【作者】 吴琼；

【导师】 陈士林；

【作者基本信息】 中国协和医科大学 ， 生药学， 2010， 博士

【摘要】 功能基因组学(functional genomics)是利用结构基因组学提供的信息,应用高通量、大规模分析方法,在基因组或系统水平全面研究基因的表达、调控与功能,并探索基因间、基因与蛋白质之间、基因及其产物与生长发育相互联系和规律的科学。功能基因组学将生物学研究从单个基因或蛋白质上升至基因组,同时对多个基因和蛋白质进行系统研究。阐明药用植物生物活性成分的次生代谢途径及其调控是药用植物功能基因组学的主要内容之一。人参、西洋参和红豆杉是三种有重大药用和经济价值的药用植物,主要生物活性成分均为萜类化合物。本课题采用目前最新的技术,结合西洋参、人参、东北红豆杉的研究现状,从微观和宏观的不同角度,采用功能基因组学研究方法和地理信息系统技术,分别从转录组分析、药用天然产物生物合成途径、关键酶基因克隆与鉴定、产地适宜性等方面,有针对性地进行了研究。西洋参(Panax quinquefolius L.)为五加科(Araliaceae)人参属(Panax)的名贵药材,以根入药,具有促进记忆、抗癌、降血压、降血脂、抗疲劳、抗糖尿病、强心、镇静等多种药理作用。但由于生长缓慢,连作障碍严重,西洋参药用资源的发展受到极大的限制。本研究以四年生西洋参根、叶、花为材料,构建了cDNA文库,通过5′端测序,获得了根、叶、花的表达序列标签(expressed sequence tag, EST),共有6,678条ESTs,序列拼接产生了3,349条unigenes,包括534条contigs和2,815条singletons。通过转录组分析,探讨了组织特异性基因表达模式。采用Gene Ontology(GO)功能分类,将基因产物划分到生物学过程,细胞成分和分子功能三大本体。BLAST比对发现了24条参与人参皂苷生物合成的关键酶,对甲羟戊酸途径中的限速酶3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)进行了克隆与鉴定。采用同源克隆的方式,克隆了达玛烷合成酶(DS)基因。克隆了人参皂苷生物合成的最后一步糖基转移酶(GT)基因。发现了63条转录因子序列,编码包括zinc finger, WRKY, homeobox和MADS-box家族蛋白。并且发现了588条SSR微卫星标记,探讨了其组成与特点。这些结果,提供了西洋参转录谱、功能基因、转录调节、花发育和分子标记方面的重要信息,对于促进西洋参及其人参属药用植物的发展具有重要意义。红豆杉是重要的木本药用植物,是紫杉醇提取和半化学合成的重要来源之一。尽管商业和医药价值巨大,关于红豆杉属的基因组或转录组研究却很少。目前,紫杉醇生物合成途径还未完全阐明,紫杉醇和紫杉烷类生物合成的调控还不清楚。本研究应用新一代高通量测序技术454 GS FLX Titanium对东北红豆杉(Taxus cuspidata)针叶转录组进行测序,共获得表达序列标签(EST)81,148条。序列拼接得到20,557条unique sequences,包括12,957条singletons和7,582条contigs。数据库中的序列同源性比较表明,68.6%(14,095)与其它生物的已知基因具有同源性。研究发现总的序列同源性在裸子植物中高于被子植物。GO分类将11,220条序列注释到45个类目上。通过生物化学途径注释,2,403条序列注释到3,821条KEGG途径中。生物信息学分析,获得了质体赤藓糖磷酸酯途径(methylerythritol phosphate pathway)的所有酶以及参与紫杉醇生物合成的部分酶的基因。发现了可能参与紫杉醇合成未阐明步骤酶的基因,包括85条CYP450,7条epoxidase,18条CoA ligases和3条N-benzoyltransferase。从数据中还获得了291个转录因子和753个微卫星标记(SSR)。这些基因的发现为紫杉醇和紫杉烷类化合物生物合成研究和代谢调控提供了有利条件,也为红豆杉属植物种质改良奠定了基础。石柱参是中国特有人参珍稀品种,具有较高的经济价值。为了对人参种植区划筛选提供科学依据,利用中药材产地适宜性分析地理信息系统(TCMGIS),以辽宁省宽甸县石柱参现代分布区最适宜生长环境因子为依据,区划石柱参在全国的适宜产地。结果显示适宜种植区(相似度为95%-100%)所在县区有15个,总面积为15,943.93平方公里,辽宁省占96.44%,吉林省占3.56%；较适宜种植区(相似度为90-95%)所在县区有22个,总面积20,070.83平方公里,辽宁省占73.88%,吉林省和秦岭中东部地区也有一定份额。研究结果覆盖了石柱参现代分布区,从一定程度上验证了TCMGIS系统用于石柱参适生地分析的科学性和准确性,预测了石柱参潜在适宜分布区。对于规划人参种植区域,保护优良品种,把人参生产规划在真正的适宜产地具有重要的指导作用。本文的研究工作,分别探讨了西洋参和红豆杉两种药用植物的基因表达谱,代谢途径相关功能基因,代谢调控机制,并预测了石柱参的适生栽培区域。这些研究工作为重要药用次生代谢产物的生物合成研究,功能基因的开发和利用,药用植物的品种改良和人工引种提供了基础数据和理论支持。更多还原

【Abstract】 Based on the information provided by structural genomics and the application of high throughput, large-scale analysis, functional genomics is a comprehensive way of study in gene expression, regulation and function, as well as to explore relationship and laws between genes, genes and proteins, and both with the growth and development at the level of genome. Biological research is increased from a single gene or protein to the genome based on functional genomics, while a lot of genes and proteins are systematically studied at the same time. Clarification the pathways and regulation of secondary metabolic pathway is one of the main contents of functional genomics in medicinal plants. Ginseng, American ginseng and yew are three medicinal plants with significantly medical and economic value and the main active ingredients are terpenoids. The three medicinal plants were selected for the study in this paper, from micro and macro perspectives, using functional genomics methods and geographic information system technology, respectively. Studies have focused on the transcriptome analysis, medicinal natural product biosynthesis, the key enzyme gene cloning and identification, and producing area suitability, respectively.American ginseng (Panax quinquefolius L.) for the Araliaceae (Araliaceae) are one of valuable medicinal herbs in the world and its roots are used as medicine, with the promotion of memory, anti-cancer, lowering blood pressure, blood fat, anti-fatigue, anti-diabetic, cardiac, sedative and other pharmacological effects. However, because of slow growth and succession cropping obstacles, the development of American ginseng as medicinal resources is extremely limited. The major bioactive phytochemicals responsible for this plant’s pharmacological features are ginsenosides. Thus far, little is known regarding the genes involved in ginsenosides biosynthesis in this species. In this study, we generated 6678 expressed sequence tags (ESTs) from the flower, leaf, and root cDNA libraries of American ginseng. Assembly of ESTs resulted in 3349 unigenes including 534 contigs (with ESTs number ranging from 2 to 52) and 2815 singletons. By analyzing the predominant transcripts within specific tissues, a gene expression pattern was obtained in a tissue-specific manner. They were assigned according to the functional classification of unigenes to broad ranges of Gene Ontology (GO) categories which include biological processes, cellular components and molecular functions. Based on BLASTX search results,24 unigenes representing candidates related to ginsenosides biosynthesis were identified. Cloning and characterization of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, EC:1.1.1.34), the rate-limiting enzyme in mevalonic acid (MVA) pathway, demonstrated that it belonged to the plant HMGR family and was highly expressed in leaves. Putative transcription factors were detected in 63 unigenes, including zinc finger, WRKY, homeobox and MADS-box family proteins. Five hundred eighty eight SSR motifs were identified, of which, dimer was the most abundant motif. These data will provide useful information on transcript profiles, gene discovery, transcriptional regulation, flower biogenesis, and marker-assisted selections. The analysis and information from this study will greatly contribute to the improvement of this medicinal plant as well as of other species in the Araliaceae family, for the purpose of ensuring adequate drug resources.Taxus species are highly valued both as renewable resources for the production of Taxol and as important trees for biodiversity conservation within the Northern Hemisphere. Thus far, few genomic or transcriptomic data are available for the members of this genus as demonstrated by only 179 expressed sequence tags (ESTs) in GenBank. In this study,81,148 high-quality reads from the needles of Taxus cuspidata were produced using the Roche GS FLX Titanium system. A total of 20,557 unique sequences were obtained, including 12,975 singletons and 7,582 contigs, with EST numbers ranging from 2 to 2,051. A total of 14,095 (68.6%) unique sequences shared a significant similarity to the sequences in public databases and have orthologs in other organisms. Gene Ontology revealed 11,220 (54.6%) unique sequences that could be assigned to 45 vocabularies. In the Kyoto Encyclopedia of Genes and Genomes mapping,2,403 transcripts were established as associated with 3,821 biochemical pathways. All enzymes in the plastidial 2-C-methyl-D-erythritol 4-phosphate pathway were well represented together with 8 of the 12 characterized enzymes involved in Taxol biosynthesis. Data mining homologues correlated with the putative remaining steps revealed candidates of cytochrome P450, epoxidase, coenzyme A ligase, and N-benzoyltransferase in the data sets. In total,291 transcripts were identified, representing putative homologues of a wide array of transcription factor families. There was also 753 simple sequence repeat motifs identified, of which hexamer was the most abundant motif. In the meantime, the results of this study revealed that a substantial number of transcripts had no obvious orthologs to known databases and might represent peculiar transcripts within the Taxus. These results provide the largest EST collections in Taxus and are essential for future efforts of gene discovery, functional genomics, and genome annotation in related species.In order to provide scinetific evidences for screening potential areas suitable for growing Chinses Shizhu Panax, adaptability and associated growing areas of its were analyzed using TCMGIS. Based on the optimum ecological data collected from Kuandian County, Liaoning Province, a geographic information system was established to analyze the adaptability of potential areas suitable for growing Chinses Shizhu Panax. Results showed that fifteen of areas under investigation enjoyed a similarity index of 95-100%, which adds up to 15943.93 km2 in area. These most adaptive areas mainly located in Liaoning Province and Jilin Province, which accounted for 96.44% and 3.56%, respectively. Results also showed that twenty two of areas under investigation having a similarity index of 90-95%, which adds up to 20070.83 km2 in area. These adaptive areas mainly located in Liaoning Province, Henan Province, Shanxi Province, and Jilin Province, which accounted for 73.88%,16.02%,9.78%, and 3.2%, respectively. The results went along with the current growing areas. Meanwhile, the potential growing regions had been identified by TCMGIS. The results in current study demonstrated the rationality and reliability of TCMGIS. The finding provides important evidances for the P. ginseng cultivation planning, protecting fine varieties, and growing the species under national standards.Data derived from present study will provide theoretical support for the research of functional genomics in medicinal plants and will lead to better utilization and development of natural medicinal resources in the future for the purpose of drug improvement.更多还原