基于转录组测序的紫外线促进鞘蕊苏有效成分isoforskolin合成机制研究

发布时间：2018-01-11 21:08

本文关键词：基于转录组测序的紫外线促进鞘蕊苏有效成分isoforskolin合成机制研究　出处：《湖北中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：鞘蕊苏药材的植物名为毛喉鞘蕊花(Coleus forskohlii(Willd.)Briq.)系唇形科(Labiatae)鞘蕊花属(Coleus Lour.)植物,主产于印度、尼泊尔、巴基斯坦、斯里兰卡、缅甸、泰国等南亚国家;我国云南、福建、台湾等地亦有分布,被植物学家界定为珍稀植物。民间将该药材用于治疗哮喘、咳嗽等疾患,疗效显著,称为“神药”。现代科学研究表明,鞘蕊苏的有效成分半日花烷型二萜类化合物,我国产鞘蕊苏的有效成分为异佛司可林(isoforskolin),并将其定为指标性成分。鉴于鞘蕊苏独特药效作用,本实验室与湖北福人药业股份有限公司实施产学研结合,以鞘蕊苏为君药,配伍前胡、甘草组方,将其开发为“鞘蕊苏胶囊”中药新品种,获得生产批号(批件号:2011S00365)。为了解决鞘蕊苏胶囊产业化所需原料药材的供求问题,实验室与湖北福人药业股份有限公司合作,从云南会泽县引种鞘蕊苏至湖北通城县规范化种植。针对通城种植鞘蕊苏药材的主要二萜类成分isoforskolin含量偏低的问题,本论文对该药用植物二萜类成分的生物合成机制进行了研究,以期为通城种植鞘蕊苏培育优良种质;并进行了紫外线照射采收的鞘蕊苏药材,诱导其主要有效成分isoforskolin增加的生物合成机制研究,以期为促进种植鞘蕊苏主要有效成分的生物合成和积累提供科学依据,并为通过遗传改良手段培育鞘蕊苏优良种质提供基础,也为二萜化合物的代谢机制的深入研究提供重要信息。本研究的主要研究结果如下:1.基于Illumina HiSeq高通量测序的鞘蕊苏转录组分析以通城县种植基地的鞘蕊苏为试验材料,于10月下旬采集该药材的叶和根作为供试品,采用HiSeq技术进行转录组测序,获得36Gb转录组数据,拼接后得到422,761条高质量序列;对组装的Unigene基因进行预测,获得CDS序列和Protein序列;采用Trinity和TGICL的方法从头组装、序列拼接和去冗余处理,得到291,061个Unigene基因,平均长度629bp;将所得Unigene基因与Nr、Swiss-Prot、KEGG和COG公共蛋白数据库进行比对,140,228(48.19%)的unigene基因能够获得注释信息。将140,228个注释信息的unigene基因进行go和cog聚类分析及kegg代谢通路分析,86,578条unigene基因获得cog功能注释,被归类为25个功能分类;共有186,957条unigene基因获得go功能注释,被归入到49个go次级功能分类;共有6,616个unigene可以分配到261个代谢通路。因此,本项研究为鞘蕊苏有效成分的萜类合成酶基因克隆,序列分析及表达研究奠定了基础,并且根与叶中23个萜类差异基因则为传统育种和转基因育种的基因调控靶点提供了研究基础。2.基于高通量测序的鞘蕊苏萜类合成酶基因克隆、序列分析及表达研究在上述高通量基因测序研究基础上,从注释信息数据中挑选基因片段,采用race技术进行序列分析,获得三个萜类合成酶的全长cdna序列,并通过blast和蛋白序列分析。结果如下:(1)cfgcs基因全长2059bp,包含235bp的5’非编码区,1632bp的开放式阅读框(orf),192bp的3’非编码区,编码544个氨基酸;对蛋白理化性质进行预测,cfgcs相对分子质量为63690.84;等电点为5.23;分子式为c2868h4438n738o848s27。qpcr表明cfgcs在鞘蕊苏叶的表达量较高。(2)cfvps基因全长1874bp,包含116bp的5’非编码区,1647bp的orf,111bp的3’非编码区;cfvps相对分子质量为63608.40,等电点为5.06,分子式为c2859h4412n744o852s24。以cfvps为目标基因,构建表达载体pet28-cfvps,将构建好的表达载体于大肠杆菌中进行原核表达,32℃时诱导6h,菌液上清中有大量表达。cfvps参与植物单萜化合物合成,而鞘蕊苏叶片表面有丰富的腺毛分布,是单萜合成的主要场所,qpcr结果也表明cfvps在叶中的表达量最高。(3)cfgas基因全长1234bp,包含72bp的5’非编码区,993bp的orf,169bp的3’非编码区;cfgas相对分子质量为36895.20,等电点为6.37,分子式为c1672h2575n437o482s12。与山茶花、丹参、番薯零、大麻、烟草、碧冬茄、节节麦、小麦进行同源性比较,发现cfcps蛋白与丹参GA蛋白的亲缘关系比较近。同时通过Gateway的方法进行GAS过量表达重组载体构建,为后续发根农杆菌侵染提供实验基础。qPCR结果表明CfGAS参与赤霉素的合成,赤霉素是二萜类植物激素,在植物的整个生长过程中发挥着重要作用,在鞘蕊苏各个组织中均有分布。3.紫外线照射提高鞘蕊苏药材有效成分含量的分子机制研究本实验前期研究表明,通过紫外线照射鞘蕊苏药材能提高其二萜类有效成分的含量。基于上述鞘蕊苏高通量转录组研究所获萜类生物合成酶关键基因的启示,本论文进行了紫外线照射诱导鞘蕊苏二萜类成分生物合成机制研究。研究结果表明,紫外线照射能够有效提高鞘蕊苏药材根部isoforskolin含量,提高幅度高达53%。通过q PCR检测紫外照射鞘蕊苏后萜类生物基因表达水平,表明TPS1、TPS2、TPS3、TPS4、TPS14表达量明显受紫外线的诱导而升高,而TPS15、赤霉素合成酶和贝壳杉烯合成酶受紫外线诱导而降低。本研究为提高种植鞘蕊苏药材有效成分含量提供了科学依据,并为提高规范化种植中药材的资源品质探索了新途径。
[Abstract]:Qiaoruisu medicinal plant named Coleus forskohlii (Coleus forskohlii (Willd.) Briq.), Labiatae (Labiatae) Coleus plant (Coleus Lour.), mainly in India, Nepal, Pakistan, Sri Lanka, Burma, Thailand and other South Asian countries; China's Yunnan, Fujian, Taiwan and other places also have the distribution is defined as a botanist of rare plants. The folk medicine for the treatment of asthma, cough and other diseases, the curative effect is remarkable, known as the "God of medicine". Modern scientific research shows that half effective components of Qiaoruisu flower type two terpenoids, effective components of China Qiaoruisu for isoforskolin (isoforskolin), and it is the index components. Given the role of Qiaoruisu unique efficacy, the laboratory and Hubei Furen pharmaceutical Limited by Share Ltd to implement the combination with Qiaoruisu Weijun medicine compatibility decursivum, licorice prescription, will be developed as "Qiaoruisu capsule "New varieties of Chinese medicine, to obtain production batches (approval number: 2011S00365). In order to solve the problem of supply and demand of Qiaoruisu capsule industry required for raw materials, laboratory in cooperation with Hubei Furen pharmaceutical Limited by Share Ltd, from the introduction of Yunnan Huize County Qiaoruisu to Hubei Tongcheng County standardized cultivation for planting. Tongcheng Qiaoruisu medicine major two low isoforskolin content of terpene components, the biosynthetic mechanism of two terpenoids in the medicinal plants were studied, in order to Tongcheng plant Qiaoruisu to cultivate excellent germplasm; and the ultraviolet irradiation to harvest Qiaoruisu herbs, on the mechanism of biosynthesis induced by the main effective components of isoforskolin increased, in order to promote the biosynthesis of plant Qiaoruisu main effective components and accumulation to provide a scientific basis, and for genetic improvement through breeding germplasm sheath Rui Su Youliang provided For the foundation, also provide important information for further study on the metabolic mechanism of two terpene compounds. The main results of this study are as follows: 1. based on the Qiaoruisu transcriptome Illumina HiSeq high-throughput sequencing analysis in Tongcheng County planting base Qiaoruisu as experimental material, in late October acquisition of the crude drug as a test for Ye Hegen product of transcriptome sequencing using HiSeq technology, 36Gb transcriptome data, obtained by splicing 422761 high quality sequences; Unigene gene of assembled prediction, CDS sequence and Protein sequence; using Trinity method and TGICL de novo assembly sequence splicing and redundant processing, 291061 Unigene genes, average length 629bp; the Unigene gene and Nr, Swiss-Prot, KEGG and COG were compared to the public protein database, 140228 (48.19%) of the UniGene gene to obtain annotation information. The 140228 letter notes Go analysis and cog cluster analysis and KEGG pathway of UniGene gene the UniGene gene was 86578 cog functional annotation, were classified into 25 functional categories; a total of 186957 UniGene go gene functional annotation, be classified into 49 sub function go classification; a total of 6616 UniGene can be assigned to 261 the metabolic pathway. Therefore, terpene synthase gene cloning for effective components of Qiaoruisu this study laid the foundation for the study of sequence analysis and expression of 23 genes and terpenoids in roots and leaves for differences in gene regulation of traditional breeding and transgenic breeding control target provides the research foundation.2. Qiaoruisu terpene synthase gene cloning based on high-throughput sequencing, sequence analysis and expression based on the high-throughput gene sequencing research, selected gene fragment from the annotation information data, sequence analysis was performed using race technology, three The full-length cDNA sequence of terpene synthases, and through blast and protein sequence analysis. The results are as follows: (1) the full-length 2059bp cfgcs gene, 235bp contains 5 'non encoding region, open reading frame of 1632bp (ORF), 192bp 3' non encoding region, encoding 544 amino acids; prediction of eggs white physicochemical properties, relative molecular mass of cfgcs is 63690.84; the isoelectric point was 5.23; the molecular formula c2868h4438n738o848s27.qpcr showed that cfgcs was highly expressed in stamen sheath sage. (2) the full-length 1874bp cfvps gene, 116bp contains 5 'non encoding region, 1647bp ORF, 111bp 3' non encoding region; cfvps the molecular weight of 63608.40 and isoelectric point is 5.06, molecular formula is c2859h4412n744o852s24. with cfvps as the target gene, construct the expression vector of pet28-cfvps, expression vector constructed in Escherichia coli for prokaryotic expression at 32 DEG 6h induced expression of.Cfvps in the supernatant of the bacteria Involved in the synthesis of monoterpene compounds in plant, and the sheath surface glandular hair core sage rich, is the main place of monoterpene synthesis, the qPCR results showed that expression of cfvps was highest in leaf. (3) the full-length 1234bp cfgas gene, 72bp contains 5 'non encoding region, 993bp ORF, 169bp 3 "non encoding region; the molecular weight of cfgas was 36895.20, the isoelectric point is 6.37, molecular formula c1672h2575n437o482s12. and camellia, salvia, sweet potato zero, hemp, tobacco, Petunia, Aegilops tauschii, wheat genetic homology, found the relationship between cfcps protein and GA protein in Salvia miltiorrhiza. At the same time by Gateway methods GAS overexpression of recombinant vector, for subsequent Agrobacterium infection experiments results show that CfGAS is involved in.QPCR synthesis of gibberellin, gibberellin is two terpenoid plant hormone, plays an important role in the whole plant growth process, The Qiaoruisu various tissues. The distribution of.3. ultraviolet irradiation on improving the preliminary study of this experiment showed that the molecular mechanism of active ingredient content of Qiaoruisu medicinal materials, through ultraviolet irradiation Qiaoruisu herbs can improve the content of the terpenes. On the sheath Rui Su Gaotong group was the amount of transcription of terpenoid biosynthesis key genes based on the enlightenment, this paper Qiaoruisu two terpenoids biosynthesis mechanism induced by UV irradiation. The results showed that UV irradiation can effectively improve Qiaoruisu medicinal root isoforskolin content increased by 53%. gene expression level of terpenoids, ultraviolet irradiation Qiaoruisu Q PCR assay showed that TPS1. TPS2, TPS3, TPS4, TPS14 expression increased significantly by UV and TPS15, gibberellic acid synthase and ENT kaurene synthase by UV induced decreases. This study provides a scientific basis for improving the content of the effective components of the Chinese medicinal herbs, and explores a new way to improve the quality of the traditional Chinese medicinal materials.

【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】：Q946

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