生物农药 8生物技术在生物农药上的应用

在生物农药上的应用生物技术ThebirthofbiotechnologyStanfordmedicalprofessorStanleyCohenandbiochemistHerbertBoyerfromtheUniversityofCalifornia,SanFrancisco,wereinHonolulutoattendameetingonplasmids,theringletsofDNAcontainedinbacteria.StanleyCohenBirthofanindustry1953DNAstructureproposedbyJamesWatsonandFrancisCrick1960ArthurKornbergsynthesizesDNAinvitro1970HamiltonSmithandKentWilcoxisolatethefirstrestrictionenzyme1971Thefirstbiologicalengineeringcompany,Cetus,founded1972PaulBergusesarestrictionenzymetoformahybridcircularmolecule1973StanleyCohenandHerbertBoyerdevelopDNAcloningandrecombinantDNA生物农药定义•化学农药专家：应包括微生物活体、昆虫天敌、部分植物源农药；不包括农用抗生素、植物生长调节剂、转基因农药。•国外：农用抗生素列为化学农药的范畴。争论点？•寡糖•丙烷脒•激活蛋白•多粘类芽孢杆菌•地衣类芽孢杆菌•海洋地衣芽孢杆菌•嗜线虫致病杆菌•绿色木霉•抑霉菌素•放线菌新菌株•植物活性物质新型生物农药品种防病防止病害生物农药的功用杀虫防止虫害除草防止草害提高自然产物的生物农药活性生物技术1AnoverviewofDNAcloningSomeconceptionneedtoknowSomeusefultechnologyimportanttogenemanipulationEnzymesforDNAcloningVectorsConceptionDNAcloningistoplacearelativelyshortfragmentofagenome,whichmightcontainthegeneorothersequenceofinterest,inanautonomouslyreplicatingpieceofDNA,knownasavector,formingrecombinantDNA,whichcanbereplicatedindependentlyoftheoriginalgenome,andnormallyinotherhostspeciesaltogether.PropagationofthehostorganismcontainingtherecombinantDNAformsasetofgeneticallyidenticalorganism,oraclone.ThisprocessiscalledDNAcloning.Hostorganism/cell:wheretheplasmidsgetmultipliedandpropagatedfaithfully,whichiscrucialforDNAcloning.HostsforDNAcloningvectorProkaryotichost:E.coli(mostcases)Eukaryotichost:Yeast(Saccharomycescerevisiae),largefragmentsofhumangenomeGeneralfeaturesofaVector1.autonomouslyreplicatingDNAindependentofhost’sgenome.2.Easilytobeisolatedfromthehostcell3.Mostarecircular,somearelinear4.Containsatleastoneselectivemarker,whichallowshostcellscontainingthevectortobeselectedamongstthosewhichdonot.5.Containsamultiplecloningsite(MCS)TypesofvectorsCloningvectors:allowingtheexogenousDNAtobeinserted,stored,andmanipulatedatDNAlevel.E.colicloningvector:plasmids,bacteriophages(λandM13),plasmid-bacteriophagelhybrids(cosmids).Yeastcloningvector:yeastartificialchromosomes(YACs)Expressionvectors:allowingtheexogenousDNAtobeinsertedandexpressed.PromoterandterminatorforRNAtranscriptionarerequired.•bacterialexpressionvectors•yeastexpressionvectors•mammalianexpressionvectorsIntegrationvectors:allowingtheexogenousDNAtobeinsertedandintegratedintoachromosomalDNAafteratransformation.Theintegrationisconductedbyhomologousrecombinationbetweenthehomologoussequencesharedbytheplasmidandthegenomeoftherecipientcells.•bacterialintegrationvectors(AgrobacteriumtumefaciensTiplasmidisusedtointegrateDNAintoplantgenome)•yeastintegrationvectors•Mammalianintegrationvector:virusbasedSubcloningTransferofafragmentofclonedDNAfromonevectortoanother.1.Enablesustoinvestigateashortregionofalargeclonedfragmentinmoredetail.2.Totransferagenefromoneplasmidtoavectordesignedtoexpressitinaparticularspecies.PreparationofplasmidscontainingaclonedDNAfragment(insert)Plasmidpreparation(vector)Restrictiondigestion(trimmingtheDNAends)Ligation(jointheinsertandthevector)Transformation&selectionoftransformants(introducetheplasmidsintohostcells)AssayoftherecombinantsDNASubcloning:aflowchartRestrictionendonucleaseDNAlibrariesaresetsofDNAclones,eachofwhichhasbeenderivedfromtheinsertionofadifferentfragmentintoavectorfollowedbypropagationinthehost.AcloneisageneticallydistinctindividualorsetofidenticalindividualsGenomiclibrariescDNAlibrariesGenomiclibrariespreparedformrandomfragmentsofgenomicDNA,whichmaybeinefficienttofindagenebecauseofthehugeabundanceofthenon-codingDNAcDNAlibrariesDNAcopies(cDNA)synthesizedfromthemRNAbyreversetranscriptionareinsertedintoavectortoformacDNAlibrary.Muchmoreefficientinidentifyingagene,butdonotcontainDNAcodingfunctionalRNAornoncodingsequence.TechnologyPlasmidpreparationRestrictiondigestsAgarosegelelectrophoresisDNAligationTransformationSelectionPreparationofplasmidDNAPlasmids:small,extrachromosomalcircularmolecules,from2to~200kbinsize,whichexistinmultiplecopieswithinthehostcells.Characters:containanoriginofreplicationandreplicateindependentlyUsuallycarryafewgenes,oneofwhichmayconferresistancetoantibacterialsubstance.Example:amprgeneencodingtheenzymeb-lactamasewhichdegradespenicillinantibioticssuchasampicillin.PlasmidminipreparationfromE.coliPlasmids•~2-20kbinlengththatmuchsmallerthanE.colichromosomalDNA(4600kb),andindependentlysupercoiled•Resistanttoshearingforceandchemicaldenaturation,thuscanbeisolatedfromthechromosomalDNAeasilysuchasalkalinelysis.Minipreparation1.Growthofthecellscontainingplasmids2.Collectthecellsbycentrifugation3.Alkalinelysisresuspensionalkalinelysisneutralization4.Phenolextractiontogetridoftheproteincontaminants5.Ethanolprecipitationtoconcentratethenucleicacidsremained.(PleasenotedthatRNaseAisverybadforthelabworkingwithRNA)Alkalinelysis•Resuspendthecellsinabuffersolution•Lysozymetodigestthecellwall(optional)•CelllysisinlysisbuffercontainingSDS(disruptscellmembraneanddenaturesproteins)andNaOH(denaturesDNA)•Neutralizationbuffercontaini