Research Of Host Factors With P22 Gene Of Cucurbit Chlorotic Yellows Virus And Production Of Antiserum Against Cucurbit Chlorotic Yellows Virus

Cucumis melo,as one important kind of Cucurbitaceae plant,is widely cultivated in tropics and temperate regin.It's also a significant commercial crop in China.Cucurbit chlorotic yellows virus(CCYV)can infect Cucumis melo and affect the quality seriously.Cucurbit chlorotic yellows virus(CCYV)is an emergent virus that belongs to genus Crinivirus in the family Closteroviridae.The virus genome consists of two single-stranded RNA components designated as RNA1 and RNA2.P22 gene,the 22-kDa protein,is encoded by 3' end on RNA1.In order to screen the host factors interacted with the P22 protein,the study is based on Yest Two-Hybrid system to illuminate the mechanism of the P22 protein in virus infection process.The main research content and result are as follows:P22 gene was amplified by RT-PCR and clone into the expression vector pGex-4T-3.After verification by PCR and sequencing,the recombinant plasmid,designated as pGexp22,was transformed into Escherichia coli strain BL21.The SDS-PAGE analyses showed that about 48 kDa specific fusion protein was produced after induction by IPTG.The expressed protein was purified from SDS-PAGE and the antiserum against the protein was raised in rabbit.The titer of antiserum was 1.28×106 estimated by ACP-ELISA.Western blot confirmed the detection specificity by polyclonal antiserum in diseased leaf.This experiment laid the foundation for both detection of CCYV and study of P22 function.Extract the total RNA from the cucumis melo leaf to build a cDNA prey libray by SMART technology.Connect the full-length P22 gene with the bait plasmid pGBKT7 to get the recombinant clone pGBKT7p22.Transcriptional activation test showed that the protein is inactive.Mating the library host strain with bait strain,we obtained about seleventeen positive clones.After sequencing analysis,we selected four genes which is with the high homology and the right reading frame for further analysis.Amplifying the whole reading frames of the four genes and build them on the pGADT7 carrier.After the validation of the Yeast co-transformation with P22,all the four genes can trigger the interaction.Devide the P22 gene into three parts after the analysis of protein founction.Build each of three parts with the bait plasmid pGBKT7 so that to get Recombinant clones pGBKT7Fl?pGBKT7F2 and pGBKT7F3.Then mate each pGADT7M2,pGADT7RPol and pGADT7M39 with the three recombinant clones.The study shows that M2 can interact with pGBKT7F2;Rpol can interact with pGBKT7Fl and pGBKT7F2,while M39 can't trigger the interaction.