Screening And Identification Of Proteins Interaction With Drosophila CG3295

Our lab cloned the Drosophila heart development gene CG3295 by bioinformatics analysis and molecular biology techniques. Our lab have successfully constructed the null mutant line of CG3295 for further study by use the P factor knockout method. In addition, CG3295 transgenic fly strains were successfully constructed by our lab. Through these research indicate that the CG3295 gene is involved in the development of Drosophila heart. This paper further screened and identified the interaction proteins of this gene, and studied the signalling pathways in the process of heart development.The Drosophila CG3295 gene （NM₁39917.2） is located on the 2nd chromosome in 57B9-57B9, and its full-length cDNA is 1853bp and ORF length is 1298bp, containing only one exon that encodes a protein of 432 amino acids, The protein includes three major domains including LisH, CTLH and CRA. The previous results from our lab suggest that the Drosophila CG3295, Dad, Mad and Smox genes could interact. In this paper, we cloned Drosophila CG3295, Dad, Mad and Smox into pMD18-T. After confirming by resteicion endonuclease reaction and sequencing, the genes were constructed the eleven plasmids pCMV-BD-CG3295, pCMV-AD-Dad, pCMV-AD-Mad, pCMV-AD-Smox, pCMV-myc-CG3295、 pCMV-tag2B-Mad, pCMV-tag2B-Smox, pGEX-4t-1-Smox, pET-28a-Dad, pET-28a-Mad. The present research aims at validating the interaction between Drosophila CG3295 and Dad, Mad, Smox by using mammalian two-hybrid, Co-Immunoprecipitation, GST pull-down. According to the result of mammalian two-hybrid, Drosophila CG3295 may exist interaction with Dad.Further validation was performed by using Co-Immunoprecipitation. The pCMV-myc-CG3295 and pCMV-tag2B-Dad were transferred into HEK293 cells and the proteins were extracted. The interaction was analyzed by Western blot with flag and myc monoclonal antibody, respectively. The results showed that CG3295 and Dad with full-length interacted.To sum up, This paper has screened and identified the interaction protein Dad of Drosophila CG3295, which establish an bases for further study on the Drosophila CG3295 signaling pathway.