利用酵母双杂交技术筛选鉴定与番茄环纹斑点病毒NSs蛋白互作的西花蓟马蛋白

Screening and identification of the protein interacting with the NSs protein of Tomato zonate spot virus in Franiklinella ocicdentalis(Thysanoptera:Thripidae)by yeast two-hybrid technique

【目的】筛选鉴定西花蓟马Franiklinella ocicdentalis体内与番茄环纹斑点病毒(tomato zonate spot virus,TZSV)NSs蛋白互作的介体因子。【方法】利用酵母双杂交技术筛选与TZSV NSs互作的西花蓟马蛋白;进行序列分析鉴定后,将捕获的蛋白与NSs基因回转到酵母细胞,利用营养缺陷型培养基鉴定蛋白互作情况。再利用GST Pull-down技术验证TZSV NSs与鉴定出的西花蓟马蛋白的体外互作关系。【结果】构建了TZSV NSs的酵母双杂交诱饵质粒pGBKT7-NSs,确定了诱饵质粒对酵母AH109细胞无毒性,并且无自激活活性。序列分析发现与TZSV NSs互作的西花蓟马蛋白为类电压依赖性阴离子通道(voltage-dependent anion-selective channel-like,VDAC)。酵母回转实验显示TZSV NSs与西花蓟马VDAC在酵母细胞内存在特异性互作。GST Pull-down结果表明TZSV NSs与西花蓟马VDAC在体外存在相互作用。【结论】通过酵母双杂交和GST Pull-down技术,分别在酵母细胞内和体外证实了TZSV NSs和西花蓟马VDAC存在特异性互作。这些结果有助于揭示西花蓟马VDAC蛋白调控西花蓟马持久传毒的机制,为虫传病毒病的防治提供理论基础。

【Aim】To screen and identify the protein interacting with the tomato zonate spot virus(TZSV)NSs protein in the western flower thrips,Franiklinella ocicdentalis.【Methods】The protein in F.ocicdentalis interacting with TZSV NSs was screened by yeast two-hybrid technique.After sequence analysis,the captured protein and NSs genes were cotransformed into yeast cells,and their interaction was identified by nutrient deficient medium.Furthermore,GST Pull-down was used to confirm the interaction of NSs and the identified protein in F.ocicdentalis in vitro.【Results】pGBKT7-NSs,a yeast two-hybrid bait plasmid of TZSV NSs,was constructed,and it showed no toxicity and no autoactivition to AH109 yeast cell.Sequence analysis showed that a voltage-dependent anion-selective channel-like(VDAC)protein of F.ocicdentalis interacted with TZSV NSs.The cotransformation test results showed that there was a specific interaction between TZSV NSs and VDAC in yeast cell.The GST Pull-down results showed that there was interaction between TZSV NSs and VDAC in vitro.【Conclusion】The specific interaction between TZSV NSs and F.ocicdentalis VDAC was confirmed in yeast cell and in vitro by yeast two-hybrid and GST Pull-down techniques,respectively.The results are helpful to reveal the mechanism of the protein VDAC regulating the transmission of persistent-propagative plant viruses in F.ocicdentalis and provide a theoretical basis for the prevention and control of insect-borne virus diseases.