白背飞虱酵母双杂交cDNA文库的构建  

Construction of yeast two-hybrid cDNA library of white-backed planthopper ( Sogatella furcifera)

作　　者：薛进 李静[2] 羊健[2] 唐彦[1,3] 梁瑶 陈剑平 张恒木[2] 

机构地区：[1]植物病虫害生物学与防控湖南省重点实验室湖南农业大学植物保护学院病毒研究所,湖南长沙410128 [2]浙江省农业科学院病毒学与生物技术研究所,浙江杭州310021 [3]湖南农业大学东方科技学院,湖南长沙410128

出　　处：《浙江农业学报》2017年第12期2060-2067,共8页Acta Agriculturae Zhejiangensis

基　　金：湖南省教育厅科学研究项目(16C0773)

摘　　要：白背飞虱(Sogatella furcifera Horváth)作为一种迁飞性害虫,不仅自身危害水稻,而且以持久性方式传播南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)。为了研究白背飞虱与SRBSDV之间的相互作用,需要构建一个高质量的酵母筛选文库,试验利用Gateway技术构建了白背飞虱cDNA酵母表达文库,检测分析显示,白背飞虱初级文库库容量约为1.72×10~7CFU,cDNA插入片段主要分布在500~2 000 bp,重组率约为95.8%;次级文库库容量约为1.73×10~7CFU,cDNA插入片段主要分布在400~2 000bp,重组率为100%;Y187酵母菌株的白背飞虱酵母双杂交cDNA文库库容量约5.09×10~7CFU,插入片段大小在750~2 000 bp,重组率100%,再随机挑选36个单克隆进行测序分析,所得结果与Gen Bank数据库比对显示36个克隆中含有22个不同的基因,长度在750~2 000 bp,这些分析表明,该文库可以用于酵母双杂交的后续筛选,为研究白背飞虱与SRBSDV之间的互作奠定了基础。White-backed planthopper( Sogatella furcifera Horváth) is a major migratory pest that feed on rice plants and transmit Southern rice black-streaked dwarf virus( SRBSDV) in a persistent manner. In order to screen interactions between the genes of WBPH and SRBSDV,a cDNA expression library of WBPH was firstly constructed into the yeast plasmids using Gateway technique in this study. The results showed that the primary library consisted of 1. 72 ×10~7 CFU( colony-forming units),the sizes of most inserts were 500-1 500 bp in length,and the recombinant frequency was about 95. 8% ; the titer of secondary cDNA library was 1. 73 × 10~7 CFU,the sizes of most inserts were 400-2 000 bp in length,and the recombinant frequency was 100% ; the titer assays showed that the cDNA library was approximately 5. 09 × 10~7 CFU for yeast two-hybrid cDNA library of Y187; PCR assays showed that the average length of insertions were 750-2 000 bp and the recombinant frequency of the cDNA library was high up to 100% ,random sequence analysis of 36 clones showed that 22 of the clones were different in Gen Bank. These results consistently indicated that the cDNA library could be used for screening interactions in the yeast two hybrid experiments,which laid the foundation for studying on the interactions between white-backed planthopper and SRBSDV.

关 键 词：白背飞虱 南方水稻黑条矮缩病毒(SRBSDV) GATEWAY技术 酵母双杂交 CDNA文库 

分 类 号：S435.11[农业科学—农业昆虫与害虫防治]