本文關(guān)鍵詞：基于轉(zhuǎn)錄組測序的紫外線促進(jìn)鞘蕊蘇有效成分isoforskolin合成機(jī)制研究　出處：《湖北中醫(yī)藥大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鞘蕊蘇 生物合成 萜類合成酶 功能基因 轉(zhuǎn)錄組

【摘要】：鞘蕊蘇藥材的植物名為毛喉鞘蕊花(Coleus forskohlii(Willd.)Briq.)系唇形科(Labiatae)鞘蕊花屬(Coleus Lour.)植物,主產(chǎn)于印度、尼泊爾、巴基斯坦、斯里蘭卡、緬甸、泰國等南亞國家;我國云南、福建、臺(tái)灣等地亦有分布,被植物學(xué)家界定為珍稀植物。民間將該藥材用于治療哮喘、咳嗽等疾患,療效顯著,稱為“神藥”。現(xiàn)代科學(xué)研究表明,鞘蕊蘇的有效成分半日花烷型二萜類化合物,我國產(chǎn)鞘蕊蘇的有效成分為異佛司可林(isoforskolin),并將其定為指標(biāo)性成分。鑒于鞘蕊蘇獨(dú)特藥效作用,本實(shí)驗(yàn)室與湖北福人藥業(yè)股份有限公司實(shí)施產(chǎn)學(xué)研結(jié)合,以鞘蕊蘇為君藥,配伍前胡、甘草組方,將其開發(fā)為“鞘蕊蘇膠囊”中藥新品種,獲得生產(chǎn)批號(hào)(批件號(hào):2011S00365)。為了解決鞘蕊蘇膠囊產(chǎn)業(yè)化所需原料藥材的供求問題,實(shí)驗(yàn)室與湖北福人藥業(yè)股份有限公司合作,從云南會(huì)澤縣引種鞘蕊蘇至湖北通城縣規(guī)范化種植。針對(duì)通城種植鞘蕊蘇藥材的主要二萜類成分isoforskolin含量偏低的問題,本論文對(duì)該藥用植物二萜類成分的生物合成機(jī)制進(jìn)行了研究,以期為通城種植鞘蕊蘇培育優(yōu)良種質(zhì);并進(jìn)行了紫外線照射采收的鞘蕊蘇藥材,誘導(dǎo)其主要有效成分isoforskolin增加的生物合成機(jī)制研究,以期為促進(jìn)種植鞘蕊蘇主要有效成分的生物合成和積累提供科學(xué)依據(jù),并為通過遺傳改良手段培育鞘蕊蘇優(yōu)良種質(zhì)提供基礎(chǔ),也為二萜化合物的代謝機(jī)制的深入研究提供重要信息。本研究的主要研究結(jié)果如下:1.基于Illumina HiSeq高通量測序的鞘蕊蘇轉(zhuǎn)錄組分析以通城縣種植基地的鞘蕊蘇為試驗(yàn)材料,于10月下旬采集該藥材的葉和根作為供試品,采用HiSeq技術(shù)進(jìn)行轉(zhuǎn)錄組測序,獲得36Gb轉(zhuǎn)錄組數(shù)據(jù),拼接后得到422,761條高質(zhì)量序列;對(duì)組裝的Unigene基因進(jìn)行預(yù)測,獲得CDS序列和Protein序列;采用Trinity和TGICL的方法從頭組裝、序列拼接和去冗余處理,得到291,061個(gè)Unigene基因,平均長度629bp;將所得Unigene基因與Nr、Swiss-Prot、KEGG和COG公共蛋白數(shù)據(jù)庫進(jìn)行比對(duì),140,228(48.19%)的unigene基因能夠獲得注釋信息。將140,228個(gè)注釋信息的unigene基因進(jìn)行g(shù)o和cog聚類分析及kegg代謝通路分析,86,578條unigene基因獲得cog功能注釋,被歸類為25個(gè)功能分類;共有186,957條unigene基因獲得go功能注釋,被歸入到49個(gè)go次級(jí)功能分類;共有6,616個(gè)unigene可以分配到261個(gè)代謝通路。因此,本項(xiàng)研究為鞘蕊蘇有效成分的萜類合成酶基因克隆,序列分析及表達(dá)研究奠定了基礎(chǔ),并且根與葉中23個(gè)萜類差異基因則為傳統(tǒng)育種和轉(zhuǎn)基因育種的基因調(diào)控靶點(diǎn)提供了研究基礎(chǔ)。2.基于高通量測序的鞘蕊蘇萜類合成酶基因克隆、序列分析及表達(dá)研究在上述高通量基因測序研究基礎(chǔ)上,從注釋信息數(shù)據(jù)中挑選基因片段,采用race技術(shù)進(jìn)行序列分析,獲得三個(gè)萜類合成酶的全長cdna序列,并通過blast和蛋白序列分析。結(jié)果如下:(1)cfgcs基因全長2059bp,包含235bp的5’非編碼區(qū),1632bp的開放式閱讀框(orf),192bp的3’非編碼區(qū),編碼544個(gè)氨基酸;對(duì)蛋白理化性質(zhì)進(jìn)行預(yù)測,cfgcs相對(duì)分子質(zhì)量為63690.84;等電點(diǎn)為5.23;分子式為c2868h4438n738o848s27。qpcr表明cfgcs在鞘蕊蘇葉的表達(dá)量較高。(2)cfvps基因全長1874bp,包含116bp的5’非編碼區(qū),1647bp的orf,111bp的3’非編碼區(qū);cfvps相對(duì)分子質(zhì)量為63608.40,等電點(diǎn)為5.06,分子式為c2859h4412n744o852s24。以cfvps為目標(biāo)基因,構(gòu)建表達(dá)載體pet28-cfvps,將構(gòu)建好的表達(dá)載體于大腸桿菌中進(jìn)行原核表達(dá),32℃時(shí)誘導(dǎo)6h,菌液上清中有大量表達(dá)。cfvps參與植物單萜化合物合成,而鞘蕊蘇葉片表面有豐富的腺毛分布,是單萜合成的主要場所,qpcr結(jié)果也表明cfvps在葉中的表達(dá)量最高。(3)cfgas基因全長1234bp,包含72bp的5’非編碼區(qū),993bp的orf,169bp的3’非編碼區(qū);cfgas相對(duì)分子質(zhì)量為36895.20,等電點(diǎn)為6.37,分子式為c1672h2575n437o482s12。與山茶花、丹參、番薯零、大麻、煙草、碧冬茄、節(jié)節(jié)麥、小麥進(jìn)行同源性比較,發(fā)現(xiàn)cfcps蛋白與丹參GA蛋白的親緣關(guān)系比較近。同時(shí)通過Gateway的方法進(jìn)行GAS過量表達(dá)重組載體構(gòu)建,為后續(xù)發(fā)根農(nóng)桿菌侵染提供實(shí)驗(yàn)基礎(chǔ)。qPCR結(jié)果表明CfGAS參與赤霉素的合成,赤霉素是二萜類植物激素,在植物的整個(gè)生長過程中發(fā)揮著重要作用,在鞘蕊蘇各個(gè)組織中均有分布。3.紫外線照射提高鞘蕊蘇藥材有效成分含量的分子機(jī)制研究本實(shí)驗(yàn)前期研究表明,通過紫外線照射鞘蕊蘇藥材能提高其二萜類有效成分的含量�；谏鲜銮嗜锾K高通量轉(zhuǎn)錄組研究所獲萜類生物合成酶關(guān)鍵基因的啟示,本論文進(jìn)行了紫外線照射誘導(dǎo)鞘蕊蘇二萜類成分生物合成機(jī)制研究。研究結(jié)果表明,紫外線照射能夠有效提高鞘蕊蘇藥材根部isoforskolin含量,提高幅度高達(dá)53%。通過q PCR檢測紫外照射鞘蕊蘇后萜類生物基因表達(dá)水平,表明TPS1、TPS2、TPS3、TPS4、TPS14表達(dá)量明顯受紫外線的誘導(dǎo)而升高,而TPS15、赤霉素合成酶和貝殼杉烯合成酶受紫外線誘導(dǎo)而降低。本研究為提高種植鞘蕊蘇藥材有效成分含量提供了科學(xué)依據(jù),并為提高規(guī)范化種植中藥材的資源品質(zhì)探索了新途徑。
[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.

【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q946

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王身昌;胡尚連;曹穎;徐剛;;梁山慈竹高通量轉(zhuǎn)錄組測序及差異表達(dá)基因分析[J];華北農(nóng)學(xué)報(bào);2016年03期

2 馬婧;成鐵龍;孫燦岳;鄧楠;史勝青;江澤平;;草麻黃高通量轉(zhuǎn)錄組分析及黃酮類代謝途徑相關(guān)基因的鑒定[J];浙江農(nóng)業(yè)學(xué)報(bào);2016年04期

3 吳欣;沈和定;王冬鳳;劉宸;楊鐵柱;刁亞;;基于Illumina HiseqTM 2000高通量轉(zhuǎn)錄組測序的里氏擬石磺SSR標(biāo)記開發(fā)[J];海洋科學(xué);2015年11期

4 姜福星;楊麗娟;陳其兵;高順;;瀘定百合轉(zhuǎn)錄組測序與特性分析[J];西北林學(xué)院學(xué)報(bào);2015年05期

5 唐亮;馬香;周志欽;;植物萜類合成酶的進(jìn)化研究[J];西南大學(xué)學(xué)報(bào)(自然科學(xué)版);2014年04期

6 呂遠(yuǎn)大;李坦;張曉林;趙涵;;利用玉米高通量RNA-Seq數(shù)據(jù)預(yù)測長鏈非編碼RNA(LncRNA)[J];江蘇農(nóng)業(yè)學(xué)報(bào);2013年06期

7 張媛;肖霞;張俊紅;齊力旺;韓素英;;落葉松干細(xì)胞發(fā)育模型研究中的高通量測序技術(shù)[J];生物產(chǎn)業(yè)技術(shù);2013年04期

8 王曉鋒;何衛(wèi)龍;蔡衛(wèi)佳;阮倩倩;潘婷;季孔庶;;馬尾松轉(zhuǎn)錄組測序和分析[J];分子植物育種;2013年03期

9 張文;郭躍偉;;中國南海軟體動(dòng)物及其食源生物的化學(xué)、化學(xué)生態(tài)學(xué)和生物活性研究[J];上海醫(yī)藥;2012年09期

10 鄭洲翔;范燕萍;周紀(jì)剛;徐平;;植物萜類合成酶1-脫氧-D-木酮糖-5-磷酸還原酶研究進(jìn)展[J];安徽農(nóng)業(yè)科學(xué);2011年10期

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