嗜人按蚊雌蚊消减cDNA文库的构建及分析

Construction and analysis of sex-differentially expressed cDNA library for Anopheles anthropophagus

目的 构建嗜人按蚊雌雄蚊差异表达cDNA文库。为进一步筛选、克隆嗜人按蚊雌雄蚊差异新基因奠定基础。方法 以嗜人按蚊为实验模型．分别将雌蚊和雄蚊作为T组和D组．采用抑制消减杂交方法和T／A克隆技术构建嗜人按蚊差异表达cDNA文库。扩增正向消减cDNA文库获得3000余个阳性克隆．随机挑取100个克隆进行PCR扩增，90％的克隆中均有200～1000bp的插入片段。测序显示．有与冈比亚按蚊未知功能基因同源的克隆，去除重复序列，得到72个EST序列．其中40与已知序列具有相似性．32个为新的EST序列。本项研究成功构建了嗜人按蚊雌蚊差异表达cDNA文库，获得了32个新的EST序列。从而为下一步筛选、克隆嗜人按蚊性别差异新基因打下了基础。

The objective of the present study was to construct a sex-differentially expressed eDNA library for female Anopheles anthropophagus, and to search for differentially expressed genes in female An. anthropophagus. A subtractive cDNA library for An. Anthropophagus was constructed by technique of suppression subtractive hybridization （SSH）. Total RNA was separately isolated from pools of adult female and male An. anthropophagus, and SSH was performed by using the PCR-select cDNA Subtraction kit （Clontech）. The forward subtracted library was constructed by using female as tester and male as driver, and the reverse-subtracted library was constructed by using male as tester and female as driver, respectively. The subtracted cDNAs were ligated into pGEM-T Easy Vector. Recombinant plasmids were transformed into competent Escherichia coli cells DHSa. One hundred randomly-selected positive clones were sequenced, and 72 valid EST sequences were analyzed using the basic local alignment search tool （BLAST） online （http.-//www. ncbi. nlm. nih. gov/BLAST）. Forty ESTs had homologous sequences in the GenBankTM database, and 32 ESTs had no homologs in the DNA database. In conclusion, sex-differentially expressed cDNA library for female An. anthropophagus was constructed successfully, and some female-specific EST sequences were identified, which provides foundation to study differentially expressed genes in female An. Anthropophagus.