The Differential Expressed Protein Profile Research Of Drosophila Melanogaster Circadian Rhythm Through ITRAQ Method

Circadian rhythms are widespread in the organic sphere.The physiological activities and metabolism of the organisms are controlled by the biological clock.Along with the alternates of daily circadia,the proteome of the body also occurs with spatial and temporal changes.Drosophila melanogaster as a model organismis widely used inthe field of life science.In order to find the circadianrhythm-related proteins in wild-type and clock gene mutant D.melanogaster,protein samples were collected on the four time serious points including ZT2,ZT8,ZT14,ZT20.By using the iTRAQ technology and TiO2 phosphorylation peptide enrichment method,the whole proteome and phosphorylated proteins in wild-type and clock gene mutant D.melanogaster were analyzed.The differential and oscillating proteins were investigated.In addition,the interactions between these related proteins andsignaling pathways were analyzed.Meanwhile,the BG2-c6 cell lines stably expressed dsCLK was constructed.The main results are as follows:1.The most commonly five kinds of one-step method of extracting the D.melanogaster head protein were compared,the results showed that 2%STD Buffer and Extraction Buffer are the most suitable Buffer for this experiment.2.iTRAQ technology was used to identify and quantitatively analyze the protein of the Drosophila head,it was shown that 3270 proteins were identified from the 8 treatments and 3 biological replicates samples.Based on the GO analysis,the results showed that the function of the total protein in the head of D.melanogaster was mainly concentrated in the metabolic processes(39%),cellular processes(19%),positioning(12%),and biological regulation(8%).46 proteins were differentially expressed between wild type and clkjrk.24 cycling proteins were identified from the wild type,and 24 from the clkjrk.A total of 12 cycling proteins were both found in the wild type and clkjrk D.melanogaster.3.A total of 1275 phosphorylated proteins were identified based on the iTRAQ experiment which carried out after the enrichment of TiO2 phosphorylated peptide.There are 8 treatments and 3 biological replicates.According to GO analysis,the functions of these proteins were mainly centered on the metabolic processes(28%),cellular processes(20%)and localization(11%).54 phosphorylated proteins were differentially expressed between wild type and clkjrk.44 and 81 was identified to cycling proteins in the wild type and the clkjrk,repectively.A total of 43 cycling proteins were both found in the wild type and clkjrk D.melanogaster.4.By pathway analysis,Phototransduction,Hippo signaling pathway,CD28 signaling,Wingless pathway has been identified.5.We aimed to establish an efficient RNA interference system in BG2-c6 cell system using dsCLK,and a reconmbinant strain that stably expressed dsCLk was obtainded.