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The Interaction between the Coat Protein of Cucumber Green Mottle Mosaic Virus and Proteins of Nicotiana Benthamiana

Author: ZhengHaiGang
Tutor: ChenQiJian
School: Fujian Agriculture and Forestry University
Course: Microbiology
Keywords: Cucumber green mottle mosaic virus Yeast two-hybrid Bimolecular fluorescence complementation Subcellular localization
CLC: S435.72
Type: Master's thesis
Year: 2011
Downloads: 40
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Abstract


Cucumber green mottle mosaic virus (Cucumber green mottle mosaic virus, CGMMV) is a member of the tobacco mosaic virus genus (Tobamovirus) , and mainly infect Cucurbitaceae , multiple outbreaks in recent years in our country , to bring huge economic losses. Currently, the CGMMV encoded protein function and disease prevention have a certain degree of understanding , but not clear interaction between the virus and the host , the interaction between the coat protein coding CGMMV host tobacco protein . This article extracted total RNA from infection CGMMV cucumber leaves obtained the target gene CGMMV -CP by RT-PCR , and successfully cloned into pMD18-T vector and verified by sequencing , the constructed recombinant yeast expression the carrier pDB - CP . Transformed yeast MaV203 yeast expression vector PDB - CP containing the PDB- CP yeast prepared yeast competent screening tobacco cDNA library , using a sequential transformation method , the conversion product of the coating of different auxotrophic medium observed yeast growth and detection of β-galactosidase activity . The results show that the CP as bait screening 14 positive clones , sequence comparison after only a clone with the correct reading frame , and tobacco ribosomal protein L14 ( RPL14 ) protein with high homology . Whether the positive clones were obtained for further determine gene encoded by the full-length protein interaction with the CP , the the RPL14 gene construct to the yeast expression vector pDEST22 co-transformed yeast MaV203 with CP , via different auxotrophic medium filtering and β galactosidase activity of the detection results show that the interaction between RPL14 and CP . Bimolecular fluorescence complementation experiments and co-immunoprecipitation experiments further confirmed CP and RPL14 existence of interaction . The subcellular localization studies show that the the RPL14 mainly localized in the nucleus and cytoplasm of small particles in CP located in the cytoplasm and the nucleus .

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Crop pests and diseases and their prevention > Economic crop pests and diseases > Tobacco pests and diseases
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