Identifying The Function Of The Protein Coded By Gene Mago Nashi In Schistosoma Japonicum

Schistosomiasis is still serious infectious diseases which is harmful to people's health and obstruct the social and economic development. The schistosome eggs are retained in the liver of the final host where they elicit inflammatory immune responses, which lead to formation of the granuloma and fibrosis, the major pathological effects of schistosomiasis. Controlling sexual dimorphism, sexual maturation and labour division may be effective in prevention of schistosomiasis.Through amino acid sequence analysis, we found the protein which was coded by Mago nashi(Genebank accession No.BM735619) has 80% homology in many animals such as drosophila, nematodes etc and there had been reported that this gene can inhibit the expression of masculinizing genes and has the function of decide the germ cell masculinize in normal condition. So we conclude the Mago nashi gene has played a key role in reproductive system. In order to research the function of the protein coded by gene of SjMago nashi, firstly we observed the morphology of the schistosomiasis japonicum which lived in final host, then leveled down the transcription of the gene of SjMago nashi by RNAi, finally wonder if the test group (RNAi) had any difference from the control group (normal) in the development of the worms (especially in reproductive systems).There is a complex procession about the development of Schistosoma japonicum in final host. It has great changes in physiology and morphology from living in nature to final host and these changes have important significance to the prevention and treatment of the schistosomiasis.Now CLSM technology is applying to many fields such as biomedicine, life sciences and etc.The morphology research of the Schistosoma Mansoni under CLSM had reported, but not about the Schistosoma japonicum.In our study, we designed mixed and unisexual infection of Schistosoma japonicum in mice. We collected adult worms which are infected 15, 20,25,30,35 days separately in mixed sexual infection and adult worms which infected 35 days in unisexual infection. Then the adult worms are fixed, stained, clarified, dehydrated, and finally observing the morphology development of the worms in different stages in mixed infection and the morphology of the worms which are infected 35 days in unisexual infection. We measure many parameters such as length, width of body; length, width, area of testicular lobes and ovary. The results of the observation and statistic analysis show every organ developed maturely, the structure of the testicular lobes,gynaecophoric canal, seminal vesicle, genital pore and the ovary,vitelline glands,vitelline duct,ootype,mehli' gland, uterus are clear under the CLSM.In each development stages, the morphology changes significantly are worms which were infected after 15-25 days. On the fifeenth day ,33% of male worms had formed a distinct gynaecophoric canal ,8% of them had testicular lobes with free areas around the few germinative cells inside. Of females, 13% had an incipient ovary lacking cellular differentiation. On the twentieth day, most worms had developed reproductive systems .Of male worms, 86% had 5-7 testicular lobes where more germinative cells could be observed, the border of the reproduction cell in this stage is not clear adequately. In female worms, vitelline glands around the digestive canal were composed of several anomalous cells which were sparsely arranged. The ovaries had more immature cells without cellular differentiation. On the twenty-fifth day, all worms had developed complete reproductive systems. In females, vitelline cell numbers were increased while the vitelline glands were irregular arranged closely. Ovaries were differentiated. 90% of them had presented few eggs in the uterus .The worms showed fully developed organs from day 30 onwards. The adult worms which were infected after 35 days in unisexual infection are smaller than in mixed infection, and develop immature especially the female. The adult worms which were infected 35 days in unisexual infection have significant differences from the mixed infection in length of body; length, width, area of testicular lobes and ovary through SPSS13.0 statistics software package.According to in vitro dsRNA synthesis kit demand, we amplified Mago nashi gene from schistosomulum cDNA library. Then we amplified the positive and anti-sense strand with promoter T7 by PCR then transcript into ssRNA and purification, finally syntheses dsRNA with a couple of ssRNA.We use the gene provided by the kit in control group. We electroporated with dsRNA to schistosomulum at 125V for 20ms,1 pulse using an Electro Square PoratorTM ECM830(BTX).Aliquots of parasites were harvested respectively in day 1,3,5 after electroporation.Total RNA,DNA and proteins were isolated using TRIZOL Reagent according to the manufacturer's guidelines. Levels of Mago nashi mRNA and proteins were determined by RT-PCR and Western blotting analysis. The results showed that SjMago mRNA levels were decreased by22%,69%,80% in test group,respectively,at day 1,3,5 after electroporation.The SjMago protein expression levels were decreased by12%,39%,56% in test group,respectively,at day 1,3,5 after electroporation.The results suggested the dsRNA could inhibit the expression of the target gene and the protein specifically and efficiently. The schistosomula which were electroporated with dsRNA were injected into the mice,and we made them into specimen through a procession of methods after 6 weeks,then oberserved the morphology characteristics of each organ and measured the length and width of the worms,the length,width and area of the testicular lobes,the length,width and area of the ovaries under the confocal laser scanning microscopy.The results show there are many spermatozoa in testicular lobes of six worms of eight male in SjMago group and no changes in ovary and vitelline gland of female. The adult worms which were infected 6 weeks in SjMago dsRNA group have significant differences from control group in width of the worms, the length, width and area of the testicular lobes, the length, width and area of the ovaries through SPSS13.0 statistics software package. So we concluded that Mago nashi is the gene associated with reproduction in schistosomiasis japonicum.