【摘要】：紫外光根據(jù)波長(zhǎng)范圍可分為三種：UV-A(320-400 nm)、UV-B(290-320nm)和UV-C(200-290 nm),作為一種非生物脅迫因子對(duì)地球生物產(chǎn)生了顯著的影響,紫外光主要通過(guò)影響生物體的DNA、蛋白質(zhì)合成以及各種信號(hào)傳導(dǎo)途徑而使生物體的生理代謝以及外部形態(tài)發(fā)生明顯變化。地球表面的植被是保持地球生物循環(huán)以及生物圈完整性的重要組成部分,植物體表面大多分布有蠟質(zhì)層、角質(zhì)層、纖毛以及其它類(lèi)型的堅(jiān)硬組織,同時(shí)植物體在感受環(huán)境脅迫時(shí)會(huì)調(diào)整自身生理及代謝來(lái)抵御外界不良因素的影響,因此植物體對(duì)于紫外光脅迫的適應(yīng)能力超過(guò)動(dòng)物及微生物。紫外光能夠?qū)χ参矬w產(chǎn)生多方面的影響,如使植株變矮、影響光合作用、影響蛋白質(zhì)合成等,此外,植物體本身可以產(chǎn)生大量的次生代謝物質(zhì)來(lái)抵御紫外光的損害,這些次生代謝產(chǎn)物多為苯丙素類(lèi)化合物、生物堿、萜類(lèi)化合物等,有些次生代謝產(chǎn)物是中藥和天然藥物活性成分,對(duì)于疾病的防治具有重要意義。 蛋白質(zhì)組學(xué)是對(duì)生物體或生物體的某種器官、組織、細(xì)胞以及相關(guān)基因所表達(dá)的所有蛋白質(zhì)進(jìn)行研究,它涉及到蛋白質(zhì)的表達(dá)、蛋白質(zhì)的氨基酸序列、蛋白質(zhì)的結(jié)構(gòu)功能、翻譯后修飾以及蛋白質(zhì)之間的相互作用等內(nèi)容,是生命科學(xué)中與基因組學(xué)密切相關(guān)的重要研究領(lǐng)域之一。蛋白質(zhì)在生物體結(jié)構(gòu)組成、生物體的生長(zhǎng)、發(fā)育、繁殖、代謝等各個(gè)環(huán)節(jié)都發(fā)揮著極其重要的作用,也是引起生物多樣性的重要因素。隨著基因組學(xué)的不斷發(fā)展以及人們對(duì)基因組研究的不斷深入,科研工作者發(fā)現(xiàn)對(duì)于生物體內(nèi)直接行使功能的蛋白及酶的研究將成為繼基因組之后的重要的研究領(lǐng)域。 圓錐鐵線蓮系毛茛科鐵線蓮屬植物,其地上部分在浙江北部地區(qū)被廣泛用于治療呼吸系統(tǒng)、泌尿系統(tǒng)炎癥以及腫瘤,其含有的主要天然活性成分(次生代謝產(chǎn)物)為黃酮及木脂素類(lèi)化合物。 本課題對(duì)離體的圓錐鐵線蓮葉片進(jìn)行不同時(shí)間以及不同波長(zhǎng)范圍的紫外光誘導(dǎo),通過(guò)誘導(dǎo)前后指紋圖譜的對(duì)比,發(fā)現(xiàn)紫外光對(duì)圓錐鐵線蓮次生代謝產(chǎn)物有顯著的影響,并對(duì)誘導(dǎo)后發(fā)生變化的次生代謝產(chǎn)物進(jìn)行分離純化以及結(jié)構(gòu)鑒定。通過(guò)我們的研究發(fā)現(xiàn),UV-A對(duì)圓錐鐵線蓮新鮮葉片次生代謝的影響并不明顯,而在120.83微瓦/平方厘米強(qiáng)度的UV-B下誘導(dǎo)一定時(shí)間后,指紋圖譜中出現(xiàn)了三個(gè)新的色譜峰,以色譜峰的峰面積為指標(biāo)進(jìn)行誘導(dǎo)條件的篩選,發(fā)現(xiàn)隨著UV-B誘導(dǎo)時(shí)間的不斷延長(zhǎng),三個(gè)色譜峰的峰面積有不斷增加的趨勢(shì),并確定最佳的誘導(dǎo)時(shí)間為4-6小時(shí)。對(duì)圓錐鐵線蓮新鮮葉片進(jìn)行大量誘導(dǎo)后烘干,對(duì)三個(gè)新峰進(jìn)行追蹤分離,采用UV、IR、NMR以及MS對(duì)分離得到的三個(gè)化合物進(jìn)行結(jié)構(gòu)鑒定,最終發(fā)現(xiàn)UV-B能夠誘導(dǎo)圓錐鐵線蓮產(chǎn)生香豆素類(lèi)化合物,它們分別是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-甲基香豆素和魯望橘內(nèi)酯。 為了揭示紫外光誘導(dǎo)圓錐鐵線蓮產(chǎn)生香豆素類(lèi)化合物生物合成的機(jī)理,本課題采用雙向電泳的方法對(duì)誘導(dǎo)前后葉片的差異蛋白進(jìn)行了研究,通過(guò)Image Master5.0軟件處理,得到七十三個(gè)差異點(diǎn),通過(guò)MALDI-TOF-MS分析對(duì)差異蛋白進(jìn)行鑒定。 綜合上述內(nèi)容,本課題研究的創(chuàng)新之處在于：①通過(guò)對(duì)照指紋圖譜發(fā)現(xiàn)了不同波段紫外光對(duì)圓錐鐵線蓮次生代謝的影響,并對(duì)誘導(dǎo)后新產(chǎn)生的色譜峰(次生代謝產(chǎn)物)進(jìn)行了分離純化和結(jié)構(gòu)鑒定；發(fā)現(xiàn)UV-B誘導(dǎo)能夠使圓錐鐵線蓮葉片中產(chǎn)生一系列香豆素類(lèi)化合物,不僅有簡(jiǎn)單香豆素,還包括線型吡喃香豆素；②發(fā)現(xiàn)UV-B對(duì)圓錐鐵線蓮的影響在誘導(dǎo)八小時(shí)之內(nèi)隨著誘導(dǎo)時(shí)間的不斷延長(zhǎng)而變得更加明顯；③采用雙向電泳技術(shù)對(duì)圓錐鐵線蓮紫外誘導(dǎo)前后的差異蛋白質(zhì)組學(xué)進(jìn)行了研究,最終得到七十三個(gè)差異蛋白點(diǎn),并對(duì)差異蛋白進(jìn)行了質(zhì)譜鑒定,從蛋白質(zhì)水平揭示了紫外光對(duì)圓錐鐵線蓮的影響。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R341

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 盧曉萍;長(zhǎng)春花葉中次生代謝產(chǎn)物響應(yīng)活性酶誘導(dǎo)的研究[D];浙江大學(xué);2013年

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本文編號(hào)：2398341