Screening Of Pod1 Interacting Proteins Using GST Pull-down Assay In Oryzias Pectoralis

As a member of important transcription factors,bHLH transcription factor family protein in eukaryotic cells,Pod1 can specifically recognize and bind DNA non-coding sequence to regulate downstream gene expression,it is also able to cooperate with other proteins to involve in differentiation of mammalian gonad and the morphogenesis of major organs such as kidney,lung,spleen,heart,as well as tumor suppression,organism immunity.In this study,taked oryzias pectoralis,a new record species of Ricefishes in China found by author,as experimental animal,Screened the proteins that interacting with Pod1 and then speculate Pod1's possible function,the main contents and results are as follows:?Using the sequence informations of Japanese medaka to design primers that modificated by restriction site.Seven genes coding sequence,pod1,sf1,bmp4,tcf3 and hdac1,ar?,ar?,were amplificated by PCR from oryzias pectoralis gonads,and cloned them to pEASY-T5 Zero plasmid vector.?Thoes seven genes coding sequence cloned in T-vector,were digested by restriction endonuclease and reconnect used T4 DNA ligase to constructed into seven recombinant prokaryotic expression plasmids,pET32a(+)-pod1 and pGEX5X1-sf1/bmp4/tcf3/hdac1/ar?/ar?.The double enzyme digestion and sequence results showed that they were successfully constructed.? Transformed pET32a(+)-pod1,pGEX5X1-sf1/bmp4/tcf3 and pGEX5X1-hdac1/ar?/ar? into E.coli BL21(DE3),BL21(DE3)pLysS and Rosetta(DE3),OrigamiB(DE3)four kinds host strains,under the different conditions that concentration of IPTG inducer,temperature,shaking speed,induction time,induce their expression of foreign proteins,sifted the optimal conditions,and verificated objective proteins use 6His-tag and GST-tag monoclonal antibody by western blot.?SDS-PAGE electrophoresis and coomassie blue staining analysis showed that Pod1 fusion protein was expressed very well,almost of the expressed proteins are soluble proteins and accounts for above 50% of the total protein,there is very few inclusion body protein;About Sf1,Bmp4,Tcf3 and Hdac1 four kinds fusion protein,the main form of expressed was soluble protein,the bands of objective protein are more bright compared with bacteria protein;In addition,Ar? and Ar? in a variety of induction conditions have not been good expression effect,the results of western blot showed that they were not expressed.?The Pod1 fusion protein was purified from bacterial lysates using Ni-NTA resin,as Prey protein respectively incubated with the GST resin that combined Sf1,Bmp4,Tcf3,Hdac1 four kinds fusion protein,after desalinated by millipore ultrafiltration centrifugal tube in GST Pull-down.The results showed that Pod1 fusion protein could interact with Tcf3 and Hdac1 fusion protein.According to the experimental results of this study,we hypothesized that in oryzias pectoralis Pod1 can binding with Tcf3,another member of bHLH transcription factor family,to involve in the regulation of cellular processes together;Pod1 may associated with Hdac1 to regulated expresion levels of downstream genes.