茯苓甾醇C-24甲基转移酶基因克隆与功能验证

发布时间：2018-07-03 01:04

  本文选题：茯苓 + 茯苓酸　； 参考：《华中农业大学》2017年硕士论文

【摘要】：茯苓为我国的传统用药,有几千年的历史,具有利水渗湿、健脾、宁心之功效,同时在食物方面也有很广的用途,比如茯苓糕,茯苓酒等。茯苓酸是茯苓的主要活性成分之一,其有抗肿瘤,抗炎抗氧化,降血糖,镇静催眠等许多功效。而目前市场上的茯苓酸主要是从茯苓中直接提取,由于茯苓栽培条件的限制,茯苓酸的提取量也会受到一定影响,未来用工程菌株来发酵茯苓酸是一种解决的办法,但是目前关于茯苓酸在茯苓体内是如何合成的,还没有弄清楚。在本研究中,首先推测了茯苓酸在茯苓体内的生物合成途径,并进行了关键酶的预测,对甾醇C-24甲基转移酶进行了克隆与功能验证。主要的研究结果如下:1预测茯苓酸的生物合成途径涉及四个关键酶基因,分别是细胞色素P450s;甾醇C-24甲基转移酶(SMT1);甾醇酰基转移酶(SOAT);固醇酯水解酶(TGL4)。由羊毛甾醇经过一系列的催化反应合成茯苓酸。2对SMT1基因进行了基因克隆,得到了两条含有完整阅读框的基因,分别是SMT1-1和SMT1-2。其中SMT1-1的开放阅读框有1038个碱基,与基因组序列对比发现,含有2个内含子,3个外显子,编码345个氨基酸,是稳定的亲水性蛋白。同时,SMT1-2基因的开放阅读框有1050个碱基,有4个内含子,5个外显子,编码349个氨基酸,也是稳定的亲水性蛋白。蛋白3D结构模拟发现蛋白SMT1-1与SMT1-2结构高度相似,系统发育分析表明SMT1的蛋白与物种Xanthophyllomyces dendrorhous中的SMT1系统发育关系很近。3构建原核表达载体pCold-SMT1,并且转化到E.coli表达菌株BL21(Pg-KJE8)里面。成功优化获得了好的原核表达条件。用获取的SMT1蛋白验证了3β-羟基羊毛甾-8,24-二烯-21-酸(trametenolic acid)生成齿孔酸(eburicoic acid)的反应。4利用荧光定量PCR讨论了SMT1基因在茯苓体表达情况。在PDA培养基上,26℃培养茯苓菌丝。在6、9、12、15、18天取样处理进行荧光定量PCR。实验结果表明SMT1基因中的SMT1-1与SMT1-2在茯苓中的表达水平不同,SMT1-1整体表达量高于SMT1-2,这样的结果说明可能SMT1-1在发挥主要的功能,而SMT1-2发挥次要功能。
[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，

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