Studies On Adult Drosophila Heart Function Of Pygo Gene And Construction Of CG7927 Gene Knockout System

Heart disease has become the first killer of human life and health. Hereditary heart disease is caused by gene mutations and expression abnormal. In this paper, Drosophila model was used to study the functional of two candidate genes involved in heart development.By regulating the differentiation of mesoderm, Wg signaling pathway is involved in the regulation of early heart development. The role of Pygo as its core members in heart development and adult heart function is unknown. We used the UAS-Gal4 system that controls the cardiac tube expression of GFP driven by the Hand-Gal4 driver to study the heart phenotype in the Pygo null mutant line. The results showed that compared with the control group, apparent heart tube defects in cardiac cardial and pericardial cells were observed in Pygo homozygous mutant. Also the heart pacing technique was used to study the adult heart physiological function of Pygo and the heart failure rate in Pygo-/+ flies was higher than that of control. When Pygo null mutant was in pair group with the null mutants of arm, mlc or ank2 in adult flies, the heart failure rate was more apparent in each dual null mutant heterozygote flies. In order to study its effect on downstream genes in Pygo null mutant heterozygous flies,1200 heart tubes from 3-day-old Pygo null mutant heterozygous flies and wild-type flies were isolated. The results obtained by real-time quantitative fluorescent PCR showed that the expression of members of Wg signaling pathways (including canonical and non-canonical) in Pygo-/+ flies was decreased, indicating that Pygo maintains adult heart function though Wg signals. The results obtained by Gene chip test showed that 182 amoung 32,447 genes detected were upregulated and 36 downregulated. These findings laid a basis for further research on Pygo.Drosophila CG7927 (vertebrates homologous gene Nulp1) is a heart development candidate gene which was cloned in our laboratory, which is located on chromosome 3L, and its mRNA is 2500 bp, encoding 702 amino acids, with a high evolutionary conservation. In order to study the biological function of this gene, Drosophila P-knockout technology was used to make CG7927 gene knockout lines. By a large scall of gene knockout screening, 32 null mutant lines were obtained. The results obtained by homozygous lethal test confirmed that ten of which were null mutant from two indepentant gene mutations. Also the results obtained by the UAS-GAL4 system with Hand-GFP showed that the ten mutant lines gave no mutation phenotype. Western blot analysis with a CG7927 polyclonal antibody showed that no changes were found with its protein expression, indicating that these 10 lines were not the CG7927 knockout lines. Homozygous lethal assay showed that the remaining 22 do not belong to the two null mutant genes analyzed and further analysis is under way.