镰形扇头蜱唾液腺抑制消减杂交文库的构建和分析

Construction and Sequence Analyse of Two Salivary Gland cDNA SSH Libraries of Rhipicephalus haemaphysaloides haemaphysaloides

【目的】寻找镰形扇头蜱吸血后较吸血前唾液腺差异表达基因。【方法】以抑制消减杂交法分别构建镰形扇头蜱半饱血雌蜱和吸血雄蜱唾液腺以pGEM-T-easy为载体的cDNA文库,并测序进行生物信息学分析。【结果】分别测得247个雌蜱有效EST序列和168个雄蜱有效EST序列,并预测雌蜱可能含有5′末端和3′末端的EST序列分别为25个和44个,雄蜱可能含有5′末端和3′末端EST序列分别为53个和74个。随机选择非重复的24个雌蜱序列和21个雄蜱序列以RT-PCR方法验证消减效果;在吸血后唾液腺中,雌蜱有13个基因表达上调或新表达,消减效率为54%,雄蜱有9个基因表达上调或新表达,消减效率为43%。对所测有效序列经BLAST分析,检索247个雌蜱序列获得141个匹配,匹配率为57%,其中有32个蜱基因,占141个匹配基因的23%;检索168个雄蜱序列获得125个匹配,匹配率为74%,其中有29个蜱基因,占125个匹配基因的23%。BLAST检索结果显示这些蛋白主要包括帮助蜱口器固定免疫调节蛋白、抗凝血蛋白、加强能量代谢的线粒体蛋白和促进基因转录的各种转录因子。【结论】这些蛋白主要是为了适应蜱吸血的生理过程及应变蜱因吸血而产生的免疫防御和排斥。

[Objective] For the purpose of screening and analyzing the differentially expressed genes from salivary gland of Rhipicephalus haemaphysaloides haemaphysaloides, [Method] Two salivary gland subtracted eDNA libraries of half-fed female and fed male ticks were constructed respectively in the clone vector of pGEM-T-easy by the way of suppression subtractive hybridization （SSH）. [Result] 247 female ESTs and 168 male ESTs were obtained from the two SSH eDNA libraries. It is forecasted that 25 female ESTs and 44 female ESTs contain 5＇ end and 3＇ end respectively and 53 male ESTs and 74 male ESTs contain 5＇ end and 3＇ end respectively. Meanwhile, in order to identify the subtraction efficiency of the two SSH eDNA libraries, RT-PCR method was used to test 24 female ESTs and 21 male ESTs selected randomly but not repeatedly. It showed that there were 13 upregulated or differently expressed genes in half-fed salivary gland of the female Rhipicephalus haemaphysaloides and revealed that the subtraction rate was 54%. Also, it indicated that there were 9 upregulated or differentially expressed genes in fed salivary gland of the male Rhipicephalus haemaphysaloides and revealed that the subtraction efficiency was 43%. Putative translations of 141（57%） female ESTs and 125（74%） male ESTs had similarity to GenBank sequences, and 32 （23%） female ESTs and 29 （23%） male ESTs exhibited similarity to tick proteins, most of which in the libraries were immune modulating proteins, anticoagulant, energy metablism proteins and transcription factors. [Conclusion] These key proteins are valuable to the ticks to adapt the feeding physiology and to accommodate the immune safeguard and repulsion.