嗜水气单胞菌FNR基因克隆表达的初步研究.docx

嗜水气单胞菌FNR基因克隆表达的初步研究
论文题目：嗜水气单胞菌FNR基因克隆表达的初步研究
摘要：FNR基因在细菌中发挥重要作用，调控着许多基因的表达。本文以嗜水气单胞菌为研究对象，通过PCR技术克隆了该菌的FNR基因，并将其表达于大肠杆菌中，并进行初步的实验分析。结果表明，通过蛋白质表达、纯化、Westernblot和Gelretardation等方法，成功获得了嗜水气单胞菌FNR蛋白，并初步探究了其对于嗜水气单胞菌的一些基因的调控作用。本研究为深入探究FNR基因在嗜水气单胞菌的调控作用提供了基础。
关键词：嗜水气单胞菌；FNR基因；表达；调控
Introduction
Understandingthemolecularmechanismsofbacterialgeneregulationisessentialtoadvanceinthefieldofmicrobiology.FNR(fumarateandnitratereductaseregulator)isatranscriptionfactorfoundinmanybacteriathatrespondstochangesinoxygenconcentration,aswellasotherenvironmentalfactors,andregulatestheexpressionofavarietyofgenes.Inthepresentstudy,weinvestigatedthecloningandexpressionoftheFNRgenefromHydrogenophilussp.inE.coli,andanalyzeditsregulationontheexpressionofsometargetgenes.
MaterialandMethods
Bacterialstrains,plasmids,andcultureconditions
Hydrogenophilussp.wasisolatedfromhydrothermalventsandculturedinanaerobicenvironmentat50°CinamodifiedWolfe’smineralnutrientsolutioncontainingNH4Cl,K2HPO4,MgSO4,CaCL2,tracemetalsandNaHCO3.E.colistrainBL21(DE3)wasusedforFNRproteinexpression.TheplasmidpET28a(+)wasusedforcloningandproteinexpression.E.coliwasculturedinLuria-Bertani(LB)mediumat37°Caerobically.
PCRamplificationandcloningofFNRgene
TheFNRgenesequencewasobtainedfromtheNCBIdatabase,andtheprimersweredesignedaccordingtothegenesequence.ThePCRreactionwasperformedusingTaqpolymerase,withthefollowingconditions:95°Cfor5min,35cyclesof95°Cfor30s,52°Cfor30s,and72°Cfor1min,withafinalextensionat72°Cfor10min.ThePCRproductwaspurifiedandclonedintotheplasmidpET28a(+)usingT4ligaseafterdigestionwithrestrictionenzymes.
ExpressionandpurificationofFNRprotein
TherecombinantplasmidwastransformedintoE.coliBL21(DE3),andproteinexpressionwasinducedbyaddingIPTG.TheproteinwasextractedfrombacterialcellsusinglysisbufferandpurifiedbyNi-NTAaffinitychromatography.ThepurifiedproteinwasanalyzedbySDS-PAGEandWesternblot.
Gelretardationassay
ToinvestigatetheinteractionbetweenFNRandDNA,agelretardationassaywasperformed.Thedigoxigenin-labeledtargetD