几种唇形科植物萜类合酶及杂萜gwa-1组合生物合成研究

	几种唇形科植物萜类合酶及杂萜gwa-1组合生物合成研究
	王安
导师	黎胜红
关键词	黄芩 Scutellaria baicalensis 半枝莲 Scutellaria barbata 萜类合酶 Terpenoid synthase β-石竹烯合酶 β-caryophyllene synthase 大麻素类 Cannabinoid derivatives
摘要	摘要本研究论文一共由四章组成。第一章对4种唇形科药用植物，包括黄芩（Scutellaria baicalensis Georgi）、半枝莲（Scutellaria barbata D.Don）、一串红（Salvia splendens）和薰衣草（Lavandula angustifolia）中的β-石竹烯合酶进行了分析和功能鉴定；第二章对黄芩和半枝莲中其它萜类合酶进行了克隆和功能鉴定；第三章利用工程大肠杆菌对“非天然的”大麻素类衍生物gwa-1进行了组合生物合成研究；第四章综述了黄芩和半枝莲的二萜类成分及其生物活性和生物合成研究进展。第一章唇形科植物中β-石竹烯合酶的多样性和功能研究唇形科植物富含挥发性芳香油，具有重要的生物功能、药理活性和经济价值。黄芩和半枝莲为唇形科（Lamiaceae）黄芩属（Scutellaria）多年生药用植物，其中黄芩干燥根和半枝莲干燥全草被《中国药典》所收录；一串红、薰衣草分别为唇形科鼠尾草属（Salvia）和薰衣草属（Lavandula）植物，具有很高的观赏和经济价值。本章对这4种唇形科植物的挥发性代谢物进行了分析，发现均含有丰富的挥发性萜类化合物，其中β-石竹烯是这4种植物共有的萜类成分；从这4种植物中各克隆了一个β-石竹烯合酶候选基因，分别为ScbTPS1、SbTPS15、SsTPS1和LaTPS1，系统进化分析显示4个萜类合酶基因均属于TPS-a家族。通过体外酶促实验结合GC-MS产物分析，鉴定ScbTPS1、SbTPS15、SsTPS1为β-石竹烯合酶，而LaTPS1为β-法尼烯合酶。荧光定量PCR分析发现β-石竹烯合酶基因在黄芩茎、叶、根中均有表达，且表达水平相当，此外还发现石竹烯在黄芩中的积累受生物节律调控。以上研究结果为β-石竹烯合酶起源进化研究以及β-石竹烯的开发利用提供了参考价值。第二章黄芩和半枝莲中其他萜类合酶的功能鉴定本章从黄芩和半枝莲中分别克隆了9个和5个萜类合酶候选基因，并运用大肠杆菌原核表达体系对它们进行了功能鉴定。体内酶活产物分析功能鉴定了黄芩中ScbTPS4为多功能的倍半萜合酶，通过跟踪分离结合NMR数据鉴定其中一个产物为(-)-δ-杜松烯，通过标品比对鉴定其中一个产物为germacrene D，通过质谱比对初步鉴定另外两个产物为cadinol类化合物，进一步跟踪分离和鉴定正在进行中；通过体内酶活产物分析鉴定半枝莲中SbTPS16为二萜柯巴基焦磷酸酯合酶。蛋白可溶性分析表明其余候选基因表达的重组蛋白可溶性良好，但运用原核表达体系通过体内和体外酶促分析均未发现特异产物，后续将通过更换表达体系对这些候选基因的功能进行研究。研究结果为黄芩和半枝莲萜类化合物的生物合成研究奠定了基础。第三章杂萜化合物gwa-1的合成生物学研究杂萜是一类由萜类生物合成途径与其它生源途径偶联生成的一类次生代谢产物，其结构丰富，活性多样。大麻萜酚酸（CBGA）属于聚酮型杂萜，是合成大麻素的前体，具有抗炎、神经保护、抗菌等多种活性。本章从土曲霉（Aspergillus terreus）、链霉菌（Streptomyces sp）和镰刀菌（Fusarium sp）中分别筛选到一个候选的异戊烯基转移酶Trt2、Orf2和AscC。运用高产萜类直链前体的大肠杆菌结合底物橄榄酸饲喂实验发现，AscC能够催化橄榄酸发生异戊烯基化反应，合成大麻素前体衍生物2,4-Dihydroxy-3-(3-methyl-2-buten-1-yl)-6-pentylbenzoic acid（gwa-1），而Trt2和Orf2均不能使橄榄酸异戊烯基化。研究结果为通过组合生物合成技术丰富大麻素类化合物的化学多样性奠定了基础。第四章黄芩和半枝莲中二萜成分的分离鉴定、生物活性及生物合成研究进展本章综述了黄芩和半枝莲两种唇形科药用植物中的二萜类次生代谢产物及其生物活性和生物合成。黄芩中一共报道了57个克罗烷型二萜；半枝莲中报道了克罗烷型二萜及克罗烷型二萜生物碱类化合物共111个。绝大部分化合物具有一定的肿瘤细胞毒活性。崔光红团队对半枝莲中的部分萜类合酶进行了功能鉴定，目前仍关于黄芩萜类合酶则鲜有报道，这两种植物萜类化合物的生物合成途径及形成机制仍需深入研究。本章为黄芩和半枝莲萜类成分的化学、生物活性及生物合成研究提供了参考。; Abstract This dissertation is made up of four chapters. In chapter 1, the function and diversity of β-caryophyllene synthases of four Labiaceae plants, including Scutellaria baicalensis, Scutellaria barbata, Salvia splendens and Lavandula angustifolia, were investigated. In chapter 2, other terpene synthases from S. baicalensis and S. barbata were cloned and functionally characterized. In chapter 3, an “unnatural” cannabinoid derivative gwa-1 was biosynthesized using engineered Escherichia coli. In chapter 4, the research progress on diterpenoids, biological activities, biosynthesis of S. baicalensis and S. barbata were reviewed. Chapter 1 Investigation on the diversity and function of β-caryophyllene synthase in Labiaceae plants Labiaceae plants are rich in volatile aromatic oils with important biological functions, pharmacological activities and economic values. S. baicalensis and S. barbata are both perennial medicinal plants, belonging to the genus Scutellaria of Lamiaceae, and the dried root of S. baicalensis and the dried whole herb of S. barbata are included in Chinese Pharmacopoeia. S. splendens and L. angustifolia belong to the genus Salvia and Lavender respectively, which have ornamental and economic values. The volatile metabolites of the above four plants were analyzed and they are particularly rich in volatile terpenoids, among which β-caryophyllene was the common component. Four β-caryophyllene candidate genes, ScbTPS1, SbTPS15, SsTPS1 and LaTPS1, were cloned from S. baicalensis, S. barbata, S. splendens and L. angustifolia, respectively. Phylogenetic analysis showed that the four terpene synthase genes all belonged to TPS-a family. By in vitro enzyme activity assay combined with GC-MS analysis, ScbTPS1, SbTPS15 and SsTPS1 were identified as β-caryophyllene synthase, while LaTPS1 was identified as β-farnesene synthase. The quantitative reverse transcription PCR analysis showed β-caryophyllene synthase gene was expressed in stem, leaf and root tissues of S. baicalensis, and the expression level was comparable in three tissues. Biorhythm analysis showed that the accumulation of β-caryophyllene was regulated by internal biological clock. These results provided reference values for investigation on the origin and evolution of β-caryophyllene synthases as well as further development and utilization of β-caryophyllene. Chapter 2 Functional identification of other terpene synthases from Scutellaria baicalensis and Scutellaria barbata Altogether nine and five candidate terpene synthase genes were cloned from S. baicalensis and S. barbata, respectively, and their functions were studied using prokaryotic Escherichia coli expression system. ScbTPS4 was identified as a multifunctional sesquiterpene synthase by in vivo enzyme activity analysis. One of the ScbTPS4 products was identified as (-)-δ-cadinene through tracking separation and NMR anaylsis, the other of the products was identified as germacrene D by authentic comparison. The other tw
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75095
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	王安. 几种唇形科植物萜类合酶及杂萜gwa-1组合生物合成研究[D]. 中国科学院大学,2022.

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