【作者】 韩慧慧；

【导师】 徐世清； 司马杨虎；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2011， 硕士

【摘要】 家蚕(Bombyx mori)是卵滞育性鳞翅目模式昆虫。滞育受亲代卵催青期温度、光照节律等环境因子诱导,并受产下后温度诱导决定表现。本文以经典的家蚕滞育卵诱导条件处理家蚕催青期蚕卵,调查家蚕促前胸腺激素基因Ptth、滞育激素—性信息素合成激活肽基因Dh-pban、羽化激素基因Eh以及垂体同源框激素基因Pitx的表达谱。主要获得了以下结果:1家蚕4个滞育相关内分泌基因的表达存在显著的组织差异性家蚕大造品种5龄幼虫期基因芯片表达谱显示,Ptth、Dh-pban、Eh以及Pitx 4个基因在家蚕头部等10个组织中的表达水平有显著差异,具有组织特异性,Ptth基因在家蚕头部表达水平最高,其它组织中几乎不表达;Dh-pban基因在家蚕头部表达水平最高,其次是后部丝腺和马氏管,其他组织中表达量很低;Eh基因在体壁的表达水平最高,其次是头部,在卵巢中几乎检测不到;Pitx基因在马氏管中表达水平最高,其次是中肠和头部,其他组织中表达水平较低,其中在血液中仅痕迹量表达。Eh和Pitx基因在不同组织中的表达有极显著的差异性。值得注意的是,卵巢这一蚕卵形成和滞育代谢的重要靶器官中,5龄幼虫期Ptth、Dh-pban、Eh以及Pitx 4个基因的表达水平都很低, Eh基因几乎检测不到有表达。2诱导子代滞育卵发生的高温与光照条件上调家蚕Dh-pban基因表达催青期在诱导子代滞育卵发生的高温25℃与持续光照(25LL)条件下,胚胎有稳定的、高水平Dh-pban基因mRNA存在,而诱导子代非滞育卵发生的低温15℃与持续黑暗(15DD)条件下,蚕卵中几乎检测不到Dh-pban基因的mRNA存在。在25LL或者20℃12h光照12h黑暗(20LD)催青环境中,胚胎发育至单眼形成后(积温高于2160℃·h),蚕卵中的Dh-pban基因表达水平开始显著上调,这与在此阶段的蚕卵胚胎对滞育诱导环境温度和光节律更加敏感相一致,但Dh-pban基因表达水平没有出现随不同滞育诱导温度或光节律变化的振荡节律,表明蚕卵胚胎的Dh-pban基因表达受到滞育诱导温度和光照这两个近日节律授时因子的影响,但不是节律授时因子直接调控的基因。从催青期开始的整个世代保护温度和光照,对家蚕胚后幼虫至成虫时期Dh-pban基因的表达水平影响,呈现高温25℃上调Dh-pban基因的表达。亲代卵孵化期的催青温度与光照,影响了子代卵的滞育性,也影响了子代卵内的Dh-pban基因mRNA水平,但这种影响与蚕卵的滞育性不完全一致。3光照与温度对家蚕Eh和Pitx基因在各个时期表达的影响不同15DD条件下,蚕卵Eh基因的mRNA水平很低,提示在非滞育卵完全诱发条件下,蚕卵胚胎可能极少翻译EH;催青期在25LL或20LD环境下,能够检测到蚕卵Eh基因的mRNA,提示在家蚕滞育卵诱发条件下胚胎期已经开始合成EH。比较25LL与20LD二种环境条件下Eh基因的表达差异,发现25LL条件下蚕卵的Eh基因表达水平总体高于20LD的蚕卵;胚胎发育后期,在积温高于2 160℃.h(单眼形成时期及单眼形成)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。亲代卵催青期25LL条件所产滞育卵中,Eh基因mRNA表达水平很低,在胚胎进入滞育后,则几乎检测不到Eh基因mRNA。而亲代15℃催青温度条件下所产子代非滞育性卵,无论是胚胎发生期间,还是其后的胚胎组织器官发育时期,都检测出一定含量的Eh基因mRNA。催青期在25LL、20LD、15DD环境下,均能检测到蚕卵Pitx基因的mRNA,且25LL条件下蚕卵的Pitx基因表达水平总体高于20LD和15DD的蚕卵。家蚕胚后时期25LL条件下Pitx的表达水平总体要高于15DD条件下的表达水平。亲代25LL催青环境下,子代滞育性卵在卵龄12-24 h胚胎发生期间,Pitx基因mRNA的含量快速增加,但随后迅速减少,至卵龄72 h后胚胎逐渐进入滞育阶段,卵内几乎检测不到Pitx基因的mRNA;而亲代15℃催青温度条件下所产子代非滞育性卵,无论是卵龄12-24 h的胚胎发生期间,还是其后的胚胎组织器官发育时期,都能检测出Pitx基因mRNA的表达;亲代20LD催青条件下所产子代卵,在12-192 h期间Pitx基因mRNA含量一直高于滞育性卵与非滞育性卵。胚胎发育时期Pitx的表达与Dh-pban的表达相一致。更多还原

【Abstract】 Bombyx mori is the model insect of Lepioloptera with diapausing during its egg stage. Diapause inducements are due to temperature and photoperiod in the incubation process of parental eggs. However, whether or not eggs at progeny enter into diapause depends on temperature after oviposition. This paper investigated the expression of diapause-related endocrine genes in Bombyx mori using the classic diapause-inducing incubation conditions, which contained prothoracicotropic hormone gene, diapause hormone and pheromone biosynthests activating neuropeptide gene, eclosion hormone gene and pituitary homeobox gene. The major results are as follows:1 Diapause-related endocrine genes had tissue specific expression charac-teristics in Bombyx moriGene microarray analysis displayed that four genes, containing Ptth、Dh-pban、Eh?and?Pitx, had issue specific expression characteristics during postembryonic stage. Ptth gene expression level in head is higher than any other tissues. Dh-pban gene expression level in head is the highest in all tissues, and then in posterior silk gland and Malpighian tubules. The expression in other tissues is very low. Expression level of Eh gene is highest in integument, and then in head. There is almost no expression in ovary. Pitx gene expression in the Malpighian tubules is the highest, followed by the midgut and the head, lower levels of other tissues, of which only traces of the blood volume in the expression.2 The temperature and photoperiod inducing diapause of offspring eggs up-regulated the expression level of Dh-pban geneEggs at progeny are completely diapause when parental eggs are under constant illumination at 25℃(25LL) incubation, and they are completely non-diapause under constant darkness at 15℃(15DD). The Dh-pban mRNA of eggs in 25LL stayed at a high level, but it was very low in 15LL. Under 25LL or 20LD, the Dh-pban level of parental eggs up-regulated in the late incubation stage, which indicated that B. mori would be sensitive to the external illumination and temperature after embryonic nervous developed. However, the mRNA level of Dh-pban was not oscillated with the periodic oscillation of incubation photoperiod and temperature. The Dh-pban expression level rose at 25℃during postembryonic developmental stage. In conclusion, the incubation temperature and photoperiod of parental eggs affected the diapause of offspring eggs, but Dh-pban was not the direct gene regulated by the zeitgebers.3 The effect of photoperiod and temperature on the expression of Eh and Pitx gene was different among different stagesThe Eh mRNA of eggs in 15DD stayed at a very low level. Under 25LL or 20LD, the Eh level of parental eggs up-regulated in the late incubation stage, which indicated that B. mori would be sensitive to the external illumination and temperature after embryonic nervous developed. In the offspring, Eh gene mRNA expression level is very low under 25LL, and in the diapause period in the embryo, the gene is almost not detected Eh mRNA. However under 15DD, the offspring Eh gene mRNA can be detected in the whole Embryonic stage.Under incubation environment of 25LL, 20LD, 15DD, Pitx mRNA can be detected in silkworm eggs, and under the conditions of 25LL Pitx gene expression is in general higher than 20LD and 15DD. In postembryonic period, Pitx mRNA level is in general higher under 25LL than 15DD. Under 25LL incubation environment, in offspring diapause eggs during the age of 12-24 h embryos, Pitx mRNA levels increased rapidly but then declined rapidly to the age of 72 h, eggs gradually into the diapause stage embryos. Pitx mRNA can not be detected. However under 15DD, the offspring Pitx mRNA can be detected in the whole Embryonic stage.更多还原