摘要
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 research the interaction between the small brown planthopper（Laodelphax striatellus Fallén,SBPH） and rice stripe virus（RSV）,yeast two-hybrid cDNA library of high-viruliferous（RSV-infected） SBPH populations was constructed.SBPH infected with RSV was maintained in our laboratory.The mRNA was isolated and purified from high-viruliferous populations,and used as the template to synthesize the double-strand cDNAs.The cDNAs were ligated with three-frame adapter and purified by the cDNA size fractionation columns.By using homologous recombination reaction,the cDNA entry library was prepared and then recombined into Gateway compatible vector pGADT7-DEST to construct yeast two-hybrid cDNA library.Detection of the library indicated that it contained 3.68×107 independent clones,and the titer of the amplified library was 2.62×1010 cfu/mL.The recombination rate was above 95%,and the average size of inserts was above 1 kb in the cDNA library.The results demonstrate that the library database meets the requirements of the standard cDNA library.The yeast two-hybrid cDNA library of high-viruliferous SBPH will be useful for the future research on the interaction between insect vector and RSV.
出处
《昆虫学报》
CAS
CSCD
北大核心
2011年第11期1324-1328,共5页
Acta Entomologica Sinica
基金
国家重点基础研究发展规划(“973”计划)项目(2010CB126203)
国家自然科学基金项目(30970132)
国家公益性行业(农业)科研专项(201003031)
江苏省自然科学基金项目(BK2010018)
关键词
灰飞虱
水稻条纹病毒
同源重组
CDNA文库
酵母双杂交cDNA文库
Small brown planthopper（Laodelphax striatellus）
rice stripe virus
homologous recombination
cDNA library
yeast two-hybrid cDNA library
