漆小泉植物代谢组学及其应用.ppt

CenterforSignalTransduction Metabolomics 漆小泉中国科学院植物研究所植物分子生理重点实验室 植物代谢组学及其应用 CenterforSignalTransduction Metabolomics Alkaloids Amines Cyanogenicglycosides Glucosinolates Monoterpenes Sesquiterpenes Diterpenes Triterpenes steroids Flavonoids Polyketides Polyacetylenes fattyacids waxes 植物合成二十多万种不同的化合物 主要为次生代谢物 CenterforSignalTransduction Metabolomics 植物次生代谢物合成的模式 CenterforSignalTransduction Metabolomics 植物产生次生代谢物适应不良自然环境 Pollinationandseeddispersal Scents Colours Flavours CenterforSignalTransduction Metabolomics 次生代谢与植物发育有着不可分割的联系 Spearmint Glandulartrichomes Thyme Trichomes monoterpenesandsesquiterpenes Lemon Secretorycavity CenterforSignalTransduction Metabolomics 次生代谢物是很多中药的主要成分 TraditionalChineseMedicines Naturalproductsasdrugs 人体需要的特殊营养物质主要来源于植物 异黄酮 植物雌激素 维生素E 叶酸 b 胡萝卜素 CenterforSignalTransduction Metabolomics 维生素A缺乏导致眼睛疾病 我国儿童VA缺乏率达9 3 维生素C缺乏导致坏血病 维生素缺乏导致人体多种疾病的发生 维生素E缺乏导致皮肤病 早衰等 叶酸缺乏导致新生儿神经系统疾病 贫血等 生长发育 适应不良环境 人体必需营养成分 药用化合物 b 胡萝卜素 GA1 Br Strigolactone 植物萜类代谢物具有重要功能和作用 CenterforSignalTransduction Metabolomics FromWink M Phytochemistry64 3 2003 Quinolizidinealkaloids Steroidalalkaloids Mono andsesqui terpenes 次生代谢多样性的特点 特定的种属合成并积累特异的代谢物 Arabidopsis Modelplant Crops Yeast Modelunicellulareukaryote Chineseherbsandotherplantspecies 250 000 Referencespecies 基因组 代谢组学研究加快代谢途径的解析 CenterforSignalTransduction Metabolomics Metabolomicsisanew omics Thenamemetabolomicswascoinedinthe1990s ThefirstpaperusingthewordmetabolomeisOliver S G Winson M K Kell D B Baganz F 1998 Systematicfunctionalanalysisoftheyeastgenome TrendsBiotechnol 16 9 373 8Thenameisgiventothevarietyoftechniquesusedtorecognisepatternsinthechemicalspresentinbiologicalsamplesinordertodeciphertheirsignificance 代谢组学 Metabolomics CenterforSignalTransduction Metabolomics Metabolomicsistheanalysisofthetotalpopulationofmetabolitesinagivensample cellortissueandtheintegrationofthedatainthecontextoffunctionalgenomics genomics transcriptomics proteomics metabolomics Combiningallthe omics datawillprovideaclearerunderstandingofcellbiology 代谢组学 Metabolomics 从组学到分子系统生物学 From漆小泉等 植物代谢组学 方法与应用 2011年 CenterforSignalTransduction Metabolomics Metabolomicsresearchisparticularlyimportantintheplantfieldbecausecollectivelyproduceahugevarietyofchemicalcompounds farmorethananimalsandevenmicroorganism Otherareasofimpact environmentalgenomicsfoodquality diagnosticsinterorganismsignallingbioactivecompounds 植物代谢组学更具有挑战性 CenterforSignalTransduction Metabolomics Nosinglemethodcanbeusedtodetectthewholepopulationofmetabolites Thechoiceofinitialextractionsolventimmediatelylimitstheclassesofmetabolitesextractedbaseduponpolarity Nospectroscopymethod currentlyavailable isideallysuitedtothedetectionofeverymetabolite Avarietyofanalyticalmethodsshouldbeusedandthedataintegratedinordertogaininformationonasmanymetabolitesaspossible Datagainedfromavarietyofmethodsneedstobeintegrated Metabolomics theneedtointegratedatafromseveralplatformstoincreasecoverage CenterforSignalTransduction Metabolomics MassSpectrometryEquipmentAvailableinTheNationalCentreforPlantandMicrobialMetabolomics UK ThermoFinniganLCQLC MS WatersGC MSAccuratemassGC MS AgilentGC MS ThermoFinniganGCQGC MS ThermoFinniganMaT95XP WatersQ TOF WatersMaldi TOF LecoPegasusTOFFastScanningGC MS boughtunderMeT RO FromMikeBeale CenterforSignalTransduction Metabolomics 600MHzNMRMagnet BACSsamplechanger BrukerEsquire3000Massspectrometer BNMIinterface DADdetector HPLC SparkIISPEUnit 2XFOXYFractionCollectors 600MHzHyphenatedLC SPE NMR MSsystemTheNationalCentreforPlantandMicrobialMetabolomics UK FromMikeBeale GC TOF MS UPLC Q TOF MS GC QQQ MS 段礼新博士 Res Assistant 漆小泉 TeamLeader 薛震 Tech Assistant 韩彬博士 Tech Assistant 样品前处理数据分析 IB CASaimstoestablishanadvanceplantmetabolomicsplatformandnetworkinChina 海量质谱数据分析软件的开发开发了Pmass质谱分析软件 用分布式并行的方式加速了GC MS数据的峰对齐和定量速度 使用目前广泛使用的GC MS数据分析软件XCMS处理1200个样本需要时近90天 而使用Pmass软件只需2 3天 杨辉华 任洪军 李灵巧 段礼新 郭拓 杜玲玲 漆小泉 2013 基于Sector Sphere平台的GC MS多样本并行对齐算法实现 计算机应用 33 1 inPress 与桂林电子科技大学的合作研究 水稻代谢物化合物库及质谱数据库的建立通用化合物纯品 230种植物甾醇及萜醇 116种质谱数据库 230 116 200 900种 IB CASaimstoestablishanadvanceplantmetabolomicsplatformandnetworkinChina Rawdataprocession MStopeaks Biologicalannotation pathwayandnetwork Multivariateanalysis findfeatureandmarkers Deconvolution Dataformatconvert Alignment Quantitative Smoothing Denoisng Peakdetection CalibrationbyISandweight PCA principlecomponentsanalysis PLS DA partialleastsquares discriminateanalysis OPLS DA OrthogonalPLS DA HCA hierarchicalclusteranalysis CA correlationanalysis ANOVA analysisofvariance T test Peakidentification Pathwayanalysis Metabolicnetwork Integratewithother omic data Normalization Metabolomicsdataanalysisworkflow Datapre procession Rawdata Deconvolution Alignment Datamatrix Quantitative Datacalibration Multivariateanalysis Non supervised Supervised PCA HCA PLS DA OPLS DA PrincipleofPCA Scoreplot Loadingplot v Pathwayandnetworkanalysis Themainmetabolicmap Metabolicnetwork Metabolomicsdatabases MetaCyc genes proteins compounds reactionandpathways Compounds MassandNMR database 植物代谢组学的应用 基因功能解析代谢途径及代谢网络调控机理植物与生物逆境和非生物逆境互作研究作物的产量作物营养成分及品质等 植物代谢组学的应用 鉴别标识化合物代谢途径的构建代谢调控网络的构建 CenterforSignalTransduction Metabolomics 中药种类繁多 资源丰富 来源复杂 品种混杂严重 2000年版药典收载的534种中药材 即有143种为多基源 二基源以上 占收载总数的27 中药材基源品种的真伪 关系到该味中药的确切疗效和疗效的重现性 进而直接影响到中药制剂的质量 是实现中药现代化的首要问题 长期的医疗实践发现即使是同种药材 由于产地不同 野生与栽培以及生长年限不同都表现出质量和疗效上的差异 这些问题为中药材鉴别方法提出了新的挑战 中药黄芪的鉴别 中药黄芪的鉴别 材料选取膜荚黄芪 Astragalusmembranaceus Fisch Bunge蒙古黄芪 Astragalusmemeranaceus Fisch Bge var mongholicus Bge Hsiao肖培根等 1965 认为蒙古黄芪是膜荚黄芪的变种 CenterforSignalTransduction Metabolomics 分析手段 分子标记技术 代谢组学分析技术 MS 材料来源 CenterforSignalTransduction Metabolomics Clusterofgenetic andmetabolicfingerprinting 蒙古黄芪 膜荚黄芪 甘肃 膜荚黄芪 吉林 蒙古黄芪 膜荚黄芪 中药黄芪的鉴别 CenterforSignalTransduction Metabolomics 中药黄芪的鉴别 OPLS分析 CenterforSignalTransduction Metabolomics 区分膜荚黄芪和蒙古黄芪标识物的选取 OPLS V plot Loadingplot 中药黄芪的鉴别 初步鉴定出21个标识物 中药黄芪的鉴别 CenterforSignalTransduction Metabolomics 产地和种植方式的差别 膜荚黄芪 产地和种植方式叠加效果 蒙古黄芪 产地因素 种植方式因素 中药黄芪的鉴别 Duanetal 2012 MolPlant5 376 CenterforSignalTransduction Metabolomics 代谢途径分析 代谢物含量比值较高代谢物含量比值较低 膜荚黄芪 蒙古黄芪 萜类 甾类 胡萝卜素 芳香化合物 木质素 黄酮 胆碱 喹啉 脂肪簇氨基酸 生物碱 Glc1P UDP Glc 多糖 脂肪酸 多胺 生物碱 中药黄芪的鉴别 Duanetal 2012 MolPlant5 376 CenterforSignalTransduction Metabolomics 代谢组学技术能够快速区分两种黄芪 能综合反映生长环境与基因相互作用的影响 利用OPLS模型鉴定了21个代谢差异物质 由于只使用了GC TOF MS分析平台 不能检测到黄芪的有效成分 如黄酮类 三萜苷类 多糖类等代谢物 应使用多平台整合分析手段 中药黄芪的鉴别 DiterpenequinonesknownastanshinonesandphenolicacidderivativessuchassalvianolicacidarethemainbioactivecomponentsofS miltiorrhiza Morethan100compoundshaveisolatedfromDanshen whilehalfofthemarediterpenes Theyhavebeenfoundtohaveavarietyofpharmaceuticalactivities includingantibacterial antiinflammatory andanticancerproperties 解析丹参二萜代谢途径 CPS1istheonlyclassIIdiTPSInvolvedinTanshinonesBiosyntheticPathwayinRoot Cuietal unpublisheddata 解析丹参二萜代谢途径 QuantificationoffiveknownmajorcompoundsinCKandCPS1 RNAilines Cuietal unpublisheddata 解析丹参二萜代谢途径 IdentificationofmetabolitesdownstreamofSmCPS1catalyticstepintanshinonespathway Cuietal unpublisheddata 解析丹参二萜代谢途径 CKirSmCPS1 DataanalyzedbyMPP hairyroot 解析丹参二萜代谢途径 6 x10 0 0 25 0 5 0 75 1 ESIEIC 277 0868 9 108min 1 1 6 x10 0 0 25 0 5 0 75 1 ESIEIC 281 1178 8 545min 1 1 6 x10 0 0 25 0 5 0 75 1 ESIEIC 279 1027 Frag 130 0VPK 1 10 plant d 1 1 min 1 2 3 4 5 6 7 8 9 10 11 12 13 14 8 178min 9 712min Trijuarone 1 2 dihydrotanshinone dihydrotanshinoneI tanshinoneI 6 x10 0 0 2 0 4 0 6 0 8 1 277 0871 9 108min 299 0683 277 0871 575 1483 335 1626 6 x10 0 0 25 0 5 0 75 1 1 25 1 5 1 75 279 1016 9 737min 281 1174 8 545min 583 2088 303 0993 235 1118 263 1070 207 1173 m z 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 2M Na 2M Na 2M Na M H M H M H 6 x10 0 0 2 0 4 0 6 0 8 1 277 0871 9 108min 299 0683 277 0871 335 1626 322 1445 249 0916 6 x10 0 0 25 0 5 0 75 1 1 25 1 5 1 75 279 1016 9 737min 301 0836 279 1016 261 0917 233 0965 324 1598 205 1012 190 0776 6 x10 0 0 25 0 5 0 75 1 1 25 1 5 281 1174 8 545min 303 0993 281 1174 235 1118 263 1070 207 1173 192 0930 319 0725 m z 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 M H M Na M H M Na M Na M H M H2O M H2O 18 0099 27 9952 27 9953 15 0236 CH3 CO CO 18 0104 27 9952 27 9945 15 0243 CH3 CO CO 18 0099 M H2O SmCPS1wastheonlyclassIIditerpenecyclaseinvovledintanshinonesbiosynthesisintherootofDanshen PeridermistheplacewheretanshinoneswerebiosynthesizedandaccumulatedTanshinonebiosynthesisisacomplexnetworkanditislikelythattanshinoneIcanbesynthesizedthroughdifferentpathways 解析丹参二萜代谢途径 CenterforSignalTransduction Metabolomics ApplicationsofMetabolomicsanalysis NatureGenetics 2006 Volume38number7July P842 849 CenterforSignalTransduction Metabolomics ApplicationsofMetabolomicsanalysis Keurentjesetal 2006 14ArabidopsisecotypesandRILsUntargetedmetabolomicsanalysis LC QTOFMS CenterforSignalTransduction Metabolomics ApplicationsofMetabolomicsanalysis Keurentjesetal 2006 CenterforSignalTransduction Metabolomics Keurentjesetal 2006 FrequencydistributionofthenumberofsignificantQTLsdetectedateachmarkerpositionatfoursignificancelevel NumberofmassesdetectedintheRIpopulationanditsparents Notdetectedintheparents Detectedinbothparents CenterforSignalTransduction Metabolomics Keurentjesetal 2006 ApplicationsofMetabolomicsanalysis Beforesidechainmodification Aftersidechainmodification CenterforSignalTransduction Metabolomics ApplicationsofMetabolomicsanalysis Keurentjesetal 2006 Largegeneticvariationsformasspeaksinthe14accessions only13 4 ofmasspeakswaredetectedincommoninall14accession 75 ofthe2000masspeakscanbeexplainedbyQTLsintheRIpopulation Many one third metabolitesareproducedasaresultoftherecombinationofthegenomesofthetwoparents sincetheyareabsentinbothparents Conclusion Insummary authorsintegratedhigh throughputmetabolomicsandgenotypingdatafromalargepopulationcohortforelucidatingthebiochemicalidentitiesofunknownmetabolites Tothisend authorsappliedmetabolomicsgenome wideassociationstudiesandGaussiangraphicalmodelinginordertolinktheseunknownmetaboliteswithknownmetabolicclassesandbiologicalprocesses Itistobenotedthatthemethoddoesnotspecificallyrequiregenotypingdata Evenmetabolomicsmeasurementsalone analyzedthroughtheGGMs mayprovidesufficientinformationfortheclassificationandevenpreciseidentityprediction Onelimitationofthisapproachistherequirementforassociationswithfunctionallydescribedlociorknownmetabolites 水稻种子低温萌发的代谢组分析 DNAmarkers Phenotypes Metabolites pQTLs mQTLs Metabolicnetwork pathwayscontrolledbyQTLs co locationcorrelation 15 10days 15 10days 萌发率分布图 RIL15MH63RIL78ZS97RIL163 水稻种子低温萌发的代谢组分析 水稻种子低温萌发的代谢组分析 珍汕97x明恢63RI群体的高密度SNP连锁图谱 Xieetal 2010 PNAS107 10578Yuetal 2011 PloSOne6 e17595 1839markers Chr5 胚根QTLs 胚芽鞘QTLs 水稻种子低温萌发的代谢组分析 低温萌发速率QTL的定位 低温胚根长度QTLs 低温胚芽鞘长度QTLs 常温胚根长度QTLs 常温胚芽鞘长度QTLs 水稻种子低温萌发的代谢组分析 种子萌发速率QTLs Chr1 Chr5 Chr11 0hour 2days 4days 6days endosperm endosperm endosperm endosperm embryo embryo embryo embryo ZS97 MH63 ZS97 MH63 ZS97 MH63 ZS97 MH63 MH63 MH63 水稻种子低温萌发的代谢组分析 亲本低温萌期间代谢谱变化 取样时间 萌发后第4天萌发温度 15oC endosperm embryo ZS97 MH63 MH63 100RILsx4 EmbryoEndosperm PolarfractionNon polarfraction PolarfractionNon polarfraction 1 600samples GC Tof MS 水稻种子低温萌发的代谢组分析 代谢组分析方法 InternalstandardcorrectionQualitycontrol mQTLanalysisforRILs GC MSofsamples XCMS Ionfeature Abundancefiltering Metaboliteinteractionnetwork Colocalizationanalysis Multiplestatisticanalysis Anovaanalysis Candidategeneprediction Molecularmechanism 水稻种子低温萌发的代谢组分析 遗传代谢组学分析流程 Chr1Chr2Chr3Chr4Chr5Chr6Chr7Chr8Chr9Chr10Chr11Chr12 GeneticPosition cM Ionfeatures 红色代表碎片离子丰度在明恢63中高于珍汕97 绿色相反 水稻种子低温萌发的代谢组分析 Lod 定位mQTLs 1417个QTLs 碎片离子丰度 3230 0 Xieetal unpublisheddata 鉴定出4个数量遗传位点 QTLs 控制种子低温和常温萌发速率 在低温 15 萌发第4天时 珍汕97和明恢63之间的代谢谱出现了明显的变化 鉴定出1417个离子碎片数量性状位点 其中 与种子萌发速率表型共定位的有405个 在控制种子淀粉成分位点出现了大量的mQTLs 去卷积分析这些离子碎片将有助于解析这些位点在种子低温萌发速率及营养成分的代谢调控机制及建立代谢调控网络 水稻种子低温萌发的代谢组分析 Methods MetabolomicsanalysisGaussiangraphicalmodeling GGM Genome wideassociationanalysis GWAS IdentificationofunknownmetabolitesbasedonGGMandGWAS GGMsarebasedonpartialcorrelationcoefficients thatispairwisePearsoncorrelationcoefficientsconditionedagainstthecorrelationwithallothermetabolites Krumsieketal BMCSystemBiology 2011 5 IdentificationofunknownmetabolitesbasedonGGMandGWAS IdentificationofunknownmetabolitesbasedonGGMandGWAS ThoughGGMscanreconstructthemetabolicpathwayreactionanddistinguishdirectfromindirectassociations itcanneverprovideaperfectreconstructionoftheunderlyingsystem A counterantagonis