葛根糖基转移酶基因的克隆与功能鉴定

发布时间：2018-03-02 23:07

本文选题：葛根素　切入点：异黄酮　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：葛根是一种药用植物,根部可作为药材,具有很高的药用价值,广泛用于治疗心脑血管疾病、糖尿病、肝病等疾病。葛根中的主要活性物质为葛根素、大豆苷等异黄酮类化合物,该类化合物具有具有抗炎、抗癌、抗氧化等特性。糖基转移酶UGT(glycosyltransferases)能将UDP活化的糖分子转移到生物大分子上,是自然界最重要的修饰基因之一。异黄酮类化合物是植物中分布最广泛的此生代谢产物之一。在葛根中,UGT对黄酮类化合物进行糖基化修饰,合成异黄酮糖苷,在黄酮的合成、转运、存储等方面发挥了重要的作用。葛根素结构的特殊性在于葡糖糖基以C-C键与母环相连,而绝大多数异黄酮糖苷都以C-O键与甘元母环相连。葛根中存在2类对应的UGT,即碳苷糖基转移酶(CGT)和氧苷糖基转移酶(OGT)。为了挖掘葛根中的UGT基因,本课题通过转录组测序找到了140多个UGT基因序列。通过与已知CGT和OGT进行序列比对分析,选定8个目标基因,分别命名为GT1、GT2、GT3、GT4、GT5、GT6、GT7、GT8,成功克隆基因,并通过蛋白原核表达及转基因大豆发根诱导技术初步验证基因功能。将目标基因构建到原核表达载体上,将载体转入原核表达菌株Rosetta(DE3)中,用IPTG诱导蛋白表达。基因GT1、GT2、GT3、GT4、GT5、GT7、GT8均成功诱导出目标蛋白(51KD左右),用超声破碎法提取蛋白,发现蛋白大部分出现在沉淀中,上清液中含量极少。在配好的反应体系中加入提取的GT蛋白上清液,发现GT4具有很强的糖基转移酶活性,能够催化大豆甘元、染料木素、芒柄花素、黄豆黄素合成对应的7-O-葡糖糖苷,且转化率很高,接近完全转化;GT4对甘草素和异甘草素无反应。由此说明GT4是特异性识别异黄酮并高效催化其合成7-O-葡萄糖苷的糖基转移酶基因。此外,发现GT1可催化芒柄花素合成芒柄花苷,但是活性很低。其他GT基因无糖基转移酶活性。将目标基因构建到超表达载体pB7WG2D,转入发根农杆菌K599并侵染大豆子叶,诱导转基因发根产生。提取发根异黄酮类化合物,经过HPLC检测,结果发现与对照相比,超表达目标基因的发根中无葛根素,且异黄酮糖苷的含量也无显著差异。本实验未能成功克隆催化葛根素合成的CGT,但是鉴定出一个新的糖基糖基转移酶基因,该基因具有很强的活性,可为后续研究及生产应用提供借鉴。
[Abstract]:Pueraria lobata is a kind of medicinal plant, the root can be used as medicinal material, it has high medicinal value, and is widely used in the treatment of cardiovascular and cerebrovascular diseases, diabetes, liver diseases, etc. The main active substance in Pueraria lobata is puerarin, the main active substance in Pueraria lobata is puerarin. Isoflavones, such as daidzein, have anti-inflammatory, anticancer and antioxidant properties. Glycosyltransferases (UGTG) can transfer carbohydrates activated by UDP onto biomolecules. Isoflavones are one of the most widely distributed metabolites in plants. UGT glycosylation of flavonoids in Pueraria lobata, synthesis of isoflavone glycosides, synthesis of flavonoids, The special structure of puerarin is that the glucosyl group is linked to the parent ring by C-C bond. However, most isoflavone glycosides are linked to the Ganyuan maternal ring by C-O bond. There are two types of UGTs in Pueraria lobata, that is, CGT) and Oxyglycosyltransferase (OGTT). In order to explore the UGT gene in Pueraria lobata, In this study, more than 140 UGT gene sequences were identified by transcriptome sequencing. Through sequence alignment analysis with known CGT and OGT, eight target genes were selected, named GT1, GT2, GT3, GT3, GT5, GT6, GT7 and GT8, respectively. The target gene was constructed into prokaryotic expression vector and transferred into prokaryotic expression strain Rosetta-DE3). Protein expression was induced by IPTG. The target protein was successfully induced by the gene GT1, GT1, GT2, GT3, GT4, GT5, GT7, GT8. The protein was extracted by ultrasonic fragmentation, and most of the proteins appeared in the precipitate. The supernatant contained very little in the supernatant. Adding the supernatant of GT protein into the reaction system, it was found that GT4 had strong activity of glycosyltransferase and could catalyze glycosyltransferase activity of soybean, genistein and amaranthin. The corresponding 7-Oglucoside was synthesized by daidzein, and the conversion rate was very high. Nearly complete transformation of GT4 did not react with glycyrrhizin and isoglycyrrhizin, which indicated that GT4 is a glycosyltransferase gene that can specifically recognize isoflavones and efficiently catalyze the synthesis of 7-O- glucoside. In addition, it was found that GT1 can catalyze the synthesis of Acanthopanthin. But the activity is very low. The other GT genes have no glycosyltransferase activity. The target gene was constructed into pB7WG2D. the target gene was transformed into Agrobacterium rhizogenes K599 and infected with soybean cotyledons to induce the production of transgenic hairy roots. The results of HPLC analysis showed that there was no puerarin in the hair root of the overexpression target gene compared with the control. There was no significant difference in the content of isoflavone glucosides. In this study, the CGT catalyzed puerarin synthesis was not successfully cloned, but a new glycosyltransferase gene was identified, which has strong activity. It can be used for reference for follow-up research and production application.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.19

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