Construction and application of anti-sclerotinia sclerotiorum gene GmPR5 and GmPR5 transgenic plant

A sclerotinia and transgenic technology, applied in the field of genetic breeding, can solve problems such as unclear function of PR5

Active Publication Date: 2020-03-06
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are few reports on the PR5 gene, and the function of PR5 is still unclear

Method used

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  • Construction and application of anti-sclerotinia sclerotiorum gene GmPR5 and GmPR5 transgenic plant
  • Construction and application of anti-sclerotinia sclerotiorum gene GmPR5 and GmPR5 transgenic plant
  • Construction and application of anti-sclerotinia sclerotiorum gene GmPR5 and GmPR5 transgenic plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Cloning of the GmPR5 gene.

[0067] 1) The soybean antibacterial sclerotinia cultivar ‘Maple Arrow’ 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 predicted GmPR5 gene sequence on Phytozome, use Primer 5 software to design gene cloning primers (primer pair 1), and use cDNA as a template to carry out RT-PCR reaction. The reaction system is as follows, and the reaction program: 94 ° C for 5 min; 37 cycles: 95°C for 30s, 60°C for 30s, 72°C for 80s; 72°C for 10min. After the reaction, the PCR product was taken for 1% agarose gel electrophoresis detection and gel recovery and purification of the target fragment ( figure 1 ).

[0069]

[0070] 3) According to the steps of the PGM-T cloning kit of TIANGEN Company, the gel recovery product obtained above was connected to the cl...

Embodiment 2

[0071] Example 2. Analysis of the expression pattern of the GmPR5 gene.

[0072] 1. The pathogen, Sclerotinia sclerotiorum, was collected from the soybean sclerotinia disease plot in Daqing, Heilongjiang Province. It was isolated and purified in the laboratory, and the bacteria were inoculated on PDA medium to multiply.

[0073] 2. Take materials.

[0074] (1) sowing "Maple Arrow" and William82 (William82 is used as the experimental reference control group), and the roots, stems, leaves, hypocotyls, cotyledons, flowers, Seed in three replicates each.

[0075] (2) Sow the disease-resistant variety "Maple Arrow" and the susceptible variety "Hefeng 25" in vermiculite, grow until the true leaves are pulled out, the leaf margins are separated, and the true leaves are fully expanded, and the hypocotyl is near the 1 / 3 of the surface. Cut a wound with a length of about 1 cm and a depth of about 1 mm, and cut the pre-populated Sclerotinia sclerotiorum (the hyphae is in the growth sta...

Embodiment 3

[0088] Example 3. GmPR5 gene subcellular localization analysis

[0089] Using pCambia1302 as the backbone vector, using Clontech's In-Fusion seamless ligation kit ( HD Cloning Kit), and completed the construction of the pCambia1302-GmPR5-GFP fusion expression vector according to the operating instructions provided by it, specifically: designing the full-length CDS primer of the GmPR5 gene, and adding a 15bp NcoI restriction site from pCambia1302 to the 5' end of the primer Point the flanking sequence as a modification (primer pair 5), with a reaction program: 98°C for 3min; 38 cycles: 98°C for 10s, 55°C for 10s, 72°C for 5s; 72°C for 10min. The PCR reaction was carried out, and the product was recovered by gel. The pCambia1302 plasmid was digested with Nco Ⅰ enzyme and then recovered by the gel. The recovered product and the vector fragment were separated according to HD CloningKit operation instructions Complete the ligation reaction, transform Escherichia coli, and finall...

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Abstract

The invention discloses construction and application of an anti-sclerotinia sclerotiorum gene GmPR5 and a GmPR5 transgenic plant, and belongs to the technical field of genetic breeding. In order to research the sclerotinia sclerotiorum resisting mechanism of soybeans and accelerate the sclerotinia sclerotiorum resisting breeding process of the soybeans, the invention provides the anti-sclerotiniasclerotiorum gene GmPR5, wherein the nucleotide sequence of the anti-sclerotinia sclerotiorum gene GmPR5 is shown as SEQ ID No. 1; and a recombinant vector and a recombinant bacterium containing the gene. According to the invention, a transgenic soybean plant is obtained by overexpression of the disease-resistant gene GmPR5. Through post-phenotypic identification, the GmPR5 is determined to participate in the sclerotinia sclerotiorum resisting reaction, a new thought is provided for obtaining a soybean variety which is completely resistant to sclerotinia sclerotiorum, a basis is provided for further researching the function and the disease resisting mechanism of the gene, and effective molecular markers and gene resources are provided for molecular design and breeding of sclerotinia sclerotiorum resisting of soybeans.

Description

technical field [0001] The invention belongs to the technical field of genetic breeding, and in particular relates to the construction and application of a soybean sclerotinia-resistant gene GmPR5 and a GmPR5 transgenic plant. Background technique [0002] Soybean (Glycine max) is an important food crop in my country and the world, and it is also one of the important sources of edible vegetable protein and edible oil. Soybean sclerotinia is a high-incidence disease with serious damage, and it is one of the three major diseases that cause the most serious loss of soybean yield in the world. Soybean sclerotinia is a fungal disease caused by Sclerotinias clerotiorum (Lib.) de Bary, which directly affects the quality and yield of soybean. When the disease is severe, the incidence rate is as high as 50% to 90%, or even extinction. Due to the limitations of traditional breeding methods, it is particularly important to use molecular methods to screen and identify effective disease...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84C12N1/21A01H5/00A01H6/54C12R1/01
CPCC07K14/415C12N15/8282
Inventor 赵雪姜海鹏韩英鹏战宇航李文滨李海燕滕卫丽
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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