GbRPS1 gene for resisting plant fusarium wilt and verticillium wilt and application thereof

A technology for verticillium wilt and plants, which is applied in the field of broad-spectrum disease-resistant proteins and can solve problems such as no significant resistance to fusarium wilt and verticillium wilt

Inactive Publication Date: 2013-05-08
左开井 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese scientists have transformed genes such as chitinase and peroxidase into susceptible varieties of cotton, which have a certain effect on improving the resistance of cotton to Fusarium wilt and Verticillium wilt, but the above genes are not effective for Fusarium wilt and Verticillium wilt No significant resistance

Method used

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  • GbRPS1 gene for resisting plant fusarium wilt and verticillium wilt and application thereof
  • GbRPS1 gene for resisting plant fusarium wilt and verticillium wilt and application thereof
  • GbRPS1 gene for resisting plant fusarium wilt and verticillium wilt and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Cloning of the GbRPS1 gene resistant to Fusarium wilt

[0071] (1) Construction of a cDNA library from the roots of Gossypium sea-island cotton

[0072] The construction method of the library is implemented by the method of the cDNA library construction kit of Stratagene Company, and the specific steps of the method are as follows.

[0073] The sea-island cotton variety Pima-90 can be used to extract the total RNA of cotton after being treated with Verticillium dahliae for 2 hours. Weigh 0.5 g of cottony root, grind it into a fine powder with liquid nitrogen, and divide it into two 1.5 ml eppendorf tubes. Add 1ml TRIZOL to each tube, shake vigorously to make the mixture even, and place at room temperature for 5min. Then centrifuge at 12,000g for 10min at 4°C, and pipette the supernatant into a clean 1.5ml eppendorf tube. Add 0.2ml chloroform to each tube, vibrate vigorously for 15s, and place at room temperature for 2-3min. Then centrifuge at 12,000 g for ...

Embodiment 2

[0084] The artificial synthesis of the GbRPS1 protein gene of embodiment 2 anti-fusarium wilt

[0085] According to the completed nucleotide sequence containing the 2712bp coding region, first divide 8 segments into 150-200bp single-stranded oligonucleotide fragments with cohesive ends according to the positive and secondary strand sequences respectively. Anneal the 8 complementary single-stranded oligonucleotide fragments in one-to-one correspondence between the positive strand and the auxiliary strand respectively to form 8 double-stranded oligonucleotide fragments with sticky ends. Mixed double-stranded oligonucleotide fragments, T 4 DNA ligase catalyzes assembly into a complete GbRPS1 gene. The synthetic DNA fragment contains the 202-2916 nucleotide sequence in SEQ ID NO: 1, and the two ends of the synthetic gene contain XbaI and SacI sites.

[0086] The above-mentioned artificially synthesized 5' and 3' end restriction sites are XbaI and SacI site GbRPS1 gene, which is ...

Embodiment 3

[0087] Embodiment 3 Construction of broad-spectrum resistance GbRPS1 gene plant expression vector

[0088] The specific method of broad-spectrum resistance GbRPS1 gene plant expression vector construction is as follows:

[0089] A. pBI121 and pCAMBIA2301 were digested with HindIII and EcoRI, and the pBI121 fragment with p35S-GUS-Nos-ter was ligated into pCAMBIA2301 to form the intermediate vector p35S-2301-GUS;

[0090] B. double-cut p35S-2301-GUS and the above-mentioned artificially synthesized GbRPS1 gene with XbaI and SacI, and replace the GUS at the corresponding restriction site of p35S-2301-GUS with GbRPS1, thereby obtaining the GbRPS1 gene plant expression vector ( figure 1 ). Then it was transformed into Agrobacterium strain LBA4404 for transforming cotton.

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Abstract

The invention provides a GbRPS1 gene which can improve the resistance of plants to fusarium wilt and verticillium wilt, GbRPS1 protein polypeptides coded by the GbRPS1 gene, and a method for producing the GbRPS1 protein by recombination technology. The invention also discloses the functions and applications of the GbRPS1 gene in broad-spectrum disease resistance in the resistance to fusarium wiltand verticillium wilt.

Description

technical field [0001] The invention relates to a broad-spectrum disease-resistant protein isolated from a constructed sea-island cotton cDNA library capable of resisting cotton fusarium wilt and Verticillium dahliae. Background technique [0002] There are many plant pathogenic microorganisms in the natural environment, and the outbreak of the diseases caused by them will lead to a significant reduction in crop yield, or even failure. Therefore, improving plant disease resistance to ensure crop yield is an important direction for crop genetic improvement. [0003] During the long-term evolution process, plants have gradually formed a series of complex and effective protection mechanisms: (1) the cell wall of the plant is an effective barrier for the plant to effectively prevent the invasion of pathogens; (2) when the pathogen passes through the plant cell wall, the plant passes through the Reciprocal recognition of physical elicitors produces pathogen-related resistance re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12P21/02A01H5/00
Inventor 左开井王劲陈继军黄逸群
Owner 左开井
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