虫生真菌分子致病机理及基因工程改造研究进展

微生物学通报MAR20,2008,35(3):443~449Microbiology©2008byInstituteofMicrobiology,[e-mail]基金项目：863(No.2006AA10A212)*通讯作者：Tel:[phone];:[e-mail]收稿日期：2007-08-07;接受日期:2007-09-13专论与综述虫生真菌分子致病机理及基因工程改造研究进展吕丁丁1,2李增智1王成树2*(1.230036)(2.200032)摘要:虫生真菌侵染寄主昆虫的复杂过程可分为体表附着、体壁穿透及体内定殖和致死等不同阶段。近年来,以金龟子绿僵菌(Metarhiziumanisopliae)和球孢白僵菌(Beauveriabassiana)为代表的基因功能研究取得了长足的进展,从不同角度阐明了虫生真菌的分子致病机理;同时,基因工程技术的应用为昆虫病原真菌的遗传改良和选育高毒力杀虫菌株开辟了新的途径。对近年来昆虫病原真菌侵染寄主的分子对策及基因工程改造的研究进展进行了综述,并就进一步研究虫生真菌的毒力基因及功能进行了探讨。关键词:虫生真菌,致病机理,毒力基因,基因工程AdvancesinMolecularPathogenesisandGeneticEngineeringofEntomopathogenicFungiLVDing-Ding1,2LIZeng-Zhi1WANGCheng-Shu2*(1.AnhuiProvincialKeyLaboratoryofMicrobialControl,AnhuiAgriculturalUniversity,Hefei230036)(2.InstituteofPlantPhysiology&Ecology,ShanghaiInstitutesofBiologicalSciences,ChineseAcademyofSciences,Shanghai200032)Abstract:Thecomplexofinsectmycosesbyentomopathogenicfungiincludes,ingeneral,sporeadhering,cuticlepenetration,hemocoelcolonizationandhostdeath.RecentgenefunctionstudiesofMetarhiziumani-sopliaeandBeauveriabassianahavebetteredtheunderstandingofthemechanismsofmolecularentomopa-thogenicitythattherearedifferentgenesinvolvedindifferentinfectionstages.Withtheidentificationsofdifferentvirulentgenesandtheexploringofexogenoustoxins,geneticengineeringworkshavebeenre-viewedinthispapertodemonstratethepotentialstoimprovethecontrolefficaciesofmycoinsecticidesbythestrategyoftoxingeneoverexpression.Futurestudiesarediscussedtofurtherexplorefungalvirulentgenesandtheirfunctionsduringinfectionprocesses.Keywords:Entomopathogenicfungi,Molecularpathogenesis,Virulentgene,Geneticengineering,,10%[1],444微生物学通报2008,Vol.35,No.3虫生真菌的侵染过程及相应的分子对策,,,31);2);3),[2](expressedsequencetag,EST)(Microarray),,,[3],,“”(re-modification)1.1体表附着阶段[4]1),,;2),,(hydrophobins)Bowden[5](Ophiostomanovo-ulmi)C-U(Cerato-ulmin)(barkbeetle),StLeger[6]ssgA,,[7],,,[4],[4],,,,[8]Wang[9](adhesin)MAD1,,,(Candidaalbicans)Int1Als1(Saccharomycescerevisiae)Flo11,MAD1,septin,,MAD2,1.2体壁穿透阶段1.2.1附着胞(appressorium)的形成：(epicuticle)(procuticle)(epidermis)(sebumlayer),,,,[10],,Wang[11](M.anisopliaevar.acridum)(Schistocercagregaria),(Magicicadaseptendecim):445[12],[13],cAMP,cAMP(cAMPdependentproteinkinase,PKA),PKA[14],(Magnaporthegrisea)MPG1[15],,,,,,Wang[16]MPL1,(perilipin),Nβ-3,,,MPL1(septum),1.2.2分解寄主外壳的水解酶：,(proteinases)(chitinase)(lipase)[10],,,,1),[17],,Suc-Ala-Pro-Phe-Nz,PR1(,subtil-isin);Ben-Phe-Val-Arg-Na,PR2()[18]Pr1A[19]PR1A11[20](1)Pr1A[21](Beauveriabrongniartii)[22](Paecilomycesfarinosu)[23],,[24,25]PR1A,PR1,PR1H,;PR1C,(horizontalgenetransfer)[20−24]2)17~50,[26],C/N[27]Dimilin()(Heliothisassulta),[10],图1金龟子绿僵菌Pr1蛋白酶种类Fig.1SubtilisinproteasesproducedbyMetarhiziumanisopliae446微生物学通报2008,Vol.35,No.3[28](VerticilliumLecanii),,,[29]5,CHIT42[30]CHI11[31]CHI2(AJ293217)CHI3(AJ293218)CHIA[32](Metarhiziumflavoviride)CHI1(AJ243014)2Bbchit1[33]Bbchit2(AY147011),,,CHIT(Manducasexta)[34],(Bbchit1),[33]:1),;2)1.3体内定殖阶段,,1.3.1免疫抵抗反应：,(cellularimmunity)(humoralimmunity),(encapsulation)(phagocytosis)(antimicrobialpeptides),,β-1,3(β-1,3glucan)(PR1),[35][36],(hyphalbody),,[37],,“”,(collagen)MCL1,“”(molecularmimicry)[38]MCL13,(GXY)n,X,Y;C(gly-cosylphosphatidylinositol)MCL1,,β-1,3-,,1.3.2分泌毒素：,,()(beauverolides)(cyclosporin)(destruxins),[36]Ca2+(programcelldeath),[39]Ca2+,,[40],,[41]1.3.3体内生长：,,(trehalose),,α-;,:447[42],α-,,[43],[3],2虫生真菌毒力基因工程的研究-,,,PEG/(PEG/Protoplasts)(restrictionenzyme-mediatedinteg-ration(Lithiumacetate)(Electroporation)(Biolistics)(AgrobacteriumtumefaciensMediatedTransformation,ATMT),[44,45]ATMT,pBENA3PrlpMAPR-11080,Prl[46]ATMTBbchitl(Myzuspersicae)[33]Fan[47](Bbchit1-BmChBD),23,Bbchit1,,,,3总结与展望,,,,,-,,,,,,,参考文献[1]JarvisP.Biopesticides:trendsandopportunities.AgrowReports.London:PJBPublicationsLtd,2001,p.97.[2]ClarksonJM,CharnleyAK.Newinsightsintothemecha-nismsoffungalpathogenesisininsects.TrendsMicrobiol,1996,4:197−203.[3]WangCS,HuG,StLegerRJ.DifferentialgeneexpressionbyMetarhiziumanisopliaegrowinginrootexudateandhost(Manducasexta)cuticleorhemolymphrevealsmechanismsofphysiologicaladaptation.FungalGenetBiol,2005,42:704−718.[4]HolderDJ,KeyhaniNO.Adhesionoftheentomopatho-genicfungusBeauveria(Cordyceps)bassianatosubstrata.ApplEnvironMicrobiol,2005,71(9):52605266.[5]BowdenCG,SmalleyE,GuriesRP,etal.Lackofassocia-tionbetweencerato-ulminproductionandvirulenceinOphiostomanovo-ulmi.MolPlantMicrobeInteract,1996,9(7):556−564.[6]StLegerRJ,StaplesRC,RobertsDW.Cloningandregu-latoryanalysisofstarvation-stressgene,ssgA,encodingahydrophobin-likeproteinfromtheentomopathogenicfungus,Metarhiziumanisopliae.Gene,1992,120(1):448微生物学通报2008,Vol.35,No.3−124.[7]FangW,BidochkaMJ.Expressionofgenesinvolvedingermination,conidiogenesisandpathogenesisinMetarhi-ziumanisopliaeusingquantitativereal-timeRT-PCR.MycolRes,2006,110(10):1165−1171.[8]WostenHA.Hydrophobins:multipurposeprote