紫外诱导对圆锥铁线莲次生代谢的影响及其差异蛋白质组学研究
发布时间：2019-01-02 09:43
【摘要】：紫外光根据波长范围可分为三种：UV-A(320-400 nm)、UV-B(290-320nm)和UV-C(200-290 nm),作为一种非生物胁迫因子对地球生物产生了显著的影响,紫外光主要通过影响生物体的DNA、蛋白质合成以及各种信号传导途径而使生物体的生理代谢以及外部形态发生明显变化。地球表面的植被是保持地球生物循环以及生物圈完整性的重要组成部分,植物体表面大多分布有蜡质层、角质层、纤毛以及其它类型的坚硬组织,同时植物体在感受环境胁迫时会调整自身生理及代谢来抵御外界不良因素的影响,因此植物体对于紫外光胁迫的适应能力超过动物及微生物。紫外光能够对植物体产生多方面的影响,如使植株变矮、影响光合作用、影响蛋白质合成等,此外,植物体本身可以产生大量的次生代谢物质来抵御紫外光的损害,这些次生代谢产物多为苯丙素类化合物、生物碱、萜类化合物等,有些次生代谢产物是中药和天然药物活性成分,对于疾病的防治具有重要意义。 蛋白质组学是对生物体或生物体的某种器官、组织、细胞以及相关基因所表达的所有蛋白质进行研究,它涉及到蛋白质的表达、蛋白质的氨基酸序列、蛋白质的结构功能、翻译后修饰以及蛋白质之间的相互作用等内容,是生命科学中与基因组学密切相关的重要研究领域之一。蛋白质在生物体结构组成、生物体的生长、发育、繁殖、代谢等各个环节都发挥着极其重要的作用,也是引起生物多样性的重要因素。随着基因组学的不断发展以及人们对基因组研究的不断深入,科研工作者发现对于生物体内直接行使功能的蛋白及酶的研究将成为继基因组之后的重要的研究领域。 圆锥铁线莲系毛茛科铁线莲属植物,其地上部分在浙江北部地区被广泛用于治疗呼吸系统、泌尿系统炎症以及肿瘤,其含有的主要天然活性成分(次生代谢产物)为黄酮及木脂素类化合物。 本课题对离体的圆锥铁线莲叶片进行不同时间以及不同波长范围的紫外光诱导,通过诱导前后指纹图谱的对比,发现紫外光对圆锥铁线莲次生代谢产物有显著的影响,并对诱导后发生变化的次生代谢产物进行分离纯化以及结构鉴定。通过我们的研究发现,UV-A对圆锥铁线莲新鲜叶片次生代谢的影响并不明显,而在120.83微瓦/平方厘米强度的UV-B下诱导一定时间后,指纹图谱中出现了三个新的色谱峰,以色谱峰的峰面积为指标进行诱导条件的筛选,发现随着UV-B诱导时间的不断延长,三个色谱峰的峰面积有不断增加的趋势,并确定最佳的诱导时间为4-6小时。对圆锥铁线莲新鲜叶片进行大量诱导后烘干,对三个新峰进行追踪分离,采用UV、IR、NMR以及MS对分离得到的三个化合物进行结构鉴定,最终发现UV-B能够诱导圆锥铁线莲产生香豆素类化合物,它们分别是2H,8H-Benzo[1,2-b:5,4-b']dipyran-2-one,10-hydroxy-8,8-dimethyl-(9CI)、4,6,7-三甲氧基-5-甲基香豆素和鲁望橘内酯。 为了揭示紫外光诱导圆锥铁线莲产生香豆素类化合物生物合成的机理,本课题采用双向电泳的方法对诱导前后叶片的差异蛋白进行了研究,通过Image Master5.0软件处理,得到七十三个差异点,通过MALDI-TOF-MS分析对差异蛋白进行鉴定。 综合上述内容,本课题研究的创新之处在于：①通过对照指纹图谱发现了不同波段紫外光对圆锥铁线莲次生代谢的影响,并对诱导后新产生的色谱峰(次生代谢产物)进行了分离纯化和结构鉴定；发现UV-B诱导能够使圆锥铁线莲叶片中产生一系列香豆素类化合物,不仅有简单香豆素,还包括线型吡喃香豆素；②发现UV-B对圆锥铁线莲的影响在诱导八小时之内随着诱导时间的不断延长而变得更加明显；③采用双向电泳技术对圆锥铁线莲紫外诱导前后的差异蛋白质组学进行了研究,最终得到七十三个差异蛋白点,并对差异蛋白进行了质谱鉴定,从蛋白质水平揭示了紫外光对圆锥铁线莲的影响。
[Abstract]:UV-A (320-400 nm), UV-B (290-320nm) and UV-C (200-290 nm) can be divided into three types: UV-A (320-400 nm), UV-B (290-320nm) and UV-C (200-290 nm). Protein synthesis and various signal transduction pathways have a significant change in the physiological metabolism and the external morphology of the organism. the vegetation of the earth's surface is an important part of the preservation of the biological cycle of the earth and the integrity of the biosphere, and the surface of the plant body is mostly distributed with a wax layer, a stratum corneum, a cilia and other types of hard tissue, at the same time, the plant body can adjust the physiological and metabolism of the plant to resist the influence of the external adverse factors when the environment stress is felt, so that the plant body can adapt to the ultraviolet light stress to exceed the animal and the microorganism. The ultraviolet light can produce various effects on the plant body, such as dwarf the plant, affect the photosynthesis, influence the protein synthesis, and the like, in addition, the plant body can generate a large amount of secondary metabolites to resist the damage of the ultraviolet light, Some secondary metabolites are the active components of traditional Chinese medicine and natural medicine, and it is of great significance to the prevention and treatment of diseases. Proteomics is a study of all proteins expressed by certain organs, tissues, cells, and related genes of an organism or organism, which involves the expression of a protein, the amino acid sequence of a protein, the structure of a protein, The interaction of function, post-translational modification and the interaction of proteins is an important field of research that is closely related to genomics in the life science One of the important roles of the protein in the structure of the organism, the growth, the development, the reproduction and the metabolism of the organism is also an important part of the biological diversity. With the development of the genomics and the further development of the research of the gene group, the researchers have found that the research of the protein and the enzyme that directly exercise the function in the organism will be an important study after the genome The present invention relates to the field of the genus Clematis, which is widely used in the treatment of the respiratory system, the inflammation of the urinary system and the tumor in the northern part of Zhejiang, and contains the main natural active components (secondary metabolites) as the flavone and the wood fat. In this paper, the effects of ultraviolet light on the secondary metabolism of the conical iron clematis were found by the comparison of different time and different wavelength range of the cone-shaped clematis leaves of the body, and by the comparison of the fingerprints of the before and after the induction. The product has a significant effect and the secondary metabolites that change after induction are isolated The results showed that the effect of UV-A on the secondary metabolism of the fresh leaves of Clematis cone was not obvious, and after a certain time was induced under UV-B of the intensity of 120. 83 microwatts/ cm2, the fingerprint of the fresh leaves was found in the fingerprint. Three new peaks were selected, and the peak area of the peak was selected as the index, and the peak area of the three peaks was continuously increased with the time of UV-B induction, and the best induction was determined. The time is 4-6 hours. After a large number of induction and drying on the fresh leaves of the conical iron clematis, the three new peaks are tracked and separated, and the three compounds obtained by separation are carried out by using the UV, IR, NMR and the MS, and finally, the UV-B can be used for inducing the production of the conical iron clematis. Coumarin compounds, which are 2H, 8H-Benzo[1,2-b: 5,4-b '] dipyrian-2-one, 10-hydroxy-8,8-dimetyl-(9CI), 4,6,7-trimethoxy-5-methylfragrance, respectively In order to reveal the mechanism of the biosynthesis of coumarin compounds induced by ultraviolet light, the differential protein of the leaves before and after induction was studied by the method of two-dimensional electrophoresis. then, seventy three difference points are obtained, and by means of MALDI-TOF-MS, The results of this study are as follows: the effect of different band ultraviolet light on the secondary metabolism of the conical iron clematis is detected by the control of the fingerprint spectrum, and the newly generated chromatographic peak (secondary metabolism) after induction is obtained. The results showed that UV-B induced the production of a series of coumarin compounds, not only was the simple coumarin, but also the linear coumarins. The effect of UV-B on the conical iron clematis was induced within eight hours after induction. The effect of two-dimensional electrophoresis on the proteomics of the differential protein before and after the ultraviolet induction of the conical iron-line lotus was studied, and the different protein points were obtained, and the difference protein was identified by mass spectrometry, and the protein was removed from the protein level.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
本文编号：2398341
[Abstract]:UV-A (320-400 nm), UV-B (290-320nm) and UV-C (200-290 nm) can be divided into three types: UV-A (320-400 nm), UV-B (290-320nm) and UV-C (200-290 nm). Protein synthesis and various signal transduction pathways have a significant change in the physiological metabolism and the external morphology of the organism. the vegetation of the earth's surface is an important part of the preservation of the biological cycle of the earth and the integrity of the biosphere, and the surface of the plant body is mostly distributed with a wax layer, a stratum corneum, a cilia and other types of hard tissue, at the same time, the plant body can adjust the physiological and metabolism of the plant to resist the influence of the external adverse factors when the environment stress is felt, so that the plant body can adapt to the ultraviolet light stress to exceed the animal and the microorganism. The ultraviolet light can produce various effects on the plant body, such as dwarf the plant, affect the photosynthesis, influence the protein synthesis, and the like, in addition, the plant body can generate a large amount of secondary metabolites to resist the damage of the ultraviolet light, Some secondary metabolites are the active components of traditional Chinese medicine and natural medicine, and it is of great significance to the prevention and treatment of diseases. Proteomics is a study of all proteins expressed by certain organs, tissues, cells, and related genes of an organism or organism, which involves the expression of a protein, the amino acid sequence of a protein, the structure of a protein, The interaction of function, post-translational modification and the interaction of proteins is an important field of research that is closely related to genomics in the life science One of the important roles of the protein in the structure of the organism, the growth, the development, the reproduction and the metabolism of the organism is also an important part of the biological diversity. With the development of the genomics and the further development of the research of the gene group, the researchers have found that the research of the protein and the enzyme that directly exercise the function in the organism will be an important study after the genome The present invention relates to the field of the genus Clematis, which is widely used in the treatment of the respiratory system, the inflammation of the urinary system and the tumor in the northern part of Zhejiang, and contains the main natural active components (secondary metabolites) as the flavone and the wood fat. In this paper, the effects of ultraviolet light on the secondary metabolism of the conical iron clematis were found by the comparison of different time and different wavelength range of the cone-shaped clematis leaves of the body, and by the comparison of the fingerprints of the before and after the induction. The product has a significant effect and the secondary metabolites that change after induction are isolated The results showed that the effect of UV-A on the secondary metabolism of the fresh leaves of Clematis cone was not obvious, and after a certain time was induced under UV-B of the intensity of 120. 83 microwatts/ cm2, the fingerprint of the fresh leaves was found in the fingerprint. Three new peaks were selected, and the peak area of the peak was selected as the index, and the peak area of the three peaks was continuously increased with the time of UV-B induction, and the best induction was determined. The time is 4-6 hours. After a large number of induction and drying on the fresh leaves of the conical iron clematis, the three new peaks are tracked and separated, and the three compounds obtained by separation are carried out by using the UV, IR, NMR and the MS, and finally, the UV-B can be used for inducing the production of the conical iron clematis. Coumarin compounds, which are 2H, 8H-Benzo[1,2-b: 5,4-b '] dipyrian-2-one, 10-hydroxy-8,8-dimetyl-(9CI), 4,6,7-trimethoxy-5-methylfragrance, respectively In order to reveal the mechanism of the biosynthesis of coumarin compounds induced by ultraviolet light, the differential protein of the leaves before and after induction was studied by the method of two-dimensional electrophoresis. then, seventy three difference points are obtained, and by means of MALDI-TOF-MS, The results of this study are as follows: the effect of different band ultraviolet light on the secondary metabolism of the conical iron clematis is detected by the control of the fingerprint spectrum, and the newly generated chromatographic peak (secondary metabolism) after induction is obtained. The results showed that UV-B induced the production of a series of coumarin compounds, not only was the simple coumarin, but also the linear coumarins. The effect of UV-B on the conical iron clematis was induced within eight hours after induction. The effect of two-dimensional electrophoresis on the proteomics of the differential protein before and after the ultraviolet induction of the conical iron-line lotus was studied, and the different protein points were obtained, and the difference protein was identified by mass spectrometry, and the protein was removed from the protein level.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
【引证文献】
相关硕士学位论文 前1条
1 卢晓萍;长春花叶中次生代谢产物响应活性酶诱导的研究[D];浙江大学;2013年
，本文编号：2398341
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