阅读说明：本技术 水稻耐盐胁迫基因OsBAG4、编码蛋白及其应用 (Rice salt-tolerant stress gene OsBAG4, encoding protein and application thereof ) 是由 徐正一 刘雨同 南楠 王婕 于 2020-07-21 设计创作，主要内容包括：本发明提供了水稻耐盐胁迫基因OsBAG4、编码蛋白及其应用,属于基因工程技术领域。本发明提供了水稻耐盐胁迫基因OsBAG4,其核苷酸序列如SEQ ID NO.1所示；本发明还提供了由OsBAG4基因编码的水稻耐盐胁迫的蛋白OsBAG4,其氨基酸序列如SEQIDNO.2所示。本发明通过构建含OsBAG4基因的植物表达载体并利用农杆菌介导的方法转化水稻品种日本晴中,经过含有NaCl的营养液进行盐胁迫处理后,测定转基因水稻的表型和测量其存活率；实验结果表明,水稻耐盐胁迫基因OsBAG4可提高水稻对盐胁迫的耐受性。(The invention provides a rice salt-tolerant stress gene OsBAG4, a coded protein and application thereof, belonging to the technical field of genetic engineering. The invention provides a rice salt-tolerant stress gene OsBAG4, the nucleotide sequence of which is shown in SEQ ID NO. 1; the invention also provides a rice salt stress resistant protein OsBAG4 coded by the OsBAG4 gene, and the amino acid sequence of the protein OsBAG4 is shown as SEQ ID NO. 2. The invention constructs a plant expression vector containing an OsBAG4 gene, transforms a rice variety Nipponbare by an agrobacterium-mediated method, and measures the phenotype and the survival rate of transgenic rice after salt stress treatment is carried out on nutrient solution containing NaCl; experimental results show that the salt-tolerant stress gene OsBAG4 of rice can improve the salt stress tolerance of rice.)

1. A rice salt-tolerant stress gene OsBAG4 is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.

2. A protein for coding the rice salt-tolerant stress gene OsBAG4 as claimed in claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. A plant expression vector comprising the rice salt tolerance stress gene OsBAG4 of claim 1.

4. The plant expression vector of claim 3, wherein the species of vector in the plant expression vector comprises pCsV 1300;

the multiple cloning site for inserting the OsBAG4 into the pCsV1300 is between XbaI and BamHI.

5. A primer pair for amplifying the rice salt-tolerant stress gene OsBAG4 of claim 1, wherein the nucleotide sequence of the upstream primer in the primer pair is shown as SEQ ID NO. 3; the nucleotide sequence of the downstream primer in the primer pair is shown as SEQ ID NO. 4.

6. A rice salt stress-resistant recombinant cell, which comprises a rice salt stress-resistant gene OsBAG4 of claim 1 or a plant expression vector of claim 3 or 4.

7. Use of the OsBAG4 of claim 1, the protein of claim 2, the plant expression vector of claim 3 or 4, the primer pair of claim 5 or the recombinant cell of claim 6 in cultivation of salt-tolerant transgenic rice or salt stress of rice.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a rice salt tolerance stress gene OsBAG4, a coding protein and application thereof.

Background

Rice (Oryza sativa L.) is one of the most important food crops in the world, more than half of the population in the world is cultivated, and the production of the rice has important significance on the global food safety. It is estimated that by 2050, the production of food will increase by about 50% to meet the demand of the growing world population^[1]. However, rice is often subjected to adverse effects of various abiotic stresses throughout its growth, such as salinity, drought, flooding, low temperature stress, and the like. Among them, salt stress is one of important abiotic stresses threatening rice production.

The salinization of soil leads to the reduction of the cultivated land area and is also one of the reasons of causing the grain crisis. According to the incomplete statistics of the United nations textbook organization and the United nations grain and agriculture organization, about 9.45 hundred million hectares of land is affected by salt damage all over the world and accounts for about 20 percent of the area of the global agricultural land^[2]. China is a big country with saline-alkali soil, and the saline-alkali area of the existing inland is nearly 1 hundred million hectares^[3]The tidal flat area is 234 hectare, and the saline-alkali land area is the third in the world and is mainly distributed in northwest, northeast, north China and coastal areas. The main form of saline-alkali soil in northeast China is soda saline-alkali soil with the area as high as 756 million hectares^[3]. Wherein, the saline-alkali soil area of 12 city and county in the west of Jilin province is more than 160 million hectares^[4]. In the face of the practical production problems that the population is continuously increased, the usable cultivated land area is limited, the secondary salinization phenomenon of soil is increasingly serious due to unreasonable irrigation and utilization of land, the rice yield is difficult to obviously improve and the like, the development and utilization of coastal mudflats and inland saline-alkali land resources are one of effective ways for guaranteeing the cultivated land area^[5]. Rice is a moderately salt-sensitive crop^[6]Growing in water environment, and the rice planted can leach soluble saline alkali of soilFunction of^[7-8]. Therefore, the rice is the first choice food crop for developing coastal mudflats and saline-alkali lands.

The improvement of the salt tolerance of the rice through genetic improvement is one of effective ways for improving the planting area and the yield of the rice^[9]. At present, salt-tolerant QTL utilized in breeding is mainly two sites of qSKC-1 and Saltol on No.1 chromosome of rice^[5]. With the development of molecular biotechnology, the mutant is used for separating and excavating the salt stress genes of the water-tolerant rice, and the mutant is used for rice genetic engineering for auxiliary breeding and alkali stress improvement, and has extremely important significance for effectively controlling the damage of the salt stress to the rice, improving the rice yield and improving the rice quality. Bcl-2associated alkane (BAG) is a kind of evolutionarily conserved multifunctional protein, and is found to be involved in various important physiological processes such as tumor generation, apoptosis, nerve cell differentiation and stress response in animals. There are 6 proteins in rice that contain conserved BAG domains, of which only OsBAG4 was found to be involved in the anti-blast response^[10]However, the improvement of salt stress resistance of rice by OsBAG4 has not been reported.

Reference to the literature

[1] Dragon of very young, breeding and genetic analysis of salt-tolerant, drought-resistant, high-yield and high-quality breeding materials of rice [ D ]. Beijing, national academy of agricultural sciences, 2017.

[2]Munns R,Tester M.Mechanisms of salinity tolerance[J].Annu RevPlant Biol,2008,59:651-681.

[3] Li Bin, Wang Shi Chun, Sun Shi Gao, etc. Chinese saline-alkali soil resource and sustainable utilization research [ J ] agricultural research in arid area, 2005,23(2):154 and 158.

[4] Liuxing soil, pine and tender plain degraded land remediation and agricultural development [ M ]. Beijing, scientific Press, 2001.

[5] The research status, problems and suggestions of saline-alkali tolerant rice [ J ] Chinese rice, 2019,25(1):1-6.

[6] Wangxing, Zhangnational people, Lijing Peng, etc. the research progress and development prospect of alkali resistance of japonica rice in cold regions [ J ] crop academic newspaper 2016(6):1-8.

[7] Discussion of problems related to planting rice in saline and alkaline land [ J ] Chinese rice, 2018,24(4):1-2.

[8] Influence of saline-alkali soil cultivation and salt washing ways on growth and yield of rice [ J ] Chinese rice, 2018,24(4):68-71.

[9] Positioning and cloning of well, Zhang Wenhua, rice salt-tolerant gene and research progress of auxiliary selection and improvement of variety salt-tolerant molecular marker [ J ] Chinese Rice science, 2017,31(2): 111-.

[10]You QY,Zhai K,Yang DL,Yang WB,Wu JN,Liu JZ,Pan WB,Wang JJ,Zhu XD,Jian YK,Liu JY,Zhang YY,Deng YW,Li Q,Lou YG,Xie Q,He ZH.An E3 ubiquitinligase-BAG protein module controls plant innate immunity and broad-spectrumdisease resistance[J].Cell Host&Microbe,2016,20:758-769.

Disclosure of Invention

In view of the above, the invention aims to provide a rice salt tolerance stress gene OsBAG4, a coding protein and application thereof.

The invention provides a rice salt-tolerant stress gene OsBAG4, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention provides a protein for coding a rice salt-tolerant stress gene OsBAG4, wherein the amino acid sequence of the protein is shown in SEQ ID NO. 2.

The invention provides a plant expression vector containing the rice salt-tolerant stress gene OsBAG 4.

Preferably, the species of vector in the plant expression vector includes pCsV 1300;

the multiple cloning site for inserting the OsBAG4 into the pCsV1300 is between XbaI and BamHI.

The invention provides a primer pair for amplifying a rice salt-tolerant stress gene OsBAG4, wherein the nucleotide sequence of an upstream primer in the primer pair is shown as SEQ ID NO. 3; the nucleotide sequence of the downstream primer in the primer pair is shown as SEQID NO. 4.

The invention provides a rice salt stress resistant recombinant cell, which comprises a rice salt stress resistant gene OsBAG4 or a plant expression vector.

The invention provides application of the OsBAG4, the protein, the plant expression vector, the primer pair or the recombinant cell in cultivation of salt-tolerant transgenic rice or salt stress of rice.

The invention provides a rice salt-tolerant stress gene OsBAG4, which is characterized in that a plant expression vector containing an OsBAG4 gene is introduced into rice Nipponbare mature embryo induced callus cells by an agrobacterium-mediated method, hygromycin is used for screening resistant callus, and a transgenic positive plant is obtained through differentiation and rooting. Obtaining homozygous T2 generation rice transgenic plant through hygromycin screening and molecular identification. The obtained homozygous transgenic rice plant is subjected to stress resistance analysis after salt stress treatment in a rice nutrient solution containing 100mM NaCl, and the result shows that the salt stress tolerance of the OsBAG4 over-expressed plant is remarkably higher than that of a wild Nipponbare.

Drawings

FIG. 1 is a schematic structural diagram of a genetic transformation vector pCsV1300 provided by the present invention;

FIG. 2 is a schematic diagram of the results of the expression level detection of the salt stress-tolerant related gene OsBAG4 of transgenic rice plants and wild type Nipponbare rice;

FIG. 3 is a graph showing the phenotypic results of transgenic rice plants and wild-type Nipponbare salt stress treated;

FIG. 4 is a statistical graph of the survival rate of transgenic rice plants after treatment with wild type Nipponbare salt stress.

Detailed Description

The invention provides a rice salt-tolerant stress gene OsBAG4, the nucleotide sequence of which is shown as SEQ ID NO.1 and is 789bp Open Reading Frame (ORF) (ATGATGAGCGGCGTTGGAGGAGGCAGATCGGGCGGGAGGGACGCGGAGGGCGAGTGGGAGGTCCGGCCTGGCGGGATGCTGGTGCAGCGCAGGGACGGCGACACGGGTCCGGCCGTCAGGCTCAGGGTCTCCCACGGCGCCTCCTTCCGCGACGTCGCCGTGCCGGCGCACTCCACCTTCGGTGAATTGAAGGGGGTCCTTACCCAGGCAACTGGCGTAGAGCCTGAAAGGCAGAGGCTCTTCTTCCGTGGGAAGGAGAAGAGTGACAATGAGTTCCTGCATACAGCTGGGGTCAAGGATGGAGCAAAACTTCTTCTACTTGAGAAGCCTGCCCCTGCCAATGTAGAGCAGAGGGCCGAGCCAGTAATTATGGATGAGAGCATGATGAAGGCTTGTGAGGCTGTTGGCCGTGTAAGAGCTGAAGTTGACAGACTCTCTGCCAAGGTATGTGATTTGGAGAAGAGTGTGTTTGCAGGGAGAAAGATTGAGGATAAAGATTTTGTTGTCTTGACGGAGCTTCTTATGATGGAGCTGCTGAAACTTGATGGCATAGAGGCAGAGGGAGAAGCAAGGGCACAAAGGAAGGCTGAGGTACGCCGTGTCCAAGGTCTTGTGGAGACGTTGGATAAGCTGAAGGCAAGAAATGCCAATCCCTTCAGCGATCAAAACAAATCTGTTTCAGTGACAACGCAGTGGGAGACGTTCGACAATGGCATGGGCAGCTTGAATGCACCCCCACCACGGGTTTCTTCCACACAAATAAACACCGACTGGGAGCAATTCGACTAG), the initiation codon is ATG, and the termination codon is TAG.

The invention provides a protein for encoding a rice salt-tolerant stress gene OsBAG4, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2 (MMSGVGGGRSGGRDAEGEWEVRPGGMLVQRRDGDTGPAVRLRVSHGASFRDVAVPAHSTFGELKGVLTQATGVEPERQRLFFRGKEKSDNEFLHTAGVKDGAKLLLLEKPAPANVEQRAEPVIMDESMMKACEAVGRVRAEVDRLSAKVCDLEKSVFAGRKIEDKDFVVLTELLMMELLKLDGIEAEGEARAQRKAEVRRVQGLVETLDKLKARNANPFSDQNKSVSVTT QWETFDNGMGSLNAPPPRVS STQINTDWEQ FD). The protein coded by the rice salt-tolerant stress gene OsBAG4 contains a BAG structural domain and consists of 262 amino acids.

The invention provides a plant expression vector containing the rice salt-tolerant stress gene OsBAG 4. The type of vector in the plant expression vector preferably includes pCsV 1300; the multiple cloning site of the OsBAG4 inserted into the pCsV1300 is between XbaI and BamHI (the structure of the plant expression vector is shown in figure 1). The preparation method of the plant expression vector preferably carries out enzyme digestion on XbaI and BamHI to obtain a linearized vector for purification and recovery, connects the recovered DNA fragment to the restriction enzyme digestion site of the genetic transformation vector pCsV1300, transforms the recombinant vector into escherichia coli, and extracts a plasmid for PCR and enzyme digestion verification.

The invention provides a primer pair for amplifying the rice salt-tolerant stress gene OsBAG4, wherein the nucleotide sequence of an upstream primer in the primer pair is shown as SEQ ID NO.3 (OsBAG 4-XbaI-F: 5-gctctagaatgatgagcggcgttgga-3'); the nucleotide sequence of the middle and the downstream primers of the primer pair is shown in SEQ ID NO.4 (OsBAG 4-BamHI-R: 5-cgggatccgtcgaattgctcccagtcg-3'). The source of the primer pair is not particularly limited in the present invention, and it can be obtained by a method well known in the art, for example, artificially synthesized by a gene synthesis company。

The invention provides a rice salt-tolerant stress recombinant cell, which comprises a rice salt-tolerant stress gene OsBAG4 or a plant expression vector. The cells in the recombinant cells comprise eukaryotic cells and prokaryotic cells, the eukaryotic cells comprise rice cells, and the prokaryotic cells comprise escherichia coli. The preparation method of the recombinant cell preferably introduces the plant expression vector into cells, the introduction method is different according to different cell types, a transformation method is preferably adopted when the cells are prokaryotic cells, and the cells are eukaryotic cells and are preferably transformed by an agrobacterium-mediated transformation method.

The invention provides application of the OsBAG4, the protein, the plant expression vector, the primer pair or the recombinant cell in cultivation of salt-tolerant transgenic rice or salt stress of rice.

In the method for cultivating salt-tolerant transgenic rice or resisting salt stress of rice, the OsBAG4 is preferably inserted into a vector to construct the plant expression vector, the positive plant expression vector is introduced into rice cells after screening and checking, and the rice plant obtained through positive screening and cultivation is the transgenic rice. Through qPCR verification, compared with wild type, the expression level of OsBAG4 in 3 independent OsBAG4 overexpression plants (OsBAG4OX-1, OsBAG4OX-2 and OsBAG4OX-3) is increased by more than 60 times.

In the invention, the transgenic rice is subjected to a salt tolerance test, and the result shows that the tolerance of the OsBAG4 overexpression plant to salt stress is enhanced compared with that of the wild Nipponbare, and the survival rate of the OsBAG4 overexpression plant is obviously higher than that of the wild Nipponbare.

The rice salt tolerance stress gene OsBAG4, the encoded protein and the application thereof provided by the invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the invention.