Half Smooth Tongue Sole (Cynoglossus Semilaevis) Gender Related Gene, Dmrt1and Csw1, Research In Medaka (Oryzias Latipe)

Medaka（Oryzias latipe）has been widelyused in basic biology,developmental biology and toxicology research field, which is a kind ofideal experiment material for scientific research to study vertebrate germcell proliferation as well as genetic mutations and disease caused bypollutants toxic effects as a good model organisms. This study describesfrom three aspects: The first is the construction of heterologous andhomologous dmrt1gene over-expression vector. The second is integrationand expression comparative analysis of heterologous and homologousdmrt1expression vector in Medaka. The third is gene-knock out analysis ofolcsw1Talen vector. The main experimental results are as follows:1. The construction of heterologous and homologous dmrt1geneover-expression vector.We extract testis tissue RNA of half smooth tongue sole and Medakaand reverse transcription cDNA. Then we combine these two cDNA withpIRES-hrGFP-1a vector by double digestion of BamHâ… å'ŒXhoâ… .Thedmrt1gene is driven by CMV promoter. The two over-expression vectorare named pIRES-Csdmrt1-hrGFP-1a and pIRES-Oldmrt1-hrGFP-1a respectively. We obtained enough vector solution by Plasmid Mini PrepKit.2. Integration and expression comparative analysis of heterologous andhomologousdmrt1expression vector in Medaka.In vivo over-expression of dmrt1in Medaka was induced by injectingthese two dmrt1gene over-expression vectors (30ng/μl) into the cytoplasmof fertilized Medaka embryos at the one cell stage. We then measured GFPexpression and hatching rate. Additionally, we quantified the level of geneintegration, expression, and the effect on cyp19a expression. GFP wasexpressed athigh levels48h after injection and the number of embryosexpressing GFP decreased with the development of embryos. The hatchingrate and integration efficiency of embryos injected with thehomologousdmrt1expression vector (69.5%,30%) was significantly higherthan those injected with the heterologous dmrt1expression vector (36.5%,30%). We observed the RNA expression of Medaka dmrt1but not halfsmooth tongue sole dmrt1. Furthermore, in fishes with dmrt1expression,the expression of cyp19a was down-regulated. Our research provides afoundation for the study of half smooth tongue sole sex differentiation andcontributes to our knowledge of sex-determining genes in marine fish.3.The gene knock-oμt analysis of olcsw1Talen vector.Sequence homology analysis result shows that it was75%homologous identity between csw1gene of half-smooth tongue sole and olcsw1ofmedaka. And semi-quantitatively characterize that they have similar geneexpression pattern, that is, abundantly expressed in ovary and almost noexpression in testis and other groups. webuiltolcsw1Talen vector by furtheranalyzing the genetic structure of olcsw1and induce into one cell stage ofmedaka(250ng/μl). Injection embryos by electrophoresis detection andsequencing analysis show that:olcsw1gene knock-out rates as high as90%above and containing a variety of different types of gene knock-out. Theproportion of the male and female phenotype has no obvious changes aftermicroinjection, being close to1:1. Through RT-PCR detection we foundthat after the injection of medaka fish gonads olcsw1expression weakly ornot. At the same time, gene cyp19a1a,foxl2, dmrt1, gsdf and so on have nobeen influenced in expression. But improve sox9b and amh expression inovarian can be improved. So we speculated that medaka olcsw1mayindirectly involve in the female gender differentiation process. We need tobe further screening of heritable F0generation of fish, and establish ahomozygousknockout strains and furtherstudy the function of the gene inthe strain.