Prokaryotic Expression And Purification Of GST-SRY Fusion Protein And The Effect Of Histone Modification In SOX9 Expression

The SRY gene is one of the key genes regulating the development of the testis,which is located on the Y chromosome.SRY is the first member of the SOX family and,like all other members,has a common feature that contains an extremely conservative DNA binding domain-HMG.So far,SRY,SOX3,SOX8,SOX9,MIS,WT-1,SF-1,DAX-1 and other genes have been found,and they have been involved in the important process of sex determination.In the chapter Two to obtain SRY,We constructed a prokaryotic expression vector harboring the SRY gene to express GST-SRY fusion protein in Escherichia coli,and then purified it by using Glutathione Sepharose.In this project,using method of RT-PCR,obtain the gene that encodes SRY,and then digested it restriction enzyme and ligated the insert to Prokaryotic expression vector pGEX-6p-1.After that,IPTG was used to induce the expression of the GST-SRY fusion protein,which was purified by Glutathione Sepharose later on.It was concluded that the successful construction of SRY gene recombinant prokaryotic expression vector and expression of SRY protein will be helpful for further studies of SRY gene function.In our preliminary study,we found that SRY activates WDR5,and they synergetically activates the expression of SOX9.H3K4me2 marks at the SOX9 promoters are increased compared to the control when WDR5 was overexpressed in LNCaP cells.In order to further verify the effect of WDR5 on SOX9,In the chapter Three,using Chromosome immunoprecipitation(ChIP)assay demonstrated the initial epigenetic regulation of WDR5 on SOX9 gene.