基因步移在分离蚊虫细胞色素P450启动子中的应用及意义  

The Application and Significance of Gene-walking in Isolating the Promoter of Cytochrome P450 from Mosquitoes

作　　者：周国理[1] 黄炯烈[1] 吴瑜[1] 王玲[1] 

机构地区：[1]中山医科大学寄生虫学教研室,广东广州510089

出　　处：《中山医科大学学报》2001年第6期406-409,413,共5页Academic Journal of Sun Yat-sen University of Medical Sciences

基　　金：广东省自然科学基金资助项目 ( 970 0 92 );"2 11工程"重点学科建设基金资助项目 ( 9812 4)

摘　　要：【目的】快速扩增出白纹伊蚊溴氰菊酯抗性株CYP 6N3基因 (CYP 6N3)上游启动子区序列。【方法】根据本室已获得的白纹伊蚊CYP 6N亚家族新成员CYP6N3全长cDNA的 5′ 末端核苷酸序列 ,设计 2个反向特异引物 (GSP1和GSP2 ) ,利用 4种限制性内切酶 DraⅠ、EcoRⅤ、Pvu Ⅱ和 StuⅠ分别消化白纹伊蚊溴氰菊酯抗性株高分子量基因组DNA ,消化产物与适配子连接产生 4种消化的DNA库 (DL1、DL2、DL3及DL4) ,并分别以此为模板 ,进行基因步移。用特异引物GSP1与适配子引物AP1进行第 1步PCR扩增 ,然后再以第 1步PCR产物为模板、用内部特异引物GSP2与内部适配子引物AP2进行第 2次PCR扩增。【结果】第 1步PCR结果显示 :在上述 4种消化文库中分别扩增出约 80 6、2 190、2 2 0 6和 3 0 76bp的特异条带 ;第 2步PCR结果与第 1次PCR相类似 ,但各泳道主带扩增效率明显升高。【结论】本实验已成功地获得了白纹伊蚊溴氰菊酯抗性株 CYP6N3上游启动子区核苷酸序列及其 4种限制性内切酶图谱。此外还对基因步移法在昆虫细胞色素P45 0多样性及其参与杀虫剂抗性分子机理研究中的意义进行了讨论。To rapidly amplify the promoter of CYP6N3 gene (CYP6N3) from deltamethrin resistant Aedes albopictus (Ae.albopictus). Two reverse specific primers (GSP1 and GSP2) were designed based on the 5′ end nucleotide sequence of the previous obtained CYP6N3 from Ae.albopictus. The genomic DNA purified from deltamethrin resistant Ae.albopictus were digested by DraⅠ, EcoRⅤ, PvuⅡ and StuⅠ, then ligated with adaptors to produce 4 digested libraries, respectively. The primary PCR was performed from the 4 digested libraries by the use of GSP1 and AP1, and its products were used for the second PCR with GSP2 and AP2. Four major bands of 806?2 190?2 206 and 3 076 bp were obtained from the 4 digested libraries, respectively. The second PCR showed a similar result to the first one, but had a stronger major band in all lanes. [Conclusions] The promoter of CYP6N3 from deltamethrin resistant Ae.albopictus and its restriction map containing 4 restriction sites were successfully obtained. The significance of gene walking method in studying the diversity of insect P450s and the molecular mechanism of insecticide resistance mediated by insect P450 enzymes was discussed.$$$$

关 键 词：基因步移 伊蚊属 细胞色素P450 CYP 6N3 杀虫药抗药性 蚊虫 

分 类 号：R384.1[医药卫生—医学寄生虫学]