细胞色素P450在植物三萜和甾醇骨架修饰中的功能研究进展

植物三萜类化合物在植物生长发育、抵御逆境胁迫与病虫害、生物间相互作用以及传递信息等方面发挥重要作用,植物甾醇具有重要的药用价值.细胞色素P450单加氧酶(CYP450)是参与植物代谢的最大酶家族,在三萜及甾醇骨架结构多样化及功能化修饰中具有关键作用.到目前为止,研究发现约有80个CYP450参与植物三萜代谢,包括亚家族CYP51H, CYP71A, D, CYP72A, CYP81Q, CYP87D, CYP88D, L, CYP93E, CYP705A, CYP708A和CYP716A, C, E, S, U, Y,它们参与包括特定病原体的化学防御功能和药理活性的三萜及其皂苷类化合物的代谢.亚家族CYP51G, CYP85A, CYP90B-D, CYP710A, CYP724B和CYP734A与甾醇和类固醇激素的生物合成有关.本文针对CYP450基因在三萜及甾醇化合物形成过程中不同位点的修饰功能进行概述,重点探讨了陆地植物CYP450基因11个家族的进化及在双子叶、单子叶植物中五环三萜物质合成过程中的功能.以期为充分利用具有重要价值的抗肿瘤、抗艾滋病的三萜物质的合成生物学的研究及其代谢调控提供进一步的理论依据.     

细胞色素P450酶系与除草剂代谢

细胞色素P450是广泛存在于动物、植物和微生物体内的一类具有混合功能的血红素氧化酶系。它不但能够催化苯丙烷类、萜类化合物和脂肪酸等内源性物质的生物合成 ,而且参与许多外源性物质包括除草剂等的生物氧化。综述了代谢除草剂的细菌、哺乳动物和植物细胞色素P450酶系 ,概述了细胞色素P450酶系参与除草剂代谢的作用方式 :脱烷基化作用、环甲基化羟基化作用和芳环的羟基化作用等。这些细胞色素P450酶系在培育除草剂抗性作物、生物安全和生物修复方面表现出了巨大的潜能     

家蚕细胞色素P450的基因组学分析

细胞色素P450参与许多基础代谢过程, 确保有机体避免外源复合物对它们的伤害. 将预测的家蚕基因同已知的P450基因进行蛋白质序列比对, 在家蚕基因组中发现86个可能的细胞色素P450基因, 初步将它们归于32个P450亚家族. 通过比较基因组学分析, 发现细胞色素P450基因在果蝇与家蚕中的分布规律具有总体上的相似性, 但在某些P450基因家族中的分布也有差异. 特别是在CYP4A, CYP4C, CYP4D, CYP6A, CYP6AE, CYP6B和CYP9A等7个亚家族中P450s差异分布更明显. 进一步将这些P450基因的DNA序列与家蚕ESTs进行核酸序列比对, 其中49个可能P450基因发现有ESTs证据, 证明了这些基因在转录水平的真实性.     

甜菊醇糖苷生物合成途径关键酶基因KA13H的克隆及序列分析

本研究利用RT-PCR方法从甜叶菊叶片中分离了一个与甜菊醇糖苷生物合成密切相关的基因KA13H,对该基因的ORF、编码产物结构、同源性比对及次级结构等进行了生物信息学预测分析,同时初步分析该基因的组织表达和原核表达。经DNAMAN6.0软件分析,该基因编码一条分子量为54.476kD的由476个氨基酸残基组成的多肽,含有典型的细胞色素P450的血红素结合位点FXXGXXXCXG,TMPRED程序预测其C-端含有一个明显的跨膜区MIQVLTPILLFLIFFVFWKVY,是一个典型的细胞色素P450基因。推断的KA13H编码产物与其它生物合成相关细胞色素P450同源比对和系统发生分析表明,该蛋白与苜蓿(Medicago truncatula)CYP716A12和云杉(Sitka spruce)CYP720B1的一致性较高,分别为51%和46%,推测KA13H可能与CYP716A12和CYP720B1具有类似的功能。KA13H次级结构分析结果表明,KA13H与CYP74A2的一致性仅为15%,但是它们却有着类似的次级结构,都含有6个基质识别位点(SRSs)。半定量RT-PCR分析表明:KA13H在根、茎、叶和花中呈组成型表达,叶和花中的表达丰度较高;将该基因融合到原核表达载体pGEX-4T-1中,在原核中得到了成功表达。根据本研究结果,我们推测KA13H可能在甜菊醇糖苷生物合成过程中发挥着重要作用,该基因的克隆和表达分析为进一步深入了解甜菊醇的生物合成过程中相关酶和基因的功能奠定了基础。     

籼稻细胞色素P450超基因家族成员及其EST证据

将预测的籼稻基因同289个已知的拟南芥细胞色素P450基因进行蛋白质序列比对, 在籼稻(Oryza sativa L. ssp. indica)工作框架草图中找到了528个可能的细胞色素P450基因, 初步认定上述籼稻P450基因来自于已在拟南芥中确定的40个P450基因家族. 比较拟南芥和籼稻的细胞色素P450s在基因家族中的分布, 发现这两种植物P450基因的家族分布规律总体相似, 但也有不同, 例如: CYP71, CYP72, CYP76, CYP89, CYP94, CYP709等家族的成员在籼稻中远远多于拟南芥的; CYP705家族的成员在籼稻中没有, 但在拟南芥中却多达33个. 另外, 发现籼稻CYP71和CYP81家族的成员在基因组中成串联重复排列, 它们可能是进化过程中基因复制的结果. 进一步将这些籼稻P450基因的DNA序列同籼稻表达序列标签(expression sequence tags, ESTs)进行核酸序列比对, 为263个可能的籼稻P450s找到了ESTs的证据, 说明这些基因在转录水平上确实有所表达.     

苹果蠹蛾细胞色素P450基因CYP332A19和CYP337B19的克隆及表达分析

【目的】本研究旨在通过克隆苹果蠹蛾Cydia pomonella细胞色素P450基因CYP332A19和CYP337B19,并对其进行序列和表达分析,以更好地了解这两个P450基因在植物次生物质解毒方面的作用,为进一步的功能研究提供依据。【方法】采用本地BLAST搜索苹果蠹蛾转录组数据库获得细胞色素P450基因cDNA序列,采用RT-PCR技术克隆目的基因的编码区。利用生物信息学软件分析目的基因的序列特征及与其他近缘物种的P450基因的系统进化关系。采用RT-qPCR技术测定目的基因在苹果蠹蛾不同发育阶段(卵、1-5龄幼虫、蛹和成虫)、4龄幼虫不同组织(头部、表皮、脂肪体、中肠和马氏管)以及4龄幼虫分别取食添加0.1%香豆素和0.5%槲皮素的人工饲料2 d后的表达水平。【结果】克隆获得苹果蠹蛾细胞色素P450基因CYP332A19(GenBank登录号: MF574708)和CYP337B19(GenBank登录号: MF574697)的全长cDNA序列,开放阅读框(ORF)分别长1 518和1 491 bp,分别编码505和496个氨基酸,其蛋白质分子量分别为58.586和57.734 kD,理论等电点分别为8.99和7.61。结构域分析显示,CYP332A19和CYP337B19中均包含包括血色素结合区在内的5个保守的细胞色素P450结构域。系统发育树显示,苹果蠹蛾CYP332A19与苹淡褐卷蛾Epighyas postvittana CYP332A9等CYP332A基因聚在一枝,而CYP337B19与稻纵卷叶螟Cnaphalocrocis medinalis CYP337B12和六星灯蛾Zygaena filipendulae CYP337B11等CYP337B基因聚在另一枝。RT-qPCR分析结果表明,CYP332A19和CYP337B19在苹果蠹蛾幼虫期的表达水平高于卵期的,分别在4龄幼虫脂肪体和中肠中的表达量最高。取食分别含0.1%香豆素和0.5%槲皮素的人工饲料2 d后,4龄幼虫体内的CYP332A19和CYP337B19相对表达量显著高于对照组(取食含2%DMSO的人工饲料)。【结论】CYP332A19和CYP332B19分别在苹果蠹蛾幼虫脂肪体和中肠中高表达,且在取食含香豆素和槲皮素的人工饲料的苹果蠹蛾幼虫体内表达量升高,说明这两个基因可能参与苹果蠹蛾对外源物质的解毒代谢过程。本研究的结果有助于我们了解苹果蠹蛾对寄主次生物质解毒代谢机理,为苹果蠹蛾防治提供新思路。     

鸡细胞色素P450 1A5的原核表达与活性重组

旨在对鸡细胞色素P450 1A5(CYP1A5)蛋白进行体外功能研究,采用大肠杆菌系统进行CYP1A5的异源表达。以鸡的cDNA为模板,扩增出CYP1A5基因,将该基因的N端编码区进行修饰,并连接到pCW载体中构建His-CYP1A5,经IPTG诱导在大肠杆菌中表达。经CO-差示光谱检测,所获得的His-CYP1A5具有典型的P450吸收峰。该蛋白与细胞色素P450还原酶(CPR)进行体外重组,构成的重组酶系表现出乙氧基试卤灵-O-脱乙基酶活性。结果表明,所采用的表达策略可以成功产生出具有催化活性的鸡细胞色素P450 1A5(CYP1A5)蛋白。     

细胞色素P450与除草剂抗性转基因植物

介绍了除草剂代谢有关的细胞色素P450基因及其应用,已从动植物体中分离具有除草剂代谢活性的细胞色素P450基因,通过转基因方法,成功培育出抗除草剂的转基因植物。     

细胞色素P450的多样性与介导抗性的研究

细胞色素单加氧酶是一类广泛分布在生物体内的重要酶系,在生物体内广泛分布,CYP450参与多种外源性化合物的代谢,内源性物质的合成,在生物体起到十分重要的作用,对P450的研究已从最开始的功能与定位深入到了它的分子机制和多样性等的研究,就P450的多样性、介导抗性的机制和分离方法作一综述。     

氯虫苯甲酰胺诱导甜菜夜蛾细胞色素P450基因上调表达

【目的】明确氯虫苯甲酰胺对甜菜夜蛾Spodoptera exigua (Hübner)细胞色素P450基因的诱导表达作用。【方法】采用O-脱乙基香豆素法研究了低剂量氯虫苯甲酰胺处理对甜菜夜蛾幼虫中肠P450s酶活性的影响,应用Real-time PCR方法测定了其对P450基因(CYP9A9, CYP4G37,CYP4S11和CYP6B)和NADPH细胞色素P450还原酶基因(HQ852049)表达的影响。【结果】氯虫苯甲酰胺对甜菜夜蛾P450酶及相关基因的诱导作用均表现出时间效应和剂量效应,。甜菜夜蛾4龄幼虫取食0.02 mg/kg氯虫苯甲酰胺饲料至5龄, 在蜕皮后6-36 h内, 其P450s酶活性增加为对照组的1.90~2.92倍, 诱导效应高于0.01 mg/kg氯虫苯甲酰胺处理组（其P450s酶活性为对照组的1.11~1.62倍）。同时, 0.02 mg/kg氯虫苯甲酰胺处理组甜菜夜蛾中肠P450基因CYP9A9, CYP4G37和CYP6B mRNA的相对表达量分别上升为对照组的1.97~3.95, 2.46~4.29及1.53~4.48倍, NADPH细胞色素P450还原酶基因 HQ852049 的相对表达量亦增加为对照的1.85~4.08倍。【结论】结果提示,氯虫苯甲酰胺可能通过诱导3种P450基因及细胞色素P450还原酶基因 HQ852049 基因mRNA的上调表达而增强了甜菜夜蛾幼虫中肠P450s酶活性。     

Forty years of cytochrome P450

The term "cytochrome P450" first appeared in literature in 1962. It was a microsomal membrane-bound hemoprotein without known physiological functions at that time and was characterized by a unique 450-nm optical absorption peak of its carbon monoxide-bound form, which was originally reported as the spectrum of a novel "microsomal carbon monoxide-binding pigment" in 1958. Elucidation of its function as the oxygenase in 1963 triggered a rapid expansion of research on this hemoprotein. Annual numbers of the published papers dealing with cytochrome P450, which were listed in Biological Abstracts, increased from 60 in 1970 to 500 in 1980, 900 in 1990, and 1500 in 1997. Cytochrome P450 is now regarded as the collective name of a large family of hemoproteins, "cytochrome P450 superfamily, "which seems to have diversified from a single ancestral protein to many forms during the course of biological evolution and is distributed widely among various forms of life from animals and plants to fungi and bacteria. Multicellular eukaryotic organisms including animals and plants have about 100 or more P450 genes in their genomes, and those many P450 genes are expressed tissue specifically and developmental stage specifically, indicating their diverse physiological functions. In mammals, various P450s participate in the biosynthesis and metabolism of sterols and steroid hormones and the metabolism of various lipid biofactors including eicosanoids, vitamin D3, and retinoids. Oxidative metabolism of foreign hydrophobic compounds as the first step of their excretion from the animal body is apparently another major function of cytochrome P450, which protects animals from noxious foreign compounds, man-created and natural.     

烟草细胞色素P450的基因组学分析

细胞色素P450是一类含血红素的单加氧酶超基因家族, 在植物多种代谢途径中起着重要作用。为了解烟草中的P450的种类和数量, 文章将植物代表性P450蛋白质序列与烟草基因组序列比对, 在烟草基因组中鉴定了44个P450家族共263个成员。将这些烟草P450基因与烟草表达序列标签(EST)比对, 发现173个成员有EST证据。通过与拟南芥中已知的P450蛋白序列比较, 分析了部分烟草P450蛋白序列的特征和二级结构。根据烟草基因芯片数据和部分基因的RT-PCR结果, 发现73个烟草P450基因能够在不同的生长发育时期表达, 其中部分基因具有组织特异性。这些研究结果为烟草P450基因功能的深入分析奠定了基础。     

A general cloning strategy for divergent plant cytochrome P450 genes and its application in Lolium rigidum and Ocimum basilicum

The investigation of plant cytochrome P450 genes and enzymes is a field of growing interest. Apparently, an even greater diversity of cytochrome P450 genes exists in plants in comparison to other eukaryotes. This may be due to their role in the biosynthesis of secondary metabolites that are present in plants in an enormous variety. Most cloning approaches are hampered by the large sequence diversity of plant cytochrome P450 genes. We present a method to clone divergent cytochrome P450 ESTs by a nested RT-PCR-strategy. These ESTs were used for the subsequent cloning of the corresponding full-size cDNAs of divergent families via cDNA-library screening. Sixteen cytochrome P450 genes belonging to different cytochrome P450-families have been identified in this way, proving the efficacy of the strategy. Received: 1 December 2000 / Accepted: 26 February 2001     

昆虫抗药性相关细胞色素P450基因的表达调控机制

杀虫剂的频繁持续使用,必然导致昆虫产生抗药性。大量研究事例表明参与杀虫剂解毒的细胞色素P450(简称P450)过量表达是昆虫对不同类型杀虫剂产生抗性的重要原因,但目前人们对P450基因过量表达机制的认识还非常有限。近十年来,随着生命科学与相关研究技术的发展,有关昆虫P450基因表达调控机制的研究取得了实质性的进展。本文综述了这一研究领域的重要发现。除了基因重复或基因扩增导致的P450基因拷贝数增加外,P450基因在转录层面的上调表达是P450介导抗药性的普遍且重要的机制。P450基因的转录上调由顺式调控元件与反式作用因子相互作用得以实现。现已发现了几种不同类型的转录因子(CncC, CREB和核受体等)对昆虫P450表达的直接调控,也鉴定了间接调控P450表达的作用因子如G蛋白偶联受体及其下游效应子。ncC:Maf/Keap1是抗药性相关P450基因表达的重要而普遍的调控途径。越来越多的事例表明小RNA在昆虫P450的表达调控中起重要作用。现有的研究结果揭示了昆虫P450基因调控因子和信号转导通路的多样性及调控机制的复杂性。     

Cytochrome P450 pathways of arachidonic acid metabolism

Cytochrome P450s metabolize arachidonic acid to hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids. These eicosanoids are formed in a tissue and cell-specific manner and have numerous biological functions. Of major interest are the opposing actions of hydroxyeicosatetraenoic and epoxyeicosatrienoic acids within the vasculature. Regio- and stereoisomeric epoxyeicosatrienoic acids have potent vasodilatory properties while 20-hydroxyeicosatetraenoic acid is a potent vasoconstrictor. Both effects are mediated through actions on large-conductance Ca2+-activated K+ channels. Cytochrome P450-derived eicosanoids are also important in the regulation of ion transport, and have recently been shown to influence a number of fundamental biological processes including cellular proliferation, apoptosis, inflammation, and hemostasis. The formation of these functionally relevant eicosanoids is tightly controlled by the expression and activity of the cytochrome P450 epoxygenases and hydroxylases. In addition, soluble epoxide hydrolase catalyzes the hydrolysis of epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids, and the activity of this enzyme is a critical determinant of tissue epoxyeicosatrienoic and dihydroxyeicosatrienoic acid levels. The intracellular balance between epoxyeicosatrienoic, dihydroxyeicosatrienoic and hydroxyeicosatetraenoic acids influences the biological response to these eicosanoids and alterations in their levels have recently been associated with certain pathological conditions. The involvement of the cytochrome P450-derived eicosanoids in a wide array of biological functions and the observation that levels are altered in pathological conditions suggest that the enzymes involved in the formation and degradation of these fatty acids may be novel therapeutic targets.     

植物细胞色素P450基因的异源表达系统研究进展

细胞色素P450氧化酶是一类具有多种催化功能含血红素的氧化酶系。由于参与多种类型的氧化反应,在植物生命活动中有重要功能,其研究一直受到重视。自1990年第一个植物P450基因成功克隆以来,到2002年年底,已有600多个P450基因被克隆,有100多个基因在细菌、酵母、杆状病毒昆虫细胞等异源表达系统中成功表达并鉴定了功能。拟对植物P450的大肠杆菌、酵母、杆状病毒昆虫细胞表达系统的特点进行比较,对在各表达系统中成功表达并进行了功能鉴定的植物P450进行归纳,并对目前植物P450异源表达的现状和应用进行概述。     

Research progress relating to the role of cytochrome P450 in the biosynthesis of terpenoids in medicinal plants

Terpenoids are an extensive and diverse group of plant secondary metabolites. To date, they have been applied in many fields including industry, medicine and health. The wide variety of terpenoid compounds cannot arise solely from simple cyclisations of a precursor molecule or from a single-step reaction; their structural diversity depends on the modification of many specific chemical groups, rearrangements of their skeletal structures and on the post-modification reactions. Most of the post-modification enzymes that catalyse these reactions are cytochrome P450 monooxygenases. Therefore, the discovery and identification of plant P450 genes plays a vital role in the exploration of terpenoid biosynthesis pathways. This review summarises recent research progress relating to the function of plant cytochrome P450 enzymes, describes P450 genes that have been cloned from full-length cDNA and identifies the function of P450 enzymes in the terpenoid biosynthesis pathways of several medicinal plants.