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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 c DNA of Anopheles stephensi, analyze the sequence of the gene, and study its transcriptional level in different mosquito stages. Methods The total RNA of Anopheles dirus was extracted and reversely transcribed into c DNA. Then, degenerate primers of insect vitellogenin were designed. The c DNA was used as a template for PCR amplification and sequencing. The obtained sequences were analyzed by bioinformatics Finally, real-time fluorescent quantitative PCR was used to detect the transcriptional level of vitellogenin genes in mosquito eggs, larvae, pupae and adult mosquito at different stages. Results A single objective band was amplified by PCR and sequenced as a 1 071 bp sequence. BLAST analysis showed that the sequence was highly homologous to the vitellogenotypes of Anopheles stephensi, Anopheles sinensis and Anopheles stephensi The homology is as high as 90%, with 89% homology with Anopheles gambiae, Anopheles minimus and An. Anopheles. The results showed that the mRNA level of vitellogenin in 4-day-old mosquitoes was 15.2-fold higher than that in 3-day-old mosquitoes, while the mRNA levels of vitellogenin were up-regulated efficiently after vasculitis. (P <0.01). The mRNA expression of vitellomala on the 6th day after vampire injection was significantly lower than that on the 4th day after vampire injection (down nearly 1 870 times) Anopheles d compared age, the difference was not statistically significant. Conclusion The study successfully cloned c DNA of Anopheles stephensi vomitus protein gene. The expression of vitellogenin gene in Anopheles stephensi after 24-hour blood meal implied that the vitellogenin gene may be involved in the mosquito gene of Anopheles stephensi development.