腰带长体茧蜂cDNA文库的构建

Construction of cDNA library of Macrocentrus cingulum Brischke

从寄生在亚洲玉米螟 (Ostriniafurnacalis)幼虫体内 6d的腰带长体茧蜂 (Macrocentruscingulum)胚胎中提取总RNA和mRNA ,用分离到的微量mRNA合成cDNA第 1链。通过长距离PCR扩增得到足够量的双链cDNA。将SfiⅠ酶切后并纯化的cDNA片段连接到经SfiⅠ酶切的噬菌体λTripIEX2上 ,并对噬菌体进行包装 ,建成cDNA的全长库。经大肠杆菌XL1 Blue平板检测 ,未扩增文库的滴度为 0 75× 10 6pfu·mL-1,克隆重组百分率为 96 77% ,扩增后文库的滴度为 0 5×10 10 pfu·mL-1。

A cDNA library of Macrocentrus cingulum embryos,excised from their host Ostrinia furnacalis six days after parasitism was constructed.After synthesizing the first strand cDNA using limited amount of mRNA isolated from total RNA,the double strand cDNA was amplified with long distance PCR method.Then the ligation of the Sfi Ⅰ digested cDNA to the Sfi Ⅰ digested vector λTripIE X2 was performed and the phage was packed to construct the full length cDNA library.It was determined that the titer of unamplified library was 0 75×10 6 pfu·mL -1 and the percentage of recombinant clones was 96 77%,and the titer of amplified library was 0 5×10 10 pfu·mL -1 by plating E coli XL1 Blue.