番茄斑萎病毒侵染的西花蓟马酵母双杂交cDNA文库的构建和分析

Construction of Yeast Two-hybrid cDNA Library on Tomato Spotted Wilt Tospovirus-infected Frankliniella occidentalis

【目的】探明番茄斑萎病毒(Tomato spotted wilt tospovirus,TSWV)和介体西花蓟马(Frankliniella occidentalis)之间的分子互作关系,探索介体因子如何参与病毒侵染过程,以期为解析西花蓟马蛋白调控介体传毒机理奠定基础。【方法】通过若虫期饲毒获得高带毒西花蓟马群体,借助SMART技术构建TSWV侵染的西花蓟马酵母双杂交三框cDNA文库。【结果】3种读码框初级cDNA文库的库容量分别为3.0×10^(6)、2.0×10^(6)和2.0×10^(6) cfu,文库实际扩增基数大于1.5×10^(6) cfu(3种读码框均大于5×10^(5))。扩增文库平均插入片段主要分布在0.5~3.0 kb。对文库随机挑选16个克隆,测序后与GenBank数据库比对,结果显示各插入片段具有同源序列。【结论】构建的cDNA文库具有较高的库容量和重组率,可用于后续筛选西花蓟马与TSWV互作的蛋白。

【Objective】A two-hybrid eDNA library was constructed for studying the interaction between tomato spotted wilt tospovirus(TSWV)and Frankliniella occidentalis to decipher the mechanism of the disease transmission by thrip and the factors involved in the virus infection.【Method】A highly infected F.occidentalis population was created by inoculating TSWV into the thrip nymphs.Thereby,a yeast two-hybrid three-frame cDNA library on TSWV-infected F.occidentalis was constructed using the SMART technology.【Result】The established 3 reading frame cDNA libraries had the capacities of 3.0×10^(6),2.0×10^(6),and 2.0×10^(6) cfu.The actual amplification base of the library was more than 1.5×10^(6) cfu or 5×10^(5) cfu each with an average insert fragment of 0.5–3.0 kb in length.From the library 16 clones were randomly selected,sequenced,and compared with the GenBank database for of homology verification on the insert fragments.【Conclusion】The established cDNA library had an ample capacity and high recombination rate adequate for studies on the interactions between TSWV and F.occidentalis.