羽化激素(EH)是调控生长发育的重要神经肽激素.本文研究了家蚕滞育发生诱导温度和光周期条件对羽化激素基因(Eh)表达的影响.结果显示,在15DD非滞育卵诱发环境下,催青期胚胎的Eh基因mRNA一直处于很低水平；而在25LL滞育卵诱发环境或20LD滞育卵与非滞育卵同时出现的诱发条件下,能够检测到Eh的高水平表达,提示胚胎期可能已经开始合成EH.其中25LL蚕卵Eh基因的表达水平高于20LD区,并在积温高于1 440℃.h(反转期)后,Eh基因表达水平显著上调,这与在此阶段的蚕卵胚胎对滞育诱导环境温度和光照节律更加敏感相一致.在胚后幼虫至成虫期维持催青期的15DD或25 LL环境中,1-4龄幼虫在食桑期Eh基因继续了胚胎后期25LL中高表达的现象,但5龄幼虫至成虫期出现15DD环境Eh基因表达水平显著高于25LL区的现象.胚后改用25℃自然光照环境保护,25LL区的子代滞育性卵和15DD区的子代非滞育性卵中,表现出15DD环境Eh基因表达水平高于25LL区的现象,在卵龄48h后更加明显.结果表明亲代卵的孵化温度与光周期在决定子代卵的滞育性同时,显著影响整个世代Eh基因的表达.

Pituitary homeobox hormone(PITX) affected directly on the secretion and the role of DH in Bombyx mori.In order to explore the signal transduction pathway of diapause,we investigated the effects of three typical diapause inducing temperature and photoperiod on expression of Pitx gene.The results showed that the constant illumination at 25℃(25LL) significantly up-regulated the expression level.The Pitx mRNA level from 1st to 3rd instar larvae and adults in 25LL were higher than the which in 15DD.Moreover,the gene expression level were significantly differences between tissues,and down-regulated during moulting period.The Pitx gene mRNA levels in progeny eggs were influenced by diapause-inducing temperature and photoperiod during the parental stage.The maternal Pitx gene mRNA had not detected from diapause eggs.Howerer,the gene up-regulated the expression level during egg age of 12h to 24h,and almost stoped the expression in diapause occuring stage elder than age 72h.The non-diapause eggs had higher level of maternal Pitx mRNA,and had higher gene expression level during embryogenesis and development period.The diapause of eggs from 20LD parental conditions were unstable.However,the Pitx gene mRNA level in those eggs were significantly higher than the level in diapause eggs and non-diapause eggs.The incubation temperature and photoperiod of parental eggs determined the diapause of progeny eggs,and also affected the Pitx gene expression level.Anyway,the gene expression level of progeny eggs might be not related to the diapause of eggs itself.

Diapause hormone (DH) is the key factor that leads to diapause in silkworm (Bombyx mori).In present study,expressional variations of silkworm diapause hormone gene (Dh) caused by various temperature and photoperiod conditions were examined to explore the regulatory mechanism of temperature and illumination to silkworm diapause.The results showed that Dh was steadily and highly expressed in developmental embryos under constant illumination at 25 ℃ (25LL),but lowly expressed under constant darkness at 15 ℃ (15DD).Under 12 h illumination and 12 h darkness cycle at 20 ℃ (20LD) or 25LL,the Dh mRNA level of embryos was up-regulated significantly at the late developmental stage (after the effective accumulated temperature was beyond 2 160 ℃·h),being consistent with the super sensitivity of embryos to external illumination and temperature at this stage.Dh was dramatically up-regulated at 25 ℃ during the postembryonic stage,and illumination could also up-regulate the Dh expression level during pupal and moth stages.And the regulation by high temperature was stronger than that by illumination.In addition,gene microarray analysis showed that Dh had sex-and tissue-specific expression characteristics on day 3 of the 5th instar larvae.The Dh gene's mRNA transcriptional levels in multiple tissues of female larvae were significantly lower than those of the male larvae,and were very low in ovary.The incubation temperature and photoperiod of parental eggs had influence on the diapause of offspring eggs,and also affected the mRNA level of Dh.However,the mRNA transcriptional level of Dh gene in offspring eggs was not the key factor to determine whether diapause of the eggs occurred.It was suggested that the sex difference of silkworm Dh gene expression and the discrepancy between the expression pattern of Dh gene in silkworm eggs and the diapause of the eggs were relaxed to splicing of precursor polypeptide of the Dh gene.

In insects,temperature and photoperiod are the two main zeitgebers,which can affect the expression of circadian genes,and also are the determinant of diapause in Bombyx mori.In order to understand the relationship between diapause and the regulation of circadian rhythm in Bombyx mori,we investigated the effects of diapause-inducing incubation temperature and photoperiod on per gene expression.The results showed that per transcriptional levels in dark environment were significantly higher than those in light environment when all developmental stages of the Bombyx mori were under high temperature(25 ℃) or its larval and pupal stages were under relatively high temperature(20 ℃).The mRNA level of per gene under high temperature was higher than that under low temperature when the larval and pupal stages were under the same photoperiod.In addition,the mRNA transcriptional level of per gene in dark environment was in the order of 20 ℃>25 ℃>15 ℃.Besides,the effect of temperature on per gene expression was more obvious than darkness.During the postembryonic developmental stage,the expression level of per gene under 25 ℃ and continuous illumination(25LL) was higher than that under 15 ℃ and continuous darkness(15DD),while the expression of per gene under 20 ℃ and 12 h light/dark photoperiod(20LD) was more sensitive,being consistent with the induction of temperature and photoperiod on Bombyx mori diapause.Both eggs from virgin moths and unfertilized eggs at 0.5 h presented a high level of maternal per mRNA.While developing embryos of the Bombyx mori maternal generation were exposed to high temperature(25 ℃) or darkness and 20LD,their progeny exhibited up regulation of per mRNA levels.However,this up regulation only occurred before egg age of 72 h,a turning time point to generate diapause or to activate other developmental events.In conclusion,the diapause-inducing incubation temperature and photoperiod directly affect the differential expression of per gene during the whole life cycle of Bombyx mori,and the expression of per gene was closely related to the difference of embryonic diapause status.

时间间隔测定酶(TIME-EA4)是近年在家蚕中首先发现的一种生物测时蛋白质,具有正常的SOD酶活性与酯酶ATPase瞬时活性,参与家蚕滞育卵的活化调控.利用RACE技术克隆了棉铃虫TIME-EA4的同源基因Haea4(GenBank登录号:GU143551),全长cDNA 799 bp,编码199个氨基酸,蛋白质等电点为5.45,相对分子质量18 768.88,含有1个Cu/Zn SOD核心结构域.RT-PCR分析发现,Haea4基因在棉铃虫的头、中肠、脂肪体、生殖腺、体壁、丝腺中均有高量表达.氨基酸遗传距离和同源性分析结果,棉铃虫的TIME-EA4与家蚕和野蚕间的遗传距离分别为0.536、0.548,同源性均为44%;分子进化分析表明,棉铃虫和家蚕、野桑蚕的TIME-EA4同源蛋白质都与Cu-Zn/SOD蛋白的进化距离较近,蛋白质二级结构分析也显示,棉铃虫与家蚕TIME-EA4基本特性有很大相似性.

In order to elucidate the control mechanism of temperature,photoperiod and other circadian rhythm zeitgebers on diapause in the silkworm (Bombyx mori) ,silkworm eggs at late embryonic development stage were treated with normal diapause-inducing incubation temperature and photoperiod to investigate the response of expression of major circadian rhythm loop genes (Cry1,Cry2,Per and Tim) against zeitgebers oscillation.The results showed that light could upregulate the expression of Cry1 gene at late development stage of silkworm embryos,and temperature could change the oscillation cycle phase of Cry1 expression.While temperature was able to change the oscillation cycle phase of Cry2 gene expression,low temperature (15 ℃) could up-regulate the expression of Cry2 gene at late development stage of silkworm embryos.Elevated temperature could shorten the oscillation cycle of Per gene expression.Continuous darkness could induce oscillation of Tim gene expression and temperature could change the oscillation cycle phase of Tim gene expression.These results suggest that silkworm embryos have a circadian clock feedback loop with CRY1 as the photoreceptor which is similar to other insect species,and Cry1,Cry2,Per and Tim are circadian clock genes that respond to temperature and photoperiod circadian rhythm zeitgebers and induce diapause at silkworm embryonic development stage.