棉铃虫中肠Cry1A受体蛋白氨肽酶N1在Tn细胞系的表达.pdfVIP

中国农业科学 2008,41(6):1667-1672 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2008.06.013 棉铃虫中肠Cry1A受体蛋白氨肽酶N1在Tn细胞系的表达 常洪雷，梁革梅，王桂荣，于宏坤，吴孔明，郭予元 （中国农业科学研究院植物保护研究所/植物病虫害生物学国家重点实验室，北京 100094 ） 摘要：【目的】将棉铃虫中肠Bt 受体蛋白在真核表达系统中成功表达。【方法】用PCR方法分别扩增出对Cry1Ac 毒素敏感和抗性棉铃虫的中肠氨肽酶 N1（APN1 ）基因的去信号肽片段，将其克隆至 pUC 19 载体，测定核酸序列 后，克隆入Bac-to-Bac表达系统中杆状病毒转移载体pFastBacHTB中多角体基因启动子的下游，筛选出重组质粒 pFastBacHTB/APN1 并转化大肠杆菌 DH10Bac，在体内进行重组，经抗性和蓝白斑筛选，获得杆状病毒重组载体 Bacmid/APN1。转染粉纹夜蛾（Trichoplusia ni ）细胞（Tn-5B1-4），获得含 APN1 基因的重组杆状病毒，重组病 毒感染 Tn 细胞后，得到表达的蛋白。【结果】SDS分析和点杂交显示，目的基因得到了成功表达。【结论】 APN1基因在Tn 细胞中成功表达，为今后继续研究其功能和与抗性的关系奠定了基础。 关键词：APN；杆状病毒表达载体；Tn；表达 Expression of Aminopeptidase N (APN1)，the Main Receptor Protein for Bacillus thuringiensis Cry1A Toxin from Helicoverpa armigera Larval Midgut in Tn Cells CHANG Hong-lei, LIANG Ge-mei, WANG Gui-rong, YU Hong-kun, WU Kong-ming, GUO Yu-yuan (State Key Laboratory for Biology of Plant Diseases and Insect Pests/Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094) Abstract: 【Objective 】 The present study was conducted to successfully express the Bt (Bacillus thuringiensis) toxin receptor protein located on internal membrane of larval midgut of cotton bollworm (Helicoverpa armigera Hübner), within eukaryotic expression system. 【Method 】 PCR method was used to separately amplify the fragments without signal peptide of Aminopeptidase N1 gene from midguts of susceptible and resistant H. armigera, and separately cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system transfer vector pFastBacHTB under