Application of OsSTA230 protein and its encoding gene in regulating salt tolerance in rice

Abstract

This invention relates to the field of rice genetic engineering, disclosing the application of the OsSTA230 protein and its encoding gene in regulating salt tolerance in rice. This invention uses CRISPR / Cas9 technology to edit the OsSTA230 protein gene, introduces it into the japonica rice variety Zhonghua 11 using Agrobacterium-mediated transformation, and screens to obtain knockout mutants; experimental results demonstrate the effectiveness of the knockout mutant. OsSTA230 The rice mutant of the gene, after being treated with 150 mM NaCl and 200 mM NaCl for 11 days at the 2-leaf-1-heart stage and then rehydrated for 7 days, showed a significantly higher survival rate than the wild-type Zhonghua 11 plants. This provides a simple and effective technical means for rapidly creating new salt-tolerant rice lines. Therefore, the OsSTA230 protein and its encoding gene of this invention can regulate the salt tolerance of rice, which is of great significance for breeding 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:

[0007] The first aspect of this invention provides the application of genes that knock out the OsSTA230 protein, said application being any of the following:

[0008] A1) Application in improving salt tolerance in rice;

[0009] A2) use in preparing a product for improving salt tolerance of rice;

[0010] A3) use in breeding salt-tolerant rice;

[0011] A4) use in preparing a product for breeding salt-tolerant rice;

[0012] A5) use in salt-tolerant breeding of rice or improvement of salt-tolerant germplasm of rice;

[0013] The amino acid sequence of the OsSTA230 protein is shown in SEQ ID NO. 3.

[0014] The second aspect of the present application provides use of a biological material related to the OsSTA230 protein, and the use is any one of the following:

[0015] B1) use in improving salt tolerance of rice;

[0016] B2) use in preparing a product for improving salt tolerance of rice;

[0017] B3) use in breeding salt-tolerant rice;

[0018] B4) use in preparing a product for breeding salt-tolerant rice;

[0019] B5) use in salt-tolerant breeding of rice or improvement of salt-tolerant germplasm of rice;

[0020] The biological material is any one of the following C1) to C3):

[0021] C1) a nucleic acid molecule that inhibits or reduces expression of a gene encoding the OsSTA230 protein;

[0022] C2) an expression cassette containing the nucleic acid molecule of C1);

[0023] C3) a recombinant vector containing the nucleic acid molecule of C1), or a recombinant vector containing the expression cassette of C2);

[0024] C4) a recombinant microorganism containing the nucleic acid molecule of C1), or a recombinant microorganism containing the expression cassette of C2), or a recombinant microorganism containing the recombinant vector of C3), and the microorganism is Agrobacterium.

[0025] Further, the nucleotide sequence of the nucleic acid molecule encoding the OsSTA230 protein is shown in SEQ ID NO. 2.

[0026] The third aspect of the present application provides a method for improving salt tolerance of rice, the method comprising knocking out the gene of OsSTA230 protein in rice, obtaining rice with improved salt tolerance, and the amino acid sequence of the OsSTA230 protein is shown as SEQ ID NO. 3.

[0027] In the above method, the expression amount of the coding gene of the OsSTA230 protein is reduced by using gene knockout technology.

[0028] In the above method, the expression amount of the coding gene of the OsSTA230 protein is reduced by using gene knockout technology, which is realized by using the CRISPR / Cas9 system containing the target site shown as SEQ ID NO. 5 to directionally knock out the gene of the OsSTA230 protein.

[0029] The fourth aspect of the present application provides a breeding method of a salt-tolerant rice strain, comprising: using the salt-tolerant rice obtained by the above method as a parent to cross with a target material, and backcrossing the obtained F1 generation with the target material to obtain a backcross progeny with the same salt tolerance as the parent.

[0030] Further, the backcross progeny with salt tolerance and the parent have the same mutant gene; the OsSTA230 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the mutant gene is OsSTA230 An "A" base is inserted at the 3750th base from the start codon ATG of the gene, and the nucleotide sequence is shown as SEQ ID NO. 4.

[0031] The beneficial effects of the present application are:

[0032] (1) The present application first discovers and confirms that OsSTA230 The gene negatively regulates the salt tolerance of rice. Inhibiting or knocking out the gene can enhance the salt tolerance of rice. The present application provides a new perspective and target for the molecular mechanism of rice response to salt stress, and enriches the theoretical basis of plant salt tolerance.

[0033] (2) The present application specifically provides a method for precisely knocking out OsSTA230 The gene of the gene editing technology of CRISPR / Cas9, including a specific target site (SEQ ID NO. 5) and a corresponding targeting vector. The method has clear target, high efficiency and short cycle, and overcomes the bottleneck of large selection difficulty and long cycle in traditional breeding.

[0034] (3) By the above method, a OsSTA230Gene knockout mutant. Strict physiological experiments prove that the survival rate (58.33%) of the mutant is significantly higher than that of the wild type (8.33%) after 150 mM and 200 mM NaCl salt stress treatment. This provides genetic material and gene resources with direct application value for rice salt-tolerant breeding.

[0035] (4) The application not only provides a method for creating a core salt-tolerant germplasm, but also clearly defines a breeding strategy for rapidly introducing the salt-tolerant trait into other excellent rice varieties through hybridization and backcrossing. This means that the technical achievement can be quickly applied to practical production to cultivate new rice varieties suitable for planting in saline-alkali land. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 For OsSTA230 Gene expression pattern analysis chart

[0037] Figure 2 For OsSTA230 Gene structure and CRISPR / Cas- OsSTA230 Vector target sequence element schematic diagram

[0038] Figure 3 For sequencing result peak chart; wherein the upper chart is the wild type ZH11 target point sequencing peak chart, and the lower chart is the mutant ossta230 Target point sequencing peak chart.

[0039] Figure 4 For ossta230 Mutant salt tolerance detection. A is the growth state of ZH11 and ossta230 Plants before and after salt treatment, bar = 5 cm; B is the survival rate of ZH11 and ossta230 Before and after salt treatment. The values shown are the mean ± standard deviation, n = 3. *, significant difference, P < 0.05; **, extremely significant difference, P < 0.01, and the statistical analysis method is one-way analysis of variance. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the embodiments in the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of the present application.

[0041] Unless otherwise specified, the following experimental methods can be performed by conventional methods in the art. Unless otherwise specified, commercially available materials can be used for the following experimental materials.

[0042] CRISPR / Cas vector BGK03: Hangzhou Baige Biotechnology Co., Ltd., product catalog number BGK03.

[0043] Example 1

[0044] This embodiment provides rice. OsSTA230 The functions and applications of genes include the following:

[0045] 1. Rice OsSTA230 Gene sequence and expression pattern analysis

[0046] The Ensembl Plants database (http: / / plants.ensembl.org / index.html) contained information on rice. OsSTA230 The 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.

[0047] 2. Rice OsSTA230 Functional verification of genes

[0048] 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 ).

[0049] (1) OsSTA230 Construction of CRISPR / Cas9 gene editing vector

[0050] 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:

[0051] i) Design of target gRNA. The gene sequence of OsSTA230 was input into https: / / zlab.bio / guide-design-resources for target design, and the PAM sequence was set as NGG. The DNA sequence of the target region selected by the present application is shown as SEQ ID NO. 5.

[0052] ii) Amplification of sgRNA expression cassette by overlap PCR and nested PCR. A primer pair containing the sgRNA target sequence described above was synthesized, and the primer pair was annealed and then ligated with the Bsa I digested 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, the vector pEGCas9Pubi-B was kindly presented by Long Tan of Hainan University) to obtain the recombinant vector pEGCas9Pubi- OsSTA230 . The recombinant vector pEGCas9Pubi- OsSTA230 was transformed into E. coli DH5a, and positive clones were selected for sequencing. The specific steps refer to the method 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 / Cas9 toolkit for multiplex genome editing in plants. BMC plant biology 14: 327."

[0053] iii) Sequencing verification.

[0054] The pEGCas9Pubi- OsSTA230 constructed successfully was verified by sequencing.

[0055] (2) Agrobacterium-mediated genetic transformation of rice

[0056] The pEGCas9Pubi- OsSTA230The vector was transformed into Agrobacterium EHA105 by heat shock method, and the bacterial solution was stored at -80°C after PCR identification. Freshly peeled 1.5 mm rice hybrid Zhonghua 11 embryo was used as the receptor material. The peeled rice embryo was placed in a 2 mL plastic centrifuge tube containing 1.8 mL of suspension, and the placement time was not more than 1 hour, about 100 embryos were placed in each centrifuge tube; the suspension was sucked off, and the embryos were washed with new suspension for 2 times, a small amount of suspension was reserved at the bottom of the tube which could cover the embryos, then 43°C heat shock for 2 minutes, followed by ice bath for 1 minute, the residual washing solution was sucked off with a pipette, and 1.0 mL of Agrobacterium infection solution was added, shaken for 30 seconds, then placed in the dark for 8 minutes. Next, the embryos and the infection solution in the centrifuge tube were poured onto the co-culture medium, shaken evenly, and the excess infection solution was sucked out with a pipette, all the embryo scutes were placed upwards, and co-cultured at 23°C in the dark for 3 days. After co-culture, the embryos were transferred to recovery medium with sterile forceps, cultured at 28°C for 7-14 days, and the young shoots growing on the embryos were removed in time during the process. After recovery culture, the embryos were placed on 1.5 mg / L Bialaphos selection medium for 3 rounds of selection, 2 weeks for each round, then transferred to 2 mg / L Bialaphos selection medium for 2 rounds of selection, 2 weeks for each round. The resistant callus was transferred to the expansion medium and cultured at 28°C in the dark for 2 weeks. Then the expanded resistant callus was transferred to the induction medium and cultured at 28°C in the dark for 2 weeks. Then transferred to the differentiation medium, 25°C, 5000 lx, light culture for 2 weeks. After culture, the differentiated seedlings were separated into single seedlings and placed in rooting medium, 25°C, 5000 lx, light culture until rooting; the seedlings were transferred to small pots for growth, and after growth and survival, they were transplanted to the greenhouse, and the offspring seeds were harvested after 3-4 months.

[0057] (3) T0 generation plant CRISPR / Cas9 mutation result detection

[0058] To determine the T0 generation plant CRISPR / Cas9 mutation results, the following steps were taken for detection:

[0059] The application first extracts rice leaf DNA by CTAB method, and the specific method is as follows: the DNA extraction method refers to the traditional CTAB method (Rogers and Bendich, 1985). Put 3 cm rice leaves into a sterilized 2 mL centrifuge tube, add 6 mm steel balls, use a cell crusher to crush the tissue, then extract by CTAB method, finally add 200 μL of sterilized water (ddH2O) to the extracted sample, dissolve the air-dried sample DNA, and wait for use. After the DNA is completely dissolved, 2 μL of sample is taken for nucleic acid OD value (A260 / A280) and nucleic acid concentration determination by ultraviolet spectrophotometer (Nanodrop 2000), and the DNA sample is diluted to 50 ng / μL for standby.

[0060] PCR uses 5 μL of 2×PCR premix (containing Mg 2+ ; Taq DNA Polymerase; 2.5 mM dNTPs; 10×PCR Buffer) of Biomiga, 1 μL of primer (containing 0.5 μL of forward and reverse primer), 1 μL of template DNA, and ddH2O to make up 10 μL. The PCR amplification program is the conventional 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 of amplification, and finally 72℃ extension for 5 min). The amplified product is electrophoresed on an 8% non-denaturing polyacrylamide gel, stained with 0.1% AgNO3, and developed with formaldehyde and NaOH for photography.

[0061] Mutant ossta230 The nucleotide sequence is analyzed to find that Figure 2 , compared with the unedited wild type (WT), the mutation of ossta230 inserts an A base at the 3750th base from the start codon ATG, and the sequence is shown as 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.

[0062] Example 2

[0063] In this embodiment, the mutant obtained in Example 1 is subjected to phenotype analysis, and the specific method is as follows: ossta230

[0064] 1. Salt tolerance identification of mutant ossta230

[0065] The parameters of light and dark alternating culture are as follows: light intensity is 120 μmol·m -2 ·s -1 , temperature is 28℃ / 25℃ (day / night), and light period is 10 h light / 14 h dark. ​​

[0066] The rice seeds to be tested are ossta230 - L1 Mutant T1 generation homozygous seeds and their background material ZH11 and empty control. The experiment is repeated 3 times to take average value, and the steps of each repeat are as follows:

[0067] 1. For each material, 12 rice seeds to be tested are taken and packed in kraft paper bags, and then soaked in water at 28-30°C for 48h.

[0068] 2. After step 1 is completed, the seeds are germinated at 28-30°C for 24h (the seeds need to be kept moist during the germination process), and germinated seeds are obtained.

[0069] 3. After step 2 is completed, 96-well plates are taken, the lower edge of each well is cut, and then 1 germinated seed is placed in each well (embryo bud upward and radicle downward).

[0070] 4. After step 3 is completed, the 96-well plates (with germinated seeds thereon) are placed on a plastic box containing Yoshida rice culture solution and the germinated seeds are immersed in the culture solution, and light and dark alternating culture is carried out for 3 weeks to obtain rice seedlings grown to the three-leaf stage. Yoshida rice culture solution needs to be replaced every 7d during the light and dark alternating culture.

[0071] 5. After step 4 is completed, the 96-well plates (with rice seedlings grown to the three-leaf stage thereon) are placed on a plastic box containing 150mM NaCl Yoshida rice culture solution and the roots are completely immersed in the culture solution, and after 3d of treatment, 200mM NaCl Yoshida rice culture solution is replaced for treatment, and after 3d, 150mM NaCl Yoshida rice culture solution is replaced for treatment, and high-salt stress is carried out for 11d under light and dark alternating culture (Yoshida rice culture solution is replaced every 2d during the high-salt stress).

[0072] 6. After step 5 is completed, the 96-well plates (with rice seedlings thereon) are placed on a plastic box containing Yoshida rice culture solution, and recovery is carried out for 7d under light and dark alternating culture.

[0073] The growth state of the rice seedlings is observed and the survival rate is counted. Survival rate = number of surviving rice seedlings / 12 x 100%.

[0074] The growth state of the rice seedlings before treatment is shown in Figure 4 A, and the growth state after treatment is shown in Figure 4 B. The survival rate counting result is shown in Figure 4 C.

[0075] The results show that, before salt treatment, ossta230Slightly shorter than ZH11; survival rate of fresh water control ZH11 and mutant ossta230 was 100%. After salt solution treatment, the survival rate of ZH11 was 8.33%, while the survival rate of the mutant ossta230 was 58.33%, and the statistical analysis result showed that ossta230 the survival rate of the mutant 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 control were basically consistent with the background material ZH11, and there was no statistical difference.

Claims

1. Application of a gene for knocking out OsSTA230 protein in improving salt tolerance of rice, wherein the amino acid sequence of the OsSTA230 protein is shown as SEQ ID NO. 3; and the nucleotide sequence of the mutated gene is shown as SEQ ID NO.

4. OsSTA230 ​ 2. Use according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the OsSTA230 protein is shown as SEQ ID NO.

2.

3. A method for improving salt tolerance in rice, the method comprising, The method comprises knocking out the gene of the OsSTA230 protein in rice, the nucleotide sequence of the mutated gene is shown as SEQ ID NO. 4, obtaining rice with improved salt tolerance, and the amino acid sequence of the OsSTA230 protein is shown as SEQ ID NO.

3.

4. The method of claim 3, wherein, The gene of the OsSTA230 protein in rice is knocked out by using a gene knockout technology to reduce the expression amount of the coding gene of the OsSTA230 protein.

5. The method of claim 4, wherein, The expression amount of the coding gene of the OsSTA230 protein is reduced by using a CRISPR / Cas9 system to achieve directional knockout of the gene of the OsSTA230 protein, and the target site of the CRISPR / Cas9 system is shown as SEQ ID NO. 5.

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