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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。采用同源重组反应制备三框型cDNA入门文库,再通过同源重组将入门文库转移到Gateway兼容载体pGADT7-DEST上,构建获得酵母双杂交cDNA文库。检测结果表明:文库库容量为3.68×107cfu,扩增文库滴度为2.62×1010cfu/mL;文库重组率大于95%,cDNA插入片段平均长度>1kb,达到了标准cDNA文库的要求。灰飞虱高带毒群体酵母双杂交cDNA文库的构建为开展昆虫介体与水稻条纹病毒互作机制的研究奠定了基础。
In order to study the interaction between Laodelphax striatellus Fallén and rice stripe virus (RSV), a yeast two-hybrid cDNA library of Laodelphax striatellus Fallén was constructed in this study. The laboratory-screened Laodelphax striatellus population was used as material to separate and purify mRNA, reverse transcribed into double-stranded cDNA, ligated into three-frame type linker, and purified by column chromatography. The triennial cDNA library was prepared by homologous recombination reaction. The library was transferred to the Gateway compatible vector pGADT7-DEST by homologous recombination to construct a yeast two-hybrid cDNA library. The results showed that the capacity of library was 3.68 × 107cfu, the titer of amplified library was 2.62 × 1010cfu / mL, the library recombination rate was more than 95%, and the average length of cDNA insert was> 1kb, which met the requirements of standard cDNA library. Construction of Yeast Two-hybrid cDNA Library of Laodelphax striatellus with High Virulence Population Layed Foundation for the Study on the Interaction Mechanism between Insect Mediators and Rice Stripe Virus.