8-王军军__组学技术及其在氨基酸营养研究中的应用.pdf

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1、Application of Omics Technology in AA Nutrition:from Development to RegulationJunjun Wang, Guoyao Wu, Defa LiState Key Laboratory of Animal NutritionCollege of Animal Science & TechnologyChina Agricultural University-Amino AcidImportant Role of Amino Acid? Synthesis of protein? Other nitrogenous sub

2、stances (NO, urea, polyamines, GSH ) ? Glucose? Fatty acids? Signaling molecularTraditional Approaches in AA Nutrition? Digestibility trials? Nitrogen balance? Assessments of performance? Isotope tracer techniques Protein turnoverAA synthesis, catabolism and fluxDNAProteinMolocular Biology Era(1953-

3、)Nutrigenomics and NutrigeneticsNutrigenomicsEmergening of Omics TechnologiesNutrigenetics? Single nucleotide polymorphisms (SNPs)? Gene copy number variants110 are from British, 1130 from SwedishUrine from British and SwedishUrine from Japanese, Chinese and American1H NMR spectra of urine from (A)

4、Japanese, (B) Chinese, and (C) American.AA transporters in duodenum, jejunum, ileum and colon and different speciesThe interactive effect of methyl diets and MTHFR polymorphismSNPs identificationDNA sequencingRestriction fragment length polymorphism, RFLPSingle-strand conformation polymorphism, SSCP

5、Denaturing gradient gel electrophoresis, DGGELow ThroughputHigh ThroughputTaqManSNP Genotyping AssayMALDI-TOF Genotyping Affymetrix 500K SNPChip ArrayAffymetrix GeneChip and Copy Number AnalyzerGene copy number analysisHigh resolution copy number estimationsAllelic imbalancesFetal programming_Intrau

6、terine growth restrictionIUGR正常正常新生仔猪新生仔猪Differentially expressed miRNA in small intestine of normal and IUGR pigletsDifferentially expressed miRNA (ratio 5)microRNA 名称名称 ID 号号IUGR/正常正常hsa-miR-29a/mmu-miR-29a/rno-miR-29a 1103911.49599 hsa-miR-218-1* 4294111.25171 hsa-miR-29b/mmu-miR-29b/rno-miR-29b

7、110409.864681 hsa-miR-29c/mmu-miR-29c/rno-miR-29c 110418.150336 mmu-miR-544 427997.334313 hsa-miR-218-2*/mmu-miR-218-2*/rno-miR-218*428157.100329 hsa-miRPlus-E1068 465047.027606 mmu-miR-705 275745.623602 hsa-miR-491-3p 179275.561725 hsa-miRPlus-E1247 464455.113036 hsa-miR-1274a 462920.190909 hsa-miR

8、Plus-E1271 465390.190615 hsa-miR-141/mmu-miR-141/rno-miR-141 109460.17717 hsa-miR-1248 464270.147373 Changes in muscle proteome after compensatory growth in pigsThe relation between the methionine cycle and methylationEffects of High Methionine Intake on DNA MethylationAvy/a Site and Coat Color of R

9、atsDietary L-Arginine Supplementation Reduces Fat Mass in Rats4w: 6%;6w: 10%;10w: 16%Fat is specifically reducedMicro-array analysis of adipose cell gene expressionEffect of Dietary Glutamine Supplementation on 21- to 28-Day-Old Weanling PigletsParameterControl Glutamine Daily weight gain, g/d146 10

10、174 13 *GSSG/GSH, nmol/nmol0.087 0.005 0.051 0.002 *Villus height, m 269 7.5 371 9.2 * P 0.05Dietary Glutamine Supplementation Affects Intestinal Dietary Glutamine Supplementation Affects Intestinal Gene Expression in Piglet Intestine (Microarray Study)Gene Expression in Piglet Intestine (Microarray

11、 Study)Down-regulated genes related to apoptosis & stressKLF-10 - 64%La protein homolog - 41%Peptidyl-prolyl isomerase- 37%MAPK-6 - 75% ICAM-1 - 38%Up-regulated genes related to proliferation, oxidative defense, and nutrient metabolismAF-p protein + 121%Heme-binding protein + 124%Myosin + 122%Signal

12、 recognition particle-72k + 121%Enhanced expression of structural proteins in microvascular cells treated with L-arginineMethods involved in transcripteomicsLow Throughput Northern Blotting Real Time Quantitative RT-PCR In Situ HybridizationMiddle Throughput DD-PCR SSH cDNA AFLP SAGEHigh Throughput

13、DNA Microarray (Genechip, Affymetrix)Proteomics Technologies_Protein SeparationMALDI-MSESI-MS/MSProteomics Technologies_Mass Spectrometry肽质量指纹谱匹配肽质量指纹谱匹配肽序列标签匹配肽序列标签匹配Proteomics Technologies_DatabaseSeparate by 2-D PAGEProtein extract 1Label with fluor 1Protein extract 2Label with fluor 2Mix labelle

14、d extractsExcitation wavelength 1Image gelExcitation wavelength 2Image analysis: overlay imagesAnalysis of differenceImage analysis:data quantitationICAT标记的标记的Cys轻轻重重合并，胰酶消化合并，胰酶消化利用生物素亲和纯化含有利用生物素亲和纯化含有Cys的肽段的肽段质谱技术质谱技术根据峰的面积计算相应蛋白质的含量根据峰的面积计算相应蛋白质的含量MS / MS 测序测序同位素标记的亲和标签技术同位素标记的亲和标签技术ICAT（isotope-

15、coded affinity tags）技术）技术ICAT试剂结构Consumption of animal and plant proteins on metabolic profilesVegetarian diet High-meat diet Creatine, Creatinine, Carnitine, Acetylcarnitine, Aurine, Trimethylamine-N-oxide, Glutamine p-hydroxyphenylacetateN,N,N-trimethyllysineMeat intake may promote higher producti

16、on of ammonia, but consumption of vegetable protein may reduce intestinal microbial activity Consumption of milk and meat protein on metabolic profiles Milk dietMeat dietHippurate (metabolite of glycine)CreatineHistidineUreaMilk diet changed the lipid profile of serum, but the meat diet had no effec

17、t on the metabolic profile of serumArg on performance of neonatal growth and gilt reproduction Metabolomics and Nutritional AssessmentAssessment of Adequacy and Safety of Amino Acid IntakeBioactive factors screening for biological fluid? NMR-MS? LC-MS? GC-MS 代谢组学研究的主要技术Two strategies of nutrigenomics researchThe smart combination of molecular nutrition and nutrigenomicsThanks for your Attention!