【作者】 郑小坚；

【导师】 贡成良；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2010， 博士

【摘要】 胰岛素/类胰岛素生长因子信号通路在所有多细胞有机体的生长和发育,繁殖,抗逆性,新陈代谢和寿命等生物学方面扮演着重要角色。昆虫类胰岛素肽(Insulin-like peptides,ILPs)种类多样,在脑和其它组织中由多基因家族编码并表达,与脊椎动物胰岛素结构组成非常相似但功能却不相同。家蚕为鳞翅目昆虫的模式种,家蚕蚕素(Bombyxin,BBX)是首次发现的昆虫类胰岛素,拥有7个(A～G)家族38个成员,是昆虫ILPs功能研究的良好材料。家蚕BBX B族的B8基因(BBX-B8),编码88个氨基酸残基,在家蚕脑神经分泌细胞高水平表达。业已明确,家蚕BBX具有促进造血器官细胞增殖、参与糖代谢、调控蓖麻蚕前胸腺的分泌活性。为进一步探讨BBX-B8基因的功能,采用RNAi和转基因技术,较系统地研究上调、下调BBX-B8表达水平对家蚕生长发育、抗逆性、化性、丝蛋白合成等方面的影响。在克隆BBX-B8 cDNA的基础上,通过RT-PCR检测到了BBX-B8在家蚕脑、卵巢、精巢、中肠、前部丝腺、马氏管、脂肪体和胚胎等组织中有不同程度的表达,而翅原基、卵黄中未检测到BBX-B8的mRNA,表明家蚕BBX-B8的表达具有组织特异性;Western boltting测定结果显示,5龄3 d脑组织中BBX-B8水平较低,5 d起明显上升。将体外转录合成的BBX-B8 dsRNA腹注5龄3 d家蚕,sqRT-PCR和Western boltting检测结果显示,注射3 d后脑中BBX mRNA水平的下降程度依赖于dsRNA的注射剂量,10μg/头注射区脑组织中BBX水平下降约30%。证实BBX-B8 dsRNA对家蚕BBX基因有表达抑制效应。以此BBX-B8 RNAi家蚕模型对BBX功能的研究结果显示,5龄3 d注射BBX-B8 dsRNA,蛹翅原基发育受阻,畸形翅增多,成虫脑的形态发育明显延缓;注射2 d后进行高温(40℃3 h)和低温(4℃3 h)冲击,化蛹率比对照区提高12.76%;注射后禁食处理,饥饿状态下的化蛹率比对照区低9.29%;注射3 d后,血淋巴中SOD活性、T-AOC分别提高17.81%、41.86%,而CAT活性与对照无显著差异;血淋巴中海藻糖和海藻糖酶的水平分别上升12.68%和68.24%。化蛹1d后的蚕蛹注射BBX-B8 dsRNA(10、15μg/10μL·头),其造卵量分别增加7.86%和12.62%。将家蚕BBX-B8w(野生型)和BBX-B8m(改造型)基因克隆进昆虫非转座子载体pIZT/V5-6His中,构建转基因重组载体pIZT/V5-B8w和pIZT/V5-B8m,分别转化家蚕BmN细胞,经Zeocin筛选和分子生物学鉴定获得转化细胞B8w和B8m;调查结果显示B8w和B8m的生长稍慢于正常BmN细胞,对杆状病毒Bm-BacPAK6的感染性B8w和B8m与正常BmN细胞之间无明显差异。采用精子介导法将转基因载体pIZT/V5-B8w导入家蚕基因组,通过荧光筛选和分子生物学方法鉴定获得了BBX-B8w转基因家蚕。研究结果显示,转基因家蚕脑提取液促进除脑蓖麻蛹羽化的活性高于正常家蚕的脑提取液;转基因家蚕的发育延迟2-8 d,蛹体重减轻,丝蛋白的合成能力下降,且对雄蚕的影响大于雌蚕;有5.56%~14.29%的转基因蛾产下非滞育卵。提示BBX-B8是家蚕的一个重要的发育调节因子,影响蚕的组织器官发育、化性、抗逆性、产卵量、个体大小、丝蛋白合成,承担多种独特的生物学功能。更多还原

【Abstract】 The insulin/IGF-like growth factor signaling pathway plays an important role in growth, development, reproduction, stress resistance, metabolism and lifespan in most multi-cellular organisms. Insect insulin-like peptides (ILPs) are a structurally diverse group encoded by a large multi-gene family and expressed in brains and other tissues,which are analogical in structure to that of vertebrate but serve different functions.As a model insect of Lepidoptera, Bombyxin (BBX) of silkworm is the first discovered insect insulin-like peptide and contains 7 gene families(A, B, C, D, E, F, G)consisting of 38 members. Accordingly, silkworm is a good material as the Lepidopteron model organism to study the function of insect ILPs. The B8 (GenBank Accession No. D00784) gene of BBX-B family encodes 88 amino acid residuces, is highly expressed in the neurosecretory cells of silkworm brain. It has been certified that silkworm BBX can promote cell proliferation of hematogenic organs, participates in saccharometabolism, regulates the secreting activity of eri silkworm pupae prothoracic gland. To further study the function of the BBX-B8 gene, the effect of up-regulation and down-regulation of BBX-B8 gene on the development, stress resistance, voltinism and fibroin synthesis were investigated by using RNAi and transgenic technology. After cloning the BBX-B8 cDNA, the expression profile was analyzed with RT-PCR, the results showed that the BBX-B8 gene was expressed in the brain, ovary, testis, midgut, anterior silk gland, malpighian tubules, fat body and embryonic, etc. in distinct expression levels, whereas the mRNA of BBX-B8 was not detected in the wing disc and the yolk, suggesting that BBX-B8 was expressed specifically in distinct tissues. The results of Western blotting showed that the expression level of BBX-B8 was lower in the brain of the third day larvae of fifth-instar, however, increased obviously from the fifth days of the fifth-instar. After injecting the synthesized BBX-B8 dsRNA in vitro into the blood of the 3-day larvae of fifth-instar, sqRT-PCR and western blotting were carried out at 3 days post injection, the results displayed that the level of mRNA declined in a dose-dependent manner, and that the level of BBX was declined strikingly by 30% in the brain after injection with BBX-B8 dsRNA (10μg per larva), indicating that the BBX expression was inhibited by injecting BBX-B8 dsRNA into silkworm. Using this BBX-B8 RNAi-silkworm model, the functions of BBX-B8 were investigated, the results indicated that the development of pupal wing disc was repressed, the proportion of deformed wing was increased and the morphological development of adult brain was significantly delayed after injecting BBX-B8 dsRNA into the 3-day larvae of fifth-instar. Compared to the control group, the pupation rate of the larvae which injected with BBX-B8 dsRNA was increased by 12.76% after injection followed by high-low temperature shock (40℃3 h, 4℃3 h) while the pupation rate of the injected larvae was decreased by 9.29% under starvation condition. Three days after the injection of BBX-B8 dsRNA into the 3-day larvae of fifth-instar (10μg per larva), the activity of SOD and T-AOC in hemolymph elevated by 17.81 % and 41.86 % respectively. The activity of CAT exhibited no discernable difference compared with control group, however, the levels of trehalose and trehalase in hemolymph increased by 12.68% and 68.24% respectively. It was also noted that the spawning number of the pupa injected with BBX-B8 dsRNA in 10 and 15μg per moth (10μL) one day after pupation increased by 7.86% and 12.62% respectively.Wild type BBX-B8 (BBX-B8w) and modified BBX-B8(BBX-B8m ) were cloned into insect non-transposon vector pIZT/V5-His to generate respectively recombination vector pIZT/V5-B8w and pIZT/V5-B8m. The BmN cells were transfected with the two respectively, after screening with Zeocin and molecular biological identification, the transformed BmN cells named B8w and B8m respectively, were obtained. Compared with normal BmN cells, the growth of transformed cells B8w and B8m was slower slightly and their infectivity to Bombyx mori nucleopolyhedrovirus was analogous to that of normal BmN cells. the vector pIZT/V5-B8w was transferred into silkworm eggs by sperm-mediated gene transfer. And after being screened for gfp gene and verified by molecular biological identification, the transgenic silkworms was obtained. The investigation demonstrated that extracts from the head of transgenic silkworms had higher activity of facilitating eclosion of brain-removed eri silkworm pupa compared with that of normal silkworm, and that the development of transgenic silkworms were delayed from 2 to 8 days, additionally, the weight of transgenic pupa were decreased, the ability of synthesizing silk protein was declined compared with normal silkworms, furthermore, in all cases influence on the male was more obvious than that on the female. It is also observed that about 5.56 % to 14.29 % transgenic moths laid non-diapausing eggs. All results mentioned above clearly indicated that BBX-B8 is an important developmental regulatory factor, serves to multiple specific biological functions and play an important role on the development of tissue and organ, voltinism, stress resistance, the amount of laying eggs, body size, synthesizing activity of silk protein.更多还原