Application and method of osari2 gene in improving salt tolerance of rice

Abstract

This invention provides the application and method of the OsARI2 gene in improving salt tolerance in rice. The application of the OsARI2 gene in improving rice salt tolerance includes the 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. The OsARI2 gene is obtained by substituting, deleting, or adding one or more nucleotides to obtain the nucleotide sequence with at least 90% sequence homology. The OsARI2 knockout lines obtained by this invention exhibit significantly better salt tolerance than the overexpression lines, indicating that this gene participates in the salt stress resistance process during rice seedling development. This provides a new gene resource for crop variety improvement and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application and method of the OsARI2 gene in improving the salt tolerance of rice. Background Technology

[0002] Rice is one of the world's most important food crops, grown in approximately 113 countries. It is a staple food for more than half the world's population and is also the most important food crop in Asia. China is the world's largest rice producer and consumer, with a long history of rice cultivation. Abiotic stress is a major environmental factor causing global crop yield reduction, and among various abiotic stresses, salt stress is the most concerning. Statistics show that there are approximately 8 billion hectares of saline-alkali land globally, of which irrigated land accounts for about 20%. In recent years, due to factors such as global climate change, inadequate irrigation systems, sea-level rise, and saline runoff, approximately 53% of rice-growing land has been affected, resulting in a reduction of rice yields 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, inhibiting plants from absorbing water from the soil, leading to harmful changes in cellular components. Ion stress is caused by salt ions (Na+). + and K + Osmotic stress and ion stress can disrupt metabolic balance, leading to oxidative stress. Oxidative stress produces large amounts of reactive oxygen species (ROS), causing metabolic disorders. ROS include singlet oxygen (1O2) and hydrogen peroxide (H2O2), which have strong oxidizing capabilities and affect various cellular components of plants (proteins, DNA, and lipids), thus significantly inhibiting plant growth.

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

[0004] The purpose of this invention is to provide the application and method of the OsARI2 gene in improving the salt tolerance of rice, thereby enhancing the salt tolerance of rice.

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

[0006] In the above applications, the OsARI2 gene is substituted, deleted, or has one or more nucleotides added to obtain the nucleotide sequence with 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 substituting, 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 application of biomaterials containing the OsARI2 gene in improving the salt tolerance of rice, wherein the OsARI2 gene is the OsARI2 gene in the above-mentioned application, and the biomaterial is a recombinant vector or recombinant cell.

[0009] The present invention also provides a method for improving the salt tolerance of rice by regulating the expression level of the OsARI2 gene described in the above-mentioned application.

[0010] In the above methods, the OsARI2 gene is regulated by means of editing, knocking out, modifying or inhibiting it.

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

[0012] This invention also provides the application of the above-described method in rice breeding.

[0013] The beneficial effects of this invention are:

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

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

[0016] The method of this invention can improve the salt tolerance of rice seedlings by regulating the expression level of the OsARI2 gene, which is of great significance for the study of the salt tolerance mechanism of rice and the cultivation of crops. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 The graph shows the MDA content detection results for each experimental group in Example 2.

[0019] Figure 2 The graph shows the H2O2 content detection results for each experimental group in Example 2.

[0020] Figure 3 The image shows the CAT enzyme activity detection results for each experimental group in Example 2.

[0021] Figure 4 The images show actual rice seedlings from each experimental group in Example 2.

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

[0023] Figure 6 The graph shows the relative water content detection results for each test group in Example 2. Detailed Implementation

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all 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 those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025]

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

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

[0028] Using wild-type Nipponbare rice (WT) as material, Baige Biotechnology Co., Ltd. transformed OsARI2-containing plasmids into wild-type Nipponbare rice (WT) plants through Agrobacterium infection, obtaining T4 generation overexpression lines OsARI2-101-1 and OsARI2-111-8 and CRISPR / Cas9 gene knockout lines osari2-141-1 and osari2-147-2 transgenic rice materials.

[0029] Example 2: Determination of physiological indicators in OsARI2 interference expression lines and overexpression lines

[0030] Select rice seeds of uniform size and plump grains from various lines, soak them in 75% ethanol for 1 minute, then disinfect them in 20% sodium hypochlorite for 20 minutes, and wash them 10 times with pure water. Soak the seeds in a temperature-controlled incubator at 28℃ for 24 hours, then germinate them at 37℃ for 24 hours. Once most seeds show signs of germination, transfer seeds with similar germination levels to a PCR plate (without a base) and place them in a plant culture incubator (16h / 8h light, 28℃ / 26℃ temperature, 70% humidity) and culture them in water for 7 days. Then, culture them in 1 / 2 IRRI nutrient solution for 3 days, and finally treat some rice seedlings with 150mM NaCl for 3 days. Figure 1 and Figure 2 As shown, the MDA and H2O2 contents increased in all lines after salt treatment, but the increase in content in the knockout lines was significantly smaller than that in the overexpression lines compared to WT. Figure 3 As shown, the CAT enzyme activity increased in all lines, but compared to WT, the knockout lines showed higher enzyme activity in rice seedlings than the overexpression lines. Figure 4 and Figure 5 As shown, in terms of relative survival rate, the survival rate of rice seedlings from the knockout lines was higher than that of the overexpression lines compared to the WT lines. Figure 6 As shown, all lines showed a decrease in relative water content, but compared to WT, the decrease in water content of rice seedlings in knockout lines was smaller than that in overexpression lines.

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

[0032] The above description is only a preferred embodiment of the present invention. Those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to 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 seedlings, characterized in that, The nucleotide sequence of the OsARI2 gene is shown in SEQ ID No.

1. Downregulating the expression of this gene can improve the salt tolerance of rice. In terms of relative survival rate, the survival rate of rice seedlings with the gene knocked out was greater than that of the overexpressing lines. In terms of relative water content, the decrease in water content of rice seedlings with the gene knocked out was less than that of the overexpressing lines.

2. Use of biological material comprising the OsARI2 gene for increasing salt tolerance in rice seedlings, characterized in that, The biological material is a recombinant vector or recombinant cell, and the nucleotide sequence of the OsARI2 gene is shown in SEQ ID No.

1. Downregulating the expression of this gene can improve the salt tolerance of rice. In terms of relative survival rate, the survival rate of rice seedlings with the gene knocked out was greater than that of the overexpressing lines. In terms of relative water content, the decrease in water content of rice seedlings with the gene knocked out was less than that of the overexpressing lines.

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