| 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. |
