THE Protein Interaction Studies Of Drosophilidae Heart Development Candidate Gene Nulp1

The heart development processes are regulated by multiple genes and the any genetic change in the precise control during development leads to aoccurrence of congenital heart disease. The new candidate gene Nulp1 for human heart development was found by our laboratory and the preliminary results obtained based on Drosophila model suggested that the homologous gene CG7927 could be involved in heart development. This paper further studied the interaction proteins of this gene and the signalling pathways in the process of heart development.The Drosophila CG7927 gene （NM₁39917.2） is located on the 3th chromosome and its mRNA length is 2500 bp,containing seven exons and six introns. This protein contains a bHLH domain and a DUF654 domain in N-terminal and C-terminal, respectively. The previous results from our lab suggest that the human Nulp1, Fhl2 andβ-catenin genes could interact. In this paper, we cloned Drosophila Nulpl, Fhl2, and Arm （β-catenin） into pMD18-T. After confirming by resteicion endonuclease reaction and sequencing, the genes were constructed the three plasmids pCMV-myc-Nulpl, pCMV-tag2B-Fhl2 and pCMV-tag2B-Arm.The pCMV-myc-Nulp1 and pCMV-tag2B-Fhl2 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 Nulp1 and Fhl2 with full-length interacted. To further analyze the specific binding sites of two proteins, six subclones for Nulpl, namely. pCMV-myc-Nulp1-P1, pCMV-myc-Nulp1-P2, pCMV-myc-Nulp1-P3 were constructed based on its domains. The Fhl2 gene was subcloned into pCMV-tag2B-Fhl2-PET and pCMV-tag2B-Fhl2-6LIM. These plasmids were in groups transferred into HEK293 cell lines, and Western blot analyses showed that the pCMV-my c-Nulp1-P1, pCMV-myc-Nulp1-P2, pCMV-myc-Nulp1-P3 interacted with pCMV-tag2B-Fhl2. The pCMV-myc-Nulp1 and pCMV-tag2b-Arm were transferred into HEK293 cell lines and Westemblot analysis showed that Nulp1 and Arm with full-length did not interacted.In order to study the distribution of Nulp1, Fhl2 and Arm proteins in the cells, these genes were constructed into the plasmids namely pEGFP-C1-Nulp1, pEGFP-N1-Fhl2 and pEGFP-N1-Arm, and the plasmids expressing red fluorescent protein, pdsRED2-N1-Fhl2 and pdsRED2-N1-Arm. And then these plasmids were in groups transferred into COS7 cells, and the results showed that the Nulpl protein was located in the cytoplasm, Fhl2 and Arm proteins in both the nucleus and cytoplasm. Both Nulp1 and Fhl2 proteins were found to co-localize in the cytoplasm, and also both Nulp1 and Arm protein were found to co-localize in the cytoplasm. While both Fhl2 and Arm proteins were co-localized in the cytoplasm and the nucleus.These results indicate that Nulp1 and Fhl2 could interact directly in cells, and Nulp1 and Arm could have an indirect interaction. Wether the three proteins are in a ternary complex remains to be elucidated.In addition, Nulp1 and Lefty2 polyclonal antibodies were prepared. Western blot analysis showed that the titer for Nulp1 polyclonal antibody is 1:2000. Drosophila embryo proteins in 1.5 hours developmental intevals were prepared and Western blot results showed that the Nulp1 protein was expressed in the embryos at stages of 0-16 h but no expression was found in the embryo of 17th hours. Also Nulp1 protein was expressed at stages of larvae and adults. The titer for the Lefty2 antibody is 1:3000, however, Western blot showed that its specificity in the embryos of zebrafish was poor that is not suitable for further applications.