RNA干扰及其在水稻抗病毒基因工程中的应用

2010,36(2):13-17PlantProtectionReviews:2009-03-27:2009-04-25:863(2007AA10Z415)*Email:[e-mail]何文,吴蓓蕾,李莉,王锡锋*(,,100193)RNA干扰(RNAinterference,RNAi)是一种基因沉默机制RNAi作为新兴的基因阻断技术具有明显的优势,已被广泛应用到动植物功能基因组和植物抗病研究中在抗病毒研究中,人为地将与病毒或宿主基因同源的双链RNA分子导入转基因植株,引起与其同源的基因发生沉默,达到抗病毒的作用本文主要综述了RNA干扰的相关知识以及在水稻抗病毒基因工程研究中的应用进展RNA干扰;基因工程;水稻病毒病害:S332.3:ADOI:10.3969/j.issn.0529-1542.2010.02.004AdvancesinRNAinterferenceanditsapplicationsingeneticengineeringagainstRicestripevirusHeWen,WuBeilei,LiLi,WangXifeng(StateKeyLaboratoryforBiologyofPlantDiseasesandInsectPests,InstituteofPlantProtection,ChineseAcademyofAgriculturalSciences,Beijing100193,China)AbstractRNAinterferenceisamechanismofgenesilencing.Asanewlydevelopedgenesilencetool,RNAipossessesdistinctadvantagesandhasbeenappliedtoplantandanimalfunctionalgenomicsandplantdiseaseresistanceresearch.Inplantantivirusresearch,doublestrandsRNAhomologoustovirusgeneorhostgeneistransferredintoplanttorepressthevirusreplicationandreduceitsdamage.ThisreviewsummarizestheadvancesinRNAinterferencetechniquesanditsapplicationsinricegeneticengineeringresearch.KeywordsRNAinterference;geneticengineering;ricevirusdiseases,()16,11(),,,,,,()t(ricestripedisease)(Ricestripevirus,RSV),[Laodelphaxstriatellus(Falln)],2080,,,,267hm2,10%~20%,50%~80%,[1],RNA(RNAinterference,RNAi)2010,,1RNA(RNAi)RNA(RNAinterference,RNAi)RNA(doublestrandedRNA,dsRNA)mRNA,1.1RNAiRNAi2090,1RNAi1928Wingard[2],,,,RNAi,RNA,1998,Fire[3]dsRNA(Caenorhabditiselegans),,RNA,RNA,20061.2RNAi1.2.1dsRNA的来源dsRNA,:1),,RNA,dsRNA;2),RNAdsRNA(hairpinRNA,hpRNA)[4-5];3)RNA(aberrantRNA),RdRP(RNAdependentRNApolymerase),dsRNA[6];4),RNAdsRNA[4-5];5)hpRNA;6)RNA,dsRNA[7];7)miRNA,miRNA(premiRNA)DCL1miRNAmiRNA*(dsRNA)[8-9]dsRNA,RNAi1.2.2RdRp介导的补充途径RNARNA,RdRp:1)RdRPsiRNAmRNA;2)RNA;3)1mRNA2siRNA,RdRPRNA;4)dsRNA,2siRNA,RdRP,dsRNADicersiRNA,[6]1.2.3RNAi的3种主要途径,,33dsRNARNaseIIIDicer21~26RNA[10]RNAsiRNA(smallinterferingRNA)miRNA(microRNA)1RNA()!14!362:RNA1.2.3.1转录后基因沉默(posttranscriptionalgenesilencing,PTGS)PTGSsiRNA[11]dsRNARNADNA,dsRNADicer(Dicerlike,DCL)dsRNAsiRNA,5∀3∀,3∀2~3[12-13]siRNA,RNA(RNAinducedsilencingcomplex,RISC),siRNA,mRNA,RISCmRNA;siRNAmRNA,mRNA21~23nt,;,siRNAmRNA,RdRp(RNAdependentRNAPolymerase)dsRNA,dsRNARNAi,mRNA[14-16],1.2.3.2转录水平的基因沉默(transcriptionalgenesilencing,TGS)TGS,TGSsiRNA,DNADNA,DNA(RNAdirectedDNAmethylation,RdDM)H39[17-21],,,DNA,,,[22]1.2.3.3翻译水平的基因沉默miRNAmiRNA70ntRNA,Dicer21~25ntRNA,miRNAmRNA,miRNA[23-24]RISC,mRNA3∀(3∀UTR),,mRNA,[25-26]miRNAsiRNA,Dicer,RNAiArgonaute(alg1alg2)RNARNAiRISCsiRNAmiRNA,,siRNA(),PTGS,miRNAmRNA[27]2RNAiRNAi1998,RNAiY,WaterhouseY(PVY)IRS(invertedrepeatsequence),[28]dsRNARNAidsRNAdsRNA2.1RNAi2.1.1靶基因序列的选择RNAiRNAidsRNARNAi,dsRNA,RNAi,RNAi,RNAi(offtargeteffect)siRNAsmRNA,siRNAs[29]siRNA[30],,RNAi,!15!2010RNAi:1)dsRNA,;2)dsRNA,,RNAidsRNA,Shuichiro[31]X(PotatovirusX,PVX)TGBp1(RNA)siRNA,,siRNAPVX,2.1.2dsRNA表达结构dsRNA,,RNARNA(hpRNA),dsRNA,hpRNA,ihpRNA(intronsplicinghpRNA),90%[32]Wesley[33],98~853bp,66%~100%(90%),(),Ubiquitin,[34]2.2RNAiRNAi,RNAiPinto[35](Riceyellowmottlevirus,RYMV),,RYMV,3[36](Ricedwarfvirus,RDV),RNA,,cpHayakawa[37][38-39]cp,RSV,RSV,,CPSP,,[40][41]CPSP,,CP,CP,SP;CP,CP,RNAicp[42]cp,pMCG161pCAMBIA1301,CaMV35SRNAi[43]siRNATargetFinder,168siRNA,BLAST,62,,pCAMBIA13012RNARNA3RNAi,Science20012002[44],!:,RNAi,RNAiRNAi,[45]siRNARNAi!16!362:RNA[46-47]RSV,,,RNAi[1],,,.[J].,2007,23(1):244-248.[2]WingardSA.Hostsandsymptomsofringspot,avirusdiseaseofplants[J].AgricRes,1928,37:127-153.[3]FireA,XuS,MontgomeryMK,etal.PotentandspecificgeneticinterferencebydoublestrandedRNAinCaenorhabditiselegans[J].Nature,1998,391:806-811.[4],,,.[J].,2003,11(1):16-19.[5]PlasterkRH.RNAsilencing:thegenomesimmunesystem[J].Science,2002,296(5571):1263-1265.[6]HutvagnerG,ZamorePD.RNAi:natureabhorsadoublestrand[J].CurrOpinGenetDev,2002,12:225-232.[7]BeclinC,BoutetS,WaterhouseP,etal.AbranchedpathwayfortransgeneinducesRNAsilencinginplants[J].CurrBiol,2002,12(8):684-688.[8]LiJ,YangZ,YuB,etal.MethylationprotectsmiRNAsandsiRNAsfroma3#enduridylationactivityinArabidopsis[J].CurrBiol,2005,15(16):1501-1507.[9]ParkMY,WuG,GonzalezSulserA,etal.NuclearprocessingandexportofmicroRNAinArabidopsis[J].ProcNatlAcadSciUSA,2005,102(10):3691-3696.[10]BaulcombeD.RNAsilencinginplants[J].Nature,2004,431:356-363.[11]HamiltonAJ,BaulcombeDC.AspeciesofsmallantisenseRNAinposttranscriptionalgenesilencinginplants[J].Science,1999,286:950-952.[12]ZamoreOD.RNAinterference:listeningtosoundofsilence[J].NatStructBiol,2001,8(9):749.[13]HarborthJ,ElbashirSM,BecheftK,etal.IdentificationofessentialgenesinculturedmammaliancellsusingsmallinterferingRNAs[J].JCellScience,2001,114(24):4557.[14]ZamorePD,TuschlT,SharpPA,etal.RNAi:doublestrandedRNAdirectstheATPdependentcleavageofmRNAat21to23nucleotideintervals[J].Cell,2000,101:25-33.[15]WinstonWM,MolodowitchC,CraigPH.SystemicRNAiinC.elegansrequirestheputativetransmembraneproteinSID1[J].Science,2002,295(5564):2456-2459.[16]JiaS,NomaK,GrewalSI.RNAiindependentheterochromatinnucleationbythestressactivatedATF/CREBfamilyproteins[J].Science,2004,304(25):1