Sclerotinia sclerotiorum resistant gene GmGST1 and the construction and application of a GmGST1 transgenic plant

A gene and plant technology, applied in the field of plant genetic engineering, can solve the problems of scarcity of genetic resources, complex anti-sclerotinia mechanism of soybeans, restrictions on the efficiency and accuracy of new variety breeding, and achieve the effect of improving resistance

Active Publication Date: 2020-03-24
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 breeding of disease-resistant varieties. efficiency and accuracy
[0003] In the study of soybean sclerotinia resistance genes, it is limited to the application of the oxalate oxidase gene Germin, oxalate decarboxylase and respiratory burst oxidase genes. There are no related reports on other types of genes. However, the mechanism of soybean anti-sclerotinia resistance is complex and there are a large number of loci and key genes to be discovered and utilized

Method used

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  • Sclerotinia sclerotiorum resistant gene GmGST1 and the construction and application of a GmGST1 transgenic plant
  • Sclerotinia sclerotiorum resistant gene GmGST1 and the construction and application of a GmGST1 transgenic plant
  • Sclerotinia sclerotiorum resistant gene GmGST1 and the construction and application of a GmGST1 transgenic plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Acquisition of anti-sclerotinia gene GmGST1.

[0031] 1) Using Maple Arrow, a soybean antibacterial sclerotinia variety, as the material, when the first group of three compound leaves grows, the material is taken, and the total RNA is extracted. The results are as follows: figure 1 shown, and reverse-transcribed to synthesize cDNA first-strand.

[0032] 2) According to the GmGST1 gene sequence on Phytozome, use Primer 5 software to design gene cloning primers,

[0033] Primer 1-S: 5'-AGATCTATGGCCGTTACCTTCTCAAAT-3', the sequence is shown in SEQ ID NO.2;

[0034] Primer 1-a: 5'-GGTTACCTTAGATTTTGTTGAATGCAAC-3', the sequence is shown in SEQ ID NO.3.

[0035] Using cDNA as a template PCR reaction, the reaction system is as follows:

[0036]

[0037] Reaction program: 94°C for 5min; 37 cycles: 94°C for 30s, 54°C for 30s, 72°C for 40s; 72°C for 10min, store at 4°C. After the reaction, the PCR products were taken for 1% agarose gel electrophoresis detection an...

Embodiment 2

[0039] Example 2. Construction of anti-sclerotinia transgenic soybean plants.

[0040] (1) Construction of overexpressed GmGST1 gene recombinant vector, the specific steps are as follows:

[0041] Plasmid pCambia3301 and pGM-GmGST1 prepared in plasmid Example 1 were double-digested with BstEII and BglII respectively, and after detection by agarose gel electrophoresis, the pCambia3301 vector backbone and the GmGST1 gene digestion fragment were recovered and purified, and the two fragments were connected and transformed. BstEII and BglII double enzyme digestion was performed on the picked single spot for identification, and the recombinant vector pCambia3301-GmGST1 carrying the GmGST1 gene was obtained. The enzyme digestion identification results are as follows: image 3 shown.

[0042] (2) Import the recombinant vector constructed in step (1) into Agrobacterium to obtain a recombinant bacterium with the GmGST1 gene, the specific steps are as follows:

[0043] Transform the pC...

Embodiment 3

[0052] Example 3. Construction of anti-sclerotinia transgenic soybean plants.

[0053] Repeat Example 2, the difference from Example 2 is that the seeds used in step (3) 2) of this example are the soybean variety Hefeng 25. In this embodiment, a total of 16 T0 generation positive plants were obtained.

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Abstract

The invention discloses a sclerotinia sclerotiorum resistant gene GmGST1 and the construction and application of a GmGST1 transgenic plant, which belong to the technical field of plant genetic engineering. The invention aims to solve the problem of few available gene resources in the aspect of soybean sclerotinia sclerotiorum resistance at present. The invention provides the sclerotinia sclerotiorum resistant gene GmGST1, wherein the sequence of the gene is shown as SEQ ID No. 1. According to the invention, the transgenic soybean plant is obtained by overexpression of the disease-resistant gene GmGST1; later representative identification determines that GmGST1 can participate in a soybean sclerotinia sclerotiorum resisting reaction; the soluble pigment level in stems of receptor soybean germplasm and the resistance of GmGST1 to sclerotinia sclerotiorum can be remarkably improved; and effective molecular markers and gene resources are provided for soybean sclerotinia sclerotiorum resisting molecular design and breeding.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to the construction and application of an antibacterial sclerotinia gene GmGST1 and a plant transfected with the GmGST1 gene. Background technique [0002] Soybean sclerotinia is a fungal disease caused by Sclerotinia sclerotiorum (Lib.) de Bary. It is a serious worldwide disease that directly affects the quality and yield of soybeans. When the disease is severe, the incidence rate is as high as 50%. % to 90%, or even extinct. 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 breeding of disease-resistant varieties. efficiency and accuracy. [0003] In the study of soybean sclerotinia resistance genes, it is limited to the application of the oxalate oxidase gene Germin, oxalate decarboxylase and respiratory burst oxidase genes....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/11C12N15/84A01H5/00A01H6/54
CPCC12N9/1088C12N15/8282C12Y205/01018
Inventor 赵雪张倩宋伟姜海鹏韩英鹏李文滨
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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