【作者】 杨靖；

【导师】 徐卫华；

【作者基本信息】 中山大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 滞育是指昆虫个体接收到环境信号后,程序性地在不良环境到来之前进入一种低代谢、低速发育甚至不发育的状态。对昆虫滞育的研究不但有助于我们了解昆虫的抗逆性,为害虫防治提供新思路,而且昆虫滞育本身也是研究动物发育调节的一个理想模型。根据不同发育阶段,昆虫的滞育可分为卵滞育、蛹滞育、幼虫滞育和成虫滞育。目前的研究结果认为不同的滞育形态分别由不同的作用机制调节的。棉铃虫Helicoverpa armiger(aHar)属鳞翅目(Lepidoptera)夜蛾科(Noctuidae)昆虫,是我国主要的农业害虫,以蛹的形式滞育。蛹滞育的经典理论认为是由于脑中促前胸腺激素(PTTH)合成的下调或者是释放受阻,导致前胸腺合成促进发育的蜕皮激素减少,最后进入滞育。蛹滞育解除时,血淋巴中PTTH滴度上升,促进前胸腺分泌蜕皮激素,使发育进程重新启动。通过给棉铃虫活体注射重组PTTH打破蛹滞育直接证明了PTTH→蜕皮激素→滞育解除这条通路。滞育激素(DH)是从家蚕促咽下神经节中分离得到的一种昆虫神经肽,能诱导家蚕卵滞育。有趣的是,DH不能诱导棉铃虫滞育,反而能打破棉铃虫蛹滞育。实验证明,DH打破滞育也是通过作用于前胸腺产生蜕皮激素这一路径。所以鉴定应答蜕皮激素的相关基因对查明滞育解除的机理是至关重要的工作。为此我们用差异展示方法(DD-PCR)对棉铃虫蛹滞育打破相关基因进行了筛选,结果得到30个有差异的cDNA片段;通过RT-PCR验证,得到三个与蛹滞育打破相关的基因。同源比较显示它们分别与番茄褐夜蛾Feltia jaculifera细胞色素氧化酶亚基1(90%)、库蚊Culex pipiens NADH dehydrogenase 1 alpha subcomplex subunit 6(ndua6)(70%)、烟草天蛾Manduca sexta V0-ATPase subunit e (92%)有较高的相似性。三个基因全长序列分别为1530 bp、567 bp和525 bp,阅读框编码510、125和85个氨基酸的蛋白。细胞色素氧化酶亚基1(Cytochrome c oxidase subunit 1, COX1)是线粒体电子传递链的末端酶COX的第一个亚基,由线粒体基因组编码,它与另外两个线粒体基因组编码的亚基COX2和COX3共同组成COX活性中心。我们获得cox1的cDNA全长,其ORF包含1530核苷酸,编码510个氨基酸,起始密码子为CGA。为弄清棉铃虫在蛹期及滞育解除时对不同能量要求的反应,我们对cox1 mRNA、蛋白水平表达、COX活性及磷酸化进行了调查。结果显示:棉铃虫cox1 mRNA和蛋白水平的表达与COX活性相关;非滞育蛹COX活性高,而滞育蛹则保持低能量代谢水平。注射20-羟基蜕皮酮打破棉铃虫蛹滞育时,cox1 mRNA及蛋白水平表达逐渐增加的同时COX活性显著提高。棉铃虫COX1的酪氨酸磷酸化与COX活性密切相关,暗示可逆蛋白磷酸化可能通过调节能量代谢的速率在昆虫滞育中起关键作用。NADH dehydrogenase 1 alpha subcomplex subunit 6 (ndua6)是线粒体电子传递链第一个酶复合物的一个附着小蛋白,由核基因编码。棉铃虫脑中ndua6基因mRNA和蛋白水平表达非滞育蛹显著高于滞育型蛹。滞育型蛹ndua6 mRNA水平表达在蛹早-中期(0-10天)和滞育维持期(10-35天)没有明显差异,蛋白表达略有下调。注射20-羟基蜕皮酮打破蛹滞育,1天时ndua6 mRNA及蛋白表达水平均上调,2-3天时,蛋白表达仍然保持高水平,而mRNA转录在2天时下调,第3天时又回调至第1天水平,表现出波动性。V0-ATPase亚基e是液泡膜上ATP水解酶的一个亚基,是一个二次跨膜蛋白。在滞育蛹脑中,V-ATPase亚基e在滞育前期及维持期都稳定表达,表达量低于非滞育蛹,注射蜕皮激素24小时后,快速上调。非滞育蛹ATPase活性高于滞育蛹,前期高,然后逐步下降。滞育蛹ATPase活性与COX活性趋势基本一致,始终维持在低水平,只是在20-羟基蜕皮酮打破滞育后会显著增高。更多还原

【Abstract】 When insects receive the environmental signals, such as temperature, photoperiod, humidity etc., they will enter a state of programmed developmental arrest called diapause which is characterized of low metabolism, low-speed development ahead of the unfavorable environment. Insect diapause is an ideal model to study animal development, it will help us to understand the resistance of insect and supply a feasible method for pest biocontrol. Diapause could occur in different stage of life cycles, such as egg diapause, larval diapause, pupal diapause, and adult diapause. Current research result demonstrates that different diapause have different mechanism.The cotton bollworm, Helicoverpa armigera (Lepidoptera noctuidae) is a major agricultural pest which belongs to pupal diapause. It is well-known that pupal diapause is regulated by prothoracicotropic hormone (PTTP) and ecdysone. Classic theory of pupal diapause think that a shut-down of prothoracicotropic hormone (PTTH) synthesis and/or release and the subsequent failure of the prothoracic glands (PGs) to synthesize the ecdysteroids needed to promote continuous development (Denlinger, 1985). Recombinant PTTH could effectively break pupal diapause in H. armigera (Wei et al., 2005). Interestingly, diapause hormone (DH), which induces embryo diapause in Bombyx mori, was found to break pupal diapause in H. armigera (Zhang et al., 2004a) and H. virescens (Xu and Denlinger, 2003) by stimulating ecdysone synthesis and release. However, the molecular mechanism of pupal diapause termination in response of ecdysone is yet unknown. With these questions in mind, we injected ecdysone into diapausing pupae to screen the related genes from pupal diapause termination by using differential display PCR (DD-PCR). Thirty cDNA fragments were isolated from brain of diapausing pupae 24 h after injection of 20-hydroxyecdysone, and three genes were identified by homology search, cytochrome c oxidase subunit 1 of Feltia jaculifera (90%) , vacuolar ATPase subunit e of Manduca sexta (92%) and NADH dehydrogenase 1 alpha subcomplex subunit 6 Culex pipiens (70%) respectively.Cytochrome c oxidase subunit 1 is the largest subunit of COX, the terminal complex of electron transfer chain (ETC), and comprises the active core of cytochrome c oxidase with COX2 and COX3. We obtained the full length of H. armigera cox1 (Hea-cox1) cDNA which has an open reading frame of 1530 nucleotides encoding a putative protein of 510 amino acid residues, with CGA as start codon. To evaluate the response to different energy demands during pupal development and at diapause termination, we assessed the expression of Hea-cox1 mRNA and protein, COX activity and its phosphorylation. The results show that Hea-cox1 expression at the mRNA and protein levels is associated with COX activity, and high levels of Hea-cox1 expression and COX activity are present in nondiapause pupae, suggesting that low energy metabolism provided by oxidative phosphorylation in mitochondria in diapause individuals is necessary. After diapause is broken by injection of 20-hydroxyecdysone, expression of Hea-cox1 mRNA and protein increases gradually and COX activity increases significantly. Furthermore, Hea-cox1 phosphorylation is closely correlated with COX activity, suggesting that reversible protein phosphorylation might play a key role in insect diapause through suppressing the rate of energy product.NADH dehydrogenase 1 alpha subcomplex subunit 6 (ndua6) is an adhere subunit of the complex I in the electron transfer chain (ETC), encoded by the nuclear genome. In the brain of H. armigera pupae, mRNA transcript of ndua6 is higher in non-diapausing than diapausing pupae. No significant difference of ndua6 mRNA transcript was found during diapause preparation and maintemance stage. When diapause was broken by injection of 20-hydroxyecdysone, mRNA transcript increase significantly within 24 h after injection. Protein expression of the ndua6 in non-diapausing pupae is higher than that of diapausing pupae. Vo-ATPase subunit e is a subunit in V0 complex of vacuolar ATPase and is a twice trans-membrane protein. In the brain of H. armigera, the same mRNA transcript was found on pupae day 0 and day 1 between non-diapausing and diapausing pupae. With the progress of diapause, mRNA transcript of diapausing pupae has the same profile as non-diapausing pupae and after pupae day 15, transcript of diapausing is significantly low than non-diapausing pupae. mRNA transcript increase significantly within 1 day after injection of 20-hydroxyecdysone. ATPase activity of nondiapause pupae is high at the early pupal stage and decrease during pupal development. the ATPase activity in diapause type pupae show a stable low level, and increases significantly within 1 day after injection of 20-hydroxyecdysone. ATPase activity in nondiapause pupae is higher than diapausing pupae.更多还原