关中奶山羊睾丸不同发育阶段代谢物的差异分析

【目的】 试验旨在从代谢组学角度探究关中奶山羊睾丸发育代谢机制。【方法】 选择1月龄断奶雄性关中奶山羊和24月龄体成熟的雄性关中奶山羊各6只,采集睾丸组织,使用液相色谱-质谱（LC-MS）技术对睾丸组织进行化学检测,通过多维和单维分析相结合的方式对两组不同代谢物进行化学检测、对比与鉴别,通过富集差异代谢物分析筛选关中奶山羊发育代谢中的潜在关键通路。【结果】 在1和24月龄关中奶山羊睾丸组织中共筛选出334个差异代谢物,其中显著上调137个,显著下调197个。对前36个差异代谢物进行比较鉴定发现,分别为脂质和类脂质分子、有机酸及其衍生物、未分类化合物和苯丙烷和聚酮化合物四大类。经相关性分析发现,在1和24月龄关中奶山羊睾丸发育过程中,脂质和类脂质分子与甘油磷脂呈最大正相关,羧基及其衍生物与甘油磷脂呈最大负相关。对不同的代谢物进行富集分析,筛选出7条潜在的重要代谢通路,分别为肿瘤中的胆碱代谢、精氨酸和脯氨酸代谢、肿瘤中的中心碳代谢、铁死亡、谷胱甘肽代谢、氨酰-tRNA生物组成以及牛磺酸和亚牛磺酸代谢。【结论】 本试验从代谢组学角度揭示了1和24月龄关中奶山羊睾丸的差异代谢物及代谢机制,为今后哺乳动物睾丸的发育研究奠定了基础。     

不同温度下褪黑素处理对‘阳光玫瑰'葡萄采后品质的影响

【目的】冷藏是提高葡萄耐储性、延长货架期的有效方法,但低温会降低糖酸和香气含量等果实品质。本研究旨在测定不同温度条件下褪黑素处理对‘阳光玫瑰'葡萄储藏期品质的影响,并探讨其调控储藏品质的代谢基础。【方法】设置室温和1℃两个储藏温度,葡萄采后利用5和50 μmol∙L^-1褪黑素浸果,于不同储藏期取样测定果实品质指标。利用HPLC-MS测定褪黑素含量,利用GC-MS测定香气组分及含量,利用毛细管电泳测定可溶性糖和有机酸含量,利用质构仪测定果实质地,利用广靶代谢组学分析褪黑素处理导致的差异代谢物。【结果】外源褪黑素处理能显著提高葡萄果实褪黑素含量;50 µmol∙L ^-1处理效果显著高于5 µmol∙L ^-1,并且低温能大幅提高褪黑素处理效果,低温、50 mol∙L^-1褪黑素处理的果实中褪黑素含量为室温同浓度处理的2.6倍。低温下,果实失水率降低;在室温和低温储藏下,褪黑素对果实失水率、果皮硬度和果肉硬度未产生显著影响。室温条件下,5 µmol∙L ^-1褪黑素处理显著提高了葡萄糖和果糖含量,而50 µmol∙L ^-1褪黑素产生了相反的结果。与室温储藏相比,低温储藏显著降低了果实糖含量;低温条件下,与对照相比,5 µmol∙L ^-1和50 µmol∙L ^-1褪黑素处理均显著提高了可溶性糖含量,储藏40 d时增幅在19.2%以上。与室温相比,低温提高了可滴定酸尤其是苹果酸含量。低温条件下,两种浓度褪黑素处理较对照显著降低了可滴定酸尤其是苹果酸含量,苹果酸含量降幅超过53.5%,对酒石酸含量影响较小。与室温储藏相比,低温储藏大幅度降低了未经褪黑素处理的果实香气含量。但是,褪黑素处理能有效增加低温条件下果实香气总量及香气组分含量,且以5 µmol∙L ^-1褪黑素处理效果较好,在处理后30和40 d,较低温对照香气总量增幅分别达2.12和1.6倍;同时显著增加了反式-2-己烯醛、里那醇、2,4-二叔丁基苯酚等特征香气含量。低温下对照和5 µmol∙L ^-1褪黑素处理的果实代谢组学分析表明,232种代谢物含量存在差异,涉及的主要代谢途径包括氨基酸生化合成、氨酰基-tRNA生化合成、精氨酸生化合成、丙氨酸、天冬氨酸和谷氨酸代谢及苯丙胺代谢。【结论】与室温储藏相比,低温冷藏降低了部分糖和绝大多数香气物质的含量。褪黑素处理能够显著提高冷藏果实的可溶性糖含量,降低有机酸水平,大幅提高香气水平,能有效提高果实品质,其中5 µmol∙L ^-1褪黑素处理效果更明显。褪黑素主要通过影响氨基酸代谢提高香气含量。     

宫内生长受限湖羊新生羔羊的血浆代谢组学研究

湖羊是我国珍贵的肉毛兼用的绵羊品种,并具有繁殖率高和宜舍饲等特点。在生产中,往往因其多胎性易导致妊娠期湖羊营养不良,造成新生羔羊宫内生长受限（intrauterine growth retardation,IUGR）。应用代谢组学一维氢谱核磁共振技术（¹H-nuclear magnetic resonance,¹H-NMR）研究新生羔羊的血浆代谢谱。根据出生体质量筛选出14只湖羊新生羔羊,分为正常出生体质量组（normal birth weight,NBW）和IUGR组（n=7）,采用¹H-NMR分析其血液样品。将代谢谱数据进行统计分析,并对鉴定的代谢物进行表达差异分析以及代谢途径归属。NBW组和IUGR组新生羔羊血浆样品共获得32种代谢物,16种代谢物在2组间差异表达,IUGR组有10个代谢物含量显著低于NBW组（P<0.05）,6个代谢物含量显著高于NBW组（P<0.05）。2组间差异显著的代谢通路主要为氨基酸、碳水化合物、脂类物质等代谢通路。应用基于¹H-NMR的代谢组学及生物信息学分析方法,确定了IUGR新生羔羊与正常羔羊血液代谢物之间存在差异,为进一步深入研究IUGR的分子机制奠定了基础。     

基于全基因关联分析的代谢组学在植物中的应用

植物代谢物是人类食物和营养物质的重要来源。代谢组学是对生物体内代谢物进行定量和定性分析,研究代谢物合成和调控机制的一门新兴学科。基于代谢组的全基因组关联分析（metabolome-based genome-wide association study,mGWAS）是将代谢组数据作为表型,与基因型数据进行关联分析的一种方法。概述了近几年利用mGWAS技术在植物代谢调控网络研究、初生和次生代谢物的形成机制、代谢物在植物生长发育和胁迫应答中的作用,同时综述了mGWAS在代谢物相关候选基因的定位和调控营养、品质相关代谢通路的挖掘,为深入了解植物代谢物合成调控的遗传机制奠定基础。     

Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library

In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]⁺ ions.     

Metabolomic Tools for Secondary Metabolite Discovery from Marine Microbial Symbionts

Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR ¹H and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening.     

代谢组学及其在动物营养中的应用

代谢组学是对内源性代谢物的综合评估,可以实现来自生物样品的代谢物及其相关代谢途径的分离、检测、表征和定量,以此来反映生物机体的营养代谢变化、营养状况甚至一些疾病的发展程度等。然而,代谢组学技术在动物营养中的研究起步较晚,目前尚处于起始阶段,不过随着代谢组学研究平台的不断完善,该技术在动物营养中的应用价值必将日渐突出。本文就代谢组学的概念特点、分析技术及其在动物营养中的应用作一综述。     

Volatile Metabolite Profiling for the Discrimination of Onion Bulbs Infected by Erwinia carotovora ssp. carotovora, Fusarium oxysporum and Botrytis allii

The volatile metabolites of the headspace gas of onion bulbs inoculated with three different pathogens, Erwinia carotovora ssp. carotovora, Fusarium oxysporum and Botrytis allii, were profiled using gas chromatography/mass spectrometry. Differences in the number and amount of volatile metabolites were observed. Two hundred and fifty three volatile metabolites were detected in bulbs inoculated with three pathogens or sterile distilled water. On day three, 202 volatile metabolites were observed, compared to 166 on day six. Of the 253 compounds, however, only 59 occurred relatively consistently over replications, of which 25 compounds were specific to one or more pathogens, including 10 that were unique to a pathogen. Metabolites such as 1-Oxa-4,6-diazacyclooctane-5-thione and 4-mercapto-3-(methylthio)--(thio-lactone)-crotonic acid were exclusive to onions inoculated with F. oxysporum. Acetone, acetic acid-hydrazide, propylcarbamate, 1-bromo-1-propene, thiirane, 1-(methylthio)-E-1-propene and 1-ethenyl-4-ethyl-benzene were specific to B. allii. 3-bromo-furan was specific to E. carotovora ssp. carotovora. Sterile water-inoculated bulbs produced 3,3-dioxy-1,2-propanediol-tetranitrate. Highest amount of sulfurs was found in pathogen-inoculated, while highest amounts of terpenes, aromatics and aliphatics were found in sterile distilled water-inoculated bulbs. The possible use of these differences in the volatile metabolites for detecting and discriminating diseases of onion in storage is discussed.     

基于GC-MS分析两地白色藜麦种子的代谢差异

为了探究四川盐源县和青海都兰县白色藜麦种子的代谢物质差异,对其进行非靶向代谢组学的分析。利用GC-MS技术以及多元统计分析对两地的白色藜麦种子进行比较,发现两地的藜麦种子代谢物质有差异,共检测到29个显著差异物质,主要包括15个氨基酸、6个有机酸和一些糖醇、胺类、多酚、生物碱。其中有22个代谢物质表达上调,7个表达下调,在上调表达代谢物中有12个氨基酸(甘氨酸、L-缬氨酸、谷胱甘肽、GABA等)和5个有机酸(乳清酸、肉桂酸、2-氧戊二酸等)。通过MetaboAnalyst进行通路分析,发现有24条代谢通路与上调表达代谢物相关,其中有7条通路影响较显著,分别为谷胱甘肽代谢,氮代谢,丙氨酸、天冬氨酸和谷氨酸代谢,氨酰基-tRNA生物合成,苯基丙氨酸代谢,硫代谢,甘氨酸、丝氨酸和苏氨酸代谢。谷胱甘肽代谢途径的影响最为显著,而此途径与植物的抗逆性密切相关,其中检测到的代谢物主要有甘氨酸和谷胱甘肽在四川盐源的藜麦中有更高的表达水平,表明四川盐源的白色藜麦较青海都兰的白色藜麦有更强的抗氧化能力,使得植物有较好的抗逆性能在逆境中生存,这为指导两地白色藜麦的栽培育种和抗逆性机理的研究提供了理论依据。