基于cDNA文库的毛喉鞘蕊花CPS基因的克隆分析及多效唑促进isoforskolin合成机制研究

发布时间：2018-04-22 02:41

本文选题：毛喉鞘蕊花 + 生物合成　；参考：《湖北中医药大学》2017年博士论文

【摘要】：毛喉鞘蕊花(Coleus forskohlii(Willd.)Briq.)系唇形科(Labiatae)鞘蕊花属(Coleus Lour.)草本植物,主产印度、巴基斯坦、斯里兰卡、阿拉伯半岛南部等热带和亚热带国家。该植物在我国云南、广东、福建、台湾等地亦稀有分布,被植物学家界定为珍稀植物。毛喉鞘蕊花药材名为鞘蕊苏,收载于《中国植物志》66卷和《云南省药品标准》,民间用于治疗哮喘、咳嗽等疾患,疗效显著,被称为“万灵药”;现代科学研究表明,该植物药中含有的半日花烷型二萜类化合物具有激活腺苷酸环化酶、强心、降压、抗肿瘤等广泛的药理作用。鉴于毛喉鞘蕊花药用植物的独特的药效作用,本课题组与湖北福人药业公司产学研结合,开发了中药新品种--“鞘蕊苏胶囊”,该药物已经获得中药新药生产批件(批件号:2011S00365)。为了保护野生毛喉鞘蕊花稀有植物的生态环境,本实验室湖北福人药业公司合作,从云南会泽县移植毛喉鞘蕊花至湖北通城县实施规范化种植研究,以期解决“鞘蕊苏胶囊”产业化的原料药材大量需求。本论文首先采用分子生物学研究方法对毛喉鞘蕊花二萜类化合物合成酶基因克隆和调控进行了研究,构建了二萜合成酶(TPS)基因研究平台,高通量筛选有效成分TPS基因,分析其结构特点与功能,阐释其表达特征;并以上述平台为基础,研究多效唑提高毛喉鞘蕊花有效成分及指标成分isofoskolin的分子机制。本论文研究可为通过植物生理学方法提高毛喉鞘蕊花有效成分提供方法,并为种植毛喉鞘蕊花主要有效成分的代谢和富集提供科学依据,以期通过遗传改良手段培育优良种质资源提供基础,进而从源头提高种植药材的品质。主要研究结果如下:1.毛喉鞘蕊花全长均一化cDNA文库的构建以毛喉鞘蕊花根、根茎、茎、叶、花为材料,采用SMART法与DSN均一化相结合构建了全长均一化cDNA文库,库容数约为1600,转化菌液体积1.5ml,计算得出菌液滴度是为1.6×106cfu/ml。以整个连接10μl计算库容大于5.2×106。从文库中随机挑取30个克隆进行菌落pcr鉴定,结果显示cdna插入片段介于1-3kb。因此,本研究构建的毛喉鞘蕊花全长均一化cdna文库质量较高。2.est测序及生物信息学分析从cdna文库中随机挑选了4224个单克隆进行sanger测序,经过组装得到2394条unigene,unigene平均长度753bp。对unigene进行nr、go及kegg功能注释。2263条unigene被nr数据库注释,在这些基因中存在有大量的涉及生长发育、物质和能量代谢等方面的基因。go功能分类显示,2100条(占87.7%)注释到了一个或多个go类别,所得go类别的总数为12121,分属于3个go主类别。kegg代谢途径分析表明,1716条(占71.7%)unigene被分配到6个主代谢途径中,其中17个unigenes被确定可能参与类萜生物合成的骨干和单萜、二萜、三萜2个二萜合成酶(柯巴烯焦磷酸合酶、贝壳杉烯氧化酶)、3个单萜合成酶(月桂烯/罗勒烯合酶、新薄荷醇脱氢酶、柠檬烯合酶)和1个三萜合成酶(β-香树脂醇合成酶)。3.毛喉鞘蕊花cfcps基因的克隆、序列分析及表达研究在est测序的基础上,从unigene中挑选柯巴烯焦磷酸合酶(cps)基因序列的片段,利用race技术得到全长cdna序列,结果表明cfcps基因全长2418bp。构建表达载体,利用大肠杆菌表达体系,获得了cfcps蛋白。对毛喉鞘蕊花cfcps进行生物信息学分析发现,该序列的全长为2418bp,编码805个氨基酸,分子量为92235,分子式为c4166h6415n1101o1219s29,pi为5.70。与迷迭香(rosmarinusofficinalis)、丹参(salviamiltiorr)、欧夏至草(marrubiumvulgare)、板栗(castananeamollissima)、桃(prunuspersica)、甘草(scopariadulcis)、番茄(solanumlycopersucum)、玉米(zeamays)植物的tps蛋白进行多重比对,发现它们具有较高的同源性性。通过构建系统进化树,发现cfcps蛋白与迷迭香、丹参tps蛋白的亲缘关系比较近。进一步研究发现,cfcps蛋白有跨膜区域和信号肽,属于可溶性蛋白。利用q PCR检测毛喉鞘蕊花Cf CPS基因在植物体的表达发现,毛喉鞘蕊花CfCPS基因在根表达量较高。4.多效唑提高毛喉鞘蕊花有效成分的分子机制在上述鞘蕊苏cDNA文库研究获得的萜类生物合成关键基因的基础上,我们又开展了多效唑处理提高毛喉鞘蕊花二萜类成分生物合成的研究。结果表明,多效唑(100mg/L)处理毛喉鞘蕊花植株,可显著改善植株株型,并显著提高毛喉鞘蕊花根、根茎和茎中isoforskolin含量,提高幅度高达53%。qPCR检测多效唑处理后叶片及根中萜类生物合成关键基因的表达水平发现,叶中ACCT、HMGR、DXR、IDI、GGPS和KAO等6个基因的表达被显著诱导,根中除CPS基因的表达量先升后降外,其余几个基因的表达量均呈下降趋势。因此,我们推测多效唑提高毛喉鞘蕊花有效成分的分子机制主要是诱导ACCT、HMGR、DXR、IDI、GGPS、KAO和CPS等基因的表达。
[Abstract]:Coleus forskohlii (Willd.) Briq.), a herb (Coleus Lour.) of the family of the family of the family of the family of the family of the family of the family (Labiatae), which mainly produces tropical and subtropical countries such as India, Pakistan, Sri Lanka, and southern Arabia peninsula. The plant is also rare in Yunnan, Guangdong, Fujian, and Taiwan in China and is defined by botanists as rare plants. The medicinal herbs of the hairy larynx are named as sheathing, contained in the Chinese flora >66 roll and the Yunnan medicine standard. The folk are used to treat asthma, cough and other diseases. The curative effect is remarkable. The modern scientific research shows that the semidiurane type two terpenes contained in the plant medicine are activated by adenylate cyclase, strong heart and descending. In view of the unique pharmacological effects of pressure and anti tumor, in view of the unique pharmacological effects of the medicinal plants of the hairy larynx flower, we have developed a new variety of Chinese medicine, the "sheath Rui Su capsule", combined with the production and research of Hubei Furen pharmaceutical company, which has obtained the production batch of Chinese traditional medicine (batch number: 2011S00365). In order to protect the wild hairy larynx sheath The ecological environment of rare plants and the cooperation of Hubei Furen pharmaceutical company in our laboratory, the standardized planting study was carried out from Huize County, Yunnan, to Hubei Tongcheng County, Hubei, in order to solve the large demand for the industrialization of the "sheath Rui Su capsule". First, the molecular biological research method was used in this paper to two The cloning and regulation of terpene synthetase genes were studied. The two terpene synthetase (TPS) gene was constructed and the TPS gene was screened by high throughput. Its structural features and functions were analyzed and its expression characteristics were analyzed. Based on the above platform, the effective components and index components isofoskol of PDF were improved. The molecular mechanism of in. This paper can provide a method for improving the effective components of the hair larynx flower through plant physiology, and provide a scientific basis for the metabolism and enrichment of the main effective components of the hairy larynx flower, in order to cultivate fine germplasm resources based on genetic improvement, and then improve the planting material from the source. The main results are as follows: 1. the total length homogenization cDNA Library of the larynx sheath is constructed with the root, rhizome, stem, leaf and flower of the larynx sheath. The full homogenization cDNA library is constructed by combining the SMART method with the homogenization of DSN. The number of the storage capacity is about 1600, the volume of the transformed bacteria is 1.5ml, and the drop of the liquid is calculated to be 1.6 * 106cfu/ml.. The total connection of 10 mu l was more than 5.2 * 106., and 30 clones were randomly selected from the library to identify the colony PCR. The results showed that the cDNA insertion fragment was 1-3kb., and 4224 single grams of single gram were randomly selected from the cDNA library for the high.2.est sequencing and the bioinformatics analysis of the full-length homogenization cDNA library of the larynx. For Sanger sequencing, 2394 UniGene were obtained by assembly, and the average length of UniGene was NR, go and KEGG functional notes were annotated by NR database. There were a large number of gene.Go functional categories involving growth and development, material and energy metabolism in these genes, and 2100 (87.7%) annotated to the genes. One or more go categories, the total number of GO categories was 12121, and 3 go main category.Kegg metabolic pathways showed that 1716 (71.7%) UniGene were assigned to 6 main metabolic pathways, and 17 of them were determined to be involved in the backbone and monoterpene, two terpenes, and three terpenoids, 2 two terpene synthetases (ocbene pyrophosphate). The clones of 3 monoterpene synthetases (laurenene / alenene synthase, new menthol dehydrogenase, limonene synthase) and 1 three terpenoid synthetases (beta arenol synthetase).3. hairy larynx cfcps gene were cloned, sequence analysis and expression studies on EST sequencing and selected cocobene pyrophosphate synthase from UniGene (CPS) the whole length cDNA sequence of the gene sequence was obtained by race technique. The result showed that the full length 2418bp. of the cfcps gene was constructed and the cfcps protein was obtained by using the Escherichia coli expression system. The bioinformatics analysis of the cfcps of the coliform flower showed that the whole length of the sequence was 2418bp, 805 amino acids were encoded, and the molecular weight was 92235. C4166h6415n1101o1219s29, PI, 5.70. and rosemary (Rosmarinusofficinalis), Salvia miltiorrhiza (salviamiltiorr), marrubiumvulgare, castananeamollissima, peach (prunuspersica), licorice (scopariadulcis), tomato (solanumlycopersucum), and maize (Zeamays) plant TPS protein multiple comparison, found that they The phylogenetic tree of the phylogenetic tree showed that the relationship between cfcps protein and rosemary and Salvia miltiorrhiza TPS protein was close. Further studies found that cfcps protein had cross membrane region and signal peptide, which belonged to soluble protein. Q PCR was used to detect the expression of Cf CPS gene of hairy larynx flower in plant body, and the CfC was found to be CfC. On the basis of the molecular mechanism of the PS gene, which is higher in the root expression of.4., the molecular mechanism of the effective components of the flower of the larynx is improved. On the basis of the key genes of terpene biosynthesis obtained by the aaaaau cDNA library, we have also carried out a study on the biointegration of two terpenoids from the flowers of the larynx. Treatment of the plant type of hairy larynx can significantly improve plant plant type and significantly increase the content of isoforskolin in the root, rhizome and stem of the hairy larynx, and increase the expression level by 53%.qPCR to detect the expression level of the key genes of terpene biosynthesis in the leaves and roots after the treatment of Paclobutrazol, and the expression of 6 genes, such as ACCT, HMGR, DXR, IDI, GGPS and KAO in leaves, are expressed. The expression of the other genes in the root was descended when the expression of CPS gene was raised first and then decreased. Therefore, we speculate that the molecular mechanism of Paclobutrazol to improve the effective components of the larynx flower is mainly to induce the expression of ACCT, HMGR, DXR, IDI, GGPS, KAO and CPS.

【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S567;Q943.2

【参考文献】