Construction and Activity Test of Bait Plasmid of RhoxF1 Gene in Yeast Two-hybrid System

Objective:To analyze the sequences of RhoxF1 protein,construct the bait plasmid of RhoxF1 in yeast two-hybrid system and test its self-activation acitivity.Method:Conserved domains were analyzed and the homology between mouse Rhox5 protein and human RhoxF1 protein was compared by bioinformatics method.PCR was used to amplify RhoxF1 gene and the fragment was inserted into the PMD 18-T vector.After verified,the fragment was subcloned into pGBKT7 vector.After that,the 1 plasmid was transformed into the yeast strain Saccharomyces cerevisiae AH109 and its toxicity and transcriptional activation was tested by both the phenotype assay and β galactosidase assays.Result:Several conserved DNA binding sites were found and the homology between Rhox5 and RhoxF1 amounted up to 30%.The bait plasmid pGBKT7-RhoxF1 was constructed correctly.Activity test of β-galactosidase revealed that pGBKT7-RhoxF1 couldn't be self-activated.Conclusion:Human RhoxF1 protein has the high homogeneous characteristics with mouse Rhox5 protein.In addition,pGBKT7-RhoxF1,as a bait plasmid of the yeast two-hybrid system,can be used to screen the protein interacting with RhoxF1.