本文選題：茯苓 + 茯苓酸�。� 參考：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：茯苓為我國的傳統(tǒng)用藥,有幾千年的歷史,具有利水滲濕、健脾、寧心之功效,同時在食物方面也有很廣的用途,比如茯苓糕,茯苓酒等。茯苓酸是茯苓的主要活性成分之一,其有抗腫瘤,抗炎抗氧化,降血糖,鎮(zhèn)靜催眠等許多功效。而目前市場上的茯苓酸主要是從茯苓中直接提取,由于茯苓栽培條件的限制,茯苓酸的提取量也會受到一定影響,未來用工程菌株來發(fā)酵茯苓酸是一種解決的辦法,但是目前關于茯苓酸在茯苓體內是如何合成的,還沒有弄清楚。在本研究中,首先推測了茯苓酸在茯苓體內的生物合成途徑,并進行了關鍵酶的預測,對甾醇C-24甲基轉移酶進行了克隆與功能驗證。主要的研究結果如下:1預測茯苓酸的生物合成途徑涉及四個關鍵酶基因,分別是細胞色素P450s;甾醇C-24甲基轉移酶(SMT1);甾醇�；D移酶(SOAT);固醇酯水解酶(TGL4)。由羊毛甾醇經(jīng)過一系列的催化反應合成茯苓酸。2對SMT1基因進行了基因克隆,得到了兩條含有完整閱讀框的基因,分別是SMT1-1和SMT1-2。其中SMT1-1的開放閱讀框有1038個堿基,與基因組序列對比發(fā)現(xiàn),含有2個內含子,3個外顯子,編碼345個氨基酸,是穩(wěn)定的親水性蛋白。同時,SMT1-2基因的開放閱讀框有1050個堿基,有4個內含子,5個外顯子,編碼349個氨基酸,也是穩(wěn)定的親水性蛋白。蛋白3D結構模擬發(fā)現(xiàn)蛋白SMT1-1與SMT1-2結構高度相似,系統(tǒng)發(fā)育分析表明SMT1的蛋白與物種Xanthophyllomyces dendrorhous中的SMT1系統(tǒng)發(fā)育關系很近。3構建原核表達載體pCold-SMT1,并且轉化到E.coli表達菌株BL21(Pg-KJE8)里面。成功優(yōu)化獲得了好的原核表達條件。用獲取的SMT1蛋白驗證了3β-羥基羊毛甾-8,24-二烯-21-酸(trametenolic acid)生成齒孔酸(eburicoic acid)的反應。4利用熒光定量PCR討論了SMT1基因在茯苓體表達情況。在PDA培養(yǎng)基上,26℃培養(yǎng)茯苓菌絲。在6、9、12、15、18天取樣處理進行熒光定量PCR。實驗結果表明SMT1基因中的SMT1-1與SMT1-2在茯苓中的表達水平不同,SMT1-1整體表達量高于SMT1-2,這樣的結果說明可能SMT1-1在發(fā)揮主要的功能,而SMT1-2發(fā)揮次要功能。
[Abstract]:Poria cocos is the traditional medicine of our country, has a history of thousands of years, has the benefit of water, seepage, spleen and heart, at the same time has a very wide use in food, such as Poria cocos cake, Poria cocos wine and so on. Poria cocos acid is one of the main active components of Poria cocos. It has many functions, such as anti tumor, anti-inflammatory and anti-oxidation, hypoglycemia, sedation and hypnosis, etc. At present, the Poria cocos acid in the market is mainly directly extracted from Poria cocos. Due to the restriction of the cultural conditions of Poria cocos, the extraction amount of Poria cocos acid will also be affected to a certain extent. In the future, it is a solution to ferment Poria acid with engineering strains. However, it is not clear how Poria cocos is synthesized in the body of Poria cocos. In this study, we first speculated the biosynthesis pathway of Poria cocos, predicted the key enzymes and cloned sterol C-24 methyltransferase. The main results are as follows: 1. The biosynthesis pathway of pachysolic acid involves four key enzyme genes: cytochrome P450 s, sterol C-24 methyltransferase (SMT1), sterol acyltransferase (SOAT) and sol-ester hydrolase (TGL4). The SMT1 gene was synthesized by a series of catalytic reactions from wool sterol, and two genes, SMT1-1 and SMT1-2, were obtained. The open reading frame of SMT1-1 contains 1038 bases, which contains 2 introns and 3 exons and encodes 345 amino acids. It is a stable hydrophilic protein. At the same time, the open reading frame of SMT1-2 gene contains 1050 bases, 4 introns and 5 exons, encoding 349 amino acids, which is also a stable hydrophilic protein. The structural simulation of protein SMT1-1 and SMT1-2 showed that SMT1-1 was highly similar to SMT1-2. Phylogenetic analysis showed that the protein of SMT1 was closely related to the phylogenetic relationship of SMT1 in Xanthophyllomyces dendrorhous. The prokaryotic expression vector pCold-SMT1 was constructed and transformed into E. coli expression strain BL21 (Pg-KJE8). Good prokaryotic expression conditions were obtained. The reaction of 3 尾 -hydroxy-fleur-824-diene-21acid (trametenolic acid) to the formation of (eburicoic acid) was verified by using the obtained SMT1 protein. The expression of SMT1 gene in Poria cocos was discussed by fluorescence quantitative polymerase chain reaction (FQ-PCR). Poria cocos were cultured on PDA medium at 26 鈩，

本文編號：2091682