亚洲璃眼蜱唾液腺差异表达基因文库的构建及分析

Construction and analysis of the libraries of differentially expressed genes in the salivary glands of the unfed and partially engorged Hyalomma asiaticum

从单雌蜱克隆群中挑选未吸血雌蜱60只,随机分成两组。未吸血组直接剖取唾液腺。半饱血组于蜱吸血第5d采集,分离唾液腺。Trizol法提取总RNA,经第一链合成、LD-PCR、RasⅠ酶切、接头连接和抑制消减杂交(SSH)等步骤,获得差异表达基因的cDNA片段。将纯化的cDNA片段与pGEMT-Easy载体连接,转化DH5a,获得204个白色菌落。扩增检查表明,136个克隆含有插入片段,片段大小为250bp～850bp。测出有效序列120个。由10个cDNA片段的RT-PCR检查结果初步断定,本研究消减效果良好。由网上资源分析得:21个片段与其他蜱的基因,19个片段与按蚊、库蚊、钩虫、奥斯特线虫等其他吸血寄生虫的基因,9个与果蝇的基因具有同源性。

60 Hyalomma asiaticum female adult ticks was selected form a laboratory colony and were divided into two groups. One group was applied to a rabbit＇s ears with the same amount of males and allowed to engorge for 4 to 5 days, the other was not. The salivary glands of the unfed and partially engorged ticks were dissected. The total RNA of the salivary glands was extracted by Trizol regents, the cDNAs were synthesized using Super SMART PCR eDNA Synthesis Kit and digested with Rsa Ⅰ. The digested tester eDNA was subdivided into two portions, and each was ligated with a diftbrent eDNA adaptor. Two hybridizations and two PCR amplifications were performed using Clontech PCR-select eDNA Subtraction Kit. The differentially expressed sequences were then inserted into pGEM T-Easy vector to generate a special subtracted eDNA library. Sequences from this library were compared with European Molecular Biology Laboratory （EMBL）/GenBank databases, which led to their classification into 3 major groups. The first group includes sequences that showed high homology to ticks genes of known function, for example, anticoagulants, inhibitors of platelet aggregation, and immunomodulatory proteins. The second group comprises cDNAs that showed due homology to other parasite genes of known function. The third group cDNAs failed to match or showed low homology to genes of known function. The RT-PCR result of the two groups RNA with 10 pairs of primer confirmed that these genes are expressed in partially engorged female ticks, but not the unfed female ticks.