【作者】 周庆祥；

【导师】 张志芳；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2007， 博士

【摘要】 翅面被覆鳞片是鳞翅目昆虫的主要特征之一；鳞片对于鳞翅目昆虫具有重要的生理意义，而鳞片构成的各种翅面图案具有特定的发育机制和进化模式，因此鳞翅目昆虫的鳞片在昆虫生理学、发育生物学以及生物进化，甚至害虫防治上都具有重要的研究价值。本文以家蚕无鳞片翅突变体scaleless(sl)为材料，从形态学、细胞生物学和分子生物学角度对突变体的产生机理进行了研究，力图阐明无鳞片翅突变表型的发生机理；并为鳞翅目昆虫鳞片的发育机制提供理论参考。首先从探明家蚕翅的生长发育机制入手，调查了五龄幼虫及蛹期翅原基(翅)的大小和形态变化，并建立了翅原基摘除和移植技术，为论文的深入研究提供实验和理论基础。翅原基在五龄前期生长缓慢，变态前生长最快；蛹期翅芽大小基本不变，但伴随有鳞片的形成和色素沉积过程。家蚕幼虫期的造血器官紧密附着在翅原基上，形成造血器官-翅原基复合体。五龄早期将4个造血器官-翅原基复合体全部摘除，家蚕幼虫仍能正常生长并发育成蛹和具有交配及产卵能力的蛾；同时其发育过程中血球总数仍呈增长趋势，但是明显低于正常蚕，由此推测化蛹变态中造血器官的崩解是变态的结果而不是其必要因素。全部造血器官-翅原基复合体摘除后配合使用MH或JHA均会增加熟蚕体内的血球数量，认为保幼激素在造血器官-翅原基复合体全部摘除的蚕体内有促进血球有丝分裂的作用。运用建立的翅原基(翅)解剖技术，分析了家蚕突变体scaleless的表型性状，发现其成虫翅面鳞片明显比野生型(WT)的少，并且所具有的少量鳞片比野生型的小且分叉少。五龄幼虫翅原基的相互移植实验证明决定这一突变性状的调控因子不存在于体液中，而是存在于翅原基自身内部。化蛹前scaleless的翅原基与WT的在形态和结构上没有明显差异，化蛹后WT家蚕的翅脉开始出现气管分叉并延伸进入翅面，2d蛹时已经可以看到翅脉之间的气管相互交织形成网状；但是scaleless家蚕蛹的翅脉中气管分叉少并且延伸不良。蛹期经过高氧分压处理可以部分挽回scaleless家蚕翅面的鳞片，使其成虫翅面鳞片明显增多。在幼虫五龄早期的翅原基表面划一道浅的刻痕使翅脉受伤，亦可形成成虫翅面鳞片少的表型。因此我们推测翅原基的翅脉中气管发育异常致使翅面细胞缺氧是导致scaleless鳞片变少的一个重要原因。进一步实验阐明了scaleless突变体家蚕细胞生物学方面的产生机理。AO/EB染色及Caspase-3/7活性分析结果显示在蛹的早期，翅面鳞片的发育伴随有细胞凋亡的参与。2d时大量翅面细胞凋亡，存活的细胞则整齐地排列在翅的表面。scaleless家蚕不同于野生型，其翅面大量细胞凋亡现象比WT出现的晚1天，且翅面细胞几乎全部死亡；并且Caspase-3/7的最大活性相当于WT的近10倍。同时有关凋亡小体和DNA片段化的实验结果也说明scaleless蛹期翅面细胞过度凋亡，从而产生了成虫翅面少鳞片的突变性状。AS-C基因与果蝇背板刚毛的发育有关；AS-C的同源体基因(ASH)B-ASH1也控制着蝴蝶翅面鳞片的形成。为了从分子生物学角度阐明家蚕scaleless突变体的产生机理，克隆并鉴定了家蚕的AS-C同源体基因。发现家蚕至少有四个ASH基因，分别命名为Bm-ASH1、Bm-ASH2、Bm-ASH3和Bm-ase；四个基因均具有明显的bHLH基序，并且前三个基因编码的蛋白质的C-端也具有16～17个氨基酸组成的保守区，这些都符合AS-C同源体基因的特点。同源性比较和进化分析说明三个原神经基因中BIn-ASH1是最早分化形成的一个；Bm-ASH2和Bm-ASH3同源性最高，推测相对于Bm-ASH1形成较晚。用RT-PCR和实时荧光定量PCR检测了家蚕ASH基因在幼虫期不同组织器官及胚胎发育过程中的表达情况，发现Bm-ASH1和Bm-ASH2表达范围最广，胚胎发育过程中也有较高的表达量；而Bm-ASH3表达特异性相对较高，因此推断其形成较Bm-ASH2为晚，即Bm-ASH3是四个家蚕ASH基因中最晚形成的一个。ase基因主要在神经前体细胞中表达，因此Bm-ase表达范围较窄，组织中总体表达量较低。但同源比较和功能分析都不能将家蚕的三个原神经基因和果蝇的一一对应，因此其命名依然按照发现的先后顺序进行。通过半定量RT-PCR发现家蚕的吐丝期和蛹期，Bm-ASH1和Bm-ASH3基因在scaleless和WT家蚕中的表达时相几乎一致；Bm-ASH2只在0 d～2 d的WT蛹翅中表达，而scaleless中没有检测到表达信号。原位杂交结果也证明Bm-ASH2在scaleless蛹翅中不能正常表达。将WT及scaleless的Bm-ASH2基因起始密码子上游的启动子序列克隆并测序，发现scaleless的ATG上游1027 bp处有一段连续26 bp的缺失突变。利用家蚕细胞瞬时表达系统分析了Bm-ASH2基因启动子活性，发现scaleless比WT低得多，只与截去了其缺失的26 bp的979 bp WT启动子片段的活性相当。EMSA结果显示scaleless缺失的26 bp序列可以特异性结合某种核蛋白。遗传学实验则证明26 bp序列缺失与无鳞片翅突变表型紧密连锁。这些都说明scaleless的Bm-ASH2基因因启动子突变而使其不能正常转录和表达。利用瞬时表达系统使Bm-ASH2基因在scaleless早期蛹翅中表达，可以使翅面鳞片明显增多，进一步说明Bm-ASH2是家蚕翅面鳞片产生的关键因子。从而初步阐明了家蚕无鳞片翅突变体scaleless产生的分子机理。更多还原

【Abstract】 The coating of scales on the surface of the wings is a basic characteristic of lepidopteran adults. Wing scales have important physical function to Lepidoptera, and patterns formed by multicolored scales not only have specifically developmental mechanisms, but also are a key innovation during lepidopteran evolution. So lepidopteran wing scales are valuable for the studies of insect physiology, developmental biology, biologic evolution, and even pest control. In this thesis, we studied the silkworm scaleless wings mutant（scaleless, sl） on morphological, cell biological and molecular biological levels, and tried to clarify the mechanism of the mutant and offer references for the studies on the development of lepidopteran wing scales.We carried out our studies beginning with the development of the wing discs/wings, in size and shape, during the 5th instar larval and pupal period. We found that the wing disc grew slowly in the prophase of the larva, while very fast just before metamorphism. We also found that pupa was a period of the wing buds perfecting, with wing scale formation and pigmentation. Hemopoietic organ is located tightly near or attach to the imaginal wing disc in the silkwom, Bombyx mori, and the two kinds of organs are customarily called by a joint name as hemopoietic organ-wing disc complex. We successfully extirpated the complexes of 5th instar larvae, and found that even when all of the complexes were extirpated, majority of the silkworms could still develop into moths normally. Using this technique, we studied the effects of the extirpation on the hemopoiesis of the silkworm, by investigating the change of density and total of hemocytes circulating in the final instar larval hemolymph. We found that mitosis could offer enough hemocytes for the development of the silkworms whose complexes were totally extirpated. And the collapse of the hemopoietic organs during wandering might be a consequence of metamorphosis, but not a prerequisite for pupation. Exogenous hormones could elevate the mitosis of circulating hemocytes in complexes totally extirpated silkworm; and during early spinning stage, complexes extirpation might cause a certain extent hemopoietic compensation of the remainders. The feasibility of the silkworm wing discs growing and developing in exotic site was proved by reciprocal transplantation technique. The results afforded us the background for the following studies.We investigated the morphology of scaleless and found that it had many fewer wing scales than wild type（WT）, and that the remaining scales were smaller in shape with fewer furcations. Reciprocal transplantation of wing discs between scaleless and WT revealed that the WT wing disc could develop into a small wing with scales after transplantation into a scaleless larva; however, the scaleless wing disc developed into a small wing without scales in a WT larva. Upon dissection of WT and scaleless wing discs at different stages from the fifth instar larva to adulthood, no obvious differences were found before pupation. However, after pupation, tracheae produced from WT wing veins extended to the lacunae between the veins and formed a network on the second day after pupation, whereas this did not happen in scaleless. At the same time, no marked difference of the adult body tracheal development was found between the mutant and wild type. Furthermore, if the surface of a wing disc was cut and its veins injured, the resulting wing also had fewer scales than that of WT. Also, we found that higher partial pressure of O₂ could rescue the loss of scales in scaleless. These data suggest that the factors affecting the growth of scales were not produced in the hemolymph, but in the wing disc itself. It is also implied that wing scale development is dependent on the correct organization of tracheal system in the wing disc.We gave data to clarify the mechanism of the mutation of scaleless at the cell biological level. The results of AO/EB dying and Caspase-3/7 analysis showed that programmed cell death participates in the wing scale development during early pupal stage. On 2d, well ranged cells could be seen on the pupal wing, surrounded with vast dead ones. There were significant differences between that of sl and the wild type（WT） at each phase. Well-differentiated scale precursor cells did not form in sl when they had formed in WT. The peak of Caspase-3/7 activity in sl occured one day later than, and ten times as much as that in WT. Apoptotic bodies and DNA ladder studies also showed that there was excessive apoptosis in sl early pupal wing.AS-C（achaete-scute Complex） genes are associated with the development of Drosophila notum bristles, and a homolog of AS-C（ASH）, B-ASH1, controls the formation of butterfly wing scales. In order to clarify the mechanism of the scaleless wings mutation of sl at the molecular level, we cloned and analyzed the AS-C homolog in Bombyx mori. Four genes, Bm-ASH1, Bm-ASH2, Bm-ASH3 and Bm-ase were cloned, and the proteins coded by the four genes had a typical bHLH motif each, and the former three also each had a 16-17 aa conservative region at the C-terminal. All of these indicated the four genes according with the characteristic of AS-C homolog. Homologous comparation and evolutional analysis showed that Bm-ASH1 formed earliest among the three preneural genes; while Bm-ASH2 and Bm-ASH3 were mostly homologous, and might both occur later than Bm-ASH1. We investigated the expression level in various larval organs and different embryo developmental ages using RT-PCR and realtime quantitative PCR methods. Bm-ASH1 and Bm-ASH2 were expressed in most of the organs and had a high expression level in proper embryo ages. Bm-ASH3 was more differential, so we deduced it formed at the most near time and gained special function. Bm-ase was also more special and the detected expression level was low. But neither homologous comparation nor functional analysis could correspond the three Bombyx preneural genes to those of Drosophila, so we still named the genes after their discoverd order.Both of the results of semi-quantative RT-PCR and in situ hybridyzation showed that Bm-ASH2 gene expressed abnomal in scaleless pupal wing. The gene expressed highly in 0 d-2 d WT pupal wing, but very low in scaleless. Sequencing results showed that there was a continuous absent mutation of 26 bp, 1 027 bp upstream of Bm-ASH2 initial code in scaleless genome. The transcriptional activity of scaleless’s, Bm-ASH2 promoter was much lower than that of the WT, just the same as the truncated 979 bp promoter segment of WT without the 26 bp. EMSA result indicated that the 26 bp had a special binding nuclear protein. Genetic experiments also showed that the 26 bp sequence absent was tightly linked with the scaleless wings mutated phenotype. All of the results proved that the mutation in scaleless’s Bm-ASH2 promoter caused the failure of Bm-ASH2 gene’s transcription and translation. If artificially made the Bm-ASH2 gene expressed in early scaleless pupal wing with immediate expression system, the wing scales on the adult wing were increased significantly. So Bm-ASH2 gene is a key regulator for the formation of silkwom wing scales.更多还原