【作者】 陈玉华；

【导师】 沈卫德；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2011， 博士

【摘要】 家蚕(Bombyx mori,L.)不仅是一种重要的经济昆虫,也是一种有中国特色的重要模式生物。家蚕基因组计划推动了家蚕模式化生物系统研究,从基因组到蛋白质组是家蚕研究的自然进程。以蛋白质组学为主要内容的家蚕功能基因组研究对于加快家蚕模式化研究进程、建立现代蚕桑产业技术体系、推动蚕丝产业发展具有重要的意义。建立家蚕蛋白组研究技术平台,利用家蚕开发实验动物模型替代,也是家蚕模式化生物系统研究的重要任务。精巢是雄蚕内部生殖器官,精子形成的场所。和大部分鳞翅目昆虫一样,家蚕具有精子二型性:上蔟吐丝前产生有核精子,之后则产生无核精子。精子形成过程,受到多种调控机制的影响:激素调控、营养调控、生态因子调控、基因调控等。研究家蚕精子形成和二型分化的分子机制,有助于搞清家蚕性别决定和分化、实现性别控制,达到提高丝质、专养雄蚕;有助于寻找优良品种选育所需要的蛋白分子标记;有利于发现农药的生殖毒性新靶标,开发新农药贡献于农业害虫控制;有助于加深对雄性生殖发生、生殖障碍、生殖调控分子机制等生殖相关基础生物学的认识。本文利用shotgun LC-MS/MS和双向电泳的方法,鉴定了家蚕5龄幼虫和蛹中期精巢蛋白质组;建立了家蚕5龄幼虫和蛹期的蛋白质标准表达谱图,并对5龄起蚕和化蛾前精巢部分差异表达蛋白点进行了鉴定;对部分精子形成相关基因进行了定量分析。建立了生精囊体外培养系,测定了双酚A对家蚕生精囊体外形成精子的影响。本研究为家蚕功能基因研究和新农药作用靶标开发提供了大量的研究对象,并对利用家蚕开发环境激素初筛、生殖毒理研究动物模型做了一些探讨。1.家蚕精巢shotgun法蛋白组表达谱鉴定利用家蚕预测蛋白数据库(silkDB:silkdatabase)和NCBI(National Center for Biotechnology Information)上已有家蚕蛋白,建立本地家蚕蛋白数据库。用shotgun LCMS/MS法鉴定了家蚕5龄幼虫和蛹中期精巢蛋白质组,分别获得753和498种蛋白质,其中二者共有蛋白为351种。建立了共有900种蛋白的家蚕精巢蛋白组数据库,为家蚕精巢功能基因组研究,新农药靶标开发提供了大量研究对象。鉴定蛋白的预测分子量、等电点分析表明,精巢中酸性蛋白稍多,蛹期以后大分子量蛋白比例减少。GO注释(Gene Ontology Annotation)分析表明,主要鉴定蛋白属于细胞组分、细胞器和大分子复合物;大部分蛋白参与代谢和细胞过程,并发现了多种与外部刺激响应有关的热激蛋白。分子功能注释表明鉴定的蛋白主要和催化、绑定功能有关。分析还发现精巢中有大量和色素沉积相关蛋白。5龄幼虫和蛹中期精巢差异表达蛋白GO分析表明,二者在细胞内部和细胞器成分上有明显差异;核苷酸、核酸、GTP结合蛋白等方面在数目和功能上差异显著;而在生物过程的差异主要表现在翻译和翻译后修饰方面。具体比较了两者之间在核蛋白上的差异。2.家蚕精巢蛋白组双向电泳研究对家蚕5龄起蚕到化蛾前家蚕精巢进行了双向电泳分析,建立了5龄幼虫和蛹期精巢蛋白质组双向电泳的标准图谱。并利用MALDI TOF/TOF质谱鉴定了22个差异或特异表达的蛋白点,获得其中9个家蚕蛋白,分别是:Ssu72蛋白(Ssu72 RNA polymerase II CTD phosphatase)、ABC转运蛋白(ABC transporter)、神经钙蛋白(海马钙结合蛋白)类似物( neurocalcin homolog ; hippocalcin )、未知蛋白(BGIBMGA014561-PA)、Tar DNA结合蛋白类似物(TBPH:TAR DNA binding protein homolog)、谷胱甘肽S转移酶ε2 (glutathione S-transferase sigma 2)、伴侣素(chaperonin)、热激蛋白19.5(19.5 kDa heat shock protein)、热激蛋白20.1 (heat shock protein 20.1)、热休克共轭蛋白(heat shock cognate protein)。3.家蚕精巢热激蛋白、细胞周期蛋白家族基因的转录表达水平分析对不同发育时相家蚕精巢中热激蛋白和细胞周期蛋白家族基因,进行了的转录水平分析。发现家蚕精巢中,热激蛋白家族hsp20.1(hsp:hot shock protein)、hsp20.4、hsp20.8相对表达量比较大,在5龄中期和化蛹前后有表达高峰,化蛾前迅速降低;hsp25.4、hsp70、hsp90表达量相对比较少。推测,小分子热激动蛋白与精子形成有密切关系。而细胞周期蛋白家族中cyclinB转录表达量最高,cyclinA非常少且随着发育进程变动不大。转录变动规律来看cyclinB最高和cyclinL1最相似,可能两者具有协同作用。4.双酚A对体外培养家蚕生精囊形成精子的影响建立了家蚕5龄起蚕到化蛾阶段精子发育进程的标准谱图,观察到有核和无核精子在分化成熟中的形态差异。建立了家蚕生精囊体外培养体系,并尝试利用双酚A作为低毒环境激素模式药物,研究了环境激素对家蚕生长发育和精子发生形成的影响。研究结果发现但家蚕生精囊体外培养时对外源物质敏感,可用作化学物质毒理初筛实验。更多还原

【Abstract】 The silkworm, Bombyx mori, is not only an important economic insect, but also a central model with Chinese characteristics. The silkworm genome project promotes Bombyx biological system research as a model. It is a natural process to study it from genomics to proteomics.Taking proteomics as the main direction in Bombyx functional genomics has an important significance to speed up the process of silkworm modeling, establish a modern technology system of sericulture, and promote the development of silk industry. Therefore, to build the technology platform of silkworm proteome research and to explore experimental animal model with the silkworm are important research tasks in the future.The testis is the reproductive organ of male silkworm to form sperms. As most of the Lepidopteran insects, the silkworm has sperm dimorphism. They generate sperm with nuclei before the silkworm spinning, after that, they produce sperm without nuclei. Spermatogenesis is affected by various regulatory mechanisms, such as hormone, ecological factors, gene regulation. To study the molecular mechanism of spermatogenesis and dimorphism is helpful to figure out the sex determination and differentiation of the silkworm, realize gender control and specially rear male silkworm to improve silk quality. It is helpful to obtain protein markers to breed excellent strains. It is helpful to find new targets to develop other new pesticides to control pests. It is helpful to deeply clarify the molecular mechanism of male gametogenesis, reproductive disorders and reproductive regulation.In this paper, shotgun LC MS / MS and two-dimensional electrophoresis methods were used to identify the proteome of testis of the fifth instar larvae and middle pupa. We established protein expression pattern of the fifth instar larvae and pupa, characterized some protein spots differentially expressed in testis between the newly moulted larvae and pupa before eclosion, and analyzed part of genes related to spermatogenesis by qRT-PCR. We also established in vitro spermatocysts culture, which was used to determine the effect of bisphenol A on spermatogenesis. Our study provides many useful materials for Bombyx function genome analysis and new pesticide exploration, also primarily discussed silkworm modeling used to screen environment hormones and research reproductive toxicology.1. Proteomics of testis identified by shotgun method in the silkwormA local database was built depending on existed protein sequences in SilkDB and NCBI. With a shotgun LC MS / MS method, we obtained 753 and 498 proteins from testes of the fifth instar larvae and middle pupa, respectively. 351 proteins were included in both of them. Bombyx proteome database of testis was established, which provided many materials for Bombyx function genome analysis and new pesticide exploration.After the analysis of predicted molecular weight and isoelectric point of proteins, we found that acidic proteins in testis were more, and high molecular weight proteins reduced after the pupal stage. GO annotation showed that most of identified proteins belonged to cellular components, organelles and macromolecular complexes, and most of them were involved in cellular metabolism. A variety of heat shock proteins responding to external stimuli were contained in them, which were related to catalytic and binding functions. We also found a large number of pigment deposition -associated proteins in testis. GO analysis of differentially expressed proteins between the fifth instar larvae and middle pupa showed that there were significant differences in cellular internal structures and organelle components. The number and function of proteins implicated in nucleotides, nucleic acids, GTP binding have obvious differences. The differences of biological processes were mainly in translation and post-translational modifications, and we compared them on the differences of the nucleoproteins.2. Proteomics of testis researched by two-dimensional electrophoresis in the silkwormIn this paper, two-dimensional electrophoresis method was used to analyze the proteome of Bombyx testis from the newly moulted larvae to pupa before eclosion, and a standard protein expression pattern of two-dimensional electrophoresis was established in testis. The MALDI TOF / TOF mass spectrometry was employed to identify 22 differentially expressed protein spots, and we got 9 Bombyx proteins, such as Ssu72 RNA polymerase II CTD phosphatase, ABC transporter, hippocalcin, BGIBMGA014561-PA, TAR DNA binding protein homolog, glutathione S-transferase sigma 2, chaperonin, 19.5 kDa heat shock protein, heat shock protein 20.1, heat shock cognate protein.3. Transcriptional level assays of Hsp and cyclin families in testis of the silkwormWe analyzed genes encoding Hsp and cyclin in Bombyx testis during the different developmental stages. It was found that small heat shock proteins like hsp20.1, hsp20.4, hsp20.8 in testis were highly expressed, and peaked at the middle of the fifth instar and before pupation, then rapidly decreased before eclosion. However, hsp25.4, hsp70, hsp90 were lowly expressed. We suggest that small heat shock proteins are closely related to spermatogenesis. In cyclin families, cyclinB mRNA was highest, and cyclinA mRNA was low and little changed. In our results, expression pattern of cyclinB was similar to cyclinL1, implying a synergistic effect may exist between them.4. The effect of bisphenol A on spermatogenesis in in vitro cultured spermatocystsA standard protein expression pattern was built from the newly moulted stage of the fifth instar to eclosion. We observed morphological differences in the differentiation of sperm with or without nuclei.To investigate the effect of environmental hormones on the growth and development of silkworm and spermatogenesis, we established in vitro spermatocysts culture system and tried to select bisphenol A, a environmental hormone, as a model drug with low toxicity. Our results showed that the spermatocysts in vitro cultured are sensitive to foreign matters, suggesting it may be useful to screen chemical substances to research toxicology.更多还原