Proteome Research On Silkworm Gonads And Embryos

Reproduction and embryo development is the important field of life science. Silkworm is an economically important insects and Lepidopteran model. The knowledge of reproduction and embryo development in silkworm will facilitate the human in utilizing the beneficial insects and controlling the pest. With the completion of silkworm genome sequence, the functional genomics become the focus. Proteins, the products of genes, are the real executor in life processes. Therefore, proteomics has become one of major methods for functional genomics. Here, we used proteomics to study the proteome profile of silkworm gonads and sex-linked embryo lethal mutant embryos. The function and regulation network of those proteins identified were revealed using bioinformatics analysis, their roles in silkworm gametogenesis and embryo development were discussed. The main results are as follows:(1) Shotgun proteomics were used to profile the proteome of silkworm larval gonads at the fifth day of the fifth instar. Totally286and205non-redundant proteins were identified from silkworm testis and ovary, respectively, with false discovery rate (FDR) of0.35%and0.49%. Of those,92proteins were detected in both tissues, while194and113proteins were sex-specific. Ribosomal proteins were the most enriched are protein family both in testis and ovary, indicating active cell growth and proliferation in gametogenesis. Apolipophorin was the abundant proteins both in testis and ovary, giving a clue that lipoprotein has a role in spermatogenesis and oogenesis. According to the spectral count, cellular retinoic acid binding protein (CRABP) showed to be very high-abundant in testis and14-3-3epsilon protein is abundant in testis.30K proteins, one of the major plasma protein groups, were identified in testis specifically. Those abundant proteins in testis may be critical for spermatogenesis. Tubulins showed to be abundant in ovary. This may be consistent with the fundamental roles of tubulins in oogenesis. Tubulins have been revealed to participate in the oocyte specification, axis formation and transport of maternal mRNA and proteins in Drosophila melanogaster.(2) Several Drosophila essential proteins for gametogenesis were also identified their homologies in silkworm, such as male meiotic arrest gene product ALY and vismay in testis, and maternal mRNA localization protein exuperantia and squid in ovary. Those identifications reveal the conservation in spermatogenesis and oogenesis between Bombyx mori and Drosophila melanogaster.(3) The bioinformatics analysis on the common and sex-specific proteins provides system-level insights into the sexual dimorphism and gametogenesis. For example, the nutrient reservoir is the ovary-specific function category, while energy generation pathway enriched in testis.(4) The correlation of expression between mRNA and protein level of eleven genes were explored. Those11proteins genes representing common and sex-specifically expression were selected for qRT-PCR analysis. The qRT-PCR results showed discrepancy of some genes between mRNA and protein level. For instance, fibroinase (Bcp) and ALY showed testis-specific expression at protein level (Figure2A), while their mRNA abundance were higher in ovary than testis. MLE protein (MLE) and cytochrome P4509G3(Cyp9g3), showed ovary-specific at protein level, while expressed higher abundance in testis than in ovary at transcriptional level. Those results suggest post-transcriptional regulation of those genes may occur. We also compared our identification with the microarray-based studies performed by Xia et al. The discrepancy between mRNA and protein level indicated the occurrence of post-transtriptional regulations on many genes.(5) The chromosome contribution of our identified proteins was investigated. All the common, testis-and ovary-specific genes were mapped onto the silkworm chromosomes. The testis-specific genes were higher than the ovary-specific genes on the sex chromosome, showed that the Z chromosome contains more testis-specific genes as revealed at transcriptional level. Furthermore, those testis-specific proteins showed to have crucial roles in spermatogenesis.(6) To gain insight into the effect of one embryo-lethal gene l2, comparative proteomic analysis was carried out between the survival embryos and lethal embryos of the sex-linked balanced lethal (SLBL) strains of silkworm before the lethal stage. The lethal stage of h was confirmed by observing the typical dead embryo morphology. The two genotype embryos before lethal stage were distinguished using polymorphic simple sequence repeats (SSR) markers closely linked to l2on the sex chromosome. Finally,11differentially expressed protein spots were successfully identified by MALDI-TOF/TOF mass spectrometry (MS). Among them, only1protein identified as heat shock protein20.4(HSP20.4) was up-regulated in the lethal embryos, while the other10were down-regulated. Small heat shock proteins act as the molecular chaperon, having a role in protein folding. They were also evidenced to have a role in association with membrane protection. The up-regulation of HSP20.4suggests that there may be abnormal polypeptides or membrane harm in the lethal embryos. The gene ontology (GO) annotation indicated those down-regulated proteins are involved in important biological processes including embryo development, nucleoside metabolism, tRNA splicing, translation and protein folding. The biological pathway analysis showed that those down-regulated proteins are mainly involved in spindle assemblage and morphogenesis. Based on our results, we suggest that the expression of l2may be has a negative effect on mitosis and morphogenesis processes.Our data enriched the information of silkworm gonad proteomics and functional genome in silkworm embryo development. Those gonad proteome data further provide valuable background for silkworm gametogenesis and sex determination.