The Research Of Sox Gene And Dmrt Gene In Rana Nigromaculata

Sox gene family and Dmrt gene family are two of the gene families which participate in the development process of animal. The Sox gene family is characterized by a highly conserved DNA-binding and bending domain known as an HMG box. They play important roles in embryonic development, sex differentiation and organism formation. The characteristic, function, classification and the molecular evolution of Sox genes were discussed in the reviews. The DMRT genes constitute a new gene family related to sex-determination. They were isolated by virtue of its homology to the double sex gene from Drosophila,Mab-3 from Caenorhabditis elegan and human DMRT1, which encode putative transcription factors with a conserved DM domain that is thought to bind specific DNA sequence in form of zinic finger. The characteristic and function of Dmrt genes were also discussed in the review. In the part of studies, the Sox genes of Rana nigromaculata were amplified by using a pair of degenerated primers which were designed based on the conservative motif (HMG-box ) of human SRY gene. The amplification band of Sox gene familays was observed in both male and female Rana nigromaculata whose length was 220bp which was consistent with that of human SRY gene and that of SOX gene; To detect the different clones, SSCP technic was used. Four different Sox genes were obtained and sequenced respectively. After sequence analysis, those four Sox genes are named afer Rana nigromaculata as rn Sox3, rn Sox21, rn Sox4, rnSox11 according to their high homology to corresponding Human SOX genes because their identities to those human SOX genes in the DNA sequence and the amino acid sequence are 86%, 83%, 82%, 89% and 94%, 93%, 78%, 98% respectively; This may be concluded that Sox genes are highly conserved in phylogeny. Similarly, the Dmrt genes of Rana nigromaculata were amplified by using another degenerated primers which were designed based on the conservative DM domain of different species. The amplification band of Dmrt gene familay was observed in both male and female Rana nigromaculata whose length was 140bp which was consistent with that of human DMRT genes. Then, PCR products were cloned. To detect the different clones, SSCP technique was used. Three different Dmrt genes were obtained and sequenced respectively. After sequence analysis, those three Dmrt genes are named afer Rana nigromaculata as rnDmrt1, rnDmrt4, rnDmrt6 according to their high homology to corresponding Human DMRT genes because their identities to those human DMRT genes in the amino acid sequence are 89%, 93%, 89% respectively; This may be concluded that Dmrt genes are highly conserved in phylogenetic. To study the function of Sox4 and Dmrt1 in development, we designed specific primers according the sequence of rnDmrt1 and rnSox4 for amplification by RT-PCR (the polymerase chain reaction with reverse transcription) of different tissues in adult Rana nigromaculata. Sox4 expression is only detectable in the heart, brain, testis and ovary. No expression was detected by RT-PCR for other tissues. Dmrt1 expression is only detectable in the testis. No expression was detected by RT-PCR for the following tissues: brain, heart, liver, muscle, ovary and kidney. This indicated that Sox4 genes may only play roles in the development of organs and Dmrt1 mainly participates in sex determining and differentiation. Our work provides molecular data for the study of the molecular evolution of Sox gene family and Dmrt gene family as well as for their function research.