Tyrosine Kinase 3 Involved In Reproduction In The Platyhelminth Parasite Schistosoma Japonicum

Background:Schistosomiasis is one of the most common parasitic diseases in the world.Schistosome egg is the key factor of schistosomiasis,and therefore,we should grasp the biological mechanism of schistosomiasis reproductive development and spawning,and find the key targets for intervention.It is very important for developing new strategies to prevent and treat these infections.Tyrosine kinase 3(TK3)is a member of the cell tyrosine kinase family.Schistosoma mansoni TK3 mainly locates in female yolk gland cells and oocytes and spermatocytes in males,suggesting that it may be involved in the reproductive activities of Schistosoma japonicum,however,the role of TK3 in the reproductive process of Schistosoma japonicum is unclear.The purpose of this study is to explore the role of TK3 in the reproductive process of Schistosoma japonicum.Methods: The full-length Sj TK3 sequence of Schistosoma japonicum was amplified according to the template of Schistosoma japonicum c DNA.DNAMAN software was used to analyze the full-length sequence and to understand the homology of Sj TK3 protein in various species.The detailed phylogenetic tree of Schistosoma japonicum Sj TK3 was established by MEGA5 software.The encoding sequence(Sj TK3)of the TK3 gene of Schistosoma japonicum was cloned.Dihydrochloride,a Src kinase inhibitor,was used in vitro in Schistosoma japonicum.7 days after dihydrochloride treatment,schistosome eggs were collected,and the number of eggs in the treatment group and the blank group were observed;meanwhile,the morphological changes of the reproductive system of these groups of parasites were observed and the morphologic pictures of Schistosoma japonicum were photographed and preserved under confocal laser scanning microscope.Using the TRIzol method to extract total RNA of Toxoplasma gondii,and fluorescence quantitative PCR was used to examine the expression of Schistosoma japonicum Sj TK3 at various stages of Schistosoma japonicum,including the stages of eggs,cercariae as well as the males and females of 16 days,24 days and 42 days respectively.qPCR was used to analyze the changes of the important genes related to the synthesis of eggs.Results: The Sj TK3 gene was obtained by PCR amplification.After sequencing and preliminary analysis,the gene was encoded by 647 amino acids,the isoelectric point was 7.01,and the relative molecular weight of 72.9 k D of Schistosoma gene.We built an evolutionary tree with several representative species of TK3.By observing it,we found that the TK3 of other species and the gene of Schistosoma japonicum was an evolutionary branch.So we confirmed that the TK3 of Schistosoma japonicum was a member of the Src family and it was named Tk3 of Schistosoma japonicum.The homology of TK3 protein was analyzed by DNAMAN software.It was found that the TK3 protein of Schistosoma japonicum was highly homologous with the TK3 protein of Schistosoma mansoni(Sj TK3 and Sm TK3),which was 91.6%.Sj TK3 m RNA showed differential expression in the life history of Schistosoma japonicum.After Dihydrochloride treatment,confocal microscopy observation showed that the schistosome reproductive organs,especially in the female ovary and vitelline gland and male testis,the morphological change can be observed with porous structure;Dihydrochloride treatment concentration increased,respectively adding 50 μM,75μM the amount in the seventh day of treatment with ordinary optical microscope to count the number of eggs,found female oviposition number two under the concentration decreased by about 18% and 31%,in addition,the Sj TK3 gene also affected the change of some important egg formation related genes such as P14,P48 and Fs800 genes in tenth days after Dihydrochloride treatment the expression level of three genes,which were compared with the blank control group changed.Conclusion: We therefore concluded that Sj Tk3 played an important role in the maturation of the reproductive organs and the development and production of the eggs.These findings helped us to understand the reproductive biology of Schistosoma japonicum.Discovery of important molecules that regulating the signal transduction pathway of Schistosoma japonicum and interfering with its key targets to inhibit its’ reproductive development were the effective ways to better control the disease.