多种材料中紫杉醇合成能力的基因证据探索

发布时间：2018-01-17 16:17

本文关键词：多种材料中紫杉醇合成能力的基因证据探索　出处：《中国林业科学研究院》2015年博士论文　论文类型：学位论文

【摘要】：紫杉醇(Taxol)是一种广泛应用于治疗肺癌、卵巢癌和乳腺癌的复杂二萜化合物,它首先从短叶红豆杉(Taxus brevifolia)树皮中分离得到。现在紫杉醇主要来源为生长缓慢的红豆杉树木,紫杉醇的供应尚严重不足。因此,寻找紫杉醇的替代来源非常重要。在过去的20多年里,人们对一些生产紫杉醇的替代方法进行了探索,其中包括产紫杉醇微生物的研究。人们在分离具有紫杉醇合成能力的内生菌(真菌和细菌)时,通常使用可培养的方法,即通过可培养的方法在培养基上分离可培养菌,但是,这种方法不能分离不可培养的内生菌,造成对菌株的筛选不全面,可能会错过高产的菌株。下一代测序技术中的宏基因组测序已经应用到对有些土壤微生物群落的物种和基因组成研究。但到目前为止,仍没有关于红豆杉根部相关微生物的菌种鉴定和基因组成进行研究的报道。在本研究中,我们使用可以靶定16S和18S r DNA的引物,利用bar-coded高通量测序技术,对红豆杉根部相关的细菌和真菌做了一个全面的调查。通过宏基因组研究调查了红豆杉根部相关微生物的物种分布和基因组成,预测了红豆杉根部相关微生物中和紫杉醇合成相关的基因,旨在通过对这些基因的研究来评估红豆杉根部相关微生物中是否存在具有紫杉醇合成能力的菌株。除产紫杉醇内生真菌以外,人们还在甜菜树(Yunnanopilia longistaminata)、粗榧(Cephalotaxus sinensis)、榛子(Corylus heterophylla Fisch)、马尾松(Pinus massoniana Lamb)、罗汉松(Podocarpus macrophyllus)等植物中发现了包括紫杉醇在内的多种紫杉烷类化合物的存在,但尚没有足够的证据证明其具有紫杉烷类化合物合成的能力。众所周知,证明紫杉二烯合酶(Taxadiene synthease,TS)基因的存在是证明这种能力存在的最好证据。所以,本研究对产紫杉醇内生真菌Penicillium aurantiogriseum NRRL 62431、甜菜树及粗榧的基因组数据或转录组数据进行了TS基因的预测,并对预测的类紫杉二烯合酶基因(Taxadiene synthease like gene,TSL)进行了功能鉴定,试图从分子水平上找到其具有合成紫杉醇能力的证据。通过实验发现,我们筛选的基因在体外都不具备紫杉二烯合酶的功能,只有粗榧中的Cs TSL能够催化GGPP合成紫杉烷类化合物的中间产物verticiol。本文主要研究结果如下:1.对南方红豆杉(Taxus chinensis(Pilger)Rehd)根部进行了微生物多样性的调查。通过16S r DNA测序共鉴定出640个细菌OTU(Operational Taxonomic Units)。本研究发现变形菌(Proteobacteria)是南方红豆杉根部最丰富的细菌种类。遗憾的是并没有找到任何报道过的能够合成紫杉醇的细菌物种。之后通过18S r DNA测序共鉴定出187个真菌OTU。发现担子菌门(Basidiomycota)是南方红豆杉根部最丰富的真菌种类。我们发现丝齿菌属(Hyphodontia)、Hemimycena属和Phialocephala属是三个南方红豆杉根部真菌优势属,这也是首次在所有红豆杉属中报道发现这3个属。本文中发现了187个真菌的OTU,这比以往报道的任何依靠培养的方法发现的菌株都要多,这样说明南方红豆杉根部寄生着多样的真菌。对南方红豆杉根部进行了宏基因组调查,经分析发现Contig主要分布在34个细菌的门中和9个真菌的门中。甲型变形菌(Alphaproteobacteria)、丙型变形菌(Gammaproteobacteria)和芽孢杆菌(Bacilli)是最占优势的细菌类群,而酵母菌(Saccharomycetes)、球囊菌纲(Glomeromycetes)和粪壳菌纲(Sordariomycetes)是最占优势的真菌类群。并发现可能存在5个具有紫杉醇合成能力的微生物物种。2.对一个产紫杉醇内生真菌P.aurantiogriseum NRRL 62431的基因组数据进行了分析,预测到一个紫杉二烯合酶和一个GGPP合酶。首先完整克隆出内生真菌P.aurantiogriseum NRRL 62431的一个TSL基因的开放阅读框(Open Reading Frame,ORF),并研究了其活性,发现其反应产物中并没有紫杉二烯,说明这个TSL基因可能不是紫杉二烯合酶基因。当然,也可能是该Pa TSL基因在体外表达的蛋白不具备紫杉二烯合酶的功能。同时完整克隆出内生真菌P.aurantiogriseum NRRL 62431的GGPPS基因的ORF序列,并用生物信息学预测和分析了Pa GGPPS的氨基酸序列、理化性质、疏水性/亲水性、功能结构域和二级结构。该基因在大肠杆菌成功进行了表达,产生了可溶性的蛋白,并通过试剂盒进行了纯化。这些工作为下一步深入研究该内生真菌的紫杉醇合成途径中此基因的作用和构建高产紫杉醇基因工程菌株提供了一定的基础。3.对含有紫杉烷类化合物的甜菜树(Y.longistaminata)进行了一系列研究。首先对其进行了转录组测序。通过转录组测序,总共得到了约4.8G的序列数据,组装得到了51,744条Unigene序列。接着,对转录组进行了KEGG代谢途径注释,共有6,513个Unigene得到注释,这些转录本主要被注释到了335条代谢途径中,其中,有41条Unigene被注释到萜类合成中,这些数据中可能含有甜菜树能够合成紫杉烷类化合物的基因证据。通过对转录组数据进行本地Blast筛选,共发现2个TSL基因片段(Yl TSL1和Yl TSL2)。然后针对Yl TSL1的功能展开了研究,利用RACE-PCR技术从甜菜树中克隆得到了Yl TSL1基因的c DNA全长,并研究了其蛋白活性,发现其并不具备二萜合酶的功能,但是却有α-法尼烯合酶的功能,所以甜菜树中得到的基因Yl TSL1并非紫杉二烯合酶基因,可以确定为α-法尼烯合酶基因。随后在展开了对Yl TSL2基因的研究,利用RACE-PCR技术从甜菜树中克隆得到了Yl TSL2基因的c DNA全长,并研究了其蛋白活性,发现Yl TSL2基因为贝壳杉烯合酶基因。4.对粗榧(C.sinensis)中的TSL基因进行了研究。通过分析Gen Bank中已有柱冠粗榧(C.harringtonia)的转录组数据(SRA:SRR064395),预测到一个TSL基因(Cs TSL),通过RACE-PCR从粗榧(C.sinensis)中克隆到了Cs TSL基因的c DNA全长,并进行了其蛋白活性的研究,发现其在体外并不具备紫杉二烯合酶的功能,但是可以催化GGPP合成verticiol和17-methyl-5a-androst-2-ene两种化合物,后者为反应的主产物,是一种激素类似物。而对于副产物verticiol,Erdtman等曾推测其是紫杉二烯合成过程中的中间化合物。Cs TSL为何不能直接催化GGPP形成紫杉二烯?可能是因其没有催化最后一步环化的活性中心,这需要在后续研究中进一步分析。
[Abstract]:Paclitaxel (Taxol) is a widely used in the treatment of lung cancer, two complex triterpenoids, ovarian cancer and breast cancer, it first from Taxus brevifolia (Taxus brevifolia) isolated from the bark. Now paclitaxel is mainly from the slow growth of Taxus trees, taxol supply is seriously insufficient. Therefore, looking for paclitaxel an alternative source of alcohol is very important. In the past 20 years, the people of some alternative methods of taxol production are explored, including the study of taxol producing microorganisms. It has the ability of taxol biosynthesis in isolated endophytic fungi (fungi and bacteria), usually using the method of culture, namely through methods cultured on the culture medium separation of culturable bacteria, however, this method cannot be separated from the non culturable endophytic bacteria, resulting in screening of strains is not comprehensive, may miss the high-yield strains. The next generation sequencing technology Macro genome sequencing has been applied to species and genes on some soil microbial community composition research. But so far, the identification and gene is still no microbial composition of Taxus roots on research reports. In this study, we can use primers targeting 16S and 18S R DNA, using Qualcomm bar-coded the amount of sequencing of Taxus root associated bacteria and fungi has done a comprehensive survey. The species distribution and composition of microbial gene of Taxus roots were investigated through metagenomic research, prediction of yew root microorganism and taxol synthesis related genes, to assess whether there has the ability of synthesis of taxol strains the root of Taxus microorganisms through the study of these related genes. In addition to Taxol producing endophytic fungi, people still beet tree (Yunnanop Ilia LONGISTAMINATA) (Cephalotaxus sinensis), Cephalotaxus sinensis (Corylus heterophylla Fisch), hazelnut, masson pine (Pinus massoniana Lamb) (Podocarpus macrophyllus), Podocarpus found a variety of taxanes including taxol in the presence of other plants, but there is not enough evidence to prove its ability of taxanes biosynthesis. As everyone knows, that of taxol diene synthase (Taxadiene, synthease, TS) gene is the best evidence of this ability exist. Therefore, the study of taxol producing endophytic fungi Penicillium aurantiogriseum NRRL 62431, genome or transcriptome data beet tree and prediction of Cephalotaxus sinensis TS gene, and to predict the taxane diene synthase gene (Taxadiene synthease like gene, TSL) of the functional analysis, trying to find the synthesis of taxol from the molecular level The ability of evidence. Through the experiment, we screened the gene does not have the taxadiene synthase function in vitro, Cs TSL can only in Cephalotaxus intermediate verticiol. catalyzed GGPP the main results of this paper are as follows: 1.. Synthesis of taxane compounds of Taxus chinensis (Taxus chinensis (Pilger) Rehd) conducted a survey of microbial diversity in root the 16S R DNA. By sequencing identified 640 bacteria OTU (Operational Taxonomic Units). The study found that the deformation of bacteria (Proteobacteria) is the most abundant species of Taxus chinensis roots. Unfortunately did not find any reported to bacterial species on the biosynthesis of taxol. After 18S R DNA sequencing Co OTU. found that identified 187 fungi Basidiomycota (Basidiomycota) is the most abundant fungal species of Taxus chinensis root. We found that hyphodontia (Hyphodontia, Hemim) Ycena and Phialocephala genus are three Taxus root fungi genera, this is the first time in all reported in Taxus found these 3 genera. Found 187 fungi OTU in this paper, the methods of cultivating it rely on any than previously reported strains are more, this shows that Taxus chinensis root parasitic a variety of fungi. The metagenomic survey of Taxus roots, the analysis shows that Contig mainly distributed in the 34 bacteria and 9 fungi door door. Alpha Proteobacteria (Alphaproteobacteria), C Proteobacteria (Gammaproteobacteria) and Bacillus (Bacilli) is the most dominant bacteria, and yeast (Saccharomycetes), glomeromycetes (Glomeromycetes) and sordariomycetes (Sordariomycetes) is the most dominant fungal taxa. And found that there are 5 has the ability of taxol biosynthesis in microorganisms Species.2. analysis of the genomic data of a taxol producing endophytic fungus P.aurantiogriseum NRRL 62431, predict a taxadiene synthase and GGPP synthase TSL gene. A first complete clone of endophytic fungus P.aurantiogriseum NRRL 62431 open reading frames (Open, Reading, Frame, and ORF) study on the activity, found that the reaction product is not taxadiene, this TSL gene may not taxadiene synthase gene. Of course, it may be the expression of Pa TSL gene in vitro protein does not have the taxadiene synthase function. The ORF sequence of GGPPS gene and cloned complete P.aurantiogriseum endophytic fungi NRRL 62431 the use of bioinformatics prediction and analysis of the amino acid sequence of Pa GGPPS, physicochemical properties, hydrophobicity / hydrophilicity, functional domains and two level structure. The gene in Escherichia coli The expression of the soluble protein, and the kit was purified. This work provides a basis for a certain.3. of the beet tree containing taxanes taxol biosynthesis pathway of this gene further study the endophytic fungi and the role of constructing high-yield paclitaxel gene engineering strain (Y.longistaminata) conducted a series of studies. Firstly, transcriptome sequencing. By transcriptome sequencing, a total sequence of data about 4.8G, assembled by 51744 Unigene sequences. Then, on the transcriptome metabolic pathways of KEGG notes, a total of 6513 Unigene notes, these major transcripts were annotated the 335 pathways, among them, 41 Unigene were annotated to terpenoid synthesis, may contain beet gene trees can evidence synthesis of taxanes through these data. Local Blast screening of transcriptome data, 2 were found in TSL gene fragments (Yl TSL1 and Yl TSL2). Then studied the function of TSL1 in Yl, cloned from beet tree obtained the full-length Yl gene of TSL1 C DNA by RACE-PCR technology, and studied its protein activity, it does not have two terpene synthase function, but alpha farnesene synthase function, so the Yl TSL1 gene of beet tree is not the taxadiene synthase gene can be identified as the alpha farnesene synthase gene. Then carries out the research on Yl TSL2 gene, cloned from sugar beet tree has been the full-length Yl TSL2 gene C DNA by using RACE-PCR technology, and studied its protein activity, found that Yl TSL2 gene for kaurene synthase gene.4. of Cephalotaxus sinensis (C.sinensis) in the TSL gene were studied. Through the analysis of the existing Bank Gen column (C.harringtonia) to the crown of Cephalotaxus sinensis Recorded data group (SRA:SRR064395), predicted that a TSL gene (Cs TSL), RACE-PCR (C.sinensis) from Cephalotaxus sinensis by cloning the full-length Cs gene of TSL C DNA, and studied its protein activity, found that it does not have the taxadiene synthase function in vitro, but can catalyze the synthesis of GGPP verticiol and 17-methyl-5a-androst-2-ene two compounds, the latter is the main reaction products, is a kind of hormone analogues. The by-products such as Erdtman verticiol, have speculated that it is why taxadiene synthesis process of the intermediate compound.Cs TSL directly catalyzed GGPP formation of taxadiene? Probably because it is not the last step of catalytic cyclization the activity center, which need further analysis in the follow-up study.

【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q933;Q943.2

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