高分辨质谱在植物代谢物分析中的应用

发布时间：2018-06-01 08:13

本文选题：植物代谢组学 + 植物激素　；参考：《中国农业大学》2017年博士论文

【摘要】：植物代谢物种类繁多、结构迥异,是植物生命活动重要的体现者。对植物代谢物进行系统深入的研究,挖掘代谢物背后蕴含的生物学意义,有利于加强对植物生命活动现象、本质与规律的理解,对增强植物对不良环境的抗性、提高作物产量与品质具有重要意义。本论文应用基于高分辨质谱的植物代谢组学技术,建立了 6种植物激素与40种游离氨基酸的检测方法;结合基因表达分析,研究了茉莉酸对拟南芥代谢组的影响;对大刍草-玉米代谢组进行了比较研究,并对玉米驯化过程中的代谢物变异进行了初步的遗传解析。植物激素检测面临着含量低、性质不稳定、基质复杂的困难,本研究应用液-液萃取与同位素稀释技术,建立了 6种植物激素的检测方法。结果表明,液-液萃取提供了较高的提取效率,同位素稀释校正了样品分析过程中的偏差,超高效液相色谱与高分辨质谱保证了方法的特异性与灵敏度。对方法的准确度、精度、灵敏度、线性和回收率进行评价,表明该方法满足实际分析的需要,同时使用该方法对拟南芥中植物激素的时间与空间分布进行了初步的分析。应用柱前衍生、反相超高效液相色谱与高分辨质谱,建立了 40种游离氨基酸的检测方法。在解决了氨基酸极性较强、缺少显色基团问题的同时,提高了分离效率与检测灵敏度。对方法的特异性、线性、灵敏度、回收率和重现性进行验证,该方法具有快速、高效和高灵敏度的优点。结合主成分分析,对多种蔬菜中的游离氨基酸含量进行了比较。结果表明,不同蔬菜中游离氨基酸的种类大致相同,但其含量存在显著差异;该方法适用于基质复杂的植物材料,也可以扩展到果汁、水果等其它相关样品的分析。对拟南芥野生型、茉莉酸合成缺失突变体opr3以及茉莉酸处理后的opr3突变体进行了代谢组分析,一共检测到超过10000个化合物离子,其中2000多个离子信号在不同的基因型或处理组间存在显著差异。统计分析结果表明拟南芥野生型与突变体opr3存在明显的代谢物差异,外源茉莉酸处理能使突变体中部分代谢物逐渐恢复到野生型的水平。本实验还建立了一个差异代谢物数据库,包含了 174个代谢物的保留时间、母离子与特征二级碎片的精确质量,以及109个代谢物的化学结构。对鉴定到的差异代谢物进行了聚类分析与通路分析,结果表明硫代葡萄糖苷与色氨酸代谢、脂类代谢、氨基酸、小肽等都受到了茉莉酸的调节。使用荧光定量反转录PCR对相关通路中的重要基因进行了表达量的检测,验证了代谢组的结果,这些结果揭示了植物激素与植物代谢物之间的相互作用与调节网络,加强了我们对茉莉酸生物功能的理解。比较了 30个大刍草和40个玉米品系的代谢组,检测了 624份玉米与大刍草重组自交系群体中的代谢物,并对挖掘到的重要代谢物进行靶标分析。结果表明玉米与大刍草代谢组存在差异,鉴定到97个玉米与大刍草间分化的代谢物。在重组自交系群体中,鉴定到了 299个有QTL定位结果的代谢物,这些代谢物种类各异,参与了多条重要的代谢通路。后续对重要差异代谢物的遗传解析,并克隆相关基因对玉米的遗传改良具有重要的指导意义。
[Abstract]:Plant metabolites are diverse and different in structure. It is an important embodiment of plant life activities. The systematic and in-depth study of plant metabolites and the biological significance behind the metabolites are beneficial to the understanding of plant life activities, the understanding of the nature and the law, the resistance to the adverse environment and the increase of crop yield. In this paper, 6 kinds of plant hormones and 40 kinds of free amino acids were established based on high resolution mass spectrometry, and the effects of jasmonic acid on the metabolic group of Arabidopsis were studied in combination with gene expression analysis. Preliminary genetic analysis of metabolite variation in the process is carried out. Plant hormone detection is faced with low content, unstable properties and complex matrix. In this study, liquid liquid extraction and isotope dilution technique were used to establish 6 methods for detecting plant hormones. The results showed that liquid liquid extraction provided high extraction efficiency and isotopic dilution. The accuracy, accuracy, sensitivity, linearity and recovery of the method were evaluated by ultra high performance liquid chromatography and high resolution mass spectrometry. The method met the needs of actual analysis, and the time and space of plant hormones in Arabidopsis were used. 40 kinds of free amino acid detection methods were established by using pre column derivatization, reversed phase ultra high performance liquid chromatography and high resolution mass spectrometry. The separation efficiency and detection sensitivity were improved. The specificity, linearity, sensitivity and recovery of the method were improved. This method has the advantages of rapid, high efficiency and high sensitivity. The content of free amino acids in various vegetables is compared with principal component analysis. The results show that the species of free amino acids in different vegetables are roughly the same, but the content of free amino acids is significantly different. This method is suitable for complex matrix plants. The material can also be extended to the analysis of other related samples, such as fruit juice, fruit, and other related samples. The metabolic group analysis was carried out on the Arabidopsis wild type, the jasmonic acid synthesis deletion mutant opr3 and the opr3 mutant after jasmonic acid treatment. More than 10000 compound ions were detected, of which more than 2000 ion signals were stored in different genotypes or treatment groups. The statistical analysis showed that there was a significant difference in metabolites between the Arabidopsis wild type and the mutant opr3. Exogenous jasmonic acid treatment could make some metabolites in the mutant gradually recover to the level of the wild type. A different metabolite database was established in this experiment, including the retention time of 174 metabolites, the mother ion and the mother ion. The exact mass of the two grade fragments and the chemical structure of the 109 metabolites were characterized by cluster analysis and pathway analysis of the identified metabolites. The results showed that glucosinolates and tryptophan metabolism, lipid metabolism, amino acids, small peptides were regulated by jasmonic acid. The fluorescence quantitative reverse transcriptional PCR was used in the related pathways. The results of the metabolic group were verified by the detection of the important genes, which revealed the interaction and regulation network between plant hormones and plant metabolites, and enhanced our understanding of the biological function of jasmonic acid. The metabolic groups of 30 rice and 40 maize lines were compared, and 624 corn and rice were detected. The metabolites in the population of the inbred lines were reorganized and the important metabolites were analyzed. The results showed that there were differences in the metabolic group between the maize and the rice. The metabolites differentiated between 97 maize and the rice were identified. In the recombinant inbred population, 299 metabolites with QTL location results were identified, and the metabolites of these metabolites were different. The genetic analysis of important differential metabolites and cloning of related genes have important guiding significance for the genetic improvement of maize.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q945

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