieties, characterized in that, include: Using the salt-tolerant rice obtained by the method of any one of claims 4-6 as the parent, hybridize it with the target material, and then backcross the F1 generation obtained with the target material to obtain backcross offspring with salt tolerance.

8. The breeding method according to claim 7, characterized in that, The salt-tolerant backcross offspring and the parent have the same... OsSTA230 Mutated genes; the aforementioned OsSTA230 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the nucleotide sequence of the mutant gene is shown in SEQ ID NO.4.

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