利用酵母双杂交系统筛选与草莓镶脉病毒P6蛋白互作的森林草莓寄主因子  

Screening of the Host Factors of Woodland Strawberry Interacting with P6 of Strawberry vein banding virus by Yeast Two-Hybrid System

作　　者：李帅[1] 蒋西子 梁伟芳 陈思涵 张享享 左登攀 胡亚会[1] 江彤[1] 

机构地区：[1]安徽农业大学植物保护学院,合肥230036

出　　处：《中国农业科学》2017年第18期3519-3528,共10页Scientia Agricultura Sinica

基　　金：国家公益性行业(农业)科研专项(201303028);国家自然科学基金(31671999;31371915)

摘　　要：【目的】草莓镶脉病毒(Strawberry vein banding virus,SVBV)是侵染草莓的主要病毒,但其侵染草莓的机制尚不清楚。论文以SVBV的P6蛋白为诱饵筛选森林草莓(Fragaria vesca)c DNA文库的寄主因子,为解析SVBV侵染草莓的分子机制提供理论依据。【方法】SVBV接种森林草莓,提取出现明显症状叶片的总RNA,Dnase I处理后,用SMART法反转录合成ds c DNA,均一化处理c DNA并酶切纯化,将<400 bp的短片段去除,其余片段连接到p GAD-T7质粒载体上,构建森林草莓初级c DNA文库。同时将SVBV P6构建到酵母双杂交诱饵载体p GBK-T7上,再将p GBK-P6和p GBK-T7分别转化酵母菌株AH109,阳性酵母菌株接种SD/-Trp液体培养基,鉴定诱饵载体对酵母细胞的毒性。将转化p GBK-P6的酵母菌分别涂布SD/-Trp、SD/-Leu-Trp和SD/-His-Trp平板,测定菌落生长情况,分析P6蛋白对酵母报告基因的自激活活性。然后用森林草莓初级c DNA文库质粒转化含有诱饵载体p GBK-P6的AH109酵母菌株,共转化子依次涂布SD/-Leu-Trp、SD/-Leu-Trp-His和SD/-Trp-Leu-His-Ade/X-α-Gal平板,最终筛选蓝色且长势较好的阳性菌落,提取酵母质粒并测序,Gen Bank中初步比对候选基因,利用Uniprot在线网站的gene ontology(GO)通路注释互作蛋白因子,分析互作蛋白的生物学功能。【结果】3种c DNA文库平均库容超过2.0×106 cfu,平均文库重组率为97%,文库插入片段平均扩增长度>1 kb,表明森林草莓c DNA文库符合试验标准。最终利用SD/-Trp-Leu-His-Ade/X-α-Gal培养基筛选得到230个酵母阳性克隆,经过序列相似性比对,除去重复序列、载体序列和移码序列,共筛得15个与SVBV P6互作的寄主因子。GO通路注释结果表明这些寄主因子参与了13种生物过程,包括泛素化、转录因子调节、防御反应、代谢过程、氧化还原和胞内氨基酸代谢等过程;这15个寄主因子的分子功能多样,包括乙酰转移酶活性、萜烯合酶活性、脱氢酶活性、�【Objective】 Strawberry vein banding virus（SVBV） is a main virus infecting woodland strawberry（Fragaria vesca）, but the SVBV infection mechanisms on woodland strawberry remains unknown. The objective of this study is to provide a theoretical basis for studying the SVBV infection mechanisms on woodland strawberry, SVBV P6 was used as a bait protein to screen the host factors from the c DNA library of woodland strawberry. 【Method】 Woodland strawberries were inoculated with SVBV, and total RNA was extracted from the leaves showed obvious disease symptoms. The total RNA was treated with Dnase I and double-stranded c DNA was synthesized using SMART technology. c DNA was treated with homogenization and enzymatic digestion, and the short fragments with length less than 400 bp were removed. Then the other c DNA fragments were ligated to plasmid vector p GAD-T7 to construct the primary c DNA library of woodland strawberry. Simultaneously, SVBV P6 was ligated into the yeast two-hybrid bait vector p GBK-T7, and the plasmids of p GBK-P6 and p GBK-T7 were transformed into AH109, respectively. The positive yeast clones were grown in the SD/-Trp liquid medium for identifying the toxicity of p GBK-P6 on the yeast AH109. The yeast transformed with p GBK-P6 was grown on the plate of SD/-Trp, SD/-Leu-Trp and SD/-His-Trp medium, respectively, and then the growth situation of the yeast was tested and the self-activating effect of p GBK-P6 on the reporter gene of yeast was analyzed. Then the AH109 containing bait vector p GBK-P6 was transformed with the primary c DNA library of woodland strawberry, the co-transformed yeasts were coated on the plate of SD/-Leu-Trp, SD/-Leu-Trp-His and SD/-Trp-Leu-His-Ade/X-α-Gal medium in turn. Finally, the blue and well grown positive clones were selected. The plasmids of positive yeast clones were extracted and sequenced. The candidate genes were preliminarily compared in the Gen Bank, and the interacted protein factors were annotated and the protein＇s biological functions were anal

关 键 词：草莓镶脉病毒 森林草莓 CDNA文库 酵母双杂交 筛选 寄主因子 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治]