草地贪夜蛾细胞系IPLB-Sf-9酵母双杂交cDNA文库的构建

Construction of a yeast GAL4 fusion cDNA library from Sf9 cells

草地贪夜蛾Spdoptera frugiperda是危害玉米、高粱等农作物的重要害虫。草地贪夜蛾卵巢细胞系IPLB-Sf-9(Sf9)广泛应用于病毒学研究和真核生物基因表达。本研究从Sf9细胞中提取总RNA,应用SMARTTM技术,构建以pGADT7-Rec为载体的Sf9细胞酵母GAL4AD融合cDNA文库。Sf9细胞总RNA的电泳谱型与哺乳动物总RNA存在差异,其28S rRNA带弱于18S rRNA带,所合成的原始ds cDNA大小为0.1～4kb。文库展现良好的多态性,载体质粒插入的cDNA片段大小大部分在0.3～2kb之间。Sf9细胞酵母GAL4AD融合cDNA文库的构建为草地贪夜蛾功能基因的识别和鉴定,为研究杆状病毒与宿主细胞的互作机制提供了重要研究工具。

IPLB-Sf-9 （sig） is a cell line derived from pupal ovarian tissue of the fall armyworm, Spo- doptera frugiperda, which is permissive to AcMNPV and other baculoviruses, and is widely used for production of functional proteins with baculovirus expression vectors. A yeast GAIA fusion cDNA library from Sf9 cells was constructed. Total RNA of Sf9 cells was purified by using Trizol reagents. The cDNAs of the mRNAs contained in the purified total RNA sample were synthesized by SMARTTM technology, which were subsequently cloned into the plasmid pGADT7-Rec. In the recombinant plasmids, the cDNA fragments were fused with the yeast GAL 4 activation domain-encoding sequence. PCR performed to examine polymorphism of the library detected cDNA inserts, which were mainly 0. 3 - 2 kb in size. The constructed library would be useful for identification and characterization of functional genes of S. frugiperda and for research in interaction between virus and host cells.