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1、Center for Signal Transduction&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,Center for Signal Transduction&Metabolomics,Alkaloids,Amines,Cyanogenic glycosides,Glucosinolates,Monoterpenes,Sesquiterpenes,Diterpenes,Triterpenes,steroids,Flavonoids,Polyketides,Polyacetylenes,fatty acids,waxes,植物合成二十
2、多万种不同的化合物（主要为次生代谢物）,Center for Signal Transduction&Metabolomics,植物次生代谢物合成的模式,Center for Signal Transduction&Metabolomics,植物产生次生代谢物适应不良自然环境,Pollination and seed dispersal,Scents,Colours,Flavours,Center for Signal Transduction&Metabolomics,次生代谢与植物发育有着不可分割的联系,Spearmint,Glandular trichomes,Thyme,Trichom
3、es monoterpenes and sesquiterpenes,Lemon,Secretory cavity,Center for Signal Transduction&Metabolomics,次生代谢物是很多中药的主要成分,Traditional Chinese Medicines,Natural products as drugs,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,Center for Signal Transduction&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
4、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,Center for Signal Transduction&Metabolomics,From Wink,M.Phytochemistry 64:3(2003),Quinolizidine alkaloids,Steroidal alkaloids,Mono-and sesqui-terpenes,次生代谢多样性的特点特定的种属合成并积累
5、特异的代谢物,Arabidopsis Model plant,Crops,Yeast-Model unicellular eukaryote,Chinese herbs and other plant species(250,000),Reference species,基因组、代谢组学研究加快代谢途径的解析,Center for Signal Transduction&Metabolomics,Metabolomics is a new“omics”.The name metabolomics was coined in the 1990s:The first paper using the
6、 word metabolome is Oliver,S.G.,Winson,M.K.,Kell,D.B.&Baganz,F.(1998).Systematic functional analysis of the yeast genome.Trends Biotechnol.16(9):373-8 The name is given to the variety of techniques used to recognise patterns in the chemicals present in biological samples in order to decipher their s
7、ignificance.,代谢组学（Metabolomics）,Center for Signal Transduction&Metabolomics,Metabolomics is the analysis of the total population of metabolites in a given sample,cell or tissue and the integration of the data in the context of functional genomics,genomics,transcriptomics,proteomics,metabolomics,Comb
8、ining all the“omics”data will provide a clearer understanding of cell biology.,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,Center for Signal Transduction&Metabolomics,Metabolomics research is particularly important in the plant field because collectively produce a huge variety of chemi
9、cal compounds,far more than animals and even microorganism.,Other areas of impact:environmental genomics food quality,diagnostics inter organism signalling bioactive compounds,植物代谢组学更具有挑战性,Center for Signal Transduction&Metabolomics,No single method can be used to detect the whole population of meta
10、bolites,The choice of initial extraction solvent immediately limits the classes of metabolites extracted based upon polarity,No spectroscopy method,currently available,is ideally suited to the detection of every metabolite.,A variety of analytical methods should be used and the data integrated in or
11、der to gain information on as many metabolites as possible,Data gained from a variety of methods needs to be integrated.,Metabolomics:the need to integrate data from several platforms to increase coverage,Center for Signal Transduction&Metabolomics,Mass Spectrometry Equipment Available in The Nation
12、al Centre for Plant and Microbial Metabolomics,UK,ThermoFinnigan LCQLC-MS,Waters GC-MSAccurate mass GC-MS,Agilent GC-MS,ThermoFinnigan GCQGC-MS,ThermoFinnigan MaT95 XP,Waters Q-TOF,Waters Maldi-TOF,Leco Pegasus TOFFast Scanning GC-MS(bought under MeT-RO),From Mike Beale,Center for Signal Transductio
13、n&Metabolomics,600MHz NMR Magnet,BACS sample changer,Bruker Esquire 3000 Mass spectrometer,BNMI interface,DAD detector,HPLC,Spark II SPE Unit,2 X FOXY Fraction Collectors,600MHz Hyphenated LC-SPE-NMR-MS system The National Centre for Plant and Microbial Metabolomics,UK,From Mike Beale,GC-TOF/MS,UPLC
14、-Q-TOF/MS,GC-QQQ/MS,段礼新博士(Res.Assistant),漆小泉(Team Leader),薛震(Tech.Assistant),韩彬博士(Tech.Assistant),样品前处理数据分析,IB-CAS aims to establish an advance plant metabolomics platform and network in China,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
15、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):in Press,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-CAS aims to establish an advance plant metabolomics platform and network in China,Raw data procession(MS to peaks),Bio
16、logical annotation(pathway and network),Multivariate analysis(find feature and markers),Deconvolution,Data format convert,Alignment,Quantitative,Smoothing,Denoisng,Peak detection,Calibration by IS and weight,PCA:principle components analysis,PLS-DA:partial least squares-discriminate analysis,OPLS-DA
17、:Orthogonal PLS-DA,HCA:hierarchical cluster analysis,CA:correlation analysis,ANOVA:analysis of variance,T-test,Peak identification,Pathway analysis,Metabolic network,Integrate with other“omic”data,Normalization,Metabolomics data analysis workflow,Data pre-procession,Raw data,Deconvolution,Alignment,
18、Data matrix,Quantitative,Data calibration,Multivariate analysis,Non-supervised,Supervised,PCA,HCA,PLS-DA,OPLS-DA,Principle of PCA,Score plot,Loading plot,v,Pathway and network analysis,The main metabolic map,Metabolic network,Metabolomics databases,MetaCyc:genes,proteins,compounds,reaction and pathw
19、ays.,Compounds(Mass and NMR)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,Center for Signal Transduction&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
20、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：Astragalus membranaceus(Fisch.)Bunge蒙古黄芪：Astragalus memeranaceus(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。,Center for Signal Transduction&Metabolomics,分析手段分子标记技术：代谢组学分析技术：-/
21、MS,材料来源,Center for Signal Transduction&Metabolomics,Cluster of genetic（）and metabolic fingerprinting（）,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,Center for Signal Transduction&Metabolomics,中药黄芪的鉴别,OPLS 分析,Center for Signal Transduction&Metabolomics,区分膜荚黄芪和蒙古黄芪标识物的选取(OPLS),V-plot,Loading plot,中药黄芪的鉴别,
22、初步鉴定出21个标识物,中药黄芪的鉴别,Center for Signal Transduction&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,Duan et al(2012)Mol Plant 5:376,Center for Signal Transduction&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
23、药黄芪的鉴别,Duan et al(2012)Mol Plant 5:376,Center for Signal Transduction&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,.,Diterpene quinones known as tanshinones and phenolic acid derivatives such as salvia
24、nolic acid are the main bioactive components of S.miltiorrhiza.More than 100 compounds have isolated from Danshen,while half of them are diterpenes.They have been found to have a variety of pharmaceutical activities,including antibacterial,antiinflammatory,and anticancer properties.,解析丹参二萜代谢途径,CPS1
25、is the only class II diTPS Involved in Tanshinones Biosynthetic Pathway in Root,Cui et al,unpublished data,解析丹参二萜代谢途径,Quantification of five known major compounds in CK and CPS1-RNAi lines,Cui et al,unpublished data,解析丹参二萜代谢途径,Identification of metabolites downstream of SmCPS1 catalytic step in tans
26、hinones pathway,Cui et al,unpublished data,解析丹参二萜代谢途径,CKirSmCPS1,Data analyzed by MPP(hairy root),解析丹参二萜代谢途径,6,x10,0,0.25,0.5,0.75,1,+ESI EIC(277.0868),9.108min,1,1,6,x10,0,0.25,0.5,0.75,1,+ESI EIC(281.1178),8.545min,1,1,6,x10,0,0.25,0.5,0.75,1,+ESI EIC(279.1027),Frag=130.0V PK-1-10-plant.d,1,1,min,
27、1,2,3,4,5,6,7,8,9,10,11,12,13,14,8.178min,9.712min,Trijuarone,1,2-dihydrotanshinone,dihydrotanshinone I,tanshinone I,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),583.2088,303.0993,23
28、5.1118,263.1070,207.1173,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(9.108 min),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(9.737m
29、in),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-
30、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,SmCPS1 was the only class II diterpene cyclase invovled in tanshinones biosynthesis in the root of Danshen.Periderm is the place where tanshinones were biosynthesized and accumulated
31、Tanshinone biosynthesis is a complex network and it is likely that tanshinone I can be synthesized through different pathways,解析丹参二萜代谢途径,Center for Signal Transduction&Metabolomics,Applications of Metabolomics analysis,Nature Genetics(2006)Volume 38 number 7 July,P842-849,Center for Signal Transduct
32、ion&Metabolomics,Applications of Metabolomics analysis,Keurentjes et al.2006,14 Arabidopsis ecotypes and RILsUntargeted metabolomics analysis(LC-QTOF MS),Center for Signal Transduction&Metabolomics,Applications of Metabolomics analysis,Keurentjes et al.2006,Center for Signal Transduction&Metabolomic
33、s,Keurentjes et al.2006,Frequency distribution of the number of significant QTLs detected at each marker position at four significance level.,Number of masses detected in the RI population and its parents.,Not detected in the parents,Detected in both parents,Center for Signal Transduction&Metabolomi
34、cs,Keurentjes et al.2006,Applications of Metabolomics analysis,Before side chain modification,After side chain modification,Center for Signal Transduction&Metabolomics,Applications of Metabolomics analysis,Keurentjes et al.2006,Large genetic variations for mass peaks in the 14 accessions(only 13.4%o
35、f mass peaks ware detected in common in all 14 accession)75%of the 2000 mass peaks can be explained by QTLs in the RI population.Many(one-third)metabolites are produced as a result of the recombination of the genomes of the two parents,since they are absent in both parents.,Conclusion,In summary,aut
36、hors integrated high-throughput metabolomics and genotyping data from a large population cohort for elucidating the biochemical identities of unknown metabolites.To this end,authors applied metabolomics genome-wide association studies and Gaussian graphical modeling in order to link these unknown me
37、tabolites with known metabolic classes and biological processes.It is to be noted that the method does not specifically require genotyping data.Even metabolomics measurements alone,analyzed through the GGMs,may provide sufficient information for the classification and even precise identity predictio
38、n.One limitation of this approach is the requirement for associations with functionally described loci or known metabolites.,水稻种子低温萌发的代谢组分析,DNA markers,Phenotypes,Metabolites,pQTLs,mQTLs,Metabolic network/pathways controlled by QTLs,co-location correlation,15,10 days,15,10 days,萌发率分布图,RIL15 MH63 RIL
39、78 ZS97 RIL163,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63 RI群体的高密度SNP连锁图谱,Xie et al(2010)PNAS 107:10578Yu et al(2011)PloS One 6:e17595,1839 markers,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,Chr11,0 hour,2 days,4 day
40、s,6 days,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：15 oC,endosperm,embryo,ZS97,MH63,MH63,100 RILs x 4,EmbryoEndosperm,Polar fractionNon-polar fraction,Polar fractionNon-polar frac
41、tion,1,600 samples,GC-Tof/MS,水稻种子低温萌发的代谢组分析,代谢组分析方法,Internal standard correctionQuality control,mQTL analysis for RILs,GC-MS of samples,XCMS,Ion feature,Abundance filtering,Metabolite interaction network,Colocalization analysis,Multiple statistic analysis,Anova analysis,Candidate gene prediction,Mol
42、ecular mechanism,水稻种子低温萌发的代谢组分析,遗传代谢组学分析流程,Chr1 Chr2 Chr3 Chr4 Chr5 Chr6 Chr7 Chr8 Chr9 Chr10 Chr11 Chr12,Genetic Position(cM),Ion features,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mQTLs1417个QTLs(碎片离子丰度),3230,0,Xie et al,unpublished data,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6
43、3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,Metabolomics analysisGaussian graphical modeling(GGM)Genome-wide association analysis(GWAS),Identification of unknown metabolites based on GGM a
44、nd GWAS,GGMs are based on partial correlation coefficients,that is pairwise Pearson correlation coefficients conditioned against the correlation with all other metabolites.,Krumsiek et al.BMC System Biology.2011,5,Identification of unknown metabolites based on GGM and GWAS,Identification of unknown
45、metabolites based on GGM and GWAS,Though GGMs can reconstruct the metabolic pathway reaction and distinguish direct from indirect associations,it can never provide a perfect reconstruction of the underlying system.A.counterantagonistic correlation-generating processes and bimolecular reactions might
46、 lead to the elimination of pairwise associationB.the respective enzyme might not be active in the current metabolic state,or its effects on the respective metabolite pools are neglectable,Identification of unknown metabolites based on GGM and GWAS,Identification of unknown metabolites based on GGM
47、and GWAS,In total,we observe 34 distinct loci that display metabolite associations at a genome-wide significance level.Out of these 34 loci,15 associate with at least one unknown compound.For 12 loci,an unknown compound constitutes the strongest association of all tested compounds.,Identification of
48、 unknown metabolites based on GGM and GWAS,GGM provides a biochemical context for unknown metabolites,Identification of unknown metabolites based on GGM and GWAS,Specific pathway annotations of unknown metabolites,Identification of unknown metabolites based on GGM and GWAS,Example 1,Example 2,Metabo
49、lite data alone(without genotypic data)may provide sufficient information for the classification and even precise identity prediction when GGM method is used.One limitation of this approach is the requirement for associations with functionally described loci or known metabolites.,Identification of unknown metabolites based on GGM and GWAS,发现新现象提供基因功能的线索具有全面性和系统性-便于网络构建,书籍及期刊,相关信息,MetabolomicsSpringerBostonISSN:1573-3882(Print)1573-3890(Online)SpringerLink Date：2005年2月8日生物医学和生命科学,SpringerVerlag，2006,化工出版社，2011,Institute of Botany,Chinese Academy of Sciences,Beijing,Thank you!,
