[Cloning of AGO2 and Dcr-2 gene fragments and analysis of their transcription level in different developmental stages of Aedes albopictus].

ABSTRACT

OBJECTIVE: To perform molecular cloning of the AGO2 and Dcr-2 gene fragments associated with RNA interference pathway of Aedes albopictus 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 DH5alpha 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 II instars larvae, III and IV 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 highest transcription level found in female Ae. albopictus, suggesting that AGO2 and Dcr-2 are the key genes of RNA interference in female mosquitoes.