SMV (soybean mosaic virus)-resistant gene GmST1, cultivation method of GmST1 transgenic soybeans and application

A technology of soybean mosaic virus and cultivation method, which can be applied in application, genetic engineering, plant genetic improvement and other directions, and can solve the problems of low transformation rate and the like

Active Publication Date: 2020-06-09
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0003] Aiming at the problems of polyploidization and low transformation rate in cultivating disease-resistant varieties by traditional

Method used

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  • SMV (soybean mosaic virus)-resistant gene GmST1, cultivation method of GmST1 transgenic soybeans and application
  • SMV (soybean mosaic virus)-resistant gene GmST1, cultivation method of GmST1 transgenic soybeans and application
  • SMV (soybean mosaic virus)-resistant gene GmST1, cultivation method of GmST1 transgenic soybeans and application

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Experimental program
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Effect test

Embodiment 1

[0065] Example 1. Obtaining the anti-soybean mosaic virus gene GmST1.

[0066] The anti-soybean mosaic virus gene GmST1 of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1, and the gene can be obtained by the following method:

[0067] 1) The soybean variety Dongnong 93-046 was used as the material, and when the first group of three compound leaves grew, the material was collected, total RNA was extracted, and the first strand of cDNA was synthesized by reverse transcription.

[0068] 2) According to the GmST1 (Glyma.13G191400) gene sequence on Phytozome, utilize Primer 5 software to design gene cloning primer (primer 1), its nucleotide sequence is as follows:

[0069] Primer 1-S: 5'-GAAGATCTATGGCTCCAACAAATGTCAC-3' (SEQ ID NO: 2);

[0070] Primer 1-A: 5'-CTTGGTTACCTTAAAATGACAAGCCTGAC-3' (SEQ ID NO: 3).

[0071] The RT-PCR reaction was carried out using cDNA as a template. The reaction system was as follows. The reaction program: 94°C for 5 minutes; 38...

Embodiment 2

[0074] Embodiment 2. The breeding method of anti-soybean mosaic virus transgenic soybean.

[0075] 1) Construct the anti-soybean mosaic virus gene GmST1 obtained in Example 1 on a plant expression vector to obtain a soybean GmST1 gene recombination vector.

[0076] 2) Introduce the above-mentioned recombinant vector into Agrobacterium, then infect soybean, and obtain transgene-positive plants after identification after culturing.

[0077] The specific method is as follows:

[0078] 1) Construction of soybean GmST1 gene recombination vector:

[0079] The pGM-T-GmST1 cloning plasmid and pCAMBIA3301 carrier plasmid carrying the target gene were double-digested with BglII and BstEII respectively. Fragments and pCAMBIA3301 vector fragments were gel recovered. use T 4 The ligase was used to connect the recovered products of the two gels, and the connected products were transformed into Top10 competent cells, and a single spot was picked for bacterial liquid PCR identification. ...

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Abstract

The invention provides an SMV (soybean mosaic virus)-resistant gene GmST1, a cultivation method of GmST1 transgenic soybeans and application, and belongs to the technical field of soybean genetic breeding. Aiming at solving the problems of polyploidization and low conversion rate of cultivation of disease-resistant varieties with a traditional method, the cultivation method of the SMV-resistant transgenic soybeans is provided and comprises steps as follows: the SMV-resistant transgenic soybean gene GmST1 with the nucleotide sequence represented as SEQ ID NO:1 is constructed to a plant expression vector, and a soybean gene GmST1 recombinant vector is obtained; the recombinant vector is introduced into agrobacterium and infects the soybeans, and after cultivation, a transgenic positive plantis obtained by identification. The SMV-resistant gene GmST1 and the cultivation method can be applied to SMV-resistant crop breeding work.

Description

technical field [0001] The invention belongs to the technical field of soybean genetic breeding, and in particular relates to a cultivation method and application of soybean mosaic virus-resistant gene GmST1 and GmST1 transgenic soybean. Background technique [0002] Soybean (Glycine max) is an important grain and economic crop in my country and even in the world. Soybean Mosaic Virus, caused by Soybean Mosaic Virus (SMV), is one of the most serious viral diseases affecting soybean yield and quality. In the main soybean producing areas in Northeast my country, N1 and N3 strains are the dominant strains, and the abnormal growth of soybean plants caused by them can cause a large-scale reduction in soybean yield by 25%-60%, and even extinction in severe cases. Due to the limitations of traditional breeding methods, it is particularly important to use molecular methods to screen and identify effective disease-resistant genes for the selection of disease-resistant varieties, whi...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/54
CPCC07K14/415C12N15/8205C12N15/8283
Inventor 韩英鹏井妍赵雪滕卫丽李文滨
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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