Construction Of Drosophila Heart Development Candidate Gene CG3295 Transgenic Flies And Its Founctional Research

RMND5A/B, previously cloned by our lab, are candidate genes of human heart development and their function have not yet been reported. In order to study the function of the human genes RMND5A/B in human heart development, we cloned the Drosophila CG3295 gene (human homologous RMND5A/B) by bioinformatics analysis and molecular biology techniques. CG3295 is located in chromosome 57B9-57B9. It contains only one exon and its full-length cDNA is 1860 bp and the ORF length is 1298bp, encoding 432 amino acids.By RT-PCR based on the cDNA transcripted from the Drosophila adult total RNA of wild type, the CG3295 cDNA was obtained and cloned into pMD18-T vector. The CG3295 gene from pMD 18-T-CG3295, identified with restriction enzymes KpnⅠand XbaⅠ, was cloned into the expression plasmid pUAST-CG3295. Then the plasmids pUAST-CG3295 and pA2-3 transposase enzyme plasmid were co-microinjected into the white-eyed Drosophila embryos fertilized Oh-0.5 h.12 transgenic strains were obtained by the progeny screening with the mini-white marker (orange eyes). In order to detect the CG3295 expression of each transgenic strain, the CG3295 homozygous transgenic flies were crossed with Twi-GAL4 and Actin5C-GAL4 lines, separately. The embryos at stage of 0h-12h were colected and the proteins were extracted. Western Blot test showed that the CG3295 protein expression in the lines 4,7 and 10 was significantly increased.In order to study whether overexpression of CG3295 could cause cardiac phenotype, the ten CG3295 transgenic homozygous strains were crossed with the Hand-GAL4:fand-GFP fly line, and the phenotypes were examined under a fluorescence microscope. The results showed that the pericardial cells of train No.4 appear significantly disordered and missing compared with those of control.In summary, total twelve CG3295 transgenic fly strains are successfully constructed. Preliminary analysis shows that the CG3295 gene integrated into the fly genome can be overexpressed, which can lead to heart mutant phenotypes, indicating that the CG3295 gene is involved in the development of Drosophila heart.