大劣按蚊卵黄蛋白原cDNA的克隆及转录水平研究

Molecular cloning and transcription research of vitellogenin c DNA in Anopheles dirus

目的克隆大劣按蚊卵黄蛋白原c DNA,进行序列分析,并研究该基因在不同蚊期的转录水平变化。方法首先提取大劣按蚊总RNA,反转录成c DNA;然后设计昆虫卵黄蛋白原简并引物,以该c DNA为模板进行PCR扩增和测序,对所获取的序列进行生物信息学分析;最后利用实时荧光定量PCR方法检测卵黄蛋白原基因在蚊卵、幼虫、蛹和成蚊各时期的转录水平变化。结果 PCR成功扩增出了清晰的单一目的条带,经测序为一段1 071 bp的序列,BLAST比对分析显示该序列与浅色按蚊、库态按蚊和斯氏按蚊的卵黄蛋白基因同源性高达90%,与冈比亚按蚊、微小按蚊和美彩按蚊的同源性达89%。对大劣按蚊各时期卵黄蛋白原转录水平的研究结果显示,4 d龄按蚊卵黄蛋白原m RNA水平是3 d龄按蚊的15.2倍,而吸血后卵黄蛋白原m RNA水平高效上调,是未吸血组的955.7倍(P<0.01);吸血组6 d龄按蚊卵黄蛋白原m RNA表达较吸血组4 d龄按蚊骤降(下降近1 870倍),而与未吸血组6 d龄按蚊相比,差异无统计学意义。结论本研究成功地克隆了大劣按蚊卵黄蛋白原基因的c DNA;大劣按蚊血餐后24 h卵黄蛋白原基因高效表达,提示卵黄蛋白原基因可能参与了大劣按蚊蚊卵的发育。

Objective To clone the vitellogenin gene of Anopheles dirus and analyze the sequence, and then study thetranscriptional levels of this gene in different stages of mosquitoes. Methods Firstly, the total RNA was extracted from An.dirus mosquitoes and reversely transcribed to c DNA. Then, PCR was performed to amplify the target gene with degenerateprimers. The products were then sent to Invitrogen Company for sequencing, and the bioinformatics analysis was carried outlater. Finally, real-time quantitative PCR was conducted to detect the transcriptional levels of vitellogenin gene in differentstages of An. dirus such as egg, larva, pupae and adult. Results A single and clear target band was successfully amplified byPCR. The length of the c DNA was 1 071 bp according to the sequencing result. By the online BLAST method, the homology wasas high as 90% with An.subpictus, An. culicifacies and An. stephensi, and 89% with An. gambiae, An. mininus and An.splendidus, respectively. The study of Ad Vg1 transcription showed that the m RNA level of 4 d Anopheles was 15.2 times as highas that of 3 d mosquitoes. After a blood meal, the relative expression level of vitellogenin was highly up-regulated, and them RNA of blood-fed Anopheles came to 955.7 times as high as that of the non-feeding controls（P〈0.01）. Conclusions In thisstudy, we successfully clone c DNA of the vitellogenin gene of An. dirus and confirm that Advg1 is highly expressed after ablood meal. The results indicate that vitellogenin gene may participate in the development of anopheles eggs.