Study On DNA Binding Sequence Of Testis-specific Transcription Factor-FANK1

IntroductionSpermatogenesis is a complex cellular process which can be divided into three phases:mitotic phase, meiotic phase and haploid phase. During spermatogenesis spermatogonia proliferate and differentiate into functionally highly specialized male gametes called spermatozoa. Testis displays transcriptional activities in the spermatogenesis and entire body in mammals, transcription factor regulates the gene expression. Fankl is a testis-specific gene encoding a nuclear protein exclusively expressed during the transition from the meiotic to the haploid phase of spermatogenesis. The researches showed it is a testis-specific transcription factor. We constructed pGEX-FnⅢconstruct which can be induced to express FnⅢdomain of FANK1 protein. Then we determined FANK1 binding sequence by cyclic amplification of sequence target assay.MethodsThe gene segment of FANK1 gene was amplified by PCR, and then inserted into the vector pGEX-4T-2. The recombinant construct was transformated into E.coli BL21. The incision enzyme assay and DNA sequencing analyze were performed to find out the right recombinant clone. The transformated bacterias induced by 0.5mM IPTG at different temperature for lh,2h,3h,4h,5h,6h,7h were collected and total protein was extracted. The protein products were analyzed throught SDS-PAGE electrophoresis to find out the best induction temperature and time under which the bacterias can obtain the highest expression efficiency. And the bacterias were induced by different concentrate of IPTG (0.5mM,0.6 mM,0.8 mM,1.0 mM) to find out the best induction concentrate of IPTG After the induction bacterias were broken by extraction reagent, the supernatant were dialysised and purified throught the GST Bind Resin to obtain the purity GST-FANK1 protein. The purity protein was detected by Western Blot. Then we analysised the consistency DNA binding sequences for FANK1 by CAST.Results1. The gene segment of FANK1 has been successfully inserted into the vector pGEX-4T-2.2. The production of the fusion protein was highter when the bacterias were induced at 30℃,5h, the protein is about 43kDa.3. Through the induction bacterias broken, dialysised and purified by the GST Bind Resin, we obtained the purity GST-FANK1 protein.4. It revealed AAAAAG was the consensus DNA binding sequence for FANK1 through alignment of 44 selected sequences obtained from CAST.Conclusion1. Constructed the construct of pGEX-FnⅢand expressed the FANK1 protein successfully. It is the foundation of research the transcription factor.2. AAAAAG may be the DNA binding sequence of FANK1.