【作者】 杨丹彤；

【导师】 赵小凡；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 棉铃虫（Helicoverpa armigera）属于鳞翅目夜蛾科（Lepidoptera Noctuidae），在世界各地广泛分布，危害棉花、谷物、玉米、土豆、大豆、高粱和烟叶等农作物达上百种，往往造成巨大的经济损失，是我国和世界农业的主要害虫之一。目前棉铃虫防治中使用的大多数化学农药对环境和人类健康有害，而对于转基因作物如Bt抗虫棉的安全性也存在争议。因此在理论研究的基础上，发展新型生物农药一直是害虫防治的研究热点。细胞凋亡，又称程序性细胞死亡（programmed cell death，PCD），系机体生长发育、细胞分化以及病理状态中细胞自主性死亡，以清除多余、受伤或有害细胞而维持机体动态平衡，是机体正常生长发育过程中所必需的。本文从棉铃虫中克隆了两个在细胞凋亡中发挥重要作用的基因，Ha caspase-1和Ha hsc70，对它们的基因及演绎蛋白的序列进行了分析，并对Ha caspase-1的表达模式、调控和功能进行了研究。一．棉铃虫Ha caspase-1基因克隆与表达Caspase（cysteinylaspartate specific proteinase）是一类具有天冬氨酸特异性的半胱氨酸蛋白酶，是细胞凋亡过程中关键的作用因子，还具有调节细胞生长、增殖和分化等非凋亡功能。Caspase家族蛋白在生物界广泛存在，进化上保守性较高，在细胞凋亡中的功能相似。参与细胞凋亡的caspase家族蛋白根据其N端结构域prodomain的序列和长度，可分为两类，具有长prodomain的caspase为initiator caspase，发挥传递凋亡信号及激活下游caspase的作用；而具有短prodomain的caspase为effector caspase，直接作用于细胞内的底物，是凋亡的执行者。本文采用同源克隆策略，从棉铃虫变态期cDNA文库中克隆到一个caspase基因，命名为Ha caspase-1。Ha caspase-1基因的cDNA全长1350bp，含有一个885bp的完整的开放阅读框，编码294个氨基酸，该演绎蛋白的理论分子量为33.5kDa，等电点为5.65。对该蛋白的结构域及活性位点分析结果显示，有caspase-p20和caspase-p10两个结构域，即大、小亚基；另外，该蛋白具有caspase蛋白酶催化作用所需的特征性的氨基酸残基His和Cys，如高度保守的QACQG五肽中的半胱氨酸残基(C₁₇₆)。Ha caspase-1与其它物种caspase氨基酸序列比对结果显示，Ha caspase-1的prodomain较短，与草地夜蛾（Spodoptera frugiperda）Sf caspase-1和果蝇（Drosophila melanogaster）drICE同源性很高，与人caspase-3的相同序列也有32％，表明Ha caspase-1是一个下游caspase，即effector caspase。采用Northern blot和半定量RT-PCR，对Ha caspase-1基因mRNA在幼虫不同组织中的表达模式进行了研究。结果显示，仅在5龄取食和6龄游走期幼虫的血细胞中检测到一条约1300 bases的杂交带，而且，6龄游走期幼虫的杂交信号略高于5龄取食幼虫。在取食和游走期幼虫的其它组织，表皮、脂肪体和中肠中均未检测到杂交信号，表明Ha caspase-1或在血细胞中特异性表达，或在血细胞中的表达显著高于其它组织中。而半定量RT-PCR结果显示，在血细胞中，Ha caspase-1在6龄游走期幼虫中的表达明显高于5龄取食幼虫，也明显高于其它组织，与Northern blot结果相符。但是，在取食幼虫的脂肪体以及游走幼虫的脂肪体和中肠中，也发现Ha caspase-1有低水平表达，这可能是由于PCR方法比Northern blot敏感性高，或因为血细胞黏附或进入脂肪体和中肠的缘故。采用Northern blot对Ha caspase-1 mRNA在棉铃虫不同发育阶段的表达进行了研究。结果显示，Ha caspase-1在胚胎期有明显表达，这与胚胎时期有活跃的细胞凋亡发生是一致的。而在1龄到3龄的取食及蜕皮期幼虫整体中无表达，可能由于Ha caspase-1在整虫中表达水平太低而未检测到。从4龄到蛹期不同发育阶段的血细胞中，Ha caspase-1的表达水平在4龄及5龄蜕皮期和变态期有两次上升，似乎与从幼虫最后一次蜕皮到蛹形成期间的两次蜕皮激素脉冲释放有关。采用原位杂交技术对Ha caspase-1在血细胞中表达定位进行了研究。结果显示Ha caspase-1仅在浆血细胞（plasmatocytes）和粒细胞（granulocytes）表达，而在原血细胞（prohaemocytes）和珠血细胞（spherulocytes）中无表达，Ha caspase-1在这两种血细胞中的表达可能与其参与幼虫变态过程有关。由于Northern blot结果发现Ha caspase-1表达在4龄及5龄蜕皮期及变态期幼虫血细胞中上升，推测Ha caspase-1可能受到蜕皮激素20E的调控。因此，采用Northern blot和半定量RT-PCR对Ha caspase-1与蜕皮激素的关系进行研究。结果显示，体内注射蜕皮激素竞争物RH-2485可以使血细胞中Ha caspase-1的表达上升；20E可以上调体外培养的血细胞中Ha caspase-1的表达，表明在发育过程中Ha caspase-1的表达与蜕皮激素的释放有关，而且可能受蜕皮激素上调。Ha caspase-1作为一种effector caspase，应该在细胞凋亡发挥作用。用苜蓿银纹夜蛾核型多角体病毒（Autographa Californica multiple nucleocapsid polyhedrovirus，AcMNPV）诱导培养的表皮细胞使之发生凋亡，观察分析了凋亡细胞的形态、DNA降解和Ha caspase-1的表达。AcMNPV诱导后表皮细胞发生了染色质疑集、胞膜皱缩、出现空泡等形态学上的变化，并解体为凋亡小体，核DNA的琼脂糖凝胶电泳显示出特异性的梯形DNA图谱。Ha caspase-1 RT-PCR的结果显示，比之未诱导的细胞，凋亡细胞中Ha caspase-1表达明显上升，表明Ha caspase-1的确在细胞凋亡中发挥作用。采用基因特异引物，PCR扩增得到含ORF的Ha caspase-1基因片断，并克隆到原核表达载体pET-30a（+）中，构建了重组表达质粒Ha casp/pET-30a（+），并成功地在大肠杆菌BL21（DE3）菌株中进行了重组蛋白的表达，为研究Ha caspase-1的功能奠定了基础。二．棉铃虫Ha hsc70基因克隆热休克蛋白（heat shock proteins，Hsps）是一类高度保守的蛋白，具有极为广泛的生物学功能，不仅在应激反应中发挥重要作用，维持细胞自稳，保护细胞生命活动，而且参与细胞凋亡、免疫调控、肿瘤及病毒感染等生理、病理过程。Hsps是一个成员众多的蛋白超家族，其中Hsp70家族是Hsps中最保守和最重要的一族。本文采用同源克隆策略，从棉铃虫变态幼虫表皮SMART cDNA中克隆了一个hsc70基因，命名为Ha hsc70。Ha hsc70基因的cDNA全长2145bp，具有一个长1965bp完整的开放阅读框，编码含654个氨基酸的Hsc70蛋白。该蛋白的理论分子量为71.6kDa，等电点为5.24。对Ha Hsc70蛋白的结构域分析结果显示，仅有一个大的HSP70结构域，无信号肽序列。比对Ha Hsc70与其它物种Hsc70的蛋白序列，结果显示Ha Hsc70与其它昆虫Hsc70的序列一致性高达90％以上，与高等哺乳动物及人Hsc70的序列一致性也达80％以上，与植物Hsc70、微生物Hsp70的序列一致性分别有70％及45％左右，说明Hsc70在进化上的确非常保守。综上所述，本文首次克隆了棉铃虫的一个caspase基因，Ha caspase-1，序列分析及比对结果显示，Ha caspase-1是一个下游的effector caspase。Ha caspase-1在棉铃虫幼虫不同发育阶段的表达模式，及其在体内、体外均可被RH-2485和20E诱导上升表达的结果，提示Ha caspase-1可能受蜕皮激素上调，在棉铃虫的蜕皮及变态发育中发挥作用。Ha caspase-1在凋亡细胞中的高表达，证实了其effector caspase的功能。本文还首次克隆了棉铃虫的一个hsp基因，Ha hscT0，序列分析及比对结果显示，Ha hsc70与其它物种hsc70高度同源，对Ha hsc70的表达及功能等尚在研究之中。细胞凋亡在生物体生长发育、细胞增殖与分化中发挥重要作用，Caspase是细胞凋亡途径中关键的作用因子，Hsps参与细胞凋亡调控，而且Caspase与Hsps之间存在相互作用。对棉铃虫Ha caspase-1和Ha hsc70基因性质、表达及功能进行研究，在此基础上研制安全性较高的抑制害虫生长发育的新型生物农药，对于发展棉铃虫防治的新方法及新途径具有重要意义。另一方面，通过对Ha caspase-1和Ha hsc70基因功能及其调控的研究，也可以进一步了解细胞凋亡发生发展的机制，从而阐明昆虫变态发育中的分子生物学机理。更多还原

【Abstract】 The cotton bollworm, Helicoverpa armigera, which belongs to Lepidoptera Noctuidae, distributes broadly in the world and harms a lot of crops including cotton, cereal, corn, potato, bean and tobacco leaf. The cotton bollworm is one of the major pests of the world agriculture, which often brings great lost in economy. At the present time, the majority chemical pesticides used to prevent cotton bollworm are harmful to human and environment, and there is controversy on the security of the transgene crops such as the Bt cotton. Therefore, development of the biological pesticides based on the theory studies is always the focus of the pest prevention.Apoptosis, or programmed cell death （PCD）, a process of cell suicide, which eliminates superfluous, damaged or deleterious cells during development, cell differentiation, and disease to maintain homeostasis, is essential to the development of organisms. In this study, two genes, Ha caspase-1 and Ha hsc70, involved in apoptosis were cloned from H. armigera. Sequences of the genes and the deduced proteins were analyzed, and the expression pattern, induction expression and function of Ha caspase-1 were investigated. 1. Cloning and expression of Ha caspaase-1 from H. armigeraCaspase （cysteinylaspartate specific proteinase） is a group of cysteine proteinase with substrate specificity at aspartate, which is the key factor in apoptosis and has non-apoptotic functions in cell survival, proliferation and differentiation. The proteins of caspase family are evolutionally conserved, executing the similar function in apoptosis. The caspases involved in apoptosis are divided into two groups according to the sequences of their N terminal domain named prodomain. The caspases with long prodomain are the initiator caspases which transfer the signals of apoptosis and activate the downstream caspases, while the caspsaes with short prodomain are the effector caspases which cleavage the substrates in cell and are the direct executioners of apoptosis.In this study, a caspase gene named Ha caspase-1 was cloned from the cDNA library of metamorphically committed H. armigera using the strategy of homologous cloning. The full-length of Ha caspase-1 cDNA is 1350 bp, including an 885 bp open reading frame （ORF） that encodes 294 amino acids. The deduced protein has a computed molecular mass of 33.5 kDa and a predicted isoelectric point of 5.65. Sequence analysis of the domain and active site shows that the protein has two domains, caspase-p20 and caspase-p10, which are the large subunit and the small subunit, respectively. In addition, the predicted protein contains the typical residues His and Cys required for catalysis by caspases and the catalytic cysteine (C₁₇₆) located in a QACQG pentapeptide is an example. Sequence alignment of amino acids of Ha caspase-1 with other known caspases revealed that it has a short prodomian and is great homologous to Sf caspase-1of Spodoptera frugiperda and drICE of Drosophila melanogaster. The homology of Ha caspase-1 to human caspase-3 is even 32%. These results suggested that Ha caspase-1 is an effector caspase in downstream of caspase cascade.Northern blot analysis and semi-quantitative RT-PCR were used to investigate the expression pattern of Ha caspase-1 in different larval tissues. The result of Northern blot showed that the Ha caspase-1 mRNA was only detected in the feeding and wandering larval haemocytes, with the concentration in haemocytes of wandering larvae slightly higher than that in haemocytes of feeding ones. There was no positive signal detected in the epidermis, midgut, fat body from both feeding and wandering larvae, suggesting that either the expression of Ha caspase-1 mRNA was probably haemocyte-specific or its mRNA concentration in haemocytes was significantly higher than that of the other tissues. The result of RT-PCR showed that the signal detected in haemocytes from wandering larvae was much stronger than that in feeding ones and other tissues, in agreement with that of Northern blot. However, in the feeding larval fat body and the midgut and wandering larval fat body, Ha caspase-1 was also found at lower levels, which is different from the result of Northern blot and may be due to the higher sensitivity of PCR compared with Northern blot, or haemocytes attachment or migration into fat body and midgut.Northern blot analysis was applied to determine the developmental profiles of Ha caspase-1 mRNA levels. The results revealed that Ha caspase-1 was expressed in embryos, which is consistent with the notion that cell death is extensive during embryogenesis, but there is no Ha caspase-1 detected in younger instar larvae （lst³rd） by whole body preparation of mRNA, which may because Ha caspase-1 mRNA level is too low to be detected in these early developmental stages. However, in later instar larval haemocytes （4th—6th instar）, Ha caspase-1 mRNA concentration was higher during 4th and 5th molting and wandering stages, which appears to be correlate with the two waves of ecdysone during the periods of last larva molting and metamoephosis.In situ hybridization was performed to locate the transcript of Ha caspaes-1 in haemocytes. Results indicated that the plasmatocytes and granulocytes in haemocytes from 6th instar wandering larvae released extensive Ha caspase-1 signal. Other cells, including prohaemocytes and spherulocytes, did not show obvious signal compared with the negative control cells. The expression of Ha caspase-1 in these two kinds of haemocytes suggested that Ha caspase-1 may be involved in the larval-pupal metamorphosis because the populations of both plasmatocytes and granulocytes increased in the late stage of the last instar larvae.Because Ha caspase-1 mRNA levels increased in haemocytes during molting and metamoephosis, it is inferred that Ha caspase-1 should be regulated by 20-hydroxyecdysone （20E）. Therefore, the relationship between Ha caspase-1 and ecdysone was measured through Northern blot analysis and RT-PCR. Results showed that Ha caspase-1 expression increased in haemocytes in vivo after ecdysone agonist RH-2485 injection and Ha caspase-1 mRNA also went up in haemocytes in vitro after treated with 20E, suggesting that Ha caspase-1 expression during development correlates to the release of ecdysone.As an effector caspase, Ha caspase-1 should play a role in apoptosis. The morphological alterations, DNA degeneration and Ha caspase-1 expression was therefore observed in the cultured epidermis cells undergoing apoptosis induced by AcMNPV （Autographa Californica multiple nucleocapsid polyhedrovirus）. After AcMNPV induction, the morphological changes on cells including chromatin condensation, membrane shrinkage and vacuole appearance and apoptotic bodies were observed. In addition, nuclear DNA presented the DNA ladder on agarose gel electrophoresis. The result of RT-PCR showed that Ha caspase-1 expression in cells induced by AcMNPV was much higher than that in the non-induced cells, indicating that Ha caspase-1 is a function factor involvd in apopotosis.In addition, a fragment containing ORF of Ha caspase-1 was subcloned into prokaryotic expression vector pET-30a（+） to construct recombined plasmid Ha casp/pET-30a（+）, and recombinant protein was highly expressed in E. coli strain BL21（DE3）, which are to be used for function study on Ha caspase-1.2. Cloning of Ha hsc70 from H. armigeraHeat shock protein is a group of greatly conserved proteins, which has a broad range of biochemical functions. Hsps not only play indispensable roles in the stress reaction to maintain the self-stabilization of cell and avoid the damage of cell, but also participate in apoptosis, immune regulation, tumor and infection. Hsps is a super family including a large number of members in which Hsp70 family is the most conserved and important.In this study, a hsc70 gene named Ha hsc70 was cloned from the SMART cDNA of epidermis from the metamorphically committedally committed H. armigera using the strategy of homologous cloning. The full-length Ha hsc70 cDNA is 2145 bp, including a 1965 bp ORF that encodes 654 amino acids. The deduced protein has a computed molecular mass of 71.6 kDa and a predicted isoelectric point of 5.24. Sequence analysis shows the protein has only one large HSP70 domain without signal peptide. Sequence alignment of amino acids of Ha Hsc70 with other known Hsc70 revealed it has the greatest homology to the Hsc70 of other insects, with an identity over 90%. The identity of Ha Hsc70 to mammal and human Hsc70s also reaches 80%. The identities of Ha Hsc70 to plant and prokaryotic Hsc70s （or Hsp70s） are even about 70% and 45%, implying that Hsc70 is greatly evolutionally conserved.In summary, we cloned a caspase gene from H. armigera , Ha caspase-1, which is a effector caspase resulted from the sequence analysis and alignment. The expression profiles of Ha caspase-1 during development and induced expression of Ha caspase-1 by RH-2485 in vivo and 20E in vitro imply that Ha caspase-lmay be up regulated by ecdysone and play a role during molting and metamorphosis. The high expression of Ha caspase-1 in the apoptotic cells approves of its function as an effector caspase. We also cloned a hsp70 gene from H. armigera, Ha hsc70, which is highly homologous to hsc70 of other species resulted from the sequence analysis and alignment. The expression and function of Ha hsc70 is under study now.Apoptosis plays an essential role in development, cell proliferation and differentiation. Caspases are the key regulator in the apoptotic pathways, Hsps also participate in apoptosis, and the interaction exists between caspases and Hsps. To develop biological pesticides with high security based on the study of the characterization, expression and function of both caspase and Hsps is important to the exploitation of new methods for pest control. On the other hand, to study the function and regulation of genes involved in apoptosis during insect development is in favor of better understanding the mechanism of apoptosis and insect development.更多还原