棉铃虫雌性成虫脂肪体cDNA文库的构建  

Construction of a cDNA Library from the Fat Body of the Female Cotton Boll Worm,WTHXHelicoverpa Armigera

作　　者：张志宏[1] 王金星[1] 赵小凡[1] 王来元[1] 

机构地区：[1]山东大学生命科学学院生物系,济南250100

出　　处：《山东大学学报（理学版）》2002年第5期449-451,457,共4页Journal of Shandong University(Natural Science)

基　　金：国家自然科学基金 ;项目编号 :3 9740 0 2 4;3 9870 0 95

摘　　要：用异硫氰酸胍 酚 氯仿 步法从棉铃虫雌性成虫脂肪体中提取总RNA ,经过Oligo(dT)纤维柱分离出mRNA .以mRNA为模板 ,Oligo(dT)为引物 ,在逆转录酶催化下合成单链cDNA(sscDNA) .然后在E .coliDNA聚合酶Ⅰ与RNaseH共同作用下 ,进一步合成双链cDNA(dscDNA) .平末端dscDNA与EcoRI/NotI接头连接 ,插入λgt11表达载体 ,经体外包装后转染Y10 90 r 宿主菌 ,构建成棉铃虫脂肪体cDNA文库 .该文库的滴度为 3.5× 10 5pfu/mL ,重组率为 10 0 % ,该文库适合于筛选低丰度mRNA的cDNA克隆 .WT5'BZTotal RNA was extracted from the fatbody of the female cotton boll worm using acid guanidinium thiocyanate phenol chloroform single step method.Poly(A) mRNA was isolated and purified through Oligo(dT) cellulose column.The mRNA template was reverse transcribted to a single strand cDNA(sscDNA) with Oligo(dT) primer using reverse transcriptase.The second strand cDNA(dscDNA)was synthesized byWTBX E.coliWTBZ DNA polymerase I in combination with RNaseH.Blunt end of dscDNA was ligased with EcoRI/NotI adapter,then inserted into a expressed vector λgt11.The recombined vector was packaged in vitro and infected a host strain,Y1090 r.The titer of the constructed cDNA library is 3.5×10 5pfu/ml and its recombination rate was 100%.The library is suitable to screen low abundant mRNA for cDNA colne.

关 键 词：棉铃虫 雌性性成虫 脂肪体 CDNA文库 cDNA库构建 基因克隆 基因表达 

分 类 号：S435.622.3[农业科学—农业昆虫与害虫防治] Q[农业科学—植物保护]