关键词: 小麦吸浆虫, 保幼激素酯酶, 保幼激素环氧水解酶, 基因克隆, 滞育, 变态, 发育

Abstract: 【Aim】 Juvenile hormone (JH) plays important roles in diapause induction, maintenance and post-diapause quiescence of the wheat midge, Sitodiplosis mosellana, an insect species undergoing obligatory larval diapause. Juvenile hormone esterase (JHE) and juvenile hormone epoxide hydrolase (JHEH) are important JH degradation enzymes that play key roles in regulating insect JH titers. This study aims to explore the potential roles of JHE and JHEH in the diapause and metamorphosis of S. mosellana. 【Methods】 The full-length cDNAs of JHE and JHEH were cloned from pre-diapause larvae of S. mosellana by RT-PCR and RACE technologies. The sequence characteristics of nucleotide and amino acid sequences of the cloned JHE and JHEH were analyzed by bioinformatics software. The 
expression levels of JHE and JHEH in the 3rd instar larvae of S. mosellana at different diapause stages (pre-diapause, diapause, post-diapause quiescent and post-diapause development) and different developmental stages from the 1st instar larva to adult  (the 1st-2nd instar larva, pre-pupa, early pupa, middle pupa, late pupa, male adult and female adult) were determined by qPCR. 【Results】 The full-length cDNA sequences of JHE and JHEH  were obtained from S. mosellana and named SmJHE and SmJHEH (GenBank accession number: MG876768 and MG876769, respectively), with the length of 3 102 and 1 980 bp, respectively. SmJHE and SmJHEH contain 1 740 and 1 371 bp ORFs, respectively, which encode the proteins of 579 and 456 amino acids with the predicted molecular weights of 65.67 and 51.65 kD, respectively. SmJHE contains five specific conserved motifs of JHE protein family, and all of the conserved domains of JHEHs including the residues Asp228, Asp404 and His431 in the catalytic triad, and two Tyr residues (Tyr299 and Tyr374) and the HGWP motif corresponding to the oxyanion hole. Multiple sequence alignment and phylogenetic analysis showed that SmJHE and SmJHEH have higher amino acid sequence identity and the closest relationship to the corresponding homologues from Nematocera insects of Diptera. The expression levels of SmJHE and SmJHEH showed no significant change at the pre-diapause stage (the 1st, 2nd and early 3rd instar larval stages), remained stable after entering diapause, but were the lowest at the post-diapause quiescence phase (from December to the next January). The expression of SmJHE increased gradually after development resumption, reached the peak in the pre-pupal stage, and was significantly lower in female adults than in male adults. The expression of SmJHEH, however, reached the highest in female adults and the lowest in pre-pupae. 【Conclusion】 SmJHE and SmJHEH are involved in diapause of S. mosellana. Their reduced expression during diapause might be related to accumulation of JH titers in post-diapause quiescence. Increased expression of SmJHE during the development process of S. mosellana might play a role in metamorphosis from larva to pupa, and its reduced expression might be related to reproductive development.

Key words:  Sitodiplosis mosellana, juvenile hormone esterase, juvenile hormone epoxide hydrolase, gene cloning, diapause, metamorphosis, development