大麦黄矮病毒外壳蛋白基因和运动蛋白基因酵母表达载体的构建

The Yeast Expression Vectors' Construction of the Barley Yellow Dwarf Virus Coat Protein Gene and the Movement Protein Gene

根据已知的大麦黄矮病毒GPV株系的外壳蛋白(CoatProteinCP)和移动蛋白(MovementProteinMP)基因序列合成了CP,MP基因的上下游引物,通过PCR扩增获得目的片段,经过SalI和PstI双酶切、连接、转化、重组质粒的酶切鉴定及基因测序,构建了酵母表达载体pGBKT7-GPV-CP和pGBKT7-GPV-MP,用于在酵母双杂交分析中表达诱饵融合蛋白,为进一步筛选小麦cDNA文库内与大麦黄矮病毒相互作用的寄主因子、克隆寄主因子,推测其种类和功能打下基础.

The primers of CP and MP gene's upstream and downstream were synthesized according to the known BYDV-GPV strain's coat protein and movement protein gene sequence.Target fragments were aquired by means of PCR amplification,which were cleaved with restrict endonuclease Sal I/Pst I,ligation,transformation,recombinant plasmids'restrict identification and gene sequencing,Yeast expression vectors of pGBKT7-GPV-CP and pGBKT7-GPV-MP were constructed,for expressing the bait fusion protein in yeast two-hybrid analysis.The solid base had been established to select the host factor which had interacted with the Barley Yellow Dwarf Virus in wheat cDNA library,then the host factor had been cloned and its specie and function had been presumed.