The Study Of Waardenburg Syndrom Pathogenic Gene MITF In Drosophila

Objectives Based on the study of pathogenic gene mutation screening in Chinese WS patients, we will construct Mitf RNA interference Drosophila model to observe the changes of phenotype and lifespan, as well as using the genechip to screen abnormally expressed genes when Mitf is silenced, aims to explore the mechanisms of Mitf gene affects Wnt pathway in vivo.Methods1. Based on the Drosophila GAL4-UAS system, observing the phenotype of Mitf RNA interference flies:use UAS-Mitf RNAi male Drosophila (experimental group) and w1118male Drosophila (control group) respectively cross elav-GAL4, ey-GAL4, DA-GAL4virgin female Drosophila, observing the changes of phenotype;2. To observe the changes of the hybrid groups’ lifespan:the hybrid progeny of Drosophila were cultured in the same conditions, recording the time of death and the mortality, to calculate the maximum life span (90%deaths), half death time, then draw the life curve;3. Using the Affymertrix GeneChip(?) Drosophila Genome2.0Array to screen the abnormally expressed genes.Results1. The experimental group Mitf RNAi>ey-GAL4Drosophila’s eyes are smaller than those of the control group w1118> ey-GAL4Drosophila, no significant gender differences; the experimental group Mitf RNAi> elav-GAL4Drosophila appers vestigial wing, while the control group w1118> elav-GAL4progeny wing develops normal; the experimental group Mitf RNAi>DA-GAL4Drosophila shows no obvious phenotypic changes compared with the control group, w1118> DA-GAL4.2. In the experimental groups Drosophila’s survival time shows no significant difference with the control group (p>0.05); compared with control group, the experimental group’s median survival time is slightly longer, the age limit is slightly shorter;3. There are15genes abnormally expressed according to the gengchip,4of their expression increased (Wnt6, Apc, wdb, fz2), the others’expression induced.Conclusions1. The F1generation of Mitf RNAi>ey-GAL4Drosophila appears smaller eyes phenotype and the lifespan of Drosophila was not significantly affected by the knockout of Mitf further confirmed that Mitf is highly conserved both in vertebrates and in invertebrates, not only in gene sequences but also in gene function;2. The F1generation of Mitf RNAi> elav-GAL4Drosophila shows vestigial wing phenotype, suggesting that Mitf may regulate the Wnt pathway function, while the specific mechanism needs further research;3. The15abnormally expressed genes indicate the mechanism of Mitf conductiong Wg/Wnt pathway, but it still need further verification.