重寄生过程中哈茨木霉转化菌株大量表达胞外碱性蛋白酶Word文档格式.docx
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重寄生过程中哈茨木霉转化菌株大量表达胞外碱性蛋白酶Word文档格式.docx
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1415-4757
Genet.Mol.Biol.
vol.21
n.3
Sã
oPaulo
Sept.
1998
MariaHelenaS.Goldman1andGustavoH.Goldman2
1DepartamentodeBiologia,FaculdadedeFilosofia,Ciê
nciaseLetrasdeRibeirã
oPretoand2DepartamentodeCiê
nciasFarmacê
uticas,FaculdadedeCiê
uticasdeRibeirã
oPreto,USP,Av.doCafé
s/n,14040-903Ribeirã
oPreto,SP,Brasil.Fax:
55-016-6331092.E-mail:
ggoldman@usp.br
SendcorrespondencetoG.H.G.
ABSTRACT
ThemycoparasiteTrichodermaharzianumproducesanalkalineproteinasethatmaybespecificallyinvolvedinmycoparasitism.Wehaveconstructedtransformantstrainsofthisfungusthatoverexpressthisalkalineproteinase.Someofthetransformantswereassessedforalkalineproteinaseactivity,andthosewithhigheractivitythanthewildtypewereselectedforfurtherstudies.Oneofthesetransformantstrainsproducedanelevatedandconstitutivepbr1mRNAlevelduringmycoparasiticinteractionswithRhizoctoniasolani.
INTRODUCTION
ThemycoparasiteTrichodermaharzianummayprovetobeaneffectivecontrolagentformanyphytopathogenicfungi.Ithasbeenproposedthatitsmycoparasiticinteractionproceedsinthreemajorsteps(Goldmanetal.,1994a,b;
Haranetal.,1996).Initially,themycoparasitehyphaegrowtowardthehosthyphae(Chetetal.,1981).Thentheparasiteattachestothetargethyphae,presumablymediatedbyahostlectin,andappressoria-likestructurescoilaroundtheattackedcells(Eladetal.,1983a,b;
Baraketal.,1985).Concurrently,degradationofβ(1,3)-glucansandchitinfromthehostcellwallhasbeenobserved(Eladetal.,1983b).Probablybothmechanicalpressureandcellwalldegradationbyhydrolyticenzymesareinvolved.Finally,themycoparasitepenetratesand/orlysesthehosthyphae(Chetetal.,1981)andreleasescellularcontents,whichprovidesnutrientstosustaingrowth.Extracellularenzymescorrespondingtothemainchemicalconstituentsofthefungalcellwall,i.e.,chitin,glucans,andproteins,havebeendetectedwhenT.harzianumisgrownonRhizoctoniasolanimyceliaorcellwallsasthesolecarbonsource(Ridoutetal.,1988;
Geremiaetal.,1993).Theenzymesappearedsequentially;
analkalineproteinasewasproducedfirst,followedbyglucanasesandchitinases(Geremiaetal.,1991,1993).Recently,wepurifiedandclonedthisalkalineproteasegene(pbr1)specificallyinducedbyRhizoctoniasolanicellwallsandchitin(Geremiaetal.,1993).Althoughtheresultssupportthehypothesisthatthisgeneisspecificallyinvolvedinmycoparasitism,thereisnodirectevidenceofitsroleinthisinteraction.
TheavailabilityofoverproducingalkalineproteasestrainsofT.harzianumwouldbeofhelptoestablishthisenzyme'
sroleinmycoparasitism.Additionally,antagonisticactivityinthesoilcouldbeimprovedbyincreasedactivityofoneormoreenzymesinvolvedincellwalldegradationofthepathogens.Thisstudydemonstratedthefeasibilityofoverexpressingpbr1geneinT.harzianumbyincreasingitscopynumberthroughtransformation.Oneofthetransformantsshowedelevatedandconstitutivepbr1mRNAexpressionduringmycoparasiticinteractions.
MATERIALANDMETHODS
Strainsandgrowthconditions
T.harzianumstrainIMI206040wasusedthroughoutthiswork.ThisstrainwasgrownasdescribedbyGeremiaetal.(1993).R.solanicellwallsandcolloidalchitinwerepreparedaccordingtoGeremiaetal.(1993).BasicproteaseactivitywasdeterminedbyincubationwithSucc-Ala-Ala-Pro-Phe-pNA(SigmaChemicalCo.,St.Louis,MO)(Geremiaetal.,1993).ProteinconcentrationwasdeterminedusingaBio-Radassay(Bio-Rad,USA).Intheantagonisticdualinteractions,simplemycelialdiscs(1.0cmindiameterfromfive-day-oldculturesofR.solaniinpotato-dextrose-agar(PDA)plates)wereinoculatedoncellophanediscs,neartheedgeofPetridishescontainingminimalmedium(Geremiaetal.,1991)plus0.5%glucose.Aftertwodays,amycelialdisc(1.0cm)ofafour-day-oldcultureofT.harzianumfromaPDAplatewasplacedoppositetothegrowingR.solanicolony,andincubatedat28oCforfourto10days.
DNA/RNAmanipulations
Restrictionenzymedigestswereperformedaccordingtomanufacturer'
srecommendations.IsolationofplasmidDNAfromEscherichiacoliandSouthernblotswasperformedusingstandardprocedures(Sambrooketal.,1989).DNAprobesweremadeusingarandomprimersystem(Boehringer).NorthernanalysiswasmadeasdescribedpreviouslybyGoldmanetal.(1992).AcellophanearearepresentingdifferentstepsoftheinteractionwascuttoobtainRNAfromthedualantagonisticinteractions.ThemyceliawerecarefullywashedwithTESbuffer,disruptedandtotalRNAextracted(Goldmanetal.,1994b;
Vasseuretal.,1995).CotransformationofthebasicproteasegenewasmadeincombinationwithplasmidspHAT-aandpBP2.2(Herrera-Estrellaetal.,1990;
Goldmanetal.,1990;
Geremiaetal.,1993).Amolarratioofapproximately1to4wasused;
afterwards,selectionwasmadeinPDAplateswith1.2MSorbitoland100µ
g/mlhygromycin.PlasmidpBP2.2wasmadeofpT3T7.lac(Boehringer)witha5.5-kbEcoRIfragmentofthepbr1gene(Geremiaetal.,1993).ThisinserthasthreeHindIIIandfourPstIsites.Furthermore,ithasafragmentofabout1.5kb,downstreamfromthestartcodon.DensitometryofthefungaltransformantswasperformedusinganLKB-Pharmacia2202UltrascanLaserDensitometer.
RESULTSANDDISCUSSION
HydrolyticenzymessecretedbyT.harzianumarebelievedtoplayanimportantroleintheparasitismofphytopathogenicfungi.Thealkalineprotease(PBR1)canbeinducedinsimulatedmycoparasiticconditions;
furthermore,colloidalchitincanalsoinducethisenzyme(Geremiaetal.,1991).Thegeneencodingthisalkalineproteinasehasbeencloned,andNorthernanalysisshowedthatinductionofthisenzymewasduetoanincreaseinmRNAlevel(Geremiaetal.,1993).Todetermineifthepbr1genewasinducedduringdualinteractionassays,totalRNAwaspreparedfromdifferentstagesofantagonismbetweenT.harzianumandR.solani(Figure1A).Asthemycoparasiticinteractionprogressedpbr1mRNAlevelsincreased(Figure1B).Goldmanetal.(1992)reportedrepressionofthepgkgeneexpressionduringasimulatedmycoparasiticstate.Infact,expressiondroppedmarkedlyduringthedualinteractionassays(Figure1C).Therewasconstantb-tubulinRNAexpressionunderalltestconditions(Figure1BandC).Theseresultsindicatethatpbr1mRNAaccumulatesprogressivelyduringantagonisticinteractionsbetweenT.harzianumandR.solani.Furthermore,simulatedmycoparasiticconditions,wheregrowthtakesplaceinminimalmediumwiththecellwallsofaphytopathogenicfungusastheonlycarbonsource,mimicantagonisticdualinteractionassays,atleastinsofaraspbr1andpgkgeneexpressionareconcerned(Figure1BandC).Thisisthefirstmoleculargeneticdemonstrationofsimulatedmycoparasiticconditionsthatreflectswhatoccursduringantagonisticdualinteractions.DuetothecomplexityoftheinvivointeractionbetweenT.harzianumandR.solaniontheplantrhizosphereorinthesoil,dualinteractionassaysprovidetheclosestandmostplausiblemethodologicalapproachforstudyingthemolecularbiologicalaspectsofmycoparasitism.
Figure1-NorthernblotofthemRNAobtainedduringspecificmycoparasiticinteractionsbetweenTrichodermaharzianumandRhizoctoniasolani.A:
Differentstagesoftheinteraction.B:
Northernanalysisofthepbr1mRNAobtainedduringdualinteractions.Theleftpanelshowshybridizationwithpbr1gene,whereastherightpanelshowshybridizationwiththetub1gene.C:
NorthernanalysisofthepgkmRNAobtainedduringdualinteractions.Theleftpanelshowshybridizationwithpgkgene,whereastherightpanelshowshybridizationwiththetub1gene.InpanelAII,T.harzianumhasgrownoverR.solanimycelia.R:
R.solani;
T:
T.harzianum;
G:
T.harzianumgrowninMMplateswith0.5%glucose;
CW:
T.harzianumgrowninMMbrothwith0.1%cellwallsofR.solani.
Therearemanyexamplesofgeneoverexpressioninfunguscausedbyanincreaseincopynumber(FowlerandBerka,1991).Consequently,wedecidedtooverexpresspbr1genebyco-transformingT.harzianumwithitsgenomicclone,plasmidpBP2.2,andaplasmidcontainingaselectedhygromycinBresistantmarker,pHAT-α(Herrera-Estrellaetal.,1990).Protoplasts(107-108ml-1)weretransformedwithbothplasmidsandselectedforhygromycinBresistance.ElectroporationofT.harzianumprotoplastsinthepresenceofthesetwoplasmidsyieldedtheexpectedhightransformationfrequency(Goldmanetal.,1990).Fifty-threetransformantswereselected,purified,andstabilizedthroughfourconsecutiveroundsoftransferinselectivemedium.Thesetransformantswereassessedforalkalineproteaseactivityandthosewithactivityhigherthanthewildtypewereselectedforfurtherstudies.
DNAfromfourtransformantsandthewild-typestrainwasisolated,digestedwithPstIorHindIII,andhybridizedwitha5.5-kbEcoRIfragmentcontainingthepbr1gene.DigestionwithHindIIIproducedhybridizingfragmentsofabout5.0and0.9kb,inthewildtypeandtransformants(Figure2A).Thisdigestionalsoshowedanadditionalfragmentofabout3.0kbinalltransformants(Figure2A).DigestionwithPstIproducedhybridizingfragmentsofabout2.3,1.7and0.6kbinthewildtypeandtransformants(Figure2B).Additionalfragmentsofabout5.5,2.8,and1.5kbcouldalsobeseeninthetransformants(Figure2B).Theadditionalfragmentspresentinbothdigestions
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- 寄生 过程 中哈茨木霉 转化 菌株 大量 表达 碱性 蛋白酶