马蓝吲哚类生物碱合成关键基因ASA和ASB的克隆与功能研究

发布时间：2018-01-28 23:57

  本文关键词： 马蓝青黛 靛玉红 吲哚类生物碱邻氨基苯甲酸合酶　出处：《华侨大学》2017年硕士论文　论文类型：学位论文

【摘要】：马蓝(Baphicacanthus cusia),是一种非常重要的爵床科药用植物,广泛分布于我国西南、华南及华东等地区。由它的茎、叶加工而成的青黛,以福建产品质最佳,被誉为“建青黛”,是福建的道地药材。马蓝的根入药即为南板蓝根,与青黛一起同为《中国药典》的药材品种。近年来,非典、甲流及禽流感等流行病频频肆虐发威,以及优质药材生境的破坏,市场上以马蓝为基源的中药材供不应求,但是马蓝的种质资源保护和驯化栽培没有得到重视,道地马蓝的优质种质资源正在面临流失、退化和灭绝的危险,因此加强对中药青黛基源植物马蓝的研究迫在眉睫。经调研发现,马蓝和青黛的主要功能性成分为靛蓝和靛玉红。特别是靛玉红已经成为青黛及其原植物马蓝的指标性成分,靛玉红为双吲哚生物碱,被认为具有抗肿瘤作用,临床上,多用于治疗慢性粒细胞白血病(chronic myel度y tic leukemia,CML),是中成药“黄黛片”及“当归芦荟丸”的主要活性成分。目前,对马蓝以及青黛的研究多集中于鉴定、加工工艺和药理活性等方面,而缺乏对马蓝药效物质生物合成途径的研究,制约了马蓝优质种质的构建,因此梳理马蓝药效物质次生代谢通路及其调控机理、挖掘马蓝药效相关关键基因,是培育优质马蓝品系的基础,也是我们研究工作的当务之急。本研究以马蓝转录组测序数据以及从微生物中预测的吲哚类生物碱合成途径为基础,分离鉴定出了吲哚类生物碱合成途径重要基因:邻氨基苯甲酸合酶α亚基(Ant hranilate Synthaseαsubunit,ASA)和邻氨基苯甲酸合酶β亚基(Anthranilate S ynthaseβsubunit,ASB),邻氨基苯甲酸合酶是具有αββα亚基结构的异质四聚体,其中α,β亚基分别由ASA和ASB基因编码。ASA具有催化分支酸生成邻氨基苯甲酸的功能,ASB具有催化裂解谷氨酰胺把形成的游离铵根离子呈递给α亚基的作用。通过生物信息学对全长cDNA进行序列特征分析和预测,对A SA和ASB基因进行一系列体内、体外生物学功能研究。本研究可为今后通过生物技术提高马蓝中吲哚类生物碱等活性物质含量以及马蓝优质品系的基因改良提供基础和保障,主要研究内容有以下几方面:(1)通过反转录扩增,得到候选基因的cDNA序列,分别命名为BcASA1、BcASA2和BcASB,经过对BcASA1和BcASA2的序列分析与gDNA序列克隆相结合表明二者为同一个基因的不同转录本。并利用多种软件对BcASA1,BcASA2,BcASB的生物信息学特征进行了分析。(2)组织特异性分析结果表明,BcASA1,Bc ASA2,BcASB在马蓝的根、茎、叶3个组织中均有不同程度的表达,其中三基因于叶片中表达量最高,根和叶片中的表达量较低。采用实时荧光定量PCR考察MeJA、SA和ABA处理后各时间点(0h、1h、2 h、4 h、8 h、12 h、24 h、36 h、48h、72h)马蓝中Bc ASA1,BcASA2,Bc ASB的表达情况,结果表明Bc ASA1,BcASA2,Bc ASB2均能被植物激素MeJA、SA和ABA诱导表达上调。通过转运肽预测和亚细胞定位实验探明三者在叶绿体中发挥作用,BcASA1在细胞质中存在少量表达。(3)原核表达诱导得到BcASA1,BcASA2,BcASB蛋白,将纯化后的BcASA1和BcASA2蛋白加入以分支酸和氯化铵为底物的混合体系中,在336nm处测定,结果显示BcASA1,BcASA2均具有催化活性,且其活性大小类似。由此可知马蓝中ASA基因的两个转录本在功能上差异较小。(4)为了验证马蓝和菘蓝吲哚类生物碱合成路径的保守性及差异,以及B cASA1,BcASA2,BcASB基因的体内功能,我们构建了BcASA1,BcASA2,BcA SB基因的单基因和双基因过表达载体,并将它们通过根癌农杆菌C58C1介导转化如入的菘蓝。目前已获得转基因毛状根,下一步将对转基因植株表型、活性成分含量进行分析。
[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. 閲忎互鍙婇┈钃濅紭璐ㄥ搧绯荤殑鍩哄洜鏀硅壇鎻愪緵鍩虹��鍜屼繚闅，

本文编号：1471917