本文選題：靈芝　切入點(diǎn)：單體靈芝酸　出處：《昆明理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：靈芝中的靈芝酸是主要抗癌、抗腫瘤轉(zhuǎn)移的活性物質(zhì)。靈芝是一種珍貴的高等藥用真菌和名貴中草藥,用它來預(yù)防和治療疾病已有幾千年歷史。靈芝酸和靈芝多糖作為靈芝的重要藥用成分,具有抗癌、抗愛滋病、提高免疫力等活性。由于其重要的藥理作用,近年來靈芝成為國內(nèi)外研究的一個(gè)熱點(diǎn)。盡管關(guān)于加速靈芝細(xì)胞生長和優(yōu)化靈芝酸與靈芝多糖生產(chǎn)有一些報(bào)道,目前二者低生產(chǎn)率仍然是制約其產(chǎn)業(yè)化生產(chǎn)的核心問題之一。先前提高靈芝酸的產(chǎn)量的報(bào)道主要集中在培養(yǎng)條件優(yōu)化,誘導(dǎo)劑添加等方面。隨著靈芝酸代謝途徑中關(guān)鍵基因被克隆,高表達(dá)靈芝酸代謝途徑基因被運(yùn)用于靈芝酸的生產(chǎn)中,通過高表達(dá)HMGR基因、SQS基因和LS基因提高了靈芝酸的產(chǎn)量,但高表達(dá)靈芝酸合成途徑中的其他關(guān)鍵酶基因還沒有相關(guān)報(bào)道。氧化鯊烯合酶(SE),在靈芝酸合成途徑中催化第一步氧化反應(yīng),將鯊烯催化生成2,3-氧化鯊烯。操縱SE基因已應(yīng)用于其他物種中次級(jí)代謝產(chǎn)物的生產(chǎn)。然而,通過高表達(dá)SE基因提高靈芝酸的報(bào)道卻未報(bào)到。本文在靈芝中克隆并高表達(dá)了靈芝酸合成途徑中的關(guān)鍵酶基因SE基因。結(jié)果顯示,高表達(dá)SE基因菌株比野生型(WT)菌株產(chǎn)總靈芝酸的產(chǎn)量提高了1.4倍。高表達(dá)SE基因菌株單體靈芝酸T,S,Mk,Me的最高產(chǎn)量分別為57.7 ± 4.2,19.1 ±2.1,4.3 ±0.2和33.6 ±2.4 μg/100 mg,這比WT菌株四種單體靈芝酸的產(chǎn)量高3.2-,,2.4-,1.8-和2.9倍。另外,SE基因的高表達(dá)導(dǎo)致了靈芝酸代謝途徑中SE基因和羊毛甾醇合酶(LS)基因表達(dá)的上調(diào)。結(jié)果暗示,在靈芝中,SE在靈芝酸的積累過程中是重要代謝途徑關(guān)鍵酶。在靈芝酸代謝途徑中,先前的研究主要僅在高表達(dá)單個(gè)基因上。因?yàn)殪`芝酸合成調(diào)控途徑的復(fù)雜性,這種策略只提高了適中的單體靈芝酸產(chǎn)量。共表達(dá)多基因已經(jīng)在其他物種中較高的提高了萜類的產(chǎn)量,而通過共表達(dá)多個(gè)基因未在靈芝中報(bào)道。3-羥基-3-甲基戊二酰輔酶A還原酶(HMGR)是靈芝酸代謝途徑MVA中的一個(gè)個(gè)限速酶。在靈芝中高表達(dá)HMGR基因提高了前體物質(zhì)的積累,SE是固醇或皂苷合成途徑中后鯊烯部分的第一個(gè)調(diào)節(jié)酶,所以同時(shí)高表達(dá)HMGR基因和SE基因可能會(huì)更高的提高靈芝酸的產(chǎn)量。因此,本文也在靈芝中共表達(dá)了 SE基因和3-羥基-3-甲基戊二酰輔酶A還原酶(HMGR)基因,結(jié)果顯示共表達(dá)SE基因和HMGR基因比單獨(dú)表達(dá)SE基因和HMGR基因靈芝酸的產(chǎn)量更高。在共表達(dá)SE基因與HMGR基因工程菌株中四種單體靈芝酸單體GA-T,GA-S,GA-Mk,GA-Me的最高含量分別為90.4±7.5.,35.9±5.4,6.2±0.5 和 61.8±5.8μg/100mg 細(xì)胞干重,分別是野生菌株(WT)中四種單體靈芝酸產(chǎn)量的5.9-,4.5-,2.4-,5.8倍。我們的結(jié)果表明,在靈芝中,操縱多個(gè)合成基因來提高靈芝酸的產(chǎn)量是一個(gè)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 鹵4.2n19.1 鹵2.1g 鹵0.2 and 33.6 鹵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 鹵7.5.35.9 鹵5.46.2 鹵5.46.2 鹵0.5 in co-expressed SE gene and HMGR gene engineering strain, respectively. And 61.8 鹵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.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S567.31

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本文編號(hào)：1614696