Reserch On The Identification Of PprM Promotor And It’s Relationship With PprⅠProtein Of Deinococcus Radiodurans

Background:Deinococcus radiodurans（DR） is a kind of extreme microorganism which has the strongest tolerance to ionizing radiation,ultraviolet radiation, drying, chemical mutagens and other DNA damages that founded to date. Ppr M（a modulator of the Ppr I-dependent DNA damage response） has discovered as a novel regulatory protein when study the relationship between ppr I and ppr A in Deinococcus radiodurans.And the deletion of ppr M contribute to extremely sensitivity to γ-rays. ppr M is a homolog of cold shock protein（Csp）,but the underlying mechanism is poorly understood yet.There has been reported that ppr M is a very important resistance gene,but the study of ppr M gene promoter is not reported till now. Ppr I is considered to be a general switch to regulate the expression of dozens of proteins from different pathways after radiation.Ppr I is a binding protein that can bind double-stranded DNA and it’s function has intimacy with ionizing radiation resistance.Based on the vitality of Ppr I protein,we speculate that Ppr I protein may play the role of enhanced ppr M radiation resistance by combining the promoter or sequence around ppr M.Objective:This study will find ppr M promoter first, determine the promoter’s function,then expression and purification of recombinant Ppr I protein.At last we use Electrophoretic Mobility Shift Assay（EMSA） to observe whether Ppr I protein combine ppr M promoter with prediction.This experiment will provide information for the status and role of ppr M gene in regulation against radiation network in D.radiodurans through the analysis and identification of ppr M promoter.The interaction with Ppr I protein contribute to important theoretical significance and application value for further exploreing the mechanism of extreme resistance and DNA repair in D.radiodurans.Methods:In this study,we use bioinformatics to predict ppr M promoter, construct p ET-28a-ppr M-e GFP combinant plamids containing the e GFP report gene to identify the function of ppr M promoter.And construct combinant plamids p ET-28a-ppr I and purificate Ppr I protein.And then use EMSA to explore whether Ppr I protein combine biotin labeled probe which to determiner the functional relationship between ppr I protein and the promoter and the sequence around ppr M.Results:We use bioinformatics to predict ppr M promoter is located at DR₀904 between DR₀906.Successfully to predict ppr M promoter through bioinformatics, construct p ET-28a-ppr M-e GFP combinant plamids containing the e GFP report gene,ppr M,ppr M promoter and identify that predicted sequence has the function of promoter.Express and purify Ppr I protein successfully,however,the results of EMSA in vitro can’t confirm that Ppr I protein can combine with forecast ppr M promoter.The analysis and identification of ppr M promoter in D.radiodurans doesn’t provide any evidence that the predicted promoter could combine the Ppr I protein which explain that this sequence of predictionis probably not be the ppr M gene promoter. The prediction of gene promote replace the promoter of p ET-28 a does not show success,and explain that the sequence of predictionis probably is not the ppr M gene promoter.Conclusions:1.Bioinformatics predicted the sequences of ppr M gene promoter is located at about 2Kbp upstream from the ppr M translation initiation site,is located at DR₀904 between DR₀906,The core sequence is on the chromosome 1 position of 911977⁹120262.The predicted sequences of ppr M promoter has promoter’s function 3.The Ppr I protein doesn’t combine with ppr M promoter in vitro...