金铁锁中三萜皂苷相关功能基因的鉴定分析与表达研究

发布时间：2018-05-26 20:53

本文选题：金铁锁关键酶基因的克隆生物信息学分析酿酒酵母工程菌的构建 + 　；参考：《云南中医学院》2017年硕士论文

【摘要】：中药材金铁锁来源于石竹科金铁锁属植物金铁锁Psammosilene tunicoides W.C.Wu et C.Y.Wu的干燥根,首载于《滇南本草》,主要用于治疗风湿痛,跌打损伤等。金铁锁的药用活性成分为齐墩果烷型三萜总皂苷类物质[1,2],此类物质主要从金铁锁植物的根中提取,但随着长期过度采挖野生资源,而人工种植中存在品种退化、人力成本等问题[3,4],金铁锁的活性成分已不能满足市场需求,亟需提供新的资源途径。在课题组前期研究基础上,本课题进行金铁锁三萜皂苷下游合成途径中关键酶基因UGT(糖基转移酶家族基因)的初步克隆鉴定。同时构建产beta-香树素的酵母工程菌,为直接生产有效成分或中间体奠定基础。主要研究结果如下:1.通过转录数据,克隆获得两条金铁锁糖基转移酶基因的全长序列。经生物信息学分析后分别命名为UGT71G1、PtT1。其中UGT71G1的序列全长为1402bp,包含一条为1107 bp的完整开放阅读框,共编码了368个氨基酸,预测相对分子质量为41.05KD,在155位到336位置处存在高度保守UDPGT结构功能域,经过多重序列比对后发现与香石竹、人参等具有较高的同源性。PtT1全长1529bp,ORF为1377bp,编码458个氨基酸,相对分子质量为51.25KD,与同科植物香石竹糖基转移酶基因DcT227具有很高的同源性。2.构建UGT71G1、PtT1、UGT1的原核表达体系,发现UGT71G1及UGT1两条基因的表达蛋白与理论蛋白大小一致,表示原核表达成功。采用实时荧光定量检测方法对UGT1的相对表达量进行分析,显示该基因在叶中的相对表达量较高。3.利用酶切连接技术将β-香树素合成酶基因与pYES2表达载体进行连接,并转入酿酒酵母细胞中构建异源表达工程菌,经分子鉴定结果显示β-香树素合成酶基因能异源表达具有活性功能的蛋白酶。本课题从金铁锁糖基转移酶基因的克隆鉴定入手,对两个可能参与到三萜皂苷合成途径的糖基转移酶基因进行克隆和生物信息学分析;成功构建了金铁锁UGT71G1和UGT1的原核表达体系;进行了β-香树素酵母工程菌的构建等工作。完善了金铁锁三萜皂苷合成途径的研究,为下一步基因的功能研究奠定了基础,同时也为今后利用合成生物学技术快速大量生产金铁锁有效成分提供基础和科学依据。
[Abstract]:The traditional Chinese medicine Jintiesin comes from the dry root of Psammosilene tunicoides W.C.Wu et C.Y.Wu, a plant of the genus Psammosilene tunicoides W.C.Wu et C.Y.Wu, which is mainly used in the treatment of rheumatism and injury. The medicinal active component of Jintiezao is oleane-type triterpenoid total saponins. This kind of substance is mainly extracted from the roots of the plant. However, with the long-term overexploitation of wild resources, there are varieties degeneration in artificial cultivation. The active components of gold and iron lock can not meet the market demand, so it is urgent to provide a new resource way. On the basis of the previous study, the key enzyme gene UGT (glycosyltransferase family) gene in the downstream synthesis pathway of triterpenoid saponins was cloned and identified. At the same time, yeast engineering bacteria producing beta-vanillin were constructed, which laid the foundation for direct production of active components or intermediates. The main results are as follows: 1. Based on the transcriptional data, the full length sequences of two gold and iron lock-glycosyltransferase genes were cloned. After bioinformatics analysis, they were named UGT71G1PtT1. The total length of UGT71G1 is 1402bp. it contains a complete open reading frame of 1107 BP, encoding 368 amino acids. The predicted molecular weight is 41.05KD. there is a highly conserved domain of UDPGT structure at the 155-336 position. After multiple sequence alignment, it was found that there was a high homology with carnation, ginseng and so on. The ORF of 1529bp was 1377bp, encoding 458 amino acids, and the relative molecular weight was 51.25kD. it had high homology with carnation glycosyltransferase gene DcT227 of the same family plant. The prokaryotic expression system of UGT71G1PtT1 and UGT1 was constructed. It was found that the expressed proteins of UGT71G1 and UGT1 were the same as the theoretical proteins, indicating that the prokaryotic expression was successful. The relative expression of UGT1 was analyzed by real-time fluorescence quantitative detection. The results showed that the relative expression of the gene in leaves was high. 3. 尾 -vanillin synthase gene was ligated with pYES2 expression vector by restriction endonuclease ligation technique, and then transformed into Saccharomyces cerevisiae cells to construct heterologous expression engineering bacteria. The results of molecular identification showed that 尾 -vanillin synthase gene was able to express protease with active function. In this paper, two glycosyltransferase genes which may be involved in the synthesis of triterpenoid saponins were cloned and analyzed by bioinformatics. The prokaryotic expression systems of UGT71G1 and UGT1 were successfully constructed, and the 尾 -vanillin yeast engineering bacteria were constructed. The study on the synthetic pathway of the triterpenoid saponins was improved, which laid a foundation for the further study of the function of the gene, and also provided the basis and scientific basis for the rapid production of the active components of the gold and iron locks by the synthetic biological technology in the future.
【学位授予单位】：云南中医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.239

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