Application of OsSAK1 gene in regulating and controlling salt stress resistance of crops

Abstract

The invention relates to the technical field of agricultural biology, in particular to application of an OsSAK1 gene to regulation and control of salt stress resistance of crops. The sequence of the OsSAK1 gene provided by the invention is as shown in SEQ ID NO. 1. It is found that crops can reduce the transcription level of the OsSAK1 gene in a salt stress environment, compared with common plants, plants with OsSAK1 gene mutation have higher survival rate and better growth vigor in the salt stress environment, and plants with OsSAK1 gene overexpression are easier to die in the salt stress environment. Therefore, the effect of the OsSAK1 gene as a negative regulatory factor in regulating and controlling the salt stress resistance of crops is verified, and a key molecular tool is provided for developing new varieties of crops with salt stress resistance. The gene has wide application potential and market value in the field of agricultural planting, especially in the aspects of improving the salt stress resistance of crops and cultivating salt stress resistant transgenic crops.

Description

Technical Field

[0001] The present invention relates to the field of crop genetic engineering technology, in particular to a OsSAK1 The application of genes in regulating crop salt stress tolerance. Background Art

[0002] Soil salinization is a major abiotic stress limiting agricultural food production, reducing crop yields and restricting land use. The surface soil in saline-alkali lands tends to harden, resulting in poor soil permeability, reduced nutrient content, and increased levels of ions harmful to crops. Consequently, crops growing in saline-alkali lands struggle to survive. Saline-alkali lands represent a potential "granary" for food production. If saline-alkali-tolerant crops are developed, they could potentially create a "second granary" within arable areas of saline-alkali land.

[0003] Rice is one of the most important food crops, and ensuring sufficient rice cultivation is crucial for food security. Rice is a salt-sensitive plant, currently ranked as the most salt-sensitive cereal crop. When the soluble salt concentration in the soil reaches 0.3%, rice begins to show symptoms, ultimately leading to plant death and a significant reduction in yield. As the area of ​​land affected by salt increases, rice yields are impacted to varying degrees. Improving rice salt tolerance through genetic modification is one of the most effective approaches to addressing this problem. Therefore, identifying and utilizing superior salt-tolerant rice genes and cultivating salt-tolerant varieties are of great significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a OsSAK1 The application of genes in regulating crop salt stress tolerance.

[0005] The present invention cloned OsSAK1 Genes related to salt stress tolerance have been found to play an important role in improving crop salt stress tolerance. OsSAK1 Gene expression can improve the tolerance of crops to salt stress. OsSAK1 The gene consists of 2154 bp of nucleotides, including 5'UTR, 2 exons, 1 intron and 3'UTR.

[0006] In a first aspect, the present invention provides a OsSAK1 Application of protein in regulating the salt stress tolerance of crops, OsSAK1 The amino acid sequence of the protein is at least one of the following: (1) As shown in SEQ ID NO.2.

[0007] (2) A protein derived from SEQ ID NO. 2 by replacing, deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2 and retaining the function of the amino acid sequence shown in SEQ ID NO. 2.

[0008] (3) An amino acid sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO. 2 and has an equivalent functional protein.

[0009] (4) An amino acid sequence obtained by connecting a tag, an enzyme cleavage site and / or a connecting peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences (1) to (3).

[0010] In a second aspect, the present invention provides a OsSAK1 Application of genes in improving salt tolerance of crops, OsSAK1 The nucleotide sequence of the gene is at least one of the following: (1) As shown in SEQ ID NO.1.

[0011] (2) A nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added to the nucleotide sequence shown in SEQ ID NO. 1 and the nucleotide sequence expresses a protein with the same functional properties.

[0012] (3) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO. 1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization in 0.1× SSPE containing 0.1% SDS or 0.1× SSC containing 0.1% SDS at 65°C and the membrane is washed with the solution.

[0013] (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and expresses the same functional protein.

[0014] above OsSAK1 The accession number of the gene is LOC_Os04g41160.

[0015] Preferably, by genetic engineering techniques OsSAK1 Protein content or activity to achieve the above applications In a third aspect, the present invention provides a method for improving the salt stress tolerance of crops, specifically, OsSAK1 Protein is not expressed or its expression level is reduced in crops, and the methods include but are not limited to: (1) Knockout through genetic engineering OsSAK1 genes; or (2) Knockout through genetic engineering OsSAK1 gene promoter; or (3) Change through genetic engineering OsSAK1 The base coding of the gene causes its protein to not be expressed or to be expressed at a reduced level; or (4) Reducing through genetic engineering OsSAK1 gene expression; (5) Screening for low expression OsSAK1 Protein wild-type plants and breeding; described OsSAK1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0016] The methods include RNA interference, CRISPR / Cas9, genome replacement, and constitutive promoter-driven expression, while the gene editing range can be OsSAK1 Genes and OsSAK1 Promoter and other related gene regulatory elements.

[0017] Preferably, the above method is to use CRISPR / Cas9 gene editing technology to target OsSAK1 The genetically designed SG sequence is connected to the gene editing vector, and then the OsSAK1 The gene editing vector of the gene SG sequence is transformed into the crop cells to obtain OsSAK1 Gene-edited mutant plants.

[0018] In a fourth aspect, the present invention provides a mutant, wherein the nucleotide sequence of the mutant is as shown in SEQ ID NO.7, but the 20th base from the 5' end is deleted; or As shown in SEQ ID NO.7, a T is inserted at the 20th base from the 5' end.

[0019] The present invention further provides the mutant OsSAK1 Application of protein inactivation to improve salt tolerance of crops.

[0020] Preferably, the recombinant gene editing vector is transformed into rice cells using any one of direct DNA transformation, microinjection, gene gun, electroporation and Agrobacterium-mediated methods, and transgenic plants are obtained by inducing callus.

[0021] The present invention also provides a method for increasing the sensitivity of crops to salt stress, wherein the method overexpresses OsSAK1 gene, the OsSAK1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0022] In a fifth aspect, the present invention provides a method for increasing OsSAK1 The method for increasing protein expression is to grow the corresponding crops in a salt stress environment. OsSAK1 The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0023] In a sixth aspect, the present invention provides the above OsSAK1 Use of a gene, or a protein encoded by the gene, or a biological material containing the gene, or a method for improving the salt stress tolerance of crops in any of the following: (1) Improve the survival rate of crops under salt stress environment; (2) Improve the growth ability of crops under salt stress environment; (3) Cultivate crops with salt stress tolerance; (4) Genetic improvement of crop germplasm resources; described OsSAK1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0024] In a seventh aspect, the present invention provides a method for reducing OsSAK1 The use of inhibitors of protein content or activity includes any of the following: (1) Improve the survival rate of crops under salt stress environment; (2) Improve the growth ability of crops under salt stress environment; (3) Cultivate crops with salt stress tolerance; described OsSAK1 The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0025] Preferably, the inhibitor in the above application is OsSAK1 The inhibitor can inhibit protein transcription, transcription processing, or function after transcription processing, and can be used as a pesticide, fertilizer, or fertilizer additive.

[0026] The above-mentioned crops include wheat, corn, rice, soybean, barley, oats, tomatoes, peppers, rapeseed, etc., preferably rice.

[0027] Beneficial effects of the present invention: The present invention found that rice will reduce the OsSAK1 The transcription level of the gene was measured and a successful construct was constructed in japonica rice ZH11. OsSAK1 Genetically mutated plants sak1-1 and sak1-2 and overexpression OsSAK1 The researchers tested the salt tolerance of rice ZH11 plants, which had the same gene as OsSAK1-OE1 and OsSAK1-OE2, after salt treatment. sak1-1 The average survival rate was 68.38%. sak1-2 The average survival rate was 66.62%, the average survival rate of OsSAK1-OE1 was 14.69%, and the average survival rate of OsSAK1-OE2 was 16.01%, indicating that OsSAK1 Genetically mutated plants sak1-1 and sak1-2Seedlings had a higher survival rate under salt stress, while overexpression OsSAK1 The opposite was true for plants with the genes OsSAK1-OE1 and OsSAK1-OE2. OsSAK1 The expression level of genes can coordinately regulate the salt stress tolerance of crops and contribute to the genetic improvement of salt-tolerant crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Picture 1 The results are as follows: OsSAK1 Changes in gene expression.

[0030] Picture 2 In Example 2 OsSAK1 Gene structure diagram and OsSAK1 Genome sequencing and identification results of gene-edited mutant plant lines.

[0031] Picture 3 The overexpression transgenic plant line in Example 3 OsSAK1 Gene expression identification results.

[0032] Picture 4 yes OsSAK1 Appearance of gene-edited mutants and overexpression plants before and after salt stress.

[0033] Picture 5 yes OsSAK1 Survival statistics of gene-edited mutants and overexpression plants after salt stress. DETAILED DESCRIPTION

[0034] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0035] Where specific techniques or conditions are not specified in the examples, all methods were performed according to conventional methods, techniques or conditions described in literature in the field, or according to product specifications. Reagents and instruments used, for which the manufacturers are not specified, are conventional products that can be purchased through regular channels.

[0036] In the following embodiments.

[0037] Example 1 Effects of salt stress on OsSAK1 Effects on gene transcription levels This example uses real-time fluorescence quantitative PCR (qRT-PCR) technology to detect OsSAK1 Gene responses to salt stress.

[0038] Select the full seeds of rice variety ZH11 and soak them in water. Germination was accelerated in a 37℃ incubator for 2-3 days. The white seeds were selected and placed in a 96-well plate with a cut edge. They were cultured in a culture box filled with pure water for 2 days. They were placed in 1 / 2 MS culture medium and continued to be cultured for about 12 days. Then they were placed in 1 / 2 MS culture medium containing 200 mM NaCl and continued to be cultured. The treatments were 0h, 0.5h, 1h, 2h, 4h, 6h, 12h, and 24h respectively. The whole rice plant was sampled, and 8 rice seedlings were taken at each time point. The samples were immediately placed in liquid nitrogen, and the total RNA was extracted and reverse transcribed by Trizol method. The qRT-PCR analysis was performed. OsSAK1 Gene responses to salt stress, qRT-PCR analysis OsSAK1 The forward and reverse specific primer sequences of the gene are SEQ ID NO.3 and SEQ ID NO.4 respectively. The reference gene used is RUBQ2, and the forward and reverse specific primer sequences of the reference gene are SEQ ID NO.5 and SEQ ID NO.6 respectively. qRT-PCR analysis showed that under salt stress conditions OsSAK1 The expression of the gene was inhibited. Picture 1 ,illustrate OsSAK 1 may negatively regulate the response of rice to salt stress.

[0039] SEQ ID NO.3: 5'-CTCGACTATCTGCACAGCCT-3' SEQ ID NO.4: 5'-GGCATGGAATCAGCAATCGT-3' SEQ ID NO.5: 5'-GAGCCTCTGTTCGTCAAGTA-3' SEQ ID NO. 6: 5'-ACTCGATGGTCATTAAACC-3'.

[0040] Example 2 OsSAK1 Construction of gene mutant plants rice OsSAK1Gene structure analysis showed that the gene contains two exons. A target site was designed on the first exon with the designed target sequence SEQ ID NO.7. The double-stranded SG sequence was connected to the vector of the CRISPR / Cas9 system. The CRISPR / Cas9 vector was transformed into the japonica rice variety ZH11 through rice transgenic technology (Agrobacterium-mediated rice genetic transformation system), and two rice mutant plants of this gene were obtained. sak1-1 and sak1-2 .

[0041] SEQ ID NO.7: 5'- GCGCCCTCGCCTCCGGAGC-3' Identification OsSAK1 The editing type primers for gene-edited mutant plants are upstream primer SEQ ID NO.8 and downstream primer SEQ ID NO.9, and the sequencing primer uses SEQ ID NO.8.

[0042] SEQ ID NO.8: 5'- TGGTCGATCGATCGATCGAT-3' SEQ ID NO. 9: 5'- GATGACGGAGTCGGAGAACATC-3'.

[0043] Identification results such as Picture 2 As shown by Picture 2 It can be seen that it was successfully built OsSAK1 Gene-edited mutant plants sak1-1 and sak1-2 ,in sak1-1 The 20th base from the 5' end of the target sequence is missing. sak1-2 A T was inserted at position 20 from the 5' end of the target sequence.

[0044] Example 3 Construction of OsSAK1 gene overexpression plants The overexpression plasmid vector used in this example is pEZR(K)-LC (Liu, Dapu, et al. "Diversification of Plant Agronomic Traits by Genome Editing of Brassinosteroid Signaling Family Genes in Rice." Plant Physiology 2021). The specific implementation steps are: RNA of japonica rice ZH11 was extracted and converted into cDNA, and the obtained cDNA was used as a template to clone the gene using SEQ ID NO.3 and SEQ ID NO.4 as primers. OsSAK1 The CDS sequence of the primers is shown in SEQ ID NO.8 and SEQ ID NO.9.

[0045] SEQ ID NO.10: 5'-ATGGCGGCGGCGCGAT-3' SEQ ID NO.11: 5'-TCAGAAAAATACGGAGAAATCGCC-3' KODFX (Toyobo) PCR enzyme was used for amplification, with an annealing temperature of 58°C, an extension time of 2 min, and 35 cycles. OsSAK1 The PCR product of the gene was recovered and connected to the expression vector pEZR(K)-LC by homologous recombination. The expression vector plasmid with correct sequencing was transformed into Agrobacterium tumefaciens EHA105. OsSAK1 The overexpression vector was transformed into japonica rice variety ZH11, and the expression of the gene was detected by real-time quantitative PCR (e.g. Picture 3 shown), obtain OsSAK1 Overexpression of OsSAK1-OE1 and OsSAK1-OE2 in plants.

[0046] Depend on Picture 3 It can be seen that the successful construction OsSAK1 The overexpression plants of genes OsSAK1-OE1 and OsSAK1-OE2, among which OsSAK1-OE1 OsSAK1 The relative protein expression level of OsSAK1-OE2 was 5.78 times that of the wild type. OsSAK1 The relative protein expression level was 17.39 times that of the wild type.

[0047] Example 4 OsSAK1 Analysis of salt stress survival rates of gene-edited mutant plants and overexpression plants This embodiment is OsSAK1 The survival rate experiment of gene-edited mutant plants and overexpression plants under salt stress is as follows: (1) The sample obtained in Example 2 OsSAK1 Gene mutant plants sak1-1 、 sak1-2 and cas9-free obtained in Example 3 OsSAK1 Seeds of OsSAK1-OE1 and OsSAK1-OE2 overexpressing plants and wild-type rice (ZH11) were germinated and co-cultured in a 96-well plate, with 40 blank and 40 experimental cells per plate. When the seedlings were two weeks old, NaCl (final concentration 200 mM) was added to the rice nutrient solution.

[0048] (2) After 4-5 days of salt treatment, observe the phenotypic differences between the mutants, overexpression materials, and wild-type rice, and then replace the culture medium with 1 / 2 MS hydroponic culture medium. Continue to grow for 5-7 days and calculate the survival rate of the mutants, overexpression materials, and wild-type rice. The rice status before and after salt treatment is as follows: Picture 4 shown.

[0049] (3) Test results are as follows Picture 5 The results showed that after salt treatment, the average survival rate of rice ZH11 was 32.5%. sak1-1 The average survival rate was 68.38%. sak1-2 The average survival rate was 66.62%, the average survival rate of OsSAK1-OE1 was 14.69%, and the average survival rate of OsSAK1-OE2 was 16.01%. This indicates that the OsSAK1 gene negatively regulates rice salt tolerance. OsSAK1 The survival rates of two mutant rice plants after salt treatment were significantly higher than those of the wild type. The survival rates of two overexpressing rice plants after salt treatment were significantly lower than those of the wild type, but the phenotypes were not significantly different. This indicates that the overexpression level has reached or exceeded the saturation point of its function, and further increasing the expression level will not significantly enhance the phenotype. The final results indicate that the OsSAK1 gene negatively regulates salt tolerance in rice.

[0050] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A OsSAK1 The application of protein in regulating the salt stress tolerance of crops is characterized in that: Said OsSAK1 The amino acid sequence of the protein is at least one of the following: (1) as shown in SEQ ID NO.2; (2) a protein derived from SEQ ID NO. 2 by replacing, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 2 and retaining the function of the amino acid sequence shown in SEQ ID NO. 2; (3) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in SEQ ID NO. 2 and has an equivalent functional protein; (4) An amino acid sequence obtained by connecting a tag, a restriction site and / or a connecting peptide sequence to the N-terminus and / or C-terminus of any of the amino acid sequences of (1) to (3).

2. A OsSAK1 The application of the gene in improving the salt stress tolerance of crops is characterized in that: Said OsSAK1 The nucleotide sequence of the gene is at least one of the following: (1) as shown in SEQ ID NO.1; (2) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence expresses a protein with the same function; (3) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO.1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization in a 0.1× SSPE solution containing 0.1% SDS or a 0.1× SSC solution containing 0.1% SDS at 65°C and the membrane is washed with the solution; (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and expresses a protein with the same function.

3. The use according to any one of claims 1 or 2, characterized in that: The application is achieved by regulating the content or activity of OsSAK1 protein in crops through genetic engineering technology.

4. A method for improving the salt stress tolerance of crops, characterized in that: make OsSAK1 The expression of a protein in a crop is reduced or eliminated, and the methods include but are not limited to: (1) Knockout through genetic engineering OsSAK1 Gene; or (2) Knockout through genetic engineering OsSAK1 Gene promoter; or (3) Changes through genetic engineering OsSAK1 The base coding of the gene causes its protein to not be expressed or to be expressed at a reduced level; or (4) Reducing through genetic engineering OsSAK1 Gene expression; (5) Screening for low expression OsSAK1 The wild-type plants of protein were bred; Said OsSAK1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. A mutant, characterized in that: The mutant nucleotide sequence is as shown in SEQ ID NO.7, but the 20th base from the 5' end is missing; or As shown in SEQ ID NO.7, a T is inserted at the 20th base from the 5' end.

6. The mutant according to claim 5 is used OsSAK1 Application of protein inactivation to improve salt tolerance of crops.

7. A method for reducing the OsSAK1 The method for measuring protein expression, characterized in that The corresponding crops are grown in a salt stress environment. OsSAK1 The amino acid sequence of the protein is shown in SEQ ID NO.

2.

8. OsSAK1 Use of a gene, or a protein encoded by the gene, or a biological material containing the gene, or a method according to any one of claims 4 to 5 in any of the following: (1) Improve the survival rate of crops under salt stress environment; (2) Improve the growth ability of crops under salt stress environment; (3) Cultivate crops with salt stress tolerance; (4) Genetic improvement of crop germplasm resources; Said OsSAK1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; or The amino acid sequence of the protein is shown in SEQ ID NO.

2.

9. Reduce the amount of OsSAK1 Use of an inhibitor of protein content or activity, characterized in that Applications including any of the following: (1) Improve the survival rate of crops under salt stress environment; (2) Improve the growth ability of crops under salt stress environment; (3) Cultivate crops with salt stress tolerance; Said OsSAK1 The amino acid sequence of the protein is shown in SEQ ID NO.

2.

10. The use according to claim 9, characterized in that: The inhibitor inhibits OsSAK1 The expression or activity of a protein can be inhibited by inhibiting transcription, inhibiting transcription processing, or inhibiting the function after transcription processing. The inhibitor can be used as a pesticide, fertilizer, or fertilizer additive.

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