灵芝中单独高表达氧化鲨烯合酶基因与共表达3-羟基-3-甲基戊二酰辅酶A还原酶基因提高灵芝酸产量

发布时间：2018-03-15 05:39

本文选题：灵芝　切入点：单体灵芝酸　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：灵芝中的灵芝酸是主要抗癌、抗肿瘤转移的活性物质。灵芝是一种珍贵的高等药用真菌和名贵中草药,用它来预防和治疗疾病已有几千年历史。灵芝酸和灵芝多糖作为灵芝的重要药用成分,具有抗癌、抗爱滋病、提高免疫力等活性。由于其重要的药理作用,近年来灵芝成为国内外研究的一个热点。尽管关于加速灵芝细胞生长和优化灵芝酸与灵芝多糖生产有一些报道,目前二者低生产率仍然是制约其产业化生产的核心问题之一。先前提高灵芝酸的产量的报道主要集中在培养条件优化,诱导剂添加等方面。随着灵芝酸代谢途径中关键基因被克隆,高表达灵芝酸代谢途径基因被运用于灵芝酸的生产中,通过高表达HMGR基因、SQS基因和LS基因提高了灵芝酸的产量,但高表达灵芝酸合成途径中的其他关键酶基因还没有相关报道。氧化鲨烯合酶(SE),在灵芝酸合成途径中催化第一步氧化反应,将鲨烯催化生成2,3-氧化鲨烯。操纵SE基因已应用于其他物种中次级代谢产物的生产。然而,通过高表达SE基因提高灵芝酸的报道却未报到。本文在灵芝中克隆并高表达了灵芝酸合成途径中的关键酶基因SE基因。结果显示,高表达SE基因菌株比野生型(WT)菌株产总灵芝酸的产量提高了1.4倍。高表达SE基因菌株单体灵芝酸T,S,Mk,Me的最高产量分别为57.7 ± 4.2,19.1 ±2.1,4.3 ±0.2和33.6 ±2.4 μg/100 mg,这比WT菌株四种单体灵芝酸的产量高3.2-,,2.4-,1.8-和2.9倍。另外,SE基因的高表达导致了灵芝酸代谢途径中SE基因和羊毛甾醇合酶(LS)基因表达的上调。结果暗示,在灵芝中,SE在灵芝酸的积累过程中是重要代谢途径关键酶。在灵芝酸代谢途径中,先前的研究主要仅在高表达单个基因上。因为灵芝酸合成调控途径的复杂性,这种策略只提高了适中的单体灵芝酸产量。共表达多基因已经在其他物种中较高的提高了萜类的产量,而通过共表达多个基因未在灵芝中报道。3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是灵芝酸代谢途径MVA中的一个个限速酶。在灵芝中高表达HMGR基因提高了前体物质的积累,SE是固醇或皂苷合成途径中后鲨烯部分的第一个调节酶,所以同时高表达HMGR基因和SE基因可能会更高的提高灵芝酸的产量。因此,本文也在灵芝中共表达了 SE基因和3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)基因,结果显示共表达SE基因和HMGR基因比单独表达SE基因和HMGR基因灵芝酸的产量更高。在共表达SE基因与HMGR基因工程菌株中四种单体灵芝酸单体GA-T,GA-S,GA-Mk,GA-Me的最高含量分别为90.4±7.5.,35.9±5.4,6.2±0.5 和 61.8±5.8μg/100mg 细胞干重,分别是野生菌株(WT)中四种单体灵芝酸产量的5.9-,4.5-,2.4-,5.8倍。我们的结果表明,在灵芝中,操纵多个合成基因来提高灵芝酸的产量是一个有效的方法。
[Abstract]:Ganoderma lucidum is the main anti-cancer, anti-tumor active substance. Ganoderma lucidum is a valuable higher medicinal fungi and valuable Chinese herbal medicine. It has been used to prevent and treat diseases for thousands of years. Ganodermic acid and Ganoderma lucidum polysaccharides, as important medicinal components of Ganoderma lucidum, have the activities of anti-cancer, anti-AIDS and enhancing immunity. In recent years, Ganoderma lucidum has become a hot topic at home and abroad. Although there are some reports on accelerating the growth of Ganoderma lucidum cells and optimizing the production of Ganoderma lucidum acid and polysaccharides, At present, their low productivity is still one of the core problems restricting their industrial production. The previous reports of increasing the yield of ganoderma acid mainly focused on the optimization of cultivation conditions. With the cloning of the key genes in the Ganodermic Acid Metabolism Pathway, the overexpression of Ganodermic Acid Metabolic Pathway genes were used in the production of Ganoderic Acid, and the production of Ganoderma lucidum was increased by overexpression of HMGR gene, HMGR gene and LS gene. However, other key enzyme genes that are highly expressed in the Ganodermic acid synthesis pathway have not been reported. The oxidation of squalene synthase, Sequene synthase, catalyzes the first oxidation reaction in the Ganodermic acid synthesis pathway. Squalene is catalysed into squalene 2zalene oxide. Manipulating the SE gene has been used in the production of secondary metabolites in other species. In this paper, we cloned and overexpressed the key enzyme gene SE gene in Ganoderma lucidum biosynthesis pathway. The results showed that the expression of SE gene in Ganoderma lucidum was higher than that in Ganoderma lucidum. The yield of total ganoderic acid was 1.4 times higher in high expression SE gene strain than in wild type WT-strain, and the highest yield was 57.7 卤4.2n19.1 卤2.1g 卤0.2 and 33.6 卤2.4 渭 g / 100 mg, respectively, which was higher than that of WT strain. The yield of ganodermic acid was 3.2-4-1.8- and 2.9 times higher. In addition, the high expression of SE gene resulted in the up-regulation of SE gene and wool sterol synthase gene expression in the metabolism pathway of Ganoderma lucidum. In Ganoderma lucidum, SE is a key enzyme in the accumulation of Ganoderma lucidum acid. In the Ganoderma lucidum metabolic pathway, previous studies mainly focused on the high expression of a single gene, because of the complexity of the regulatory pathway of ganodermic acid synthesis. This strategy only increases the yield of monomeric ganodermic acid. Co-expression of polygenes has increased terpene production in other species. However, the co-expression of several genes has not been reported in Ganoderma lucidum. 3 hydroxy-3-methylglutaryl CoA reductase (HMGR) is a rate-limiting enzyme in Ganoderma lucidum acid metabolism pathway MVA. The overexpression of HMGR gene in Ganoderma lucidum enhances the accumulation of precursor substances. Se is the first regulating enzyme in the post-squalene part of the steroid or saponins synthesis pathway. Therefore, high expression of HMGR gene and SE gene may increase the yield of ganodermic acid. Therefore, we also expressed SE gene and 3-hydroxy-3-methyl-glutaryl coenzyme A reductase gene in Ganoderma lucidum. The results showed that coexpression of SE gene and HMGR gene was higher than that of single expression of SE gene and HMGR gene Ganoderic acid. The highest content of four monomers GA-TGA-MK GA-Me was 90.4 卤7.5.35.9 卤5.46.2 卤5.46.2 卤0.5 in co-expressed SE gene and HMGR gene engineering strain, respectively. And 61.8 卤5.8 渭 g / 100 mg cell dry weight, The yield of four monomers Ganoderma lucidum was 5.9-4. 5-4. 4-5. 8 times higher than that of wild strain WT. our results showed that it is an effective method to manipulate multiple synthetic genes to increase the yield of Ganoderma lucidum in Ganoderma lucidum.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.31

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