感染甘蔗花叶病毒甘蔗叶片cDNA文库的构建及评价

Construction and Evaluation of Yeast Two Hybrid cDNA Library for Sugarcane Leaf Infected with Sugarcane mosaic virus(SCMV)

为了研究甘蔗(Saccharum officinarum L.)与甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)的互作机制,构建感染SCMV的甘蔗叶片的酵母双杂交cDNA文库。以热带种Badila为供试材料,提取感染SCMV植株的叶片总RNA,分离纯化获得mRNA,通过反转录反应合成3种读码框的双链cDNA。用Gateway技术,通过BP和LR反应,将cDNA迅速重组到酵母双杂交载体pGADT7-DEST上,构建酵母双杂交文库。结果表明,文库容量为2.0×107cfu,随机挑取24个克隆检测,重组率为100%,cDNA插入片段长度主要分布在1kb^2kb,文库质量较好。说明该文库的构建为筛选与SCMV互作的甘蔗寄主因子基因,研究甘蔗与SCMV的互作机制及甘蔗抗花叶病育种奠定基础。

In order to investigate the interaction between sugarcane and Sugarcane mosaic virus（SCMV）,ayeast two hybrid cDNA library of Badilaleaves infected by SCMV was constructed.The mRNA was isolated and purified from total RNA,and used as a template to synthesize the double-stranded cDNA.Gateway technology was applied to construct the cDNA library.The cDNA was ligated with a three-frame adapter.BP reaction was performed to generate a corresponding entry library and LR reaction was performed to transfer the cDNA into a pGADT7-DEST vector.The cDNA library contained 2.0×10^7 cfu with a 100%recombination rate based on 24 randomly selected clones.Most insert sizes were from 1kb-2kb.These results indicate that the library is in high-quality and will benefit the isolation of sugarcane genes that interact with SCMV.This will aid in understanding the mechanisms underlining the interaction.Thus,this study will facilitate sugarcane breeding and germplasm identification that focuses on sugarcane mosaic disease resistance.