白纹伊蚊CYP6N3基因启动子序列的分子克隆与功能分析

MOLECULAR CLONING AND FUNCTIONAL ANALYSIS OF PROMOTER SEQUENCES OF AEDES ALBOPICTUS CYP6N3

根据本室已获得的白纹伊蚊CYP6N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、PvuⅡ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 ,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。将扩增产物进行T A克隆及测序。结果显示 :分别以DraⅠ、EcoRⅤ、PvuⅡ和StuⅠ消化的DNA库为模板而获得 4个长短不一的DNA序列 :80 6bp、 2 190bp、 3 0 76bp和 2 2 0 6bp ,它们均包括有CYP6N3全长cDNA5′ 非翻译区序列及氨基端开头 10个氨基酸的编码序列 ;在其ATG上游序列中均存在有若干个典型的TATA盒、Barbie盒和 或CCAAT盒、抗氧化剂反应元件或外源化合物反应元件。表明本实验已成功地获得了白纹伊蚊溴氰菊酯抗性株CYP6N3基因 4个上游启动子区序列 ,并分析、讨论了它们在蚊虫中细胞色素P45

Two reverse specific primers (GSP1 and GSP2) were designed based on the 5′ end nucleotide sequence of the previous obtained CYP6N3 from Ae.albopictus. The genomic DNA purified from deltamethrin resistant Ae.albopictus were digested by DraI,EcoRV,PvuII and StuI, 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.The amplified products were cloned by the method of T A clone and sequenced.The results showed that four different sequences of 806bp、2 190bp、3 076bp and 2 206bp were obtained from the 4 digested libraries,respectively;they all include 5′ untranslated region(UTR),10 codens region at the amino terminus of CYP6N3,several TATA boxes,Barbie boxes and/or CCAAT boxes,and/or xenobiotic responsive elements or antioxidant responsive elements.It was suggested that the promoters of CYP6N3 gene from deltamethrin resistant Ae.albopictus were successfully obtained.The role(s) of these promoters in the diversity of cytochrome P450s and in their responsibility for insecticide resistance of mosquitoes were discussed.