| Hemocyanin is a kind of respiratory protein except for hemerythrin and hemoglobin in nature. Hemocyanin is widely distributed in the hemolymph in many arthropods and molluscs, with abundant concentration. The function of hemocyanin is as follows: oxygen transport and osmotic regulation in curing, molting and skin conditioning, metal ion transport, protein storage, antibacterial, antivirus and so on. Recent studies have focused on the protein structure and function of hemocyanin. However, studies regard to the genes structure of hemocyanin, especially the gene subtype of molluscs hemocyanin as well as the links between the gene subtype of molluscs hemocyanin and its functions were rarely reported. In this study, bioinformatic analyses, fluorescent quantitative PC R(qPC R) and fluorescence in situ hybridization(FISH) assay were used to study hemocyanin genes of Haliotis diversicolor(Hd Hc). The results contributed to explore the mollusc hemocyanin gene and its immune related function based on experiment level.Firstly, bioinformatics analysis software was adopted to analyze the full- length cDNA sequence of Hd Hc1, HdHc2-1 and Hd Hc2-2 which were cloned previously in our lab. The results showed that Hd Hc clustered into one group with hemocyanin from gastropods H.tuberculate and M.C renulata. The nucleotide sequence similarity between HdHc1 and Ht Hc1 was 88%. And the similarity between Hd Hc2-1, Hd Hc2-2 and Ht Hc2 was 89% and 90%, respectively. However, the similarity between Hd Hc2-1 and HdHc2-2 was 98%. The gene structure of hemocyanin of H.diversicolor was similar to that of H.tuberculata, which possessed 8 function units(FU-a~FU-h).Secondly, abalone pathogens Vibrio harveyi and Abalone herpesvirus was injected into H. diversicolor, hemolymph was sampled at 3, 6, 12, 24, 48, 72 h after injection. The concentration of total soluble protein, the activity of SOD, ACP, AKP and the antibacterial activity of plasma without cells were detected. The results showed that(1) the concentration of total soluble protein changed obviously. The protein concentration of stimulated group increased or decreased significantly compare to control group at different time course;(2) the activity of SOD, ACP and AKP from stimulated hemolymph were significantly different compared to normal hemolymp h;(3) the antibacterial activity of hemolymph without cells agaimst V. harveyi, Vibrio vulnificus and Vibrio coralliiluyitcus were enhanced at different levels.Thirdly, the relative expression level of HdHc1, HdHc2-1 and HdHc2-2 was evaluated by fluorescent quantitation PCR. The results showed that HdHc1 and HdHc2-1 expressed at development stage of H. diversicolor. Moreover, there was a significant difference beween HdHc1 and Hd Hc2-1 at the same development stage. The relative exression level of HdHc2-1 was much higer than that of Hd Hc1. However, HdHc2-2 could not be detected at these stages. The relative expression level of HdHc1 and HdHc2-1 was changed after the abalones were stimulated by V. harveyi, Ab HV and Poly(I:C) in different tissues. And the levels changed markedly at different time.At last, according to qPC R results, in situ hybridization was adopted to detect the expression of hemocyanin from H. diversicolor in mussle. The results indicated the positive signals of Hd Hc1 centralized in the margin of mussle. But the HcHc1 signals were few in inside area of mussle. However, there were fewer HdHc2-1 signals in the margin and inside of mussle.In summary, the antibacterial activity of plasma without cells from H. diversicolor was increased marked ly after bacteria and virus stimulation. The genetic relationship of hemocyanin from H.diversicolor was closest to hemocynin from H.tuberculata. The evolution of different mollusc hemocyanin subunits was earlier than evolution of the genus. HdHc1 and Hd Hc2-1 were inducible expression genes, and played important roles in antibacterial and antiviral immune responses. HdHc2-2 may be a kind of pseudogene. |
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