灰飞虱高带毒(RSV)群体酵母双杂交cDNA文库的构建

为了研究灰飞虱Laodelphax striatellus Fallén与水稻条纹病毒(rice stripe virus,RSV)互作机制,本研究构建了灰飞虱高带毒群体酵母双杂交cDNA文库.以实验室筛选的灰飞虱高带毒群体为材料,分离纯化mRNA,反转录合成双链cDNA,并连接三框型接头,层析柱分级纯化.采用同源重组...

Construction of yeast two-hybrid cDNA library of high-viruliferous(RSV-infected) populations of the small brown planthopper,Laodelphax striatellus(Hemiptera:Delphacidae)

 In order to research the interaction between the small brown planthopper (Laodelphax striatellus Fallén, SBPH) and rice stripe virus (RSV), yeast two-hybrid cDNA library of high-viruliferous (RSV-infected) SBPH populations was constructed. SBPH infected with RSV was maintained in our laboratory. The mRNA was isolated and purified from high-viruliferous populations, and used as the template to synthesize the double-strand cDNAs. The cDNAs were ligated with three-frame adapter and purified by the cDNA size fractionation columns. By using homologous recombination reaction, the cDNA entry library was prepared and then recombined into Gateway compatible vector pGADT7-DEST to construct yeast two-hybrid cDNA library. Detection of the library indicated that it contained 3.68×10⁷ independent clones, and the titer of the amplified library was 2.62×10¹⁰ cfu/mL. The recombination rate was above 95%, and the average size of inserts was above 1 kb in the cDNA library. The results demonstrate that the library database meets the requirements of the standard cDNA library. The yeast two-hybrid cDNA library of high-viruliferous SBPH will be useful for the future research on the interaction between insect vector and RSV.