关键词: 含硫脂蛋白, 大劣按蚊, 约氏疟原虫, RNA干扰

Abstract: Objective   To study the role of TEP1 gene from Anopheles dirus during Plasmodium yoelii infection by RNA interference.  Methods   TEP1 primers with T7 promoter were designed based on the sequence of An. dirus TEP1 gene from GenBank database. PCR amplification of TEP1 gene was completed with An. dirus cDNA as template. The AdTEP1 double-stranded RNA was synthesized by using in vitro transcription kit with purified PCR products. Female An. dirus emerged for 1-2 days were divided into three groups each with 200 mosquitoes: TEP1 interference group，EGFP interference group and control. Mosquitoes in TEP1 and EGFP interference groups were microinjected in chest with 147 ng of AdTEP1 and EGFP double-stranded RNA, respectively, while those of control group were untreated. Effect of TEP1 interference on P. yoelii in An. dirus was estimated through semi-quantitative PCR with internal reference AdS7 at 3 d after injection. On 4 d after injection, mosquitoes were infected by EGFP-expressing P. yoelii BY265. The infection rate and infectiosity of mosquitoes were observed through anatomizing 25 midguts from each group at 9 d post-infection.  Results   The AdTEP1 double-stranded RNA did well in the interference of TEP1 expression in An. dirus. The infection rate in the groups of control, EGFP and TEP1 interference was （24±2.83）%, （24±0.71）%, and （80±3.54）%, respectively；and the infectiosity of the three groups was 0.32±0.7, 0.44 ± 0.85, and 5.52 ± 4.84, respectively.   Conclusion  AdTEP1 interference increases the infection rate and infectiosity of An. dirus by P. yoelii, and raises the susceptibility of An. dirus to P. yoelii significantly. TEP1 plays a critical role in the process of P. yoelii infection.

Key words: Thioester-containing protein, Anopheles dirus, Plasmodium yoelii, RNAi