环境医学文献介绍

PerceptionofUV-BbytheArabidopsisUVR8Protein拟南芥UVR8蛋白应答UV-B,SCIENCEVOL3321APRIL2011,2012.11.1,IntroductionResultsConclusion,地表的UV-B辐射量伴随着大气平流层臭氧层的变薄而不断增强，给地球生态系统带来严重影响。UV-B主要是抑制植物光合作用、伤害生物膜及DNA等生物大分子，从而影响其生长发育，最终导致生物量及产量降低，甚至致死。植物在进化过程中形成了自我防护及防御机制，如DNA损伤的自我修复，活性氧自由基的酶促及非酶促清除机制，以及紫外吸收物质的诱导合成等；同时，在植物中也有许多物质及不同途径来感受和应答UV-B胁迫。,Introduction,植物对光的感受和应答叫光受体，光受体主要包括:(1)吸收红光/远红光(波长为600-750nm)的光敏色素(phytochrome)；(2)吸收蓝光/UV-A(波长为300-500nm)的隐花色素(eryptoehrome)和向光素(phototropin)；(3)UV-B(波长为282-320nm)受体，但此受体很长时间一直未分离得到。2011年4月份Science杂志报道：LucaRizzini及其同事鉴定了拟南芥紫外线抗性基因（UVresistancelocus8，uvr8，表达一种-螺旋蛋白UVR8蛋白)，UVR8蛋白在UV-B波长中充当一种光受体。,Results,一、检测UVR8和COP1在UVB照射下发生相互作用,Fig.1.UVR8interactswithCOP1inaUV-Bdependentmannerinplantproteinextractsandyeast.,(A)co-IPofUVR8withYFP-COP1fromproteinextractsexposed,UV-B(21molm-2s-2),indicatedtimes.,(B)ProteinextractsweretreatedseparatelywithUV-B(+,21molm-2s-2)ornot(-).,二、感应UVB光辐射的UVR8而不是COP1。,(C)Yeasttwo-hybridinteractionofUVR8andCOP1isUV-Bdependent(16hours,1.5molm-2s-2)andisimpairedinnonfunctionalUVR8andCOP1mutantproteins.,三、异源系统中UVR8和COP1在UVB照射下也能发生相互作用。,四、检测UVB照射下UVR8二聚体的活力,Fig.2.UVR8formsdimersintheabsenceofUV-BandmonomerizesinaUV-Bdependentmanner,(A)Co-IPofendogenousUVR8withCFP-UVR8fromCol/Pro35S:CFP-UVR8wasreducedwhenseedlingsweretreatedwithUV-B(1hour,21molm-2s-2).,(B)UVR8dimersweredetecta-bleinnon-denaturedsamplesofArabidopsisproteinextractsnottreatedwithUV-B(-),butonlyiftheproteingelwasirradiatedbyUV-Bafterthegelrun(15min,21molm-2s-2)andbeforetransfertoamembrane(lowerpanel).,五、非变性电泳验证UVR8在UVB照射下发生解聚，单体化。,(C)TimecourseanalysisofUVR8monomerizationincell-freeplantextractsafterexposuretoUV-B(21molm-2s-2)onicefortheindicatedtimes.ThegelblotofdenaturedproteinsshowsequalUVR8proteinamounts.,六、检测发现UVR8在UVB照射下快速解聚。,(D)UVR8dimersarecross-linkedbyDSP,whichisrever-siblebyreducingagents(-ME),showingequalproteinamounts,UV-B(5min,21molm-2s-2).,七、COP1对UVR8在UVB照射下发生单体化有无影响。,Fig.3.Tryptophan-285isrequiredforUV-BdependentmonomerizationofUVR8inyeastandhumancells.,LexA-UVR8proteinexpressedinyeastmonomerizedinresponsetoUV-B(5min,21molm-2s-2).Mutationsrepresentingnonfunctionalalleles(UVR8G145S,uvr8-15;andUVR8G202R,uvr8-9)didnotdimers.,八、在异源系统中证明UVR8具有UVB光受体特性。,(B)GlutathioneS-transferase(GST)UVR8andUVR8proteinsexpressedinHEK293TcellextractsmonomerizedinresponsetoUV-B(60min,1.5molm-2s-2).,FigureS3.The14tryptophansofUVR8arealllocatedatthetopofthepredictedUVR8propellerstructure.The14tryptophansarehighlightedandshowninyellow.,九、UVR8二聚体的结构和关键氨基酸位点,(C)PredictedstructureofUVR8,includinginparticularthetryptophanclusterwiththetryptophan-233,-285,and-337residues.,(D)Dependenceontryptophan-285forUV-BmediatedUVR8monomerizationinyeast.UVR8W285Adidnotformdimers,whereasUVR8W285FformeddimersbutdidnotmonomerizeinresponsetoUV-B(5min,21molm-2s-2).,十、UVB照射下关键氨基酸残基与COP1的相互作用,(E)UVR8W285AinteractedconstitutivelywithCOP1inayeasttwo-hybridassaywhereasUVR8W285FwasimpairedinitsinteractionwithCOP1(16hoursofUV-B,1.5molm-2s-2).,Conclusion一、在UVB照射下UVR8与COP1快速地相互作用，这种相互作用是U