| DNA methylation is one of the most common epigenetic phenomenon in eukaryotes, is a process of reversible, can directly affect the activity of the gene. More and more experiments have proved that DNA methylation play a decisive role in aquatic animal’s growth and development. Therefore, research on DNA methylation is the key to study from the molecular perspective of aquatic animal’s physiological phenomenon. The feature of Monopterus albus is sex reversal. For the sex related genes of Monopterus Albus we select Dmrt gene, Sox gene and Vasa gene. Dmrt2, Dmrt3, Dmrt4 gene of Monopterus albus respectively encoding full-length mRNA is 1680bp,1413bp and 1772bp,respectively, amino acids encoded 526,470,420; while the full-length Sox9a1 and Sox17 gene mRNA is 1625bp and 1775bp,the number of amino acids encoded 482 and 399;Vasa gene mRNA is 2267bp,coding the number of amino acids is 618.The three genes have been confirmed expression differences between male and female gonads in fish. Because the gene methylation opened or closed the gene expression, we compared the gene methylation in male and female gonads and other tissues can simplely understand these genes switching conditions and the appropriate switch.This experiment using methylation sensitive restriction enzyme Hpa â…¡ cutting the DNA of Monopterus Albus’s gonad and other tissues. Finding a suitable amplification conditions, each Vasa gene, Sox gene (Sox9a1, Sox17) and Dmrt gene (Dmrt2ã€Dmrt3〠Dmrt4) of enzyme digested products are amplified by each primer which according to the known mRNA sequences, at the same time, the control group with no enzyme were compared, analysis of different bands, obtain the site which may occur methylation in different tissues, at the same time, the differential bands were sequencing.Finally,obtained the different sites by comparing each gene sequences, looking for the amplification conditions suitable for the follow-up study, and also provides a method of molecular perspective for the related research of Monopterus Albus’s sex control.Through this experimental study, we found that the methylation status in different tissues are different:Dmrt3-FR2 amplified sequences have no methylation sites in the testis, but ovarian and other organizations have.so this site is probably related to sex control; there was no difference methylation sites in Dmrt4-FR2, Dmrt4-FR3, Sox9a1-FR2, Sox9a1-FR4, Sox17-FR1 and Vasa-FR5 amplification of each organization,all exist sequence methylation sites. At the same time, through the analysis of the CG site which may occur methylation,and The sequencing of Dmrt3-FR2 in ovary and testis to find out the possible site related to sex control. |
