最新全国医师定期考核业务水平测评人文医学模拟试题500题.

Chapter3ProtoplastCulture&SomaticHybridizationWenwuGuoApplicationFusionandcultureDevel.process&hybridverificationDefinitionPart2Somatichybridization（体细胞杂交）1:DefinitionofSH•SH,i.e.protoplastfusion,aprocessofPPsfromtwodifferentparentswereinducedtofuseandthenregenerateintoplantlets,alsonamedasSH(A+B)•Severaldifferentexpressions:体细胞杂交：Somatichybridization细胞融合：Cellfusion原生质体融合：Protoplastfusion无性杂交：asexualhybridization超性杂交：Parasexualhybridization超性融合：Parasexualfusion细胞操作：Cellmanipulation细胞工程:CellengineeringComparisonofSHwithSexualcrossingItemsSomaticfusionSexualcrossTime时间NoseasonlimitOnlybloomseason(花期限制，母本花期)Phylogeny亲缘关系Cancircumventgraft/sexualincompatibility克服嫁接/有性不亲和性CannotResults结果nuclear,cytoplasmicgenomesrecombinedPloidylevelincreaedDoublefertilization,norecombinationandploidylevelunchangedPossibleFusionResultsofTwoGeneticallyDifferentProtoplasts=chloroplast=nucleusFusionheterokaryoncybridcybridhybridhybrid=mitochondriaTerminology-1对称杂种(symmetrichybrid)（双亲给杂种贡献的遗传物质均为100%）非对称杂种(asymmetrichybrid)（双亲给杂种贡献的遗传物质不等（相对值）–胞质杂种（cytoplasmichybrid,Cybrid）：细胞质重组，细胞核未重组对称融合（symmetricfusion）也称标准融合（Standardfusion）非对称融合（asymmetricfusion）：融合前对一方进行处理，使其染色体丢失一部分Terminology-2供-受体融合：Donor-recipientfusion体配融合（gameto-somaticfusion）（性细胞与体细胞融合）亚原生质体-原生质体融合：Subprotoplast-protoplastfusion小原生质体：Miniprotoplast微原生质体：Microprotoplast胞质体：Cytoplast胞质体-原生质体融合:Cytoplast-protoplastfusionTechnicaldevelopmentalhistory1970s,establishandoptimizeofPPtechnique–1972,tobaccointerspecificsomatichybrid–1975,highCa-highpHPEGfusion;electro-fusion–Numerousreports,somewerenotrealisticEarly1980s,modelplantstostaple/economiccrops,symmetrictoasymmetric,morerealisticLate1980s,manyreportsonperennialwoodyplants1990stodate,aconventional&safe‘biotech’forplantbreedingincitrus,rapeseed,wheatetc.Potato+Tomato=Pomato?2:protoplastfusionFusionmethodsFusionprocessFactorsaffectingfusionFusionmethodsPEG-mediatedfusionElectro-fusionPolyethyleneglycol（PEG，聚乙二醇）Electrofusion,Electricallyinducedfusion1.PEG-mediatedfusionCharacteristics:1)Meritsinclude:lowinput,highfrequencyofhetero-karyons,nospecieslimit;2)Defectsinclude:tediousfusionprocedure,toxicitytocellsWorkingmechanism:PEG分子具有轻微的负极性，可与具有正极性基团的水、蛋白质和碳水化合物等形成H键，从而在原生质体之间形成分子桥，从而使原生质体发生粘连进而促进原生质体融合；PEG能增加类脂膜的流动性，也使原生质体的核、细胞器发生融合成为可能Fusionsol:CaCl22H2O8-10mmolKH2PO40.7mmolMannitol0.5-1.0molpH5.6Inducingsol:Fusionsol+PEG20-45％Dilutingsol:solA(g/100ml)pH6.0solB(g/100ml)pH10.5Glucose7.21Glycin0.375CaCl22H2O0.79NaOH0.169SeveralstocksolutionstoprepareP1P2P1P2Mix,keepintact1minFusionFusionsolAddPEGDilutionWashAdddilutingsolMixsolsA&BAddmediumCultureScreeningPEGinducedfusionprocedureNote:MixsolutionsA&B(9:1)tomakeworkingsolution2．Electrofusion,ElectricallyinducedfusionThreeadvantagescomparedwithPEGfusionNotoxicitytoprotoplastsHigherfusionfrequencySimplermanipulationElectro-fusion:AhighfrequencyACfieldisappliedbetween2electrodesimmersedinthesuspensionofprotoplasts-thisinduceschargesontheprotoplastsandcausesthemtoarrangethemselvesinlinesbetweentheelectrodestoformpearl-chain.Theyarethensubjecttoahighvoltagedischargewhichcausestheirmembranestofusewheretheyareincontact.Electro-fusioninstrumentinNKL,HZAUTheprocessofelectricallyinducedfusionFusionparametersAlternatecurrentvoltage(交流电压)TimedurationofAC(交变电场处理时间)Directcurrentvoltage(直流高频电压)Pulsewidth(脉冲宽度)No.ofPulses(脉冲次数)FusionprocessMembranefusion(膜融合)Cytoplasmfusion(细胞质融合)Nucleusfusion(核融合)FactorsaffectingPPfusionPPquality(intact,viabilityetc)Fusionmethod(PEGorelectro-fusion)Fusionparameters(电融合参数、溶液浓度等)