RPA文献解读

复制蛋白A（RPA1a）是水稻减数分裂和体细胞DNA修复所必需的，但不是DNA复制和同源重组所必需华中农业大学作物遗传改良国家重点实验室和国家植物基因研究中心（武汉）摘要ReplicationproteinA(RPA),ahighlyconservedsingle-strandedDNA-bindingproteinineukaryotes,isastablecomplexcomprisingthreesubunitstermedRPA1,RPA2,andRPA3.RPAisrequiredformultipleprocessesinDNAmetabolismsuchasreplication,repair,andhomologousrecombinationinyeast(Saccharomycescerevisiae)andhuman.Mosteukaryoticorganisms,includingfungi,insects,andvertebrates,haveonlyasingleRPAgenethatencodeseachRPAsubunit.Arabidopsis(Arabidopsisthaliana)andrice(Oryzasativa),however,possessmultiplecopiesofanRPAgene.RicehasthreeparalogseachofRPA1andRPA2,andoneforRPA3.Previousstudieshaveestablishedtheirbiochemicalinteractionsinvitroandinvivo,butlittleisknownabouttheirexactfunctioninrice.WeexaminedthefunctionofOsRPA1ainriceusingaT-DNAinsertionalmutant.Theosrpa1amutantshadanormalphenotypeduringvegetativegrowthbutweresterileatthereproductivestage.CytologicalexaminationconfirmedthatnoembryosacformedinfemalemeiocytesandthatabnormalchromosomalfragmentationoccurredinmalemeiocytesafteranaphaseI.Comparedwithwildtype,theosrpa1amutantshowednovisibledefectsinmitosisandchromosomepairingandsynapsisduringmeiosis.Inaddition,theosrpa1amutantwashypersensitivetoultraviolet-CirradiationandtheDNA-damagingagentsmitomycinCandmethylmethanesulfonate.Thus,ourdatasuggestthatOsRPA1aplaysanessentialroleinDNArepairbutmaynotparticipatein,oratleastisdispensablefor,DNAreplicationandhomologousrecombinationinrice.结果1、osrpa1a突变体的观察2、OsRPA1a基因的分离和描述3、遗传互补和通过RNA干扰检测OsRPA1a4、osrpa1a中雌配子体发育在大孢子母细胞减数分裂时中断5、osrpa1a中花粉母细胞减数分裂受影响6、osrpa1a对DNA诱变剂高度敏感7、osrpa1a突变体中有丝分裂不受影响Figure1.Phenotypiccharacterizationoftheosrpa1amutant.A,Awild-typeplant(left)andosrpa1amutant(right)atmaturity.BandC,Spikeletsfromwild-type(B)andosrpa1amutant(C)plantsaremorphologicallyidentical.DandE,Viabilityofmaturepollengrainsofwildtype(D)andtheosrpa1amutant(E)asassessedbyI2-KIstaining.Bars=100mm.FandG,Structureofmatureembryosacsfromwildtype(F)andosrpa1amutant(G).AN,Antipodals;PN,polarnucleus;SY,synergids.Figure2.AnalysisofT-DNAtaggingofOsRPA1aandtheexpressionofOsRPA1a.A,StructureofOsRPA1aandthepositionoftheT-DNAinsertion(invertedfilledtriangle).Theintron(line),openreadingframe(blackbox),and5#and3#untranslatedregions(whiteboxes)areindicated.Thepositionsoftheprimers(RTLandRTRusedinBandC;O1,O2,andLBT3usedinD)areindicatedbyarrows.B,SpatialandtemporalexpressionpatternofOsRPA1ainleaf(L),sheath(Sh),stem(S),root(R)fromabootingplantandpaniclesatvariousdevelopmentalstagesaccordingtotheirlength(P1–P9).P1:approximately0.5cm;P2:approximately1.0cm;P3:approximately2.0cm;P4:approximately3.5cm;P5:approximately4.5cm;P6:approximately11cm;P7:approximately16.5cm;P8:approximately19cm;P9:approximately22cm.C,RT-PCRanalysisofOsRPA1aexpressioninpaniclesfromwildtype(WT)andosrpa1amutant(M).ActinisusedasthereferenceforthemRNAlevelinBandC.D,GenotypingoftheOsRPA1aT-DNA-taggingprogeny.Figure3.Transgeniccomplementationoftheosrpa1amutants.A,Fertilityofthe17complementedosrpa1alinesoftheT0generation.B,Fertilityoftheprogeniesofasingle-copyinsertioncomplementedosrpa1alineoftheT1generation.Awild-typeplantwasusedascontrol(CK).Theaveragefertility(mean6SE)wasbasedonthreepaniclesselectedrandomlyfromeachplantinAandB.C,GenotypingoftheplantsusedtoobtainthedatashowninB.Figure4.Formationanddevelopmentoftheembryosacinwild-type(A–J)andosrpa1amutants(K–O).A,Archesporialcell(arrowhead)formationstage.B,Megasporocyte(arrowhead)formationstage.C,Megasporocytemeiosisstage.Themegasporocyteundergoestwomeioticnucleardivisionstoformalineartetradofmegaspores(arrowheads).D,Functionalmegasporeformationstage.Threemegasporesnearestthemicropyle(arrowheads)degeneratetopreservethechalazalmegasporeasthefunctionalmegaspore.E,Mononucleateembryosacformationstage.FtoH,Embryosacmitosisstage.Thefunctionalmegasporeundergoesthreemitoticdivisionstoyieldatwo-nucleate(arrowheads)embryosac(F),four-nucleate(arrowheads)embryosac(G),andeight-nucleateembryosac(H),respectively.I,Eight-nucleateembryosacdevelopmentstage.J,Matureembryosacstage.Thepolarnucleus(PN),synergids(SY),andantipodals(AN)areindicatedbyarrowheads.表皮下的珠心组织细胞→孢原细胞→大孢子母细胞→细胞减数分裂→4个大孢子→3个大孢子退化，只有一个形成有功能的大孢子→3次有丝分裂→1个卵细胞、2个极核、2个助细胞、3个反足细胞（七胞八核）K,Megasporocyteformationstageofosrpa1amutant.Inthisstage,themegasporocyteformsnormallyasinwildtype.LandM,Megasporocytemeiosisstage.Themegasporocyteislikelytoundergoitsfirst(L)andsecond(M)meioticnucleardivision,butthenormaldyadandlineartetradofmegasporesdonotform.Inaddition,thedyadandtetradofmegasporesbegintodegenerate.N,Functionalmegasporeformationstage.Alloftheabnormalmegasporesdegenerate,andnofunctionalmegasporeisformed.O,Matureembryosacstage.Thematureembryosacisalmostemptyexceptforasmallfootprintofthedegeneratednuclei.细线期：染色质浓缩呈细线状偶线期：同源染色体配对、联会粗线期：完成联会，染色体进一步浓缩呈粗线状双线期：同源染色体分离终变期：染色体进一步凝集，形成短棒状结构，形成12个二价体中期I：二价体排列在赤道板上后期I：二价体分离末期I：二价体移向细胞的两极第二次减数分裂产生4个单倍体核末期I不同程度的DNA断裂MMC:丝裂霉素CFigure6.osrpa1amutantsarehypersensitivetoMMC,MMS,andUV-Cirradiation.AandD,Leafgrowthperplantofwild-type(WT)andosrpa1amutantplantsdu