Application and method of OsARI2 gene in improving salt tolerance of rice

Abstract

The invention provides application of an OsARI2 gene in improvement of salt tolerance of rice and a method. The application of the OsARI2 gene in improving the salt tolerance of the rice comprises the application of the OsARI2 gene and a nucleotide sequence with at least 90% of sequence homology in improving the salt tolerance of the rice, and the nucleotide sequence of the OsARI2 gene is shown as SEQ ID No.1. The OsARI2 gene is subjected to substitution, deletion or addition of one or more nucleotides to obtain the nucleotide sequence with at least 90% of sequence homology. The salt tolerance of the OsARI2 knockout strain obtained by the invention is obviously better than that of an overexpression strain, which indicates that the gene participates in the salt stress resistance process of rice seedling development, provides a new gene resource for crop variety improvement, and has great application value.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to an application and method of an OsARI2 gene in improving salt tolerance of rice. Background Art

[0002] Rice is one of the most important food crops in the world. It is grown in about 113 countries. It is one of the staple foods for more than half of the world's population and the most important food crop in Asia. China is also the world's largest rice producer and consumer, and has a long history of rice cultivation. Abiotic stress is the main environmental factor causing global crop yield reduction. Among various abiotic stresses, salt stress is the issue that people are most concerned about. According to statistics, there are about 8 billion hectares of saline-alkali land in the world, of which irrigated land accounts for about 20%. In recent years, with the influence of global climate change, unreasonable irrigation systems, rising sea levels, and salt runoff from strata, about 53% of the rice cultivated area has been affected, resulting in a reduction in rice production of about 23 million hectares. Salt stress can cause osmotic stress, ion stress, and oxidative stress. Osmotic stress is caused by high salt concentrations in the soil, which inhibits plants from absorbing water from the soil, resulting in harmful changes in cell components. Ionic stress is the accumulation of salt ions (Na + and K + ) enter plant cells and cause ion imbalance. Osmotic stress and ion stress will disrupt metabolic balance and then produce oxidative stress. Oxidative stress will produce a large amount of reactive oxygen species (ROS), causing reactive oxygen metabolism disorders. Reactive oxygen includes singlet oxygen (1O 2 ), hydrogen peroxide (H 2 O 2 ) etc., have strong oxidative ability, affecting various cell components of plants (proteins, DNA and lipids), thereby significantly inhibiting plant growth.

[0003] Therefore, it is proposed to use genetic engineering and molecular biology to create rice varieties resistant to salt stress. Summary of the invention

[0004] The purpose of the present invention is to provide an application and method of OsARI2 gene in improving the salt tolerance of rice, so as to improve the salt tolerance of rice.

[0005] The present invention provides an application of OsARI2 gene in improving rice salt tolerance, including application of the OsARI2 gene and a nucleotide sequence with at least 90% sequence homology in improving rice salt tolerance. The nucleotide sequence of the OsARI2 gene is shown in SEQ ID No.1.

[0006] In the above application, the OsARI2 gene is substituted, deleted or added with one or several nucleotides to obtain the nucleotide sequence having at least 90% sequence homology.

[0007] The amino acid sequence of the protein encoded by the OsARI2 gene is shown in SEQ ID NO. 2. The protein can be obtained by replacing, deleting or adding one or more amino acids to obtain an amino acid sequence with at least 90% sequence homology.

[0008] The present invention also provides the use of a biological material containing the OsARI2 gene in improving the salt tolerance of rice. The OsARI2 gene is the OsARI2 gene in the above application, and the biological material is a recombinant vector or a recombinant cell.

[0009] The present invention also provides a method for improving the salt tolerance of rice, which comprises regulating the OsARI2 gene in the above application to change its expression level.

[0010] In the above method, the manner of regulating the OsARI2 gene includes editing, knocking out, modifying or inhibiting the OsARI2 gene.

[0011] The above method includes transforming the recombinant vector into a plant to edit, knock out, modify or inhibit the OsARI2 gene.

[0012] The invention also provides application of the method in rice breeding.

[0013] Beneficial effects of the present invention:

[0014] The present invention applies rice E3 ubiquitin ligase OsARI2 to plant genetic engineering to obtain rice transgenic plants with overexpression and knockout of OsARI2. The salt tolerance of the knockout strain is significantly better than that of the overexpression strain, indicating that the gene is involved in the salt stress resistance process of rice seedling development, providing a new gene resource for crop variety improvement and having great application value.

[0015] The biological material of the invention can efficiently regulate the expression of the OsARI2 gene in plants.

[0016] The method of the invention can improve the salt tolerance of rice seedlings by regulating the OsARI2 gene to change its expression level, which plays an important role in the research of the salt tolerance mechanism of rice and the cultivation of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1This is a graph showing the MDA content detection results of each test group in Example 2.

[0019] Figure 2 H for each test group in Example 2 2 O 2 Content test result diagram.

[0020] Figure 3 This is a graph showing the results of CAT enzyme activity detection in each test group of Example 2.

[0021] Figure 4 This is a physical picture of rice seedlings in each test group of Example 2.

[0022] Figure 5 This is a statistical chart of the survival rate of rice seedlings in each test group in Example 2.

[0023] Figure 6 This is a graph showing the relative water content detection results of each test group in Example 2. DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but those skilled in the art will understand that the embodiments described below are part of the embodiments of the present invention, rather than all of the embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025]

[0026] The protein encoded by the OsARI2 gene is used to improve the salt tolerance of rice. The amino acid sequence of the protein is shown in SEQ ID NO. 2. The protein can be obtained by replacing, deleting or adding one or more amino acids to obtain an amino acid sequence with at least 90% sequence homology.

[0027] Example 1 Construction of OsARI2 gene overexpression and knockout strains

[0028] Using wild-type Nipponbare rice WT as the material, Biotech Co., Ltd. transformed the plasmid containing OsARI2 into wild-type Nipponbare rice WT plants through Agrobacterium infection, and obtained the T4 generation overexpression strains OsARI2-101-1, OsARI2-111-8 and CRISPR / Cas9 gene knockout strains osari2-141-1, osari2-147-2 transgenic rice materials.

[0029] Example 2 Determination of physiological indicators of OsARI2 interference expression strains and overexpression strains

[0030] Select rice seeds of uniform size and full grains from each strain, soak them in 75% ethanol for 1 min, then sterilize them in 20% sodium hypochlorite for 20 min, and wash them 10 times with pure water. Soak the seeds in a temperature-controlled incubator at 28°C for 24 h, and then germinate them at 37°C for 24 h. When most of the seeds turn white, take seeds with similar germination degrees and transfer them to a PCR plate with the bottom removed, place them in a plant incubator (lighting 16h / 8h, temperature 28°C / 26°C, humidity 70%), and culture them in clean water for 7 days. After changing to 1 / 2IRRI nutrient solution for 3 days, treat some rice seedlings with 150mM NaCl for 3 days. Figure 1 and Figure 2 As shown, from the MDA content and H 2 O 2 The content of all strains increased after salt treatment, but compared with WT, the increase in the knockout strain was significantly smaller than that in the overexpression strain. Figure 3 As shown in the figure, from the perspective of CAT enzyme activity, the enzyme activity of all strains increased, but compared with WT, the enzyme activity of rice seedlings of the knockout strain was greater than that of the overexpression strain. Figure 4 and Figure 5 As shown in Figure 2, from the perspective of relative survival rate, compared with WT, the survival rate of rice seedlings in the knockout strain is greater than that in the overexpression strain. Figure 6 As shown, in terms of relative water content, all lines decreased, but compared with WT, the decrease in water content in rice seedlings of the knockout line was smaller than that of the overexpression line.

[0031] The above results indicate that the OsARI2 gene is involved in the resistance to salt stress during the development of rice seedlings and is an important gene for the resistance of rice seedlings to salt stress. At the same time, downregulating the expression of this gene can improve the salt tolerance of rice seedlings, which is important for the study of the mechanism of rice salt tolerance and crop breeding.

[0032] The above description is only a preferred implementation mode of the present invention. Those skilled in the art should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace part or all of the technical features therein with equivalents, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of OsARI2 gene in improving salt tolerance of rice, characterized in that: The invention comprises the use of the OsARI2 gene and a nucleotide sequence with at least 90% sequence homology in improving the salt tolerance of rice. The nucleotide sequence of the OsARI2 gene is shown as SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The OsARI2 gene is substituted, deleted or added with one or several nucleotides to obtain the nucleotide sequence with at least 90% sequence homology.

3. Use of a biological material containing the OsARI2 gene in improving the salt tolerance of rice, characterized in that: The OsARI2 gene is the OsARI2 gene in the use according to claim 1, and the biological material is a recombinant vector or a recombinant cell.

4. A method for improving salt tolerance of rice, characterized in that: The expression level of the OsARI2 gene is changed by regulating the OsARI2 gene in the use according to claim 3.

5. The method according to claim 4, characterized in that The methods of regulating the OsARI2 gene include editing, knocking out, modifying or inhibiting the gene.

6. The method according to claim 5, characterized in that The method comprises transforming the recombinant vector into a plant to edit, knock out, modify or inhibit the OsARI2 gene.

7. Use of the method according to claim 4 in rice breeding.

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