病毒诱导的烟草DHS基因的沉默

论文第50卷第21期2005年11月∗∗(,100089;,100094.∗,E-mail:[e-mail],[e-mail])(Nicotianabenthamiana)DHS(deoxyhypusinesynthase,)cDNAX(PVX),PVX-NbDHS.(virus-inducedgenesilencing,VIGS),DHS..RT-PCRNorthernblot,PVX-NbDHS-VIGSDHSmRNA.Westernblot,DHSeIF-5ADHS.,DHS.DHSVIGSNicotianabenthamiana(deoxyhypusinesynthase,DHS),eIF-5A(eukaryoticinitiationfactor-5A).DHSeIF-5A50,——(deoxyhy-pusine)[1∼4],(DHH),,(hypusine)[3,4].eIF-5A.eIF-5ADHS,DHSeIF-5A.eIF-5A[5].,eIF-5A.[6].,eIF-5A30%,eIF-5AmRNA[4].eIF-5A,mRNA[4,7,8].DHS1999[1],[2,9,10].DHS,[9],.(Nicotianatabacum)NtDHS,DHS[1].(VIGS),(Nicotianabenthamiana)NbDHS,.DHS.1()cDNA.TRIzol(Invitrogen)RNA.1μgRNA,oligo(dT)20,cDNA(Thermo-Script™RT-PCRsystem,Invitrogen).DHS(GenBank:AF296078,AJ242017):5′-TTCA-CTTCAAACCTTGTTTCTTC-3′5′-CCATGATAC-AGCTTCATCAGG-3′.cDNA,PCRDHScDNA,PCR.()X(PVX).PVXGV3101(pJICSa-rep)SainsburyBaulcombe.PCRDHScDNA5′3′,PVXClaSal,PVX-NbDHS.(BIO-RAD)GV3101.PVX.().,,.24,16h;208h.35%∼75%.5~6(25d).第50卷第21期2005年11月论文(agroinfiltration)[11].:LB(50mg/LKan,50mg/LRif,5mg/LTet,10mmol/L2-N-(MES)20μmol/L(AS))28,150A6001.0.,(10mmol/LMgCl2,10mmol/LMES100μmol/LAS).2~3h.5mL,.,(22,75%,)2d,,75%.15d,(,CHKAWAI,DH-505B)35%.75%,.()RT-PCR.RNAcDNA.2×TaqPCRMaster-Mix(,KT201)PCR,0.5μmol/L,25μL.Liu[12].3,PCR(ImageMaster®VDS,PharmaciaBiotech),mRNA.3-(glyceral-dehyde3-phosphatedehydrogenase,GAPDH),Burton[13].DHS5′-CTTGGTGACGCCATTCAAATTG-3′5′-CC-TCAATACCACCAGCTGTAG-3′;CN748029ESTeIF-5A1:5′-AG-AAGCTTGAAGATATTGTTC-3′5′-ATGCTTAACT-TAATTCAGATTCAC-3′.GAPDH,DHSeIF-5APCR479,238596bp.RT-PCR,.()Northernblot.RNA.RNA.30μgRNA1.2%,20×SSC.α-32P-dCTP(),PromegaPrime-a-GeneLa-bellingSystemKit.GAPDH.(Storm-820,MolecularDy-namic).().eIF-5A,,hypusine12,eIF-5A.eIF-5A.Chamot[14].12%20mA1.5h.,70V,2h(Trans-Blot®,BIO-RAD).[6].().910,[15].22.1DHScDNART-PCR,868bpDHScDNA.NtDHS,DNA95%,97%(),cDNANbDHS.3′516bp,DHS+412~+927bpcDNA,PVX,PVX-NbDHS(1).2.2PVX-NbDHSDHSeIF-5A35d,(35%),9,1011DHS.VIGS,.,RT-PCRNorthernblot[13].868bpDHScDNA5′238bpcDNANorthernblot.RT-PCR,1PVX-NbDHST-DNA2360(a)RT-PCR.MarkerPVX,PVX-NbDHS.1~518,21,24,27,30PCR.M1kbDNALadder.(b)RT-PCR.ImageMaster®VDS(Pharmacia),3.(c)Northernblot.12PVX-NbDHS;34PVX.GAPDH.(d)eIF-5A.12PVX,3PVX-NbDHSPVX-NbDHSDHSmRNA70%(2(a)(b)).Northernblot,DHS(2(c)),GAPDHmRNA(2(a)~(c)).RT-PCR,PVX-NbDHSeIF-5A165%(2(a)(b)).,DHSeIF-5A(2(d)).VIGS,[16],[17].30d,3∼5,.,9,1011,.,VIGS-DHS,DHSeIF-5A.2.3DHS()DHS.PVX-NbDHS35d,(PVX),.DHS9,10217%(3(a)).,.959%56%;1063%55%(3(b)).100,85%∼90%,(4(a)(b)).60d,DHS(5(b)).DHS.75%,DHS,(4(d)),3PVXPVX-NbDHS3;(a)(∼35%),.PVX-NbDHS,PVX.(b).PVX-NbDHS11,.(c)75%,.2PVX-NbDHS,PVX.(d)75%,.PVX-NbDHS11,,,5DHS(a)60d,DHS()().DHS,.(b)60d,DHS()().10,12~14,1617.DHS14,1617,10,1213,DHS.(c)120d,DHS()()(4(c)).()DHS.60d,14,DHS,1030d(5(a)(b)),DHS.35∼45d,(4(c)),.DHS60d,,(5(a)).80d,DHS,,40d.5(100d),DHS,2.(5(c)).DHS,,25%.1∼2.,DHS72%,75%(1).,DHS,,.()DHS.35d,DHS(4(a),(b)(d)).9102362)(n)//g·−1PVX-NbDHS271.70±1.59(−72%)0.02±0.02(−75%)PVX296.07±2.100.08±0.03);DHS,,DHS28%26%(6).6PVXPVX-NbDHS3,50d,,(7)..,[18],,.VIGS-PDS(phytoenedesatu-rase,),,().[13].7DHS(a)PVX-NbDHS,;(b)PVX,3VIGS[19].VIGS,DHS,DHS,.DHS,.()DHS,,.DHS[2],.Wang[9],DHS2,114d,;235d.,DHS1;2.VIGSDHS,30d,.,DHS.Wang[9],.,[20].DHS[9].,[9,21].,,,.,..DHS,,,,.,,DHS,[21].(50%),.,DHS,,,,(34).(75%),DHS,(4(c)(d)).DHS[9],(),DHS第50卷第21期2005年11月论文2364[14]3eIF-5A,eIF-5A,,.eIF-5A1,eIF-5A2,eIF-5A.,DHSeIF-5A,VIGS.()DHS.Wang[9],DHS,,40%∼60%,.DHS,,,30%;22%∼73%.,PVX-NbDHS217%,;1∼2,PVX-NbDHS72%75%(1).,DHSVIGS.DHS,Wang[9],,DHS,.,,DHS.,DHS,,.(),DHS,;,.,DHS.,DHS.(:5032008).1OberD,HartmannT.Deoxyhypusinesynthasefromtobacco.JBiolChem,1999,274:32040∼32047[DOI]2WangTW,LuL,WangD,etal.Isolationandcharacterizationofsenescence-inducedcDNAsencodingdeoxyhypusinesynthaseandeucaryotictranslationinitiationfactor5Afromtomato.JBiolChem,2001,276:17541∼17549[DOI]3ParkMH,WolffEC,FolkJE.Hypusine:Itspostranslationalformationineukaryoticinitiationfactor5Aanditspotentialroleincellularregulation.BioFactors,1993,4(2):95∼1044ParkMH,LeeYB,JoeYA.Hypusineisessentialforeukaryoticcellproliferation.BiolSignals,1997,6:115∼1235KemperWM,BerryKW,MerrickWC.Purificationandproper-tiesofrabbitreticulocyteproteinsynthesisinitiationfactorsM2BαandM2Bβ.JBiolChem,1976,251(18):5551∼55576,,,.5AG1-S.,2003,11(4):325∼3287ParkMH,JoeYA,KangKR.DeoxyhypusinesynthaseactivityisessentialforcellviabilityinyeastSaccharomycescerevisiae.JBiolChem,1998,273:1677∼1683[DOI]8DorianB,HerbertJ,MartinO,etal.Inducedgeneexpressionofthehypusine-containingproteineukaryoticinitiationfactor5AinactivatehumanTlymphocytes.Science,1996,271(5257):1858∼18609WangTW,LuL,ZhangCG,etal.Pleiotropiceffectsofsup-pressingdeoxyhypusinesynthaseexpressioninArabidopsisthaliana.PlantMolBiol,2003,52:1223∼1235[DOI]10AnkeS,NiemullerD,MollS,etal.Polyphyleticoriginofpyrrol-izidinealkaloidswithintheAsteraceae.Evidencefromdifferentialtissueexpressionofhom