The Explore Of Interaction Between SPAG6and TAC1/TCTE3Proteins

Objective：To explore if there are interactions between SPAG6and Tac1or Tcte3.Methods: Reversing transcription cDNA of eight tissues by extracting the RNA of thetesticle, brain, liver, kidney, heart, lung, spleen, muscle of healthy, adult male mice (18-20g). And then designing the specific primers of Tac1and Tcte3in mice, observe theexpression in different tissues of Tac1and Tcte3by PCR. Base on the testis cDNA of mice,amplify Tac1and Tcte3sequence by PCR. And Constructing the recombinant plasmids ofTac1/pGADT7、Tac1/pEGFPN2、Tcte3/pGADT7、Tcte3/pEGFPN2. The plasmids withpEGFP-N2were transfected into CHO cells and COS-1cells, using immunofluorescentstaining and western blot to detect the location in cells. Using yeast two hybrid ofTac1/pGADT7、Tcte3/pGADT7and SPAG6/PGBKT7, extracting the protein of yeast,and detecting if there are any interactions between SPAG6and TAC1or TCTE3.Results: Tac1was expressed in the testicle, brain and heart, and Tcte3was expressed inthe testicle, brain, liver, kidney, lung, spleen and muscle. The results of restriction enzymedigestion and sequencing indicate the recombinant plasmids of Tac1/pGADT7、Tac1/pEGFPN2、Tcte3/pGADT7、Tcte3/pEGFPN2construct successfully. In vitro，TAC1locate in the whole-cell and TCTE3locate in the cytoplasm. When cotransfectionof SPAG6, the location of TAC1and TCTE3changed to microtubule. In yeast two hybrid,the group of TAC1/SPAG6and TCTE3/SPAG6grow colonies in SD-Leu-Trp-His, andTAC1, TCTE3and SPAG6proteins express in colonies by Western Blot.Conclusion: By vitro text and yeast two hybrid, we verify there are directly interactionsbetween SPAG6and TAC1or TCTE3.