The Function Study Of FADD Phosphorylation In Non Apoptosis

With a death domain of Fas-associated protein (Fas-associating protein with a novel death domain,FADD), was first known as an adapter in the apoptotic process. With the further research, it was found that FADD also involved in a variety of non-apoptotic associated processes. Hua Zaichun group made a number of progress about FADD new biological functions inNanjing University.On this basis, we set the construction of six kinds of over-expression vector as a starting point and D.melanogaster as a model animal, using standard microinjection techniques, the construction of transgenic model and further researching non-apoptotic function of phosphorylated FADD.First,we constructed a full-length mouse FADD (mFADD), sustained phosphorylation mFADD (S191D), sustained non-phosphorylated mFADD (S191 A), C-terminal knockout mFADD, BG4, and a selective marker c-myc (control) of six kinds of over-expression vector:we adopted gene splicing by overlap extension PCR according to the gene of murine FADD and obtained phosphorylated mFADD, non-phosphorylated mFADD, knockout C-terminal mFADD and BG4.Each of FADD fragment connectd to the vector of pUAST attB after the PCR amplification.Secondly, we used the standard microinjection techniques and injected into 24872 (site-directed mutagenesis of chromosome 2) Drosophila embryo,we picked the orange or red eye as a sign of transgenic phenotypes, obtaining the transgenic flies of overexpressing mFADD, C-terminal knockout,pFADD, npFADD, BG4, c-myc.Third, the preparation dFADD knockout Drosophila:we utilized CRISPR/ Cas9 technology, using the standard microinjection techniques and injected into 54591 (transgenic flies of continuous expression Cas9) Drosophila embryos, individually identified by hybridization after eclosion.Fourth, the analysis of genetic traits of the six overexpressing transgenic Drosophila:(1) The analysis of transgenic flies heritable phenotypes:the eyes and wings phenotypic analysis showed that the wings were partly crimped in FADD-D, but there were no significant impact on the eyes.(2) The effect of Cell Cycle:RT-PCR to detect the expression of associated cyclin such as cyclinA, cyclinE, observing whether expression disorder or not;(3) The role in innate immunity analysis:RT-PCR to detect the expression level of Dredd protein participating Imd pathway;(4) The expression of glucose metabolism-related enzymes:RT-PCR detected that sugar metabolizing enzymes G6pc, Pepck expression instance in Drosophila;(5) The analysis of climbing ability and survival:the statistical results showed that the climbing and survival was reduced in FADD-D,and both in each transgenic flies was significantly decreased than control;Finally, we gained FADD overexpression transgenic flies based on dFADD knocking down (RNAi):we hybridized dFADD knocking down flies, overexpression flies with transgenic flies containing double balancer respectively. According to the phenotypic characteristics of balancer,we selected first generation and then hybridized from generation to generation,obtaining the desired transgenic flies.Based on the above findings, we believe that FADD phosphorylation impact on the survival and climbing in Drosophila.