PCRDGGE用于油脂废水处理系统中酵母菌群落结构解析吕文洲

微生物学通报AUG20,2008,35(8):1198~1202Microbiology©2008byInstituteofMicrobiology,[e-mail]基金项目：(No.Y507701);(No.20070970);(No.2006B100071);(No.2007A610057);*通讯作者：Tel:[phone];:[e-mail]收稿日期：2008-03-01;接受日期：2008-05-23研究报告PCR-DGGE用于油脂废水处理系统中酵母菌群落结构解析吕文洲*刘英朱建林(315211)摘要:本研究建立了适合于PCR-DGGE分析的环境样品中酵母菌基因组DNA的快速提取方法并利用PCR-DGGE对酵母菌处理油脂废水系统中的酵母菌群落结构进行了解析。结果表明,并非所有可以降解含油废水中污染物的菌株都可以稳定存在于系统中;系统对于菌株的选择开始于菌株的扩大培养阶段;系统从连续运行开始到稳定的过程中,G1、O2或W1成为优势菌株并稳定存在于系统中。关键词:酵母菌,DNA提取,PCR-DGGE,群落结构AnalysisofYeastCommunityStructureinaSaladOil-containingWastewaterTreatmentSystembyPCR-DGGELÜWen-Zhou*LIUYingZHUJian-Lin(DepartmentofEnvironmentalEngineering,CollegeofArchitecturalCivilEngineeringandEnvironment,NingboUniversity,Ningbo315211)Abstract:ArapidextractionofyeastgenomicDNAsuitableforPCR-DGGEanalysiswasestablishedandtheyeastcommunitystructureinasaladoil-containingwastewatertreatmentsystemwasinvestigated.Theresultsshow,notallyeaststrainscapableofdegradingpollutantsinwastewatercanbeplantedbysystem;theselectionofsystemtoyeaststrainsstartsfromtheperiodofyeastbiomasscultivation;frombeginningtostablestatusofcontinuouswastewatertreatment,G1,O2orW1arethedominantyeaststrainsandsurviveinselectionofsystem.Keywords:Yeast,DNAextraction,PCR-DGGE,CommunitystructureMuyzer[1]1993(DGGE),,[2−7],[8,9][10−13][14][15],,,DOI:10.13344/j.microbiol.china.2008.08.018:PCR-DGGE1199[16,17],Prakitchaiwattana[18]Nielsen[19],,10SBR,,,,,PCR-DGGE,PCR-DGGEDNA1材料与方法1.1酵母菌菌株10,,5(O);3(G)2(W)10,COD8111.2废水连续处理及环境样品采集1.2.1废水连续处理：10YPD48h,表1试验中所用的酵母菌菌株编号及名称Table1YeaststrainsandcorrespondingcodesinexperimentsStraincodeStrainO2CandidatropicalisO3CanidaboidiniiO3iRhodotorulaglutinisO4CandidautilisO5TrichosporoncutaneumG1CandidalipolyticaG2CandidaintermediaG3CandidapseudolambicaW1CandidahalophilaW2Trichosporonasteroids(SBR),,,BOD0.5kg/(kg·d)1SBR(1h),(9h),(2.5h),(0.5h),1/21.2.2环境样品的采集：,0d6d9d18d25d32d44dSBR2~3,8000r/min,-20;DNA,G3G31.5108/mL1.3酵母菌基因组DNA的快速提取1.3.1实验设计：DNA[20]DNA,,DNAPCR-DGGEDNA,10~11DNA()2表2DNA提取条件实验设计表Table2DesignofDNAextractionexperiments实验编号Experiments样品前处理Pretreatmentofsample裂解液量Volumeoflysissolution冻融次数Freezingtimes1不处理20022不处理30033不处理40044石油醚清洗20035石油醚清洗30046石油醚清洗40027玻璃珠20048玻璃珠30029玻璃珠400310试剂盒//11石油醚清洗＋试剂盒//1.3.2样品预处理：,,0.25g;DNA,,1mL31200微生物学通报2008,Vol.35,No.8提取：200µL~400µL−80(NU-6511E,NUAIRE,)5min,952min30s;200µL-(1:1,V/V),2min16000r/min3min;,1mL5min,16000r/min5min;;0.5mL70,,,;20µLTE,,DNADNA(i-Mupid@J,)DNADNAmarkerλDNA/HindIII1.3.4试剂：2TritonX-100,1SDS,100mmol/LNaCl,10mmol/LTris-HCl(pH8.0),1mmol/LEDTA(pH8.0);-(1:1);100;TE10mmol/LTris,1mmol/LEDTA(pH8.0)1.4PCR1.4.1仪器和引物：(1):PCR(IcyclerThermalCycler,Bio-rad,);(2):PCRNL-1(5′-GCATATCAATAAGCGGAGGAAAAG-3′)NL-4(5′-GGTCCGTGTTTCAAGACGG-3′)PCRNL1-GC(5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCATATCAATAAGCGGAGGAAAAG-3′)LS2(5′-ATTCCCAAACAACTCGACTC-3′)[18]1.4.2操作条件：(1):50µL0.2mLPCR:10×PCR5µL;10nmolDNTP4µL;10pmol1.5µL;DNA1µL,Taq2.5U,50µL;(2)PCR955min;9545s5545s7250s,36;7210min1.4.3PCR产物检测：PCRPCR600bp300bp100bpDNAladdermarker1.5DGGE1.5.1试剂药品：DGGE8(/37.5:1,V/V),30~50(1007mol/L40)1.5.2操作条件：PCR50µL6×loadingbuffer10µL,,30µLDcodeTM,Bio-Rad,:60140V4h~6h,,1EB20min,2,5min,(UNIVERSALHOOD-S.N.75S/02564,Bio-Rad,)1.5.3DGGE阳性参照体系：DNADNA,PCR;10PCR1µLPCR,DGGEDGGE,22,2结果与讨论2.1酵母菌基因组DNA的快速提取1DNA,DNAMarker,1~11,(1011),,168,,;,,DNA,DNA,2,DNA;DNA,,;200µL2.2PCR扩增23DNAPCRMDNAMarker,PCR,DNA600bp,1:PCR-DGGE1201图1不同实验下DNA提取结果的电泳图谱Fig.1ElectrophoreticimageofDNAextractionindiffer-rentexperiments图2第一次PCR扩增结果Fig.2ElectrophoreticimageofthefirstPCR图3第二轮PCR扩增结果Fig.3ElectrophoreticimageofthesecondPCR2.3DGGE分析10DNAPCR-DGGE,,4,10,DGGE7,O2W1O3G3O4O5SBRDGGE,5,19,SBR1,G1O2W1,(6d~10d);,,,O2W1DGGE,O2W1O2W1图4系统中10株酵母菌的DGGE图谱Fig.4DGGEimageof10yeaststrains’referencesystem1~11:O2O3O4O3iG2W2O5G1G3W1DGGE1~11:RepresentDGGEimageofO2,O3,O4,O3i,G2,W2,O5,G1,G3,W1andallstrains图5SBR系统连续运行中酵母菌DGGE分析图谱Fig.5DGGEimageof10-yeast-straincomplexincon-tinuouswastewatertreatment1,9:DGGE;2~8:0d6d9d18d25d32d44dDGGE1,9and2~8:RepresentDGGEimageofallstrainsandyeastsinSBRafteroperationof0d,6d,9d,18d,25d,32d,44d,respec-tively6DNAPCR-DGGEDGGE,M,1-11DNADGGE,G3,,DNAPCR-DGGE,,DNADNA,,2h1202微生物学通报2008,Vol.35,No.8图6不同DNA提取条件下的酵母菌DGGE图谱Fig.6DGGEimageunderdifferentDNAextractioncon-ditions3结论(1)PCR-DGGEDNA;(2)PCR-DGGE,G1O2W1;(3);参考文献[1]MuyzerG,EllenCDW,AndreGU.Profilingofcomplexmicrobialpopulationsbydenaturinggradientgelelectro-phoresisanalysisofpolymerasechainreactiongenescodingfor16SrRNA.ApplEnvironMicrobiol,1993,59:695−700.[2]KreuzingerN,FarnleitnerA,WandlG,etal.Molecularbiologicalmethods(DGGE)asatooltoinvestigatenitri-ficationinhibitioninwastewatertreatment.WaterSciTechnol,2003,47(11):165−172.[3]LaParaTM,NakatsuCH,PanteaLM,etal.Stabilityofthebacterialcommunitiessupportedbyaseven-stagebiologicalprocesstreatingpharmaceuticalwastewaterasrevealedbyPCR-DGGE.WaterRes,2002,36(3):638−646.[4]