埃及伊蚊电压依赖性钠离子通道3′端cDNA的克隆及序列分析

Cloning and sequence analysis of the voltage-dependent sodium channel gene 3'end cDNA from Aedes aegypti

目的扩增埃及伊蚊电压依赖性钠离子通道基因3′末端,并对其核苷酸序列进行分析。方法提取埃及伊蚊雌蚊总RNA,以Trsa为反转录引物反转录成单链cDNA,继而设计阳性与阴性对照,采用巢式聚合酶链反应(PCR)和快速扩增cDNA3′末端(3′RACE)技术,对埃及伊蚊电压依赖性钠离子通道基因3′末端进行PCR扩增、克隆与测序。结果获得钠离子通道基因的3′端核苷酸序列共809bp,包括编码区和非编码区poly(A)尾,编码区共编码208个氨基酸。相似性分析结果显示编码区氨基酸序列与家蝇钠离子通道基因3′端氨基酸序列相似性为65%左右。结论成功获得埃及伊蚊钠离子通道基因3′端,为进一步研究该基因的分子生物学特性奠定了基础。

Objective To amplify the 3′end gene sequence of voltage-dependent sodium channel from Aedes aegypti. Methods Total RNA was extracted from the female adult of Ae. aegypti, and a single stranded cDNA was reverse transcfipted with Trsa, then to design the positive and negative control, a 3′end cDNA was amplified by nested PCR and 3′RACE technique, and cloning and sequence analysis were performed. Results A total of 809 bp PCR product was obtained, including the coding sequence with 208 amino acids and non-coding sequence with poly（A） tail. Similarity analysis by DNAstar 5.0 showed high similarity （about 65 % ） comparing to the same region of amino acid sequence from Musca domestica. Conclusion The 3′end cDNA sequence of voltage-dependent sodium channel from Ae. aegypti was successfully obtained.