本文關(guān)鍵詞： 馬藍(lán)青黛 靛玉紅 吲哚類生物堿鄰氨基苯甲酸合酶　出處：《華僑大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：馬藍(lán)(Baphicacanthus cusia),是一種非常重要的爵床科藥用植物,廣泛分布于我國(guó)西南、華南及華東等地區(qū)。由它的莖、葉加工而成的青黛,以福建產(chǎn)品質(zhì)最佳,被譽(yù)為“建青黛”,是福建的道地藥材。馬藍(lán)的根入藥即為南板藍(lán)根,與青黛一起同為《中國(guó)藥典》的藥材品種。近年來(lái),非典、甲流及禽流感等流行病頻頻肆虐發(fā)威,以及優(yōu)質(zhì)藥材生境的破壞,市場(chǎng)上以馬藍(lán)為基源的中藥材供不應(yīng)求,但是馬藍(lán)的種質(zhì)資源保護(hù)和馴化栽培沒(méi)有得到重視,道地馬藍(lán)的優(yōu)質(zhì)種質(zhì)資源正在面臨流失、退化和滅絕的危險(xiǎn),因此加強(qiáng)對(duì)中藥青黛基源植物馬藍(lán)的研究迫在眉睫。經(jīng)調(diào)研發(fā)現(xiàn),馬藍(lán)和青黛的主要功能性成分為靛藍(lán)和靛玉紅。特別是靛玉紅已經(jīng)成為青黛及其原植物馬藍(lán)的指標(biāo)性成分,靛玉紅為雙吲哚生物堿,被認(rèn)為具有抗腫瘤作用,臨床上,多用于治療慢性粒細(xì)胞白血病(chronic myel度y tic leukemia,CML),是中成藥“黃黛片”及“當(dāng)歸蘆薈丸”的主要活性成分。目前,對(duì)馬藍(lán)以及青黛的研究多集中于鑒定、加工工藝和藥理活性等方面,而缺乏對(duì)馬藍(lán)藥效物質(zhì)生物合成途徑的研究,制約了馬藍(lán)優(yōu)質(zhì)種質(zhì)的構(gòu)建,因此梳理馬藍(lán)藥效物質(zhì)次生代謝通路及其調(diào)控機(jī)理、挖掘馬藍(lán)藥效相關(guān)關(guān)鍵基因,是培育優(yōu)質(zhì)馬藍(lán)品系的基礎(chǔ),也是我們研究工作的當(dāng)務(wù)之急。本研究以馬藍(lán)轉(zhuǎn)錄組測(cè)序數(shù)據(jù)以及從微生物中預(yù)測(cè)的吲哚類生物堿合成途徑為基礎(chǔ),分離鑒定出了吲哚類生物堿合成途徑重要基因:鄰氨基苯甲酸合酶α亞基(Ant hranilate Synthaseαsubunit,ASA)和鄰氨基苯甲酸合酶β亞基(Anthranilate S ynthaseβsubunit,ASB),鄰氨基苯甲酸合酶是具有αββα亞基結(jié)構(gòu)的異質(zhì)四聚體,其中α,β亞基分別由ASA和ASB基因編碼。ASA具有催化分支酸生成鄰氨基苯甲酸的功能,ASB具有催化裂解谷氨酰胺把形成的游離銨根離子呈遞給α亞基的作用。通過(guò)生物信息學(xué)對(duì)全長(zhǎng)cDNA進(jìn)行序列特征分析和預(yù)測(cè),對(duì)A SA和ASB基因進(jìn)行一系列體內(nèi)、體外生物學(xué)功能研究。本研究可為今后通過(guò)生物技術(shù)提高馬藍(lán)中吲哚類生物堿等活性物質(zhì)含量以及馬藍(lán)優(yōu)質(zhì)品系的基因改良提供基礎(chǔ)和保障,主要研究?jī)?nèi)容有以下幾方面:(1)通過(guò)反轉(zhuǎn)錄擴(kuò)增,得到候選基因的cDNA序列,分別命名為BcASA1、BcASA2和BcASB,經(jīng)過(guò)對(duì)BcASA1和BcASA2的序列分析與gDNA序列克隆相結(jié)合表明二者為同一個(gè)基因的不同轉(zhuǎn)錄本。并利用多種軟件對(duì)BcASA1,BcASA2,BcASB的生物信息學(xué)特征進(jìn)行了分析。(2)組織特異性分析結(jié)果表明,BcASA1,Bc ASA2,BcASB在馬藍(lán)的根、莖、葉3個(gè)組織中均有不同程度的表達(dá),其中三基因于葉片中表達(dá)量最高,根和葉片中的表達(dá)量較低。采用實(shí)時(shí)熒光定量PCR考察MeJA、SA和ABA處理后各時(shí)間點(diǎn)(0h、1h、2 h、4 h、8 h、12 h、24 h、36 h、48h、72h)馬藍(lán)中Bc ASA1,BcASA2,Bc ASB的表達(dá)情況,結(jié)果表明Bc ASA1,BcASA2,Bc ASB2均能被植物激素MeJA、SA和ABA誘導(dǎo)表達(dá)上調(diào)。通過(guò)轉(zhuǎn)運(yùn)肽預(yù)測(cè)和亞細(xì)胞定位實(shí)驗(yàn)探明三者在葉綠體中發(fā)揮作用,BcASA1在細(xì)胞質(zhì)中存在少量表達(dá)。(3)原核表達(dá)誘導(dǎo)得到BcASA1,BcASA2,BcASB蛋白,將純化后的BcASA1和BcASA2蛋白加入以分支酸和氯化銨為底物的混合體系中,在336nm處測(cè)定,結(jié)果顯示BcASA1,BcASA2均具有催化活性,且其活性大小類似。由此可知馬藍(lán)中ASA基因的兩個(gè)轉(zhuǎn)錄本在功能上差異較小。(4)為了驗(yàn)證馬藍(lán)和菘藍(lán)吲哚類生物堿合成路徑的保守性及差異,以及B cASA1,BcASA2,BcASB基因的體內(nèi)功能,我們構(gòu)建了BcASA1,BcASA2,BcA SB基因的單基因和雙基因過(guò)表達(dá)載體,并將它們通過(guò)根癌農(nóng)桿菌C58C1介導(dǎo)轉(zhuǎn)化如入的菘藍(lán)。目前已獲得轉(zhuǎn)基因毛狀根,下一步將對(duì)轉(zhuǎn)基因植株表型、活性成分含量進(jìn)行分析。
[Abstract]:Malan (Baphicacanthus cusia), is a very important medicinal plant Acanthaceae, widely distributed in Southwest China, Southern China and East China. From its stem, leaf processing of indigo in Fujian, the best quality products, known as "Jian Qingdai", is the genuine medicinal materials in Fujian. Malan is the root medicine Banlangen, and indigo together with varieties of Chinese herbal medicines "China Pharmacopoeia >. In recent years, SARS, swine flu and avian flu epidemics such as frequently raging angry, and the destruction of habitat quality medicines, on the market in Malan is based in Chinese demand, but the protection of germplasm resources and Malan domestication and cultivation did not get attention, high-quality germplasm resources are facing the loss of genuine Malan, risk of degradation and extinction, so to strengthen the research on Chinese medicine source plant indigo acanthaceous indigo imminent. The research found that the main function and the composition of natural indigo Malan For indigo and indirubin. Especially the indirubin has become the index component of indigo and its original plant acanthaceous indigo, indirubin - indole alkaloid, is believed to have anti-tumor effects, clinical practice, for the treatment of chronic myeloid leukemia (chronic myel y tic leukemia, CML), is the main active ingredient Chinese medicine "Huangdai tablets" and "aloe Angelica pill". At present, the research of Malan and indigo are more concentrated in the identification, process and activity of pharmacology, and the lack of research on the biosynthesis of Malan medicinal substances, restricts the construction of good germplasm Malan, thus combing Malan pharmacodynamic material secondary metabolism pathway and its regulation the mechanism of mining Malan efficacy genes, is the basis for developing high-quality Malan lines, is a pressing matter of the moment of our research. In this study, Malan transcriptome sequencing data to and from microorganisms Indole alkaloid biosynthesis prediction based isolation and identification of important genes of indole alkaloid biosynthesis: anthranilate synthase alpha subunit (Ant hranilate Synthase subunit alpha, ASA) and anthranilate synthase beta subunit (Anthranilate S Ynthase beta subunit, ASB), anthranilic acid four heterogeneous synthase is a dimer, with alpha beta beta alpha subunit structure of the alpha, Yaki respectively by ASA and ASB gene encoding.ASA with catalytic chorismate formation of anthranilic acid, ASB has the catalytic cracking of glutamine free ammonium ion was formed to alpha subunits. By bioinformatics study of sequence analysis and prediction of the length of cDNA, a series of A in SA and ASB gene and biological function study in vitro. The study can be used for the future through biotechnology to improve the indole alkaloids in active substances containing Malan etc. 閲忎互鍙?qiáng)椹摑浼樿川鍝伨p葷殑鍩哄洜鏀硅壇鎻愪緵鍩虹鍜屼繚闅，

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