影响酿酒酵母电击转化率的条件.pdf

•YSõžÈÄq¥Hq*秦玉静* * 金建玲 鲍晓明 高 东(öêvб3þ/ŒSE×ÄLi)K1 以酿酒酵母为材料,用穿梭质粒pYES2进行电击转化的条件实验.实验表明电击中电压大小a细胞的生长状态a处理条件和电击后细胞的培养时间以及质粒浓度等对电击转化率均有影响.取对数生长期酵母细胞,用二硫苏糖醇(DTT)处理以疏松细胞壁,加0.7 Lg/mL质粒DNA,5kV电压电击,转化后细胞在完全培养基中培养60min后,涂布选择培养基,转化率可达4.8@ 105个转化子/Lg质粒DNA,细胞的存活率为39.2%,比完整细胞转化的效率高,存活率偏低.1oM Sõž;ÈÄ;ÄqÏms Ë| Q933Sõž(Saccharomyces cerevisiae)^†%‡‚3þ,ñ‚Ç µ3É y,L¿¡!„L. T¥ªÄ,7O µ1vËÆ )÷›!¥yVrØ eÅ„VrÁþFý©e#sߥ? ï,yN,SõžT¹yýñ¥Vr"d°msž×j.“÷yÉÆž )BĥZE,[1~ 5]y 랥ÚrÄ"d'ƹùîž )s03þÐ µ×1il.1978MnQž )ð3É8ÄE,[1,2]ƒÕZEÄq Ú,Œð3É8¥Å!#3¥„ç1 Në,‚ æ¿Ä0¥ yÎÅ!.Ûªy 뛜žÄE,[3]ZEe®,ŒÄqÊ®.1985M,Karube©¦[4] æ¨È, dEÄžð3É8#›œ%¤îÿª,ÈÄE[ÚÄq, TZL©ªÄ Ÿsž×j.C,ÈÄ/Œ ؂‚4Ú,X$†¯ð.Èª,ž%YPD›†¡!AÏ3ÉBHWPÉ›ù3É,Ä^µ æ¥,ÂT°¤|Èª¥ž%pƒÊ4¡!,®¿Ê4¡!îsö1¹í÷,‚ æ¿%¥ yÎ3É„ë¯,V7•YÄ¥²T.’Ä%YPDÏ¡!60minª,ÄqüA4Ú,Ù–?á! V[ÉB„4ÚÄq,Œ^rTi‚üA,yN InHW„rqZëyÍ,Ê460min¥¡!HW^1  ØX¥.2.5 ÈÄ„›œ%Ä¥1 ÈÄ^ æ¨È, d/Œ, æ¨Ú¥Èʵž%É›Ä,7›œ%Ä^‚åÊ%C¥Hq/,¨ÄÐk4) Øž%,PVÛ¶ÌâÏ|“÷DNA.L²TÜ9²,|KDÈÄHq:”3Éù%a30mmol/LDTT) Øa0.7 Lg/mlÉ ìDNAa5kVÈâÈ,ÈÄa›œ%ÄÉ›1 L(V2).239»2ù UÖá©:•YSõžÈÄq¥Hq V2 ‚]ÄHqžÄ¥•YTable2 Transformationefficiency ondifferent conditionsÈ  ÄElectrotrans›œ%ÄIntactcelltransÄq(105/LgDNA)Transformationefficiency 4.79 0.09iq(%)Viability 39.2 70.8²TAU,ÈÄ1›œ%Ä¥Äq1Ú,¥Ä0”›†?¡@òÕžs03þÐÄL¥³p,Œ®¿%¥T¨1  ,%iq ®.• IÓD1 HinnenA,HicksJB,FinkJR.TransformationofYeast.ProcNatlAcadSciUSA, 1978;75:1929~19332 BeggsJD.TransformationofYeastbyaReplicationHybridPlasmid.Nature(London),1978;275:104~ 1093 ItoH,FukudaY,MurataK, et al.Transformationof IntactYeastCellsTreatedwithAlkaliCations.JBacteriol,1983;153:163~ 1684 KarubeI,TamiyaE,MatsuokaH.Transformationof Saccharomyce cerevisiae SpheroplastsbyHighElectricPulse.FEBSLetts.,1985;182:90~ 945 HashimotoH,MorijawaH,YamataY,etal ANovelMethodforTransformationofIntactYeastCellsby ElectroinjectionofPlasmidDNA.AppliedMicrobiolBiotechnol,1985;2:336~ 3396 âo(,Zÿ B,Úê©.±3þL/Œ.62:öêvÐñ,1987.7 UÖá,>W æ,kü©.Úr¥ž›œ%ÄE.3þ/Œ,1997;7(3):37~ 388 MeihocE,MassonJM,TeissieJ.HighEfficiencyTransformationofIntactYeastCelllsbyElectricFieldPulses.Biotech-nol.1990;8:223~2279 HicksJB,HinnenA,FinkGP.PropertiesofYeastTransformation,ColdSpringHarborSymp.Quant Biol.1978.TRANSFORMATION OF S. CEREVISIAEBYELECTROPORATIONQinYujing,JinJianling,Bao Xiaoming,Gao Dong(State Key Lab. of Microbiol. Technol., Shandong Univ .,Jinan)ABSTRACTElectrotransformationwasperformed to transfer shuttle plasmid pYES2 into S. cere-visiae.Voltageacellgrownaincubation time andplasmidconcentration affect transformationefficiency.Thebestconditionsarethecellharvestedattheexponentialgrowthphasea5kVa0.7 Lg plasmidDNA and1h incubation time,transformationefficiency isup to 4.8@ 105transformantsper Lg plasmidDNA,whichishigher than intactcelltransformation.Thecellviabilityisabout39.2%.Key words S. cerevisiae;electrotransformation;transformationefficiency240 ö ê v Ð Ð (1– SÐñ) »34 。