| Reproductive biology is one of the important basic subject of aquatic economic animals aquaculture,and it is the important foundation of the artificial breeding.All the time,the gonad development and the mechanism of immune defense are the hot topics of the reproduction and immunology,respectively.Chinese mitten crab(Eriocheir sinensis)is an important aquatic economic animal in China.This paper is based on vitellogenin in Chinese mitten crab.And the expression pattern of vitellgenin and its receptor and antibacterial mechanism of vitellogenin were discussed.In this study,Chinese mitten crab was selected as the research object.The fulllength of cDNA of vitellogenin gene(Es-vtg1)was cloned,and the full-length was 7939 bp.The ORF domain contains 2567 animo acids.Three functional domains of the Esvtg1 were found by bioinformatics: LPD_N domain,DUF1943 domain and VWD domain.According to sequence analysis,the structure of Es-vtg1 is highly conservative in crustaceans.The results of real-time quantitative PCR and Western Blot showed that Es-vtg1 was mainly expressed in ovaries,hepatopancreas and hymocyte,but was slitghtly expressed in other tissues.The result of immunofluorescence experiment showed that the Es-vtg1 in hymocyte was mainly expressed in cytoplasm,and Es-vtg1 in ovary was mainly expressed in cyteblast.Besides,Es-vtg1 was accumulated during the ovarian development.Then,the partial cDNA of vitellogenin receptor gene(Es-vgr)was cloned and analyzed by bioinformatics.The results of real-time quantitative PCR and Western Blot showed that Es-vgr was only expressed in ovary,which implied that Es-vgr was participated in the development of ovary.Besides,we found that the expression of Esvgr was increased in the early stage of ovarian development.But its expression was decreased in the post ovarian development.The tendency of expression of Es-vgr was similar as that of Es-vtg1 expression,and this is implied that Es-vgr and Es-vtg1 were involved in the regulation of ovarian development.We cultured the ovarian tissue in vitro,and interfered the Es-vtg1 of ovarian tissue.The expression of Es-vgr was detected by qRT-PCR.And the result shows that the expression of Es-vgr was decreased significantly.This result implied that Es-vtg1 might have regulated the expression of Es-vgr.In the end,we cultured the hemocytes of Chinese mitten crab and got a high-level of mRNA expression of Es-vtg1 in hemocyteinduced by bacteria Staphylococcus aureus and Vibrio parahemolyticus for a 48-hour challenge stimulation(P < 0.01).Antibacterial assay showed that the Es-vtg1 protein containing VWD domain by prokaryotic expression could inhibit the growth of bacteria.The expression of some antibacterial peptides was down-regulated after the cultured hemocyte was stimulated by bacteria or interfered by dsRNA of Es-vtg1.These results showed that Es-vtg1 had played an important role in antibiosis. |
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