实时荧光定量PCR检测毕赤酵母基因组中外源基因拷贝数

2009122212ChinJBiologicalsDecember2009Vol.22No.12。1。。Southernblot2。DNA。PCR3。。GAPGlycer－aldehyde-3-phosphatedehydrogenaseGFPPCR。1.1.1Top10、GS115pPIC3.5KInvitrogenGFPGS115-KDR-PpMD19-TSimpleTaKaRa。[e-mail]789A1004-5503200912-1236-05【】PCR【】PCR。GAPGFPPCR。GFPPCRGFP。GAPCty=-3.612x+39.28GFPCty=-3.544x+37.99GAPGFP0.890.900.999。10GFP9GFP1GFP。PCR。【】PCRDeterminationofCopyNumberofForeignGeneinGenomeofPichiapastorisbyReal-timeFluorescentQuantitativePCRXUANYao-ji,ZHOUXiang-shan,ZHANGYuan-xingStateKeyLaboratoryofBioreactorEngineering,EastChi－naUniversityofScienceandTechnology,Shanghai200237,China【Abstract】ObjectiveTodevelopamethodfordeterminationofthecopynumberofforeigngeneingenomeofPichiapastorisbyreal-timefluorescentquantitativePCR.MethodsThestandardcurvesofGAPandGFPgenesweregeneratedwiththeplasmidscontainingGAPandGFPgenesrespectively,andthegenomicDNAofP.pastoristransformantscontainingGFPgenewasanalyzedbyreal-timefluorescentquantitativePCR.ThecopynumberofGFPgeneineachtransformantwascalculatedwiththeCtvalueofP.pastorisgenomicDNAandthestandardcurve.ResultsTheregressionequationofCtvalueandlogarithmoforiginalcopynumberofGAPgenewasy=-3.612x+39.28,whilethatofGFPgenewasy=-3.544x+37.99.ThereactionefficienciesofGFPandGAPgeneswere0.89and0.90respectively.However,boththecorrelationcoefficientsofstandardcurvesofthetwogeneswere0.999,andboththecurvesshowedgoodreproducibility.OfthetenP.pastoristransformantstested,ninecontainedonecopyandonecontainedmultiplecopiesofGFPgene.ConclusionAreal-timefluorescentquantitativePCRmethodwassuccessfullydeveloped,whichmightbeusedforscreeningofP.pastoristransformantscontainingvariouscopiesofforeigngenes.【Keywords】Real-timefluorescentquantitativePCR;Pichiapastoris;Genecopynumber1236··2009122212ChinJBiologicalsDecember2009Vol.22No.12LigationKitVer.2.0TIANprepMiniPlasmidKit、TIANgelMidiPurificationKitTIANampYeastDNAKitPCRSYBR誖GreenRealtimePCRMasterMixPCRAppliedBiosystems2720ThermalcyclerABIFTC-2000PCRND-1000NanoDropThermoFisher。1.3LB1%0.5%1%100g／ml2%YPD1%2%2%MGY1.34%YNB1%2%1mol／L4×10-5%。1.4NCBIGAPU62648GAP-1GAP-2GFPGS115-KDR-PGFPGFP-1GFP-21。1Tab1.Primersusedinthisstudy5′-3′GFP-1ACCATGGGTTCTAAAGGTGAAGAATGFP-2TTTGTACAATTCATCCATACCATGGGAP-1CACAATGGCTATCACTGTCGGAP-2GACACACTACAGCCCGCATTRTGFP-1GGCTGACAAACAAAAGAATGGTARTGFP-2GATAAGGCAGATTGAGTGGATAARTGAP-1GGTATTAACGGTTTCGGACGTATTGRTGAP-2GATGTTGACAGGGTCTCTCTCTTGG1.5GS115GAP-1GAP-2PCRGAPGFPGS115-KDR-PGFP-1GFP-2PCRGFP。GAPGFP16℃pMD19-TSimpleGAPGFPTop104LB37℃。37℃PCRM13、ExTaqPCR94℃30s55℃30s72℃1min30。PCR。DNAT-GAPT-GFP。1.6GFPGS115-KDR-PGFP-1GFP-2PCRGFP720bpDNASnaBpPIC3.5K16℃Top1050μg／mlLB37℃。5’AOX1GFP-2PCRDNApP-GFP。1.7pP-GFPSalGS1155MGY30℃72～96hYPD30℃AOX1PCRGFPDNAPCR。1.8PCR1.8.120μlSYRB誖GreenRealtimePCRMaster10μlDNA1μl0.6μl10μmol／LddH2O7.8μl。37℃2min94℃4min94℃30s50℃30s72℃1min30。1.8.2T-GAPT-GFP6.02205×1023。104、105、106、107、108／μlDNA1μlRT－GAP-1／RTGAP-2RTGFP-1／RTGFP-2PCR。333、、PCR。PCRCt。1.8.3GFP10GFPDNA1μlRTGAP-1／RTGAP-2RTGFP-1／RTGFP-2PCR333PCR。Ct1237··2009122212ChinJBiologicalsDecember2009Vol.22No.12DNAGAPGFP。GAP6GAP。GFPGAPGFP。2.2.1GAPGFPGAPGFP123。GAPy=-3.612x+39.28R=0.999GFPy=-3.544x+37.99R=0.999。GAPGFP0.890.90。GAPGFP1PCRPCR。Ct0.03~0.230.22%~1.85%2。1～5DNA108、107、106、105、104／μl1T-GFPAT-GAPBFig1.AmplificationcurvesofT-GFPAandT-GAPBplasmids2.2GFP109GFP6GFPCt0.33。764。10105。2T-GFPAT-GAPBPCRFig2.MeltingcurvesofT-GFPAandT-GAPBplasmids3GAPGFPFig3.StandardcurvesofGAPandGFPgenesDRnDRn-di／dt℃-di／dt℃LogCt57911131517192123253456798GAPGFP2Tab2.ReproducibilityofstandardcurvesCt123GAPGFPGAPGFPGAPGFP10810.649.7610.639.7410.589.440.030.180.311.8510714.0613.1513.9713.1013.7512.900.120.140.891.0410617.8716.6617.4516.6517.4916.510.230.081.320.5010521.5520.2921.1920.3121.2320.140.200.060.940.4610424.7223.9624.6623.8624.9323.880.140.050.580.22DNA／μlSDCV%GAPGFPGAPGFP1238··2009122212ChinJBiologicalsDecember2009Vol.22No.123.PCRPCRSYBRGreenTaqmanCt。CtPCRCt3。PCRSYBRGreenPCRTaqmanPCR。SYBRGreen。TaqmanSYBRGreen。PCR。。72-ΔΔCt4。。12433PCRGAPGFPTab3.CopynumbersofGAPandGFPgenedetectedbyreal-timefluorescentquantitativePCRCt10nGFPGAPGFPGAP118.19±0.1019.00±0.065.59±0.035.56±0.021.06±0.06217.37±0.1318.52±0.295.82±0.085.75±0.131.17±0.31315.72±0.1016.72±0.216.28±0.036.25±0.061.09±0.11415.29±0.0616.27±0.086.41±0.026.37±0.021.08±0.05515.62±0.1916.54±0.206.31±0.056.30±0.061.04±0.13617.98±0.1220.09±0.115.65±0.035.31±0.022.14±0.13719.28±0.0820.22±0.125.28±0.025.28±0.031.00±0.08816.28±0.1917.31±0.296.13±0.066.08±0.111.11±0.24917.87±0.1918.90±0.145.68±0.055.64±0.081.08±0.181019.79±0.1620.76±0.125.14±0.055.13±0.031.02±0.1147A6BFig4.AmplificationcurvesofDNAfromsamplesNo.7AandNo.6BART-GFP-1／RT-GFP-2BRT-GAP-1／RT-GAP-2510PCRFig5.MeltingcurvesofgenomicDNAamplifiedfrom10strainsbyreal-timefluorescentquantitativePCRGFPGFP／GAPDRnDRn℃-di／dt-di／dt℃1239··2009122212ChinJBiologicalsDecember2009Vol.22No.121239PCR。2-ΔΔCtCtΔΔCt=ΔCts-ΔCtcal。ΔCtsCtCtΔCtcalCtCt1。PCR。。SYBRGreenPCR。GAPGAPGFP。GAPGFPPCR104~108／μl0.999PCR。Ct0.33Ct0.3。GFPGFP。SouthernblotSouthernblot。1CereghinoJL,CreggJM.Heterologousp