白纹伊蚊AGO2和Dcr-2基因片段克隆及各发育期转录水平分析

Cloning of AGO2 and Dcr-2 Gene Fragments and Analysis of Their Transcription Level in Different Developmental Stages of Aedes albopictus

目的克隆白纹伊蚊(Aedes albopictus)RNA干扰通路相关AGO2和Dcr-2基因片段,并分析该蚊种不同发育阶段这2个基因片段的转录水平。方法根据埃及伊蚊(Ae.aegypti)AGO2和Dcr-2蛋白的氨基酸序列保守区设计简并引物,经RT-PCR从白纹伊蚊雌蚊总RNA中分别扩增AGO2和Dcr-2 cDNA,并与载体pMD18-T连接,转染大肠埃希菌(E.coli)后,筛选阳性克隆,测序并做Blastx分析。根据获得的白纹伊蚊AGO2和Dcr-2基因片段设计特异性引物,采用半定量RT-PCR分析白纹伊蚊卵、Ⅰ/Ⅱ龄幼虫、Ⅲ/Ⅳ龄幼虫、蛹、雄蚊和雌蚊中这2个基因的mRNA水平。结果获得的AGO2和Dcr-2 cDNA片段大小分别为326 bp和491 bp,登录号分别为JQ764670和JQ764671。Blastx分析结果显示,该2个序列编码的氨基酸序列与埃及伊蚊的AGO2和白纹伊蚊的Dcr-2氨基酸序列的同源性分别为91%和98%。AGO2和Dcr-2基因在白纹伊蚊不同发育阶段中均有转录,于雌蚊中的mRNA水平最高,分别为雄蚊中mRNA的3.1倍和15.5倍,显著高于其他各阶段的mRNA水平(P<0.05)。结论获得了白纹伊蚊AGO2和Dcr-2基因部分cDNA片段,并发现这2个基因在雌蚊中转录水平最高。

Objective To perform molecular cloning of the AGO2 and Dcr-2 gene fragments associated with RNA interference pathway of Aedes aIbopictus and characterize the transcription level of the two genes across all life stages of the mosquito. Methods The degenerate primers were designed based on the conserved regions.of AGO2 and Dcr- 2 amino acid sequences, and the AGO2 and Dcr-2 cDNA fragments were amplified from total RNA of a female mosquito by RT-PCR. The PCR products were cloned into pMD18-T vector and transformed into E. coli DHSct strain, and the positive clones were selected and sequenced, with the results for homology analysis by Blastx. The specific primers were designed according to the sequences of AGO2 and Dcr-2 from Ae. albopictus, which were used to investigate the transcription levels of these two genes from eggs, I and 11 instars larvae, II1 and 1V instars larvae, pupa, male and female mosquitoes by semi-quantitative RT-PCR. Results The AGO2 and Dcr-2 cDNA fragments obtained were 326 bp and 491 bp in length, with the Accession number of JQ764670 and JQ764671, respectively. The Blastx analysis showed that the AGO2 and Dcr-2 amino acid sequences shared 91% similarity to AGO2 of Ae. aegypti and 98% to Dcr-2 of Ae. albopictus. The transcription of AGO2 and Dcr-2 genes was detected in all life stages of Ae. albopictus, with the highest level of mRNA in female mosquitoes, which was 3.1 times and 15.5 times higher for AGO2 and Dcr-2 than in male mosquitoes, respectively, and significantly higher than other developmental stages （P〈0.05）. Conclusion The AGO2 and Dcr-2 cDNA sequences have been partially obtained and the hightest transcription level found in female Ae. albopictus, suggesting that AGO2 and Dcr-2 are the key genes of RNA interference in female mosquitoes.