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Screening and application of plant antiviral new target PsbO1

An anti-virus, plant technology, applied in applications, plant peptides, plant products, etc., can solve problems such as loss

Inactive Publication Date: 2017-05-24
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a synergy between Potavirus Y virus and other viruses of other genera. Once combined infection will cause more serious symptoms and cause greater losses

Method used

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  • Screening and application of plant antiviral new target PsbO1
  • Screening and application of plant antiviral new target PsbO1
  • Screening and application of plant antiviral new target PsbO1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Immunoprecipitation

[0026] Take N. benthamiana leaves 7 days after infection with TVBMV-GFP or TVBMV-6K1GFP, add liquid nitrogen to a mortar, grind them thoroughly to powder, and add Extraction Buffer to extract total plant protein.

[0027] The composition of Extraction Buffer is as follows:

[0028] 50mM Tris-Cl, pH 7.5,

[0029] 150mM NaCl,

[0030] 10% [v / v] glycerol,

[0031] 0.5% [v / v] Nonidet P-40,

[0032] 1 tablet protease inhibitor cocktail

[0033] Centrifuge at 20,000 g for 10 min at 4°C, add the supernatant to GFP antibody-coupled agarose beads, incubate at 4°C for 3 hours, wash 5 times with Extraction Buffer without Nonidet P-40, add 2×SDS loading buffer, Cook in boiling water for 10 minutes, and separate protein bands by SDS-PAGE electrophoresis.

[0034] The protein band was excised and sent to Shanghai Jiyun Biotechnology Company for mass spectrometric identification. The results showed that there were 4 peptides homologous to PsbO1,...

Embodiment 2

[0035] Example 2: Gene Amplification

[0036] The PsbO1 gene was amplified by reverse transcription PCR using total plant RNA as a template. The reverse transcriptase used was M-MLV, and the DNA polymerase was Phusion high-fidelity polymerase.

[0037] The reverse transcription reaction system is as follows:

[0038]

[0039] The PCR reaction system is as follows:

[0040]

[0041] The obtained PsbO1 gene is 999bp in length, its sequence is shown in Seq ID No.7, and the encoded amino acid sequence is shown in Seq ID No.8.

Embodiment 3

[0042] Embodiment 3: Verification of the interaction between PsbO1 and TVBMV 6K1, 6K2

[0043] 6K1 and 6K2 can interact. PsbO1 in the 6K1GFP complex may directly interact with 6K1, or directly interact with 6K2, and indirectly interact with 6K1 through 6K2. PsbO1, TVBMV 6K1 and 6K2 genes were cloned into co-immunoprecipitation vectors pCam35S:HA and pCam35S:GFP, bimolecular fluorescent complementation vectors pYN and pYC, respectively.

[0044] After the recombinant co-immunoprecipitation vector was transformed into Agrobacterium, the leaves of Nicotiana benthamiana were infiltrated. After 3 days, the total plant protein was extracted and added to GFP antibody-conjugated agarose beads for immunoprecipitation. Protein interaction was detected by Western blot using HA antibody. PsbO1 co-immunoprecipitated with 6K2 and co-immunoprecipitated with 6K1 ( figure 1 , a), indicating that PsbO1 can directly interact with 6K2, but not with 6K1.

[0045] After the recombined bimolecu...

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Abstract

The invention relates to the field of plant antiviral genetic engineering, and provides a plant antiviral new target. On the basis of tobacco vein banding mosaic virus infection cloning, photosystem II oxygen-evolving complex protein PsbO1 is screened through immunoprecipitation and mass spectrography, and a PbsO1 gene is obtained through reverse transcription PCR amplification. Immunoprecipitation and bimolecular fluorescence complementation prove that the PsbO1 can interact with the tobacco vein banding mosaic virus 6K2. Reduction in expression of the PsbO1 gene enables an infected plant to generate resistance to the tobacco vein banding mosaic virus and potato virus Y.

Description

technical field [0001] The invention relates to the field of plant anti-virus genetic engineering, in particular, the invention relates to the screening of new target PsbO1 for resistance to tobacco vein mosaic virus and potato virus Y and its application in anti-virus infection. Background technique [0002] Viral diseases are important diseases on crops, causing huge losses to agricultural production. Planting disease-resistant varieties is the most cost-effective way to control crop viral diseases. [0003] Recent studies have found that plants also have "Achilles' ankles." Some plant proteins play a key role in virus infection. If the genes encoding these proteins are knocked out, plants will acquire resistance to viruses. [0004] Potyvirus Y (Potyvirus) is the largest genus of plant viruses, accounting for about 30% of known plant virus species, with a wide range of hosts and serious damage. There is synergy between Potavirus genus virus and multiple viruses of othe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29A01H5/00
CPCC07K14/415C12N15/8218C12N15/8283
Inventor 李向东耿超李亦晴田延平
Owner SHANDONG AGRICULTURAL UNIVERSITY
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