Application of tify8 gene and protein in salt tolerance of rice seedling stage

Abstract

The application belongs to modern agriculture, and particularly relates to TIFY8 Application of the gene and the protein in salt tolerance of rice seedlings. The application screens the knockout mutant material with salt stress tolerance by subjecting the single gene knockout mutant material library constructed in the laboratory to salt stress treatment. After salt treatment and identification, tify8 The knockout mutant has higher salt stress tolerance and survival rate than the wild type. Therefore, the application aims to cultivate the salt tolerance line of rice by using TIFY8 the functional characteristics of the gene and tify8 the knockout mutant system. The application can significantly shorten the breeding period compared with the traditional breeding method.

Description

Technical Field

[0001] The present invention belongs to modern agriculture, and specifically relates to TIFY8 Application of genes and their proteins in salt tolerance at the rice seedling stage. Background Art

[0002] As global environmental issues become increasingly prominent, soil salinization has received widespread attention. According to statistics, approximately 8% of the world's land is threatened by varying degrees of salinization. As a salt-sensitive crop, rice has various growth and development phenotypes, such as plant height, number of tillers, number of green leaves per plant, and biomass, inhibited by salt stress, leading to a decrease in yield. Therefore, the discovery and utilization of rice salt-tolerant genes, as well as the screening and creation of salt-tolerant rice germplasm resources, are of great significance to rice production. Furthermore, the application of genetic engineering methods to the cultivation of high-quality rice varieties with good adaptability to salt-stress environments will also provide ideas and references for the breeding of other crops.

[0003] Plants resist salt stress in various ways, including through transcription factors that regulate the expression of key stress-resistance genes. TIFY family transcription factors play a crucial role in enhancing plant resistance to abiotic stresses and improving their environmental adaptability. TIFY11a By regulating potassium ion homeostasis, it affects Na + / K + Balance and positively regulate rice salt tolerance. TIFY11b The expression of OsPP2C is induced by a variety of abiotic stresses and negatively regulates OsPP2C by inhibiting ABA signaling, enhancing SnRK2 kinase activity, and promoting the expression of downstream salt-tolerance genes. LEA3, Rab16A expression, thus improving the tolerance of rice to salt stress. TIFY10c Plants overexpressing TIFY have higher tolerance to salt stress. However, the role and mechanism of TIFY transcription factors in rice salt tolerance need further exploration and analysis. Summary of the Invention

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0005] The first aspect of the present invention provides a knockout TIFY8 Application of genes in improving salt tolerance in rice.

[0006] In some embodiments of the present invention, the TIFY8 The genomic sequence of the gene is shown in SEQ ID NO: 1.

[0007] In some embodiments of the present invention, the TIFY8 The coding region sequence of the gene is shown in SEQ ID NO: 2.

[0008] In some embodiments of the present invention, the TIFY8 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 3.

[0009] The second aspect of the present invention provides a knockout TIFY8 Application of genetic reagents in breeding rice with increased salt tolerance.

[0010] In some embodiments of the present invention, the TIFY8 The coding region sequence of the gene is shown in SEQ ID NO: 2;

[0011] In some embodiments of the invention, the reagent does not include propagation material.

[0012] In some embodiments of the present invention, the reagent comprises:

[0013] 1) Nucleic acid molecules;

[0014] 2) A CRISPR / Cas9 vector comprising the nucleic acid molecule described in 1);

[0015] 3) Cells containing the CRISPR / Cas9 vector described in 2).

[0016] In some embodiments of the invention, the nucleic acid molecule comprises an inhibitory TIFY8 microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotides targeting gene function.

[0017] In some embodiments of the present invention, the sequence of the sgRNA is shown in SEQ ID NO: 4:

[0018] CTCCGGGAACTTGTAACGTC.

[0019] In some embodiments of the present invention, the vector comprises a CRISPR / Cas9 vector.

[0020] In some embodiments of the present invention, the sgRNA is used in conjunction with a CRISPR / Cas9 vector to achieve the purpose of gene knockout.

[0021] In some embodiments of the present invention, the CRISPR / Cas9 vector further includes an expression vector containing the sgRNA, and of course may also include a Cas9 protein or an expression vector for expressing the Cas9 protein.

[0022] In some embodiments of the present invention, the vector includes but is not limited to CRISPR / Cas9 vectors and other common vectors in the art.

[0023] In some embodiments of the present invention, the cell comprises at least one of Escherichia coli and Agrobacterium tumefaciens, wherein Escherichia coli is a common host cell for constructing vectors and plasmids in the art, and Agrobacterium tumefaciens is a common tool for delivering DNA molecules to plants in the art.

[0024] In some embodiments of the invention, the cell is Agrobacterium.

[0025] In some embodiments of the present invention, the Agrobacterium is EHA105 (competent cell).

[0026] The third aspect of the present invention provides a method for cultivating a rice variety with increased salt tolerance, comprising knocking out a TIFY8 genetic steps;

[0027] The rice variety with increased salt tolerance comprises the following characteristics: salt tolerance is increased relative to a reference level; the reference level is the level of the wild type;

[0028] The knockout rice TIFY8 The step of knocking out the gene is to TIFY8 Gene-related biological materials are introduced into rice tissues or rice cells.

[0029] In some embodiments of the invention, the biological material does not include propagation material.

[0030] In some embodiments of the present invention, the introduction method comprises using at least one of Ti plasmid, Ri plasmid, plant virus vector, microinjection, and electroporation.

[0031] In some embodiments of the present invention, the steps of knocking out the TIFY8 protein in rice are specifically as follows:

[0032] (1) Design TIFY8 Gene target sequence sgRNA, construct rice TIFY8 CRISPR / Cas9 vectors for gene editing.

[0033] (2) The CRISPR / Cas9 vector described in step (1) is transformed into Agrobacterium EHA105 competent cells to obtain Agrobacterium containing the CRISPR / Cas9 vector.

[0034] (3) Cultivate rice (Zhonghua 11) callus tissue, infect the callus tissue with the Agrobacterium infection solution obtained in step (3), obtain seedlings again through tissue culture, and screen rice TIFY8 A stably inherited mutant strain with a gene mutation, no exogenous Cas9 protein, and a target sequence mutation.

[0035] In some embodiments of the present invention, the vector is a CRISPR / Cas9 vector.

[0036] In some embodiments of the present invention, the host cell is an Agrobacterium EHA105 competent cell.

[0037] In some embodiments of the present invention, the rice variety is Zhonghua 11.

[0038] The beneficial effects of the present invention are:

[0039] This project screened for salt stress-tolerant knockout mutants by subjecting the laboratory-established single gene knockout mutant material library to salt stress treatment. Tify8 The knockout mutant has higher salt stress tolerance and survival rate than the wild type. TIFY8 Functional properties of genes and Tify8 Knockout mutation system, cultivate rice salt-tolerant lines. Compared with traditional breeding methods, this invention can significantly shorten the breeding cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0041] 图1 Identification of mutation sites of salt-tolerant knockout mutants, including: (A) TIFY8 Gene structure and mutation types. Black boxes represent UTRs, gray arrows represent exons, and black lines represent introns. (B) Sequencing alignment results displayed by snapgene software. Horizontal lines represent missing bases.

[0042] 图2 Wild-type plants and Tify8 Phenotypic results of knockout mutant plants: WT is wild type, tify8-1 / 2 Knockout mutant.

[0043] 图3 After treatment with 150 mM salt, the wild-type plants and Tify8 Knockout mutant plant survival results: WT is wild type, tify8-1 / 2 For knockout mutants, t test was used Tify8 ** indicates extremely significant difference in survival rate compared with the wild type (p < 0.01). DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are conventional reagents and materials in the art and can be obtained through conventional procurement.

[0046] Example 1 Construction of knockout mutant material library

[0047] A knockout target site was designed in the first exon of the gene using the website: http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR.

[0048] The target sequence was constructed in a CRISPR / Cas9 vector. Transgenic rice plants were constructed through genetic transformation. The knockout vector was transferred into Agrobacterium tumefaciens EHA105 competent cells by freeze-thaw method, and positive plaques were identified by PCR. Callus tissue was induced by infecting rice seeds with Agrobacterium containing the knockout vector. Calli were screened for hygromycin resistance, and transgenic plants were obtained through differentiation and rooting culture (a single-gene mutation library, each plant has only one gene mutation).

[0049] Example 2 Screening of salt-tolerant knockout mutants

[0050] The knockout mutant materials constructed in the laboratory are cultured in an incubator.

[0051] The incubator parameters were set as follows: 30,000 Lx of light, 28°C of temperature, 60% RH of humidity, for 16 h; 0 Lx of light, 28°C of temperature, 60% RH of humidity, for 8 h. After about 14 days of normal culture, when the seedlings reached the three-leaf, one-heart stage, salt stress treatment with 150 mM NaCl was applied. After 5 days of treatment, the seedlings were returned to the normal nutrient solution. The mutant lines with significantly different phenotypes and survival rates compared to the wild-type material (Zhonghua 11) were screened. Tify8 The numbered knockout mutant materials have higher salt stress tolerance than the wild type.

[0052] in, TIFY8 The genome sequence is:

[0053]

[0054] TIFY8 The gene coding region sequence of is as follows:

[0055] ATGCCGCCGCCGGCCGCCGTCGCAAGCCTGACGTTACAAGTTCCCGGAGGAGCTCACGATGTCACTTCTCTGGCTACTAGTCCAAGGACCATGGCCGTGCCTGGAACGACTGAACAGCTGACGATTTTCTACTCCGGGTCTATGGTGAAGTTCGACAACGTCCCAAGGGAGAAGATTCGCTATGCGTGCAGGCTGAGGAGGTTATATTCTTCGCTGCAAAGAAGTCTCCAGACGCAGGACACCAGCATGTTCCCCAGCAGCAGCAGCCTGCATATCCAAACAAAAAGAAGAGGATATTCTGTTATCAGGCTCCTGAGAGAGATGCTGATGGTTTGTTCATCCACGAGAACAAAGCCGATGCTTGTTCACAGCGACAGCATCGGAGCCCAGAGGACGGGTACGCCACCATCAAGGAGACGAATCCATGCTCGCGGCAAATCCAGATCGTGCCAAGAGATGTCTCATTGCTGGTGA (SEQ ID NO: 2).

[0056] TIFY8 The amino acid sequence obtained by gene coding of is as follows:

[0057] MPPPAAVASLTLQVPGGAHDVTSLATSPRTMAVPGTTEQLTIFYSGSMVKFDNVPREKIRYACRLRRLYSSLQRSLQTQDTSMFPSSSSLHIQTKRRGYSVIRLLREMLMVCSSTRTKPMLVHSDSIGAQRTGTPPSRRRIHARGKSRSCQEMSHCW* (SEQ ID NO: 3).

[0058] Knockout TIFY8 The sgRNA sequence used for knocking out the gene is as follows: CTCCGGGAACTTGTAACGTC (SEQ ID NO: 4).

[0059] Example 3 Tify8 Verification of mutant salt tolerance

[0060] Screening Tify8Different genotype mutants were used for salt stress treatment. DNA was extracted from leaves of knockout mutant materials using the CTAB method.

[0061] PCR was performed using laboratory-specific primers ONE215: AGCCAGGTTCGTGTCCAG (SEQ ID NO: 5) and ONE216: TGCATCCCATCCGTCAAA (SEQ ID NO: 6). The target bands were detected by agarose gel electrophoresis. The remaining PCR product was sequenced and aligned with the genomic sequence using snap gene software.

[0062] The results showed that the knockout mutant genotype was tify8-1 With 1 bp & 9 bp deletion, tify8-2 With a 1 bp deletion ( 图1 ).

[0063] The knockout mutants were cultured in an incubator. The incubator parameters were: 30,000 lux of light, 28°C, and 60% humidity for 16 hours; 0 lux of light, 28°C, and 60% humidity for 8 hours. After approximately 14 days of normal incubation, when the seedlings reached the three-leaf, one-heart stage, they were treated with salt stress using 150 mM NaCl. After 5 days of treatment, the seedlings were returned to their normal nutrient solution.

[0064] according to 图2 The experimental results show that knocking out rice TIFY8 The degree of leaf wilting in rice plants with the gene is lower than that in wild type, indicating that TIFY8 It can negatively regulate rice's ability to resist salt stress.

[0065] according to 图3 The survival rates of wild-type plants and knockout mutant plants were statistically analyzed, and it was found that after salt treatment tify8-1 and tify8-2 The survival rate of the knockout rice was significantly higher than that of the wild type. TIFY8 The gene, its encoded protein and recombinant vector can be used to enhance the salt stress tolerance of crops.

[0066] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Knockout TIFY8 The use of genetic reagents in breeding rice with increased salt tolerance; described TIFY8 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO: 2; The amino acid sequence of the protein encoded by the TIFY8 gene is shown in SEQ ID NO: 3; The reagents include: 1) Nucleic acid molecules; The nucleic acid molecule includes a CRISPR / Cas9 vector and sgRNA; The sequence of the sgRNA is shown in SEQ ID NO: 4; 2) A cell comprising the nucleic acid molecule described in 1).

2. The use according to claim 1, characterized in that: The cells include at least one of Escherichia coli and Agrobacterium.

3. A method for breeding rice varieties with increased salt tolerance, comprising knocking out TIFY8 genetic steps; in, The rice variety with increased salt tolerance comprises the following characteristics: salt tolerance is increased relative to a reference level; the reference level is the level of the wild type; Among them, knockout rice TIFY8 The step of knocking out the gene of any one of claims 1 to 2 TIFY8 Gene reagents are introduced into rice tissues or rice cells.

4. The method according to claim 3, wherein: The introduction method includes using at least one of Ti plasmid, Ri plasmid, plant virus vector, microinjection, and electroporation.

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