Identification Of Drosophila C(2)M Interacting Proteins By Yeast Two-hybrid Screening

As a classic model organism, Drosophila has not only been widely used in classical genetics, but many of its molecular mechanisms on evolution are highly conserved in mammals, especially in this oogenesis system. Compared with other model organisms, Drosophila has a simple developmental model. We hope that through the study of the reproductive system of Drosophila melanogaster, to reveal the early meiotic prophase events and improve our understanding for meiosis.Synaptonemal complex (SC) is a super-complex structure which assembles by LEs, TFs and CE components in meiotic prophase I. It begins at zygotene, and starts to disintegrate in diplotene.. The normal assembly and disassembly of SC is essential for homologous chromosome pairing, synapsis, cross and the separation of sister chromatid.As a meiosis-specific protein, C(2)M is involved in the formation of SC. According to molecular experiments and bioinformatics analysis, with C3G together C(2)M participates in the formation of the LE components and influences C3G’s positioning; C(2)M also has the similar function with α-kleisin proteins, which can connect SMC1 and SMC3, hold sister chromatids together and play an important role in the formation of the eggs. Since synapsis only occurs in meiosis prophase and it’s a tiny structure that we can not observe it at the current resolution. Because C(2)M is a member of SC, we hope to explore with C(2)M interacting proteins, try to reveal the SC complex composition, assembly and the molecular processes of meiosis.We choose C(2)M as a bait protein, design primers to amplify CDS of C(2)M and constructe the expression vector pGBKT-7-C(2)M with Nco I and Xho I Transfecting the plasmid into yeast cells mediated by LiAc solution, detecting the expression of bait protein with Western blot, and plating transformants with recombinant bait plasmid on the SD/-Ade/-Leu/-Trp and SD/-His/-Ade/-Leu/-Trp synthetic dropout minimal base to test the bait protein for automatic transcriptional activation of the bait protein. Then we transfect the cDNA library into AH 109 which contain the bait plasmid, and identify C(2)M interacting proteins by SD/-His/-Ade/-Leu/-Trp synthetic dropout minimal base. The vectors containing objected gene were extracted from yesat and transformed into E.coli DH5 a. To verify interaction, we transformed the positive plasmids back to AH 109 which contained pGBKT-7-C(2)M, to test the automatic transcriptional activation of positive plasmids, we transformed it into AH109 which contained pGBKT-7. Finally, we send these plasmids to biotechnology company for sequencing. Using bioinformatics analysis to exclude the interference of impurity protein. Combined with literature and existing data, we chose two genes to construct transgenic flies to knockdown the RNA levels in germ cell, and observed the impact on the meiosis.By yeast two-hybrid experiments, we picked out 154 clones, we have screened 40 proteins that are interacted with C(2)M, which includes many of the chromosomal binding proteins. Combined with literature and expression characteristics of the these genes, we elected Wech and Psfl to constructe transgenic flies, then knocked down the RNA levels in germ cells. Immunofluorescence results showed that, the association of chromatin was disturbed, wech and Psfl, may delay the disappearance of SC in meiosis. Such results suggest that Wech and Psfl have the founction that promote SC’s depolymerization.