【作者】 穆大帅；

【导师】 赵明文；

【作者基本信息】 南京农业大学 ， 微生物学， 2013， 博士

【摘要】 灵芝(Ganoderma lucidum)是著名的大型药用真菌,具有极高的营养价值和药用价值,在我国和东南亚国家有着悠久的应用历史。灵芝三萜类化合物是灵芝的主要药用成分之一,具有抗氧化、抗病毒、降血压和抗肿瘤等活性。灵芝三萜含量的多少是目前衡量灵芝质量高低的重要指标。最近灵芝基因组测序工作已经完成,为从分子遗传学水平研究灵芝三萜生物合成等灵芝基础生物学研究提供数据支持,有利于推动灵芝成为一种新型模式药用真菌,增加其科研和商业价值。但是,灵芝中缺乏简便有效的反向遗传学体系,限制了人们对灵芝功能基因组学研究的进展,影响了其药用价值的开发。因此,本研究将从灵芝基因沉默体系的建立,并应用该体系对NADPH氧化酶基因家族功能研究来探讨灵芝三萜调控机制。主要分为以下几个方面：(一)灵芝沉默体系建立：RNA干扰技术(RNAi)作为一种常见的反向遗传学基因操作技术被广泛应用在动物、植物和微生物功能基因组学研究领域。在本课题研究中,首先通过基于电击转化不完全酶解灵芝原生质体的方法建立了灵芝中简便快捷的转基因技术,成功的将外源绿色荧光蛋白转入灵芝中。通过抗生素抗性传代、标记基因PCR检测、绿色荧光检测等手段确定了该方法能够进行外源基因表达,为灵芝RNAi技术的建立提供转基因操作平台。进一步在灵芝中通过BLAST对比分析发现具有Dicer同源蛋白,暗示灵芝可能具有RNAi的机制,利用灵芝内源乳清酸核苷-5’-单磷酸脱羧酶基因(ura3)作为反向筛选标记,构建四种沉默载体(颈环结构沉默载体,正向沉默载体,反向沉默载体以及正反双向启动子沉默载体)并基于此转基因方法实现了对灵芝内源基因的沉默。通过系统的比对以上四种沉默方法,发现四种沉默方法都不同程度的下调了ura3基因的表达,其中双向启动子沉默不仅载体构建方便并且能够获得最高的沉默效率(达到81.9%)。利用这种双向启动子沉默载体以及ura3的反向筛选特性,进而建立了灵芝的共沉默体系,发现共沉默基因(ura3和laccase1)转录水平的下调是有一定相关性的,这提高了基因沉默的筛选效率。该反向遗传体系的建立有助于深入研究灵芝中单基因或多基因的功能,为本课题接下来对NADPH氧化酶基因家族的功能研究提供技术支持。(二)对NADPH氧化酶功能的分析：NADPH氧化酶(Nox)是一类与活性氧(ROS)合成有关的酶,最近有研究表明其在植物与动物中有多种重要的生物学作用,但是,在丝状真菌特别是大型担子菌中Nox的具体功能还没有完全被了解。在本课题研究中,根据灵芝基因组测序序列,鉴定出NADPH氧化酶基因的两种Nox亚型(NoxA和NoxB)和一种Nox调控基因NoxR。采用正反双启动子RNA沉默的方法研究Nox家族功能。通过沉默NoxA,NoxB和NoxR基因的表达,并对其表型研究,发现这个基因家族在ROS的合成,调控灵芝三萜的生物合成、子实体发育以及抗氧化胁迫等方面有重要作用。Nox沉默菌株表现出ROS积累降低,灵芝三萜生物合成下调,子实体发育受阻以及对H202耐受性降低等表型。(三)Nox对钙信号的调控机制研究：动植物中,ROS被报道能够与钙信号通路进行互作,激活钙通道,增加钙离子向胞内的流入进而影响钙信号传递。其中,钙离子作为生物体内重要的第二信使参与多种生物学功能,但是此类信号调控机制在真菌中鲜有报道。为了验证灵芝中Nox产生的ROS能否对钙信号产生影响,本课题检测了Nox沉默菌株中胞内钙离子含量,发现Nox沉默转化子中胞内钙离子水平显著降低。对其调控机理研究发现,灵芝中ROS影响胞内钙离子水平是通过激活钙通道增加钙流入实现的,同时结合转录水平分析了灵芝中37个钙信号基因响应ROS的表达情况,发现大多数钙离子信号基因在ROS刺激早期被激活表达,而在刺激后期多数被下调表达。通过对Nox沉默转化子中钙信号基因的转录进一步分析,发现钙信号基因转录能够受到Nox沉默影响。进一步研究发现Nox调控灵芝三萜生物合成及菌丝分叉是通过钙信号实现的,阻断钙信号将导致ROS的调控效果消失。综上所述,本研究突出了在灵芝中NADPH氧化酶在灵芝ROS和钙信号交互中的作用,与动物植物中一样,在细胞的生理过程中Nox产生的ROS可以激活钙信号。这些结果为发现鉴定真菌中ROS信号和Ca2+信号网络交互的潜在机制提供了有利的机会,暗示了在动物、植物和微生物共同享有一个保守的信号交互机制。更多还原

【Abstract】 Ganoderma lucidum is one of the most important medicinal mushrooms in China and Southeast Asia. Modern researches showed that the chemical composition of G. lucidum complex have a wide range of pharmacological activities. Triterpenes in G. lucidum, also called ganoderic acid, which is one kind of the secondary metabolites, are considered as the main active ingredient. They have important medicinal values, such as:anti-tumor inhibits histamine release, anti-HIV and absorption effects and so on. Ganoderic acids (GAs) have the most important pharmacological activity, as a results, their contents have been taken as an important measurement of the quality of G. lucidum. Currently, the completed genome sequence for Ganoderma lucidum has generated widespread interest in investigating this organism, which is becoming a potential model system for the study of complicated growth patterns and the regulation of secondary metabolic pathways in medicinal mushrooms. Nonetheless, the scarcity of basic biology studies has hindered the further development of its commercial value.RNA interference (RNAi) is a convenient reverse genetic method that has been widely used in several types of organisms for studying functional genomics.. In this study, we established the transformation procedure based on the electroporation of mycelial fragments, which is a convenient method for introducing transgenes into G. lucidum and results in hygromycin B resistance and GFP expression in the transformants. The endogenous orotidine5’-monophosphate decarboxylase gene (ura3) was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of ura3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of ura3silencing compared with other vectors (up to81.9%). To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing ura3and laccase1in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species for our further studies.Reactive oxygen species (ROS) have long been considered to be deleterious by-products of aerobic metabolism. However, recent studies on NADPH oxidases (Noxs), a large family of enzymes dedicated to ROS production, have highlighted the many important biological roles of ROS. NADPH oxidases have recently been highlighted due to the many important biological roles in plants and animals; however, the exact functions of Nox are still not fully understood in fungi. In this study, based on the completed genome sequence of G. lucidum, we identified two Nox isoforms (NoxA and NoxB) and a regulator, NoxR. RNA interference (RNAi) was used to examine the function of the Nox family, and silencing of the Nox isoforms and NoxR expression indicated a central role for this gene family in ROS generation to regulate ganoderic acid biosynthesis and hyphal growth. Additionally, the Nox genes are required for fruiting body development and ROS resistance.Oxidants are known to trigger the generation of Ca2+signals, in part through the activation of calcium channels in mammal cells and plants to activate a series of biological and physiological processes. However, this mechanism in fungi remains to be determined. To explore whether Nox-generated ROS could activate Ca2+signaling in G. lucidum and whether this activation would regulate GA biosynthesis. We detected the intracellular Ca2+levels of the Nox silenced strains using the Ca2+-fluorescent probe Fluo-3AM. The NoxAB silenced strains, NoxR silenced strains and DPI treatment caused a significant reduction in the cytosolic Ca2+levels. Further mechanistic investigation revealed that Nox-generated ROS elevated cytosolic Ca2+levels by activating a plasma membrane Ca2+influx pathway, thereby inducing the Ca2+signal pathway to regulate GA biosynthesis and hyphal branching. On transcription level, most of the Ca2+signaling genes exhibited an early response to ROS at30min, with some being up-regulated at3h and most genes down-regulated at12h. These results suggest a Ca2+-signal pathway in G. lucidum, and that the signaling genes respond to H2O2. Importantly, our results highlight the Nox functions in the Basidiomycetes G. lucidum, and we provide evidence that Noxs, as in plants and animals, might activate Ca2+signaling during cell physiological processes in this fungus. These findings provide an excellent opportunity to identify the potential pathway linking ROS networks to calcium signaling in fungi and suggest that plants, animals, and fungi share a conserved signal-crosstalk mechanism.更多还原