关键词: 中华按蚊, 保幼激素受体基因, 分子特性, 表达模式, 生殖调控

Abstract:  【Aim】 To clarify the spatiotemporal expression pattern of the juvenile hormone receptor methoprene-tolerant gene AsMet in Anopheles sinensis and explore its influence on the reproductive regulation and development of An. sinensis.【Methods】 Based on the transcriptome data of An. sinensis, the full-length cDNA sequence of AsMet was cloned by RACE and its molecular characteristics were analyzed. qPCR was used to analyze the expression levels of AsMet in different developmental stages (pupa and female adult) and different tissues ［head, throax, anterior part of abdomen (the first 3 segments of abdomen), posterior part of abdomen (the remaining part of abdomen), midgut, Malpighian tubules, fat body, ovary and integument］ of the 3-day-old female adults. dsAsMet was microinjected into the last instar female pupa for RNAi, and the expression levels of AsMet, AsKr-h1 and AsVg, the development of ovaries of female adults, emergence rate, number of eggs laid and egg hatching rate were observed and detected.【Results】 The full-length cDNA sequence of AsMet of An. sinensis (GenBank accession no.: OR783325) was 6 841 bp with the open reading frame (ORF) of 3 159 bp in length, encoding 1 052 amino acids with the predicted molecular weight of 114.46 kD and the isoelectric point of 6.63. AsMet had four conserved domains, including one helix-loop-helix domain, two PAS-binding domains, and one C-terminal conserved motif. AsMet clustered with Mets of An. gambiae, Aedes aegypti and Culex pipiens. AsMet was significantly highly expressed at 30 h after pupation and at most stages of adults, significantly highly expressed in the head and thorax of female adults, and lowly expressed in the midgut, Malpighian tubules and ovary. The expression levels of AsMet were reduced by 70.05%, 41.05% and 68.64%, respectively, at 24, 48 and 72 h after dsAsMet microinjection into the last instar female pupa as compared with those in the control group microinjected with dsEGFP. The emergence rate in microinjection group with dsAsMet was lower than that in microinjection group with dsEGFP, and after mating and blood-feeding the ovaries were agenesia, and the number of eggs laid decreased by 67.58% as compared with that in microinjection group with dsEGFP, and the egg hatching rate in microinjection group with dsAsMet was reduced by 93.10% compared with that in microinjection group with dsEGFP.【Conclusion】The decreased expression of AsMet can reduce the normal development of ovary, and decrease the number of eggs laid and egg hatching rate significantly. The results lay a foundation for further research on the mechanism of JH regulation of reproductive development of An. sinensis, and provide a theoretical basis for understanding the signaling pathway of juvenile hormone and the molecular mechanism of insect reproductive regulation.

Key words: Anopheles sinensis, juvenile hormone receptor gene, molecular characteristics, expression pattern, reproductive regulation