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Soybean GmST1 gene mutant plant and preparation method thereof

A mutant, soybean technology, applied in the field of plant biology, can solve problems such as difficulty, low transformation rate, and increased functional analysis of disease resistance genes

Active Publication Date: 2020-06-09
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the large soybean genome and the polyploidization phenomenon and low transformation rate of soybean, the functional analysis of disease resistance genes has increased the difficulty

Method used

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  • Soybean GmST1 gene mutant plant and preparation method thereof
  • Soybean GmST1 gene mutant plant and preparation method thereof
  • Soybean GmST1 gene mutant plant and preparation method thereof

Examples

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Embodiment 1

[0032] Example 1. Preparation method of soybean GmST1 gene mutant plant.

[0033] This embodiment takes the first target as an example (the nucleotide sequence of the target is 5'AGAAAGGGGAGGAAATAA3') located at bases 47 to 64 of the GmST1 coding region to describe the preparation method of soybean GmST1 gene mutant plants.

[0034] 1) Design gRNA single target primers based on soybean GmST1 gene.

[0035] Firstly, the soybean GmST1 gene was amplified, and the soybean variety Dongnong 93-046 was used as the material. When the first group of three compound leaves were grown, the total RNA was extracted and the first strand of cDNA was synthesized by reverse transcription. According to the sequence of the GmST1 gene (Glyma.13G191400) on Phytozome, the primer 5 for gene cloning (primer 1) was designed using Primer 5 software. The nucleotide sequence is as follows:

[0036] Primer 1-S: 5'-GAAGATCTATGGCTCCAACAAATGTCAC-3' (shown in SEQ ID NO: 20);

[0037] Primer 1-A: 5'-CTTGGTTACCTTAAAATGAC...

Embodiment 2

[0066] Example 2. Preparation method of soybean GmST1 gene mutant plant.

[0067] In this embodiment, the second target (the nucleotide sequence of the target is 5'CTTCCTAGGGAGAGAGGT 3') is located at base 115 to 132 of the GmST1 coding region to describe the preparation method of soybean GmST1 gene mutant plants.

[0068] 1) Design a gRNA single target primer based on soybean GmST1 gene.

[0069] According to the GmST1 gene sequence (shown in SEQ ID NO:1), the method for obtaining the gene is the same as that described in Example 1. Design gRNA single target primers. The forward primer is GmST1-2-S, 5'-ttgCTTCCTAGGGAGAGAGGT-3' (shown in SEQ ID NO: 4), and the reverse primer is GmST1-2-A, 5'-aacACCTCTCTCCCTAGGAAG-3' (SEQ ID NO: 5 Shown), the above-mentioned primers were synthesized by the primer synthesis company.

[0070] 2) Preparation of gRNA single target primer dimer.

[0071] Mix the above-mentioned forward and reverse primers in the following proportions to form a dimer. GmST...

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Abstract

The invention provides a soybean GmST1 gene mutant plant and a preparation method thereof and belongs to the technical field of plant biology. The method for preparing the soybean GmST1 gene mutant plant to define the disease resistance function of genes related to anti-mosaic virus of soybeans comprises the following steps: designing a gRNA single target primer according to a soybean GmST1 gene;preparing a gRNA single target primer dimer; inserting the primer dimer into a Cas9 / gRNA vector to construct a soybean GmST1 gene knock-down vector; transforming soybeans with the GmST1 gene knock-down vector constructed in the step (3), performing screening to obtain the soybean GmST1 gene mutant plant. The method determines that the GmST1 can participate in the resistance reaction of the anti-mosaic virus of soybeans and provides theoretical basis to researches on anti-mosaic virus related genes of soybeans.

Description

Technical field [0001] The invention relates to the technical field of plant biology, in particular to a soybean GmST1 gene mutant plant and a preparation method thereof. Background technique [0002] Soybean mosaic virus disease is caused by Soybean Mosaic Virus (SMV), which is one of the most serious viral diseases affecting soybean yield and quality. In the main soybean producing areas of 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 area of ​​soybean production to decrease by 25%-60%, or even stop production in severe cases. Due to the limitations of traditional breeding methods, the use of molecular methods to screen and identify effective disease-resistant genes is particularly important for the selection of disease-resistant varieties. This improves the efficiency and accuracy of the selection of disease-resistant varieties and speeds up the selection. The process of breeding re...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113C12Q1/6895A01H5/00A01H6/54
CPCC12N15/8205C12N15/8213C12N15/113C07K14/415C12Q1/6895C12N2310/10C12N2310/20C12Q2600/158
Inventor 韩英鹏井妍赵雪滕卫丽李文滨
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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