Screening Classic Proteins Interacting With The Intraflagellar Transport Protein 20 Using Yeast Two Hybrid Assay

Objective:To analyze IFT20 protein expression in various tissues in male mouse,and screen potential proteins associated with IFT20 using Yeast-2-Hybrid assay,and provide basal foundation for its role in mammalian sperm flagella formation.Methods: Adult male mice were selected as materials,total proteins of brain,lung,kidney,liver,spleen,testis and testis were extracted,Western blot assay and immufluorescence staing were used to detect level and location of IFT20 protein.Then,we designed a pair of specific primers based on the coding sequence of mouse Ift20 in the PubMed database.The total RNA of mouse testis was extracted as a template for amplification of Ift20 cDNA by RT-PCR.The Ift20 cDNA was then used to construct the recombinant plasmid of Ift20/pGBKT7 with pGBKT7 vector after TA cloning,and the recombinant plasmid was verified by enzyme digestion and sequencing.We prepared the AH109 competent cells for the transformation of Ift20/pGBKT7 recombinant plasmid in Yeast-2-Hybrid(Mating)assay,then transformed the mouse Library which was provided by Clontech into Y187 competent cells;we selected 800 colonies for sequencing after yeast plasmid extraction,and analyzed the proteins detected more than 8 times.Results: The results of Western Blot showed that IFT20 was expressed in various tissues including the brain,lung,kidney,liver,spleen and testis,with IFT20 protein was robustly expressed in testis.Next we examined the testicular expression levels during germ cell development during the first wave of spermatogenesis,the expression of IFT20 protein was first visible at postnatal day 16 and increased significantly at days 30 and 42,when germ cells enter the condensation/elongation stage.Immunofluorescence staining showed robust IFT20 signals in both testicular sections and location at manchete region in isolated germ cells.The results of restriction enzyme digestion and sequencing showed that Ift20/pGBKT7 plasmid was successfully constructed.The Yeast-2-Hybrid(Y2H)assay showed that a total of 248 proteins identified to be potential IFT20 binding partner.SPATA1 appeared most frequently with 46 times,followed by IFT81,WAC,HAUS1,TEX12,SNAPIN andso on.Similar to IFT20,SNAPIN is also highly expressed at postnatal day 30 in male mice,and is localized in the manchette during spermiogenesis.Y2 H assay,cell transfection assay,and Co-Immunoprecipitation experiment confirmed SNAPIN interacts with IFT20.Conclusion: Y2 H identified several proteins,including IFT81,SPATA1,WAC,HAUS1,TEX12,and SNAPIN to be potential IFT20 binding partners.Interaction between IFT20 and SNAPIN was further confirmed.