發(fā)布時間：2018-04-22 02:41

  本文選題：毛喉鞘蕊花 + 生物合成　； 參考：《湖北中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：毛喉鞘蕊花(Coleus forskohlii(Willd.)Briq.)系唇形科(Labiatae)鞘蕊花屬(Coleus Lour.)草本植物,主產(chǎn)印度、巴基斯坦、斯里蘭卡、阿拉伯半島南部等熱帶和亞熱帶國家。該植物在我國云南、廣東、福建、臺灣等地亦稀有分布,被植物學(xué)家界定為珍稀植物。毛喉鞘蕊花藥材名為鞘蕊蘇,收載于《中國植物志》66卷和《云南省藥品標(biāo)準(zhǔn)》,民間用于治療哮喘、咳嗽等疾患,療效顯著,被稱為“萬靈藥”;現(xiàn)代科學(xué)研究表明,該植物藥中含有的半日花烷型二萜類化合物具有激活腺苷酸環(huán)化酶、強(qiáng)心、降壓、抗腫瘤等廣泛的藥理作用。鑒于毛喉鞘蕊花藥用植物的獨特的藥效作用,本課題組與湖北福人藥業(yè)公司產(chǎn)學(xué)研結(jié)合,開發(fā)了中藥新品種--“鞘蕊蘇膠囊”,該藥物已經(jīng)獲得中藥新藥生產(chǎn)批件(批件號:2011S00365)。為了保護(hù)野生毛喉鞘蕊花稀有植物的生態(tài)環(huán)境,本實驗室湖北福人藥業(yè)公司合作,從云南會澤縣移植毛喉鞘蕊花至湖北通城縣實施規(guī)范化種植研究,以期解決“鞘蕊蘇膠囊”產(chǎn)業(yè)化的原料藥材大量需求。本論文首先采用分子生物學(xué)研究方法對毛喉鞘蕊花二萜類化合物合成酶基因克隆和調(diào)控進(jìn)行了研究,構(gòu)建了二萜合成酶(TPS)基因研究平臺,高通量篩選有效成分TPS基因,分析其結(jié)構(gòu)特點與功能,闡釋其表達(dá)特征;并以上述平臺為基礎(chǔ),研究多效唑提高毛喉鞘蕊花有效成分及指標(biāo)成分isofoskolin的分子機(jī)制。本論文研究可為通過植物生理學(xué)方法提高毛喉鞘蕊花有效成分提供方法,并為種植毛喉鞘蕊花主要有效成分的代謝和富集提供科學(xué)依據(jù),以期通過遺傳改良手段培育優(yōu)良種質(zhì)資源提供基礎(chǔ),進(jìn)而從源頭提高種植藥材的品質(zhì)。主要研究結(jié)果如下:1.毛喉鞘蕊花全長均一化cDNA文庫的構(gòu)建以毛喉鞘蕊花根、根莖、莖、葉、花為材料,采用SMART法與DSN均一化相結(jié)合構(gòu)建了全長均一化cDNA文庫,庫容數(shù)約為1600,轉(zhuǎn)化菌液體積1.5ml,計算得出菌液滴度是為1.6×106cfu/ml。以整個連接10μl計算庫容大于5.2×106。從文庫中隨機(jī)挑取30個克隆進(jìn)行菌落pcr鑒定,結(jié)果顯示cdna插入片段介于1-3kb。因此,本研究構(gòu)建的毛喉鞘蕊花全長均一化cdna文庫質(zhì)量較高。2.est測序及生物信息學(xué)分析從cdna文庫中隨機(jī)挑選了4224個單克隆進(jìn)行sanger測序,經(jīng)過組裝得到2394條unigene,unigene平均長度753bp。對unigene進(jìn)行nr、go及kegg功能注釋。2263條unigene被nr數(shù)據(jù)庫注釋,在這些基因中存在有大量的涉及生長發(fā)育、物質(zhì)和能量代謝等方面的基因。go功能分類顯示,2100條(占87.7%)注釋到了一個或多個go類別,所得go類別的總數(shù)為12121,分屬于3個go主類別。kegg代謝途徑分析表明,1716條(占71.7%)unigene被分配到6個主代謝途徑中,其中17個unigenes被確定可能參與類萜生物合成的骨干和單萜、二萜、三萜2個二萜合成酶(柯巴烯焦磷酸合酶、貝殼杉烯氧化酶)、3個單萜合成酶(月桂烯/羅勒烯合酶、新薄荷醇脫氫酶、檸檬烯合酶)和1個三萜合成酶(β-香樹脂醇合成酶)。3.毛喉鞘蕊花cfcps基因的克隆、序列分析及表達(dá)研究在est測序的基礎(chǔ)上,從unigene中挑選柯巴烯焦磷酸合酶(cps)基因序列的片段,利用race技術(shù)得到全長cdna序列,結(jié)果表明cfcps基因全長2418bp。構(gòu)建表達(dá)載體,利用大腸桿菌表達(dá)體系,獲得了cfcps蛋白。對毛喉鞘蕊花cfcps進(jìn)行生物信息學(xué)分析發(fā)現(xiàn),該序列的全長為2418bp,編碼805個氨基酸,分子量為92235,分子式為c4166h6415n1101o1219s29,pi為5.70。與迷迭香(rosmarinusofficinalis)、丹參(salviamiltiorr)、歐夏至草(marrubiumvulgare)、板栗(castananeamollissima)、桃(prunuspersica)、甘草(scopariadulcis)、番茄(solanumlycopersucum)、玉米(zeamays)植物的tps蛋白進(jìn)行多重比對,發(fā)現(xiàn)它們具有較高的同源性性。通過構(gòu)建系統(tǒng)進(jìn)化樹,發(fā)現(xiàn)cfcps蛋白與迷迭香、丹參tps蛋白的親緣關(guān)系比較近。進(jìn)一步研究發(fā)現(xiàn),cfcps蛋白有跨膜區(qū)域和信號肽,屬于可溶性蛋白。利用q PCR檢測毛喉鞘蕊花Cf CPS基因在植物體的表達(dá)發(fā)現(xiàn),毛喉鞘蕊花CfCPS基因在根表達(dá)量較高。4.多效唑提高毛喉鞘蕊花有效成分的分子機(jī)制在上述鞘蕊蘇cDNA文庫研究獲得的萜類生物合成關(guān)鍵基因的基礎(chǔ)上,我們又開展了多效唑處理提高毛喉鞘蕊花二萜類成分生物合成的研究。結(jié)果表明,多效唑(100mg/L)處理毛喉鞘蕊花植株,可顯著改善植株株型,并顯著提高毛喉鞘蕊花根、根莖和莖中isoforskolin含量,提高幅度高達(dá)53%。qPCR檢測多效唑處理后葉片及根中萜類生物合成關(guān)鍵基因的表達(dá)水平發(fā)現(xiàn),葉中ACCT、HMGR、DXR、IDI、GGPS和KAO等6個基因的表達(dá)被顯著誘導(dǎo),根中除CPS基因的表達(dá)量先升后降外,其余幾個基因的表達(dá)量均呈下降趨勢。因此,我們推測多效唑提高毛喉鞘蕊花有效成分的分子機(jī)制主要是誘導(dǎo)ACCT、HMGR、DXR、IDI、GGPS、KAO和CPS等基因的表達(dá)。
[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.

【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S567;Q943.2

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