Proteomic Analysis On Accessory Gland And Functional Characterization Of Specific Genes In Male Silkworm Moth, Bombyx Mori

Silkworm, an economically important insect for secreting silk, is widely researched as a model for lepidopteran. Silkworm has a very strong reproductive potential for propagating themselves. After mating, female moths show their differences in physiology and behavior, and the foundation what makes for such changes is silkworm male accessory glands. The male accessory glands of moth secrete a variety of substances which would mix with sperms and form the seminal fluid, Moreover, these substances have influence on mating activities between male and female. However, the components and functions of silkworm moth male accessory gland are still unclear. So, we collected silkworm moth male accessory glands and extracted the proteins of the glands. The protein components of accessory gland were identified by LC-MS/MS, and the proteins were subsequntly analyzed by GO categories and function categories.Combined with the results of two-dimensional electrophoresis(2-DE) for silkworm moth male accessory glands by Zhang et al., we select BGIBMGA001815(BmAGSP3) and BGIBMGA014604(BmAGSP4) as the candidate genes for futher study. The main results are as follows:1、Proteomic analysis of on male accessory glands of male silkworm mothThe male accessory glands from the first day of the moth were collected for extracting proteins. Totally,1657proteins were identified by LC-MS/MS. These proteins are mainly10kDa-100kDa of the molecular weight (MW) and4-10of the isoelectric point (pI). Comparison of GO categories between the proteins from male accessory glands and the components of male testis showed that the protein components of the two tissues are very similar. And accessory gland proteins includes two kinds of special proteins related with energy metabolism:NAD or NADH binding, oxidoreductase activity. According to functional annotation of accessory glands proteins, these proteins can be divided into ten categories. including reproductive related proteins, growth and evolution associated proteins, energy metabolism related proteins, chaperones associated proteins, glycoprotein associated proteins, immune related proteins, protease inhibitors, proteases, other enzymes and other proteins,unknown proteins. 2、Cloning, prokaryotic expression and purification of BmAGSP3, BmAGSP4Based on the results of proteomic analysis and2-DE map for silkworm moth male accessory glands, there are two candidate genes were choosed, BmAGSP3and BmAGSP4. The open reading frame of BmAGSP3and BmAGSP4were amplified by PCR and cloned into a pET28a-derived vector (p28). The constructs of BmAGSP3-p28and BmAGSP4-p28were transformed into E. coli for prokaryotic expression. The recombinant proteins with an N-terminal hexahistidine-tag were purified through Ni-NTA affinity chromatography. Finally, we generated polyclonal antibodies of BmAGSP3and BmAGSP4, which are critical for functional analysis.3、The expression pattern analysis and tissue localization of BmAGSP3, BmAGSP4The expression patterns of BmAGSP3and BmAGSP4characterized by RT-PCR and Western blotting showed that BmAGSP3, BmAGSP4were highly expressed in the male accessory glands on the first day of moth. Then, immunofluorescence was used to determine the localization of BmAGSP3and BmAGSP4in male moth accessory gland. We found that BmAGSP4was highly expressed in the lumen and cytoplasm of luminal cells. However, the expression of BmAGSP3was lower than that of BmAGSP4. Therefore, we conjectured that the two proteins are probably produced by the gland cells and secreted into the lumen.4、Functional analysis of BmAGSP4To explore whether BmAGSP3and BmAGSP4were transported into female moth after mating, we detected the distribution of the two proteins in female moth by western blotting. Results showed that only BmAGSP4was found in the reproductive systems of female moth. Interestingly, after being transported into the reproductive systems of female moth, BmAGSP4was degraded into different fragments with various molecular weight.Based on the above results, we introduced RNA interference (RNAi) for BmAGSP4. After the injection of dsRNA, the male moths mated with normal female moths and the numbers of egg were counted. Compared to the control group, the numbers of eggs laid by female moths mating with the treatment group were decreased significantly. It is reasonable for hypothesizing that BmAGSP4may have an effect on laying eggs of female moths.