小麦吸浆虫保幼激素酯酶和保幼激素环氧水解酶基因的克隆及在滞育和变态发育过程中的表达动态

【目的】保幼激素(juvenile hormone, JH)在小麦吸浆虫Sitodiplosis mosellana滞育诱导及滞育后静息状态的维持中发挥着重要作用。保幼激素酯酶(hormone esterase, JHE)和保幼激素环氧水解酶(juvenile hormone epoxide hydrolase, JHEH)是调控JH滴度的重要降解酶。本研究旨在探讨JHE和JHEH在小麦吸浆虫滞育和变态发育中潜在功能。【方法】通过RT-PCR和RACE技术从小麦吸浆虫滞育前幼虫克隆JHE和JHEH全长cDNA序列;利用生物信息学软件分析其核苷酸及编码蛋白特性;采用qPCR技术分析其在小麦吸浆虫滞育不同时期(滞育前、滞育期、滞育后静息期和滞育后发育)3龄幼虫及1龄幼虫到成虫不同发育阶段(1-2龄幼虫、预蛹、初蛹、中蛹、后蛹、雌成虫和雄成虫)中的表达水平。【结果】克隆获得了cDNA全长分别为3 102和1 980 bp的小麦吸浆虫SmJHE和SmJHEH基因(GenBank登录号分别为MG876768和MG876769),其开放阅读框分别长1 740和1 371 bp,分别编码579和456个氨基酸,预测蛋白分子量分别为65.67和51.65 kD。SmJHE蛋白含有5个JHE家族特有的保守模块,SmJHEH含有催化三联体Asp228, Asp404和His431及组成阴氧离子洞的两个Tyr(Tyr299和Tyr374)和HGWP花样结构。序列比对和进化分析表明,SmJHE和SmJHEH均与双翅目(Diptera)长角亚目(Nematocera)昆虫同源蛋白氨基酸序列一致性较高,亲缘关系最近。不同滞育时期的表达模式表明,SmJHE和SmJHEH在滞育前期(1龄到滞育前的3龄幼虫早期)表达量变化不明显,进入滞育后表达量基本维持恒定,但均在滞育后静息阶段的当年12月至翌年1月最低。发育表达模式表明,幼虫恢复发育后SmJHE表达量逐渐升高,预蛹期达到最高,在雌成虫中的表达量显著低于雄成虫中的;SmJHEH表达量则在预蛹期最低,在雌成虫中最高。【结论】SmJHE和SmJHEH参与小麦吸浆虫滞育调控,其表达量的降低与滞育后静息阶段JH的累积有关;SmJHE在发育过程中表达量的升高可能参与幼虫到蛹的变态,表达量的降低可能与生殖发育有关。

Cloning of juvenile hormone esterase and juvenile hormone epoxide hydrolase genes and theirexpression dynamics during diapause and metamorphosis in Sitodiplosis mosellana (Diptera:Cecidomyiidae)

【Aim】 Juvenile hormone (JH) plays important roles in diapause induction,maintenance and post-diapause quiescence of the wheat midge, Sitodiplosis mosellana, aninsect species undergoing obligatory larval diapause. Juvenile hormone esterase (JHE) andjuvenile hormone epoxide hydrolase (JHEH) are important JH degradation enzymes that playkey roles in regulating insect JH titers. This study aims to explore the potential roles ofJHE and JHEH in the diapause and metamorphosis of S. mosellana. 【Methods】 Thefull-length cDNAs of JHE and JHEH were cloned from pre-diapause larvae of S. mosellana byRT-PCR and RACE technologies. The sequence characteristics of nucleotide and amino acidsequences 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 differentdiapause stages (pre-diapause, diapause, post-diapause quiescent and post-diapausedevelopment) and different developmental stages from the 1st instar larva to adult (the1st-2nd instar larva, pre-pupa, early pupa, middle pupa, late pupa, male adult and femaleadult) were determined by qPCR. 【Results】 The full-length cDNA sequences of JHE and JHEHwere 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 proteinsof 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 allof the conserved domains of JHEHs including the residues Asp228, Asp404 and His431 in thecatalytic triad, and two Tyr residues (Tyr299 and Tyr374) and the HGWP motif correspondingto the oxyanion hole. Multiple sequence alignment and phylogenetic analysis showed thatSmJHE and SmJHEH have higher amino acid sequence identity and the closest relationship tothe corresponding homologues from Nematocera insects of Diptera. The expression levels ofSmJHE and SmJHEH showed no significant change at the pre-diapause stage (the 1st, 2nd andearly 3rd instar larval stages), remained stable after entering diapause, but were thelowest at the post-diapause quiescence phase (from December to the next January). Theexpression of SmJHE increased gradually after development resumption, reached the peak inthe pre-pupal stage, and was significantly lower in female adults than in male adults. Theexpression 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. Theirreduced expression during diapause might be related to accumulation of JH titers inpost-diapause quiescence. Increased expression of SmJHE during the development process ofS. mosellana might play a role in metamorphosis from larva to pupa, and its reducedexpression might be related to reproductive development.