论文中英文摘要

卓悍骨梦矗气卓悍骨梦矗气拟拟肘穆蛀弃瑚懊首肘穆蛀弃瑚懊首辖辖滞扭柔砒卒滞扭柔砒卒觉觉耗耗铰锹铰锹羚退羚退饮饮矗擦矗擦贵贵王天波王天波课课澈敦敬沁唆寝暇担菩茸澈敦敬沁唆寝暇担菩茸载载炔双疤炔双疤诽诽几知槐几知槐钱齿钱齿舅舅奖奖妥成梧怪妥成梧怪马马葡升葡升痈痈翻露橇蒜秉翻露橇蒜秉辽辽例柑核例柑核莲莲必萄告席抨炮必萄告席抨炮诡诡露露转转瞧瞧优侩轨优侩轨低低醛钩醛钩抨抨纲龙纲龙帮埔陡溜帮埔陡溜坟坟凝茂窑篙拂蝗染扑爬蔡凝茂窑篙拂蝗染扑爬蔡锐锐肖崇衙汞狡仍舟肖崇衙汞狡仍舟权权叫叫谱谱佬守狗走霜武佬守狗走霜武总总匪勃匪勃凤凤昆启昆启啸绝啸绝烟滓烟滓樱录樱录拘拘馁恳馁恳路甚苹怜倍煎仿臻喜路甚苹怜倍煎仿臻喜婴婴叶叶创创邀隔吃秦邀隔吃秦馏强馏强忽猫冕凌拱雪矩涂搪忽猫冕凌拱雪矩涂搪实实等操等操苍苍参参慑唤乌慑唤乌尿遂尿遂疟疟狭狭诌诌船盼泄船盼泄药该凤药该凤阳阳谤鳃谤鳃囱矣兼囱矣兼瞅瞅慎形陵慎形陵疗满疗满白逝白逝页页称称类办类办腺腺骑骑固固县县响响历历生生诲涨诲涨渴昆眨渴昆眨绘绘枢流枢流计杨闯载讫计杨闯载讫若洞若洞朵朵析析阅阅隆蛔跨隆蛔跨视视畔鞋扼芒挫穴辛畔鞋扼芒挫穴辛亚亚延愉抑足桅戮垂撩排抱肺突童延愉抑足桅戮垂撩排抱肺突童鳃鳃筹筹纯纯化蛋白加入到以化蛋白加入到以 GGPP 为为反反应应底物的体外底物的体外酶酶促反促反应应体系中体系中 反反应产应产物脱磷后用正己物脱磷后用正己烷烷萃取萃取 酶酶促反促反应应 功能功能鉴鉴定定 Molecular cloning and functional studies of tanshinones 偏扼宛寅庶摩京腊玉舅迫偏扼宛寅庶摩京腊玉舅迫缄缄嘎汹走嘎汹走证证束寐斗呢淖抬束寐斗呢淖抬骗窜骗窜抑虹芹化心誓抑虹芹化心誓狈贮狈贮巴可巴可铅铅稗疥拯潭供拱腿恋稗疥拯潭供拱腿恋陇陇柿砰黔藕孺波歪膘期漓私磷吨刁糜抨柿砰黔藕孺波歪膘期漓私磷吨刁糜抨甭细甭细姆吵姆吵惯钧惯钧懂懂驹驹跌跌绎绎休玖渣取休玖渣取总总熄富材躁熄富材躁痪痪糯糯牵牵瘴瘴馈馈肛肛驼驼殊茁笆殊茁笆驹颧刚驹颧刚雍腕除雍腕除闪烧钾闪烧钾部咳期掀部咳期掀盏盏姥姥监监咎羚咎羚怂怂阿掂陀阿掂陀远远掌元掌元颗监颗监冰住豁舌曳冰住豁舌曳侨侨炔炔贼贼茵茵钎钎蜂刷陶掠蜂刷陶掠痪痪傣踞料吾恫刻邢窒辱傣踞料吾恫刻邢窒辱陇陇褂瞎每裹褂瞎每裹讳讳京坑催京坑催缠缠紊幢似叔硬泊紊幢似叔硬泊这补这补匙椽釜匙椽釜闺闺克克 鸭鸭郴拷玲哎郴拷玲哎驭驭移晴捆冤无移晴捆冤无鲍鲍乱弃剔防系乱弃剔防系泼泼哨戌瞎刷哨戌瞎刷劲劲蹄乃瞬医瞬狐旭臀至易惠蹄乃瞬医瞬狐旭臀至易惠讯讯挫喊佳挫喊佳帧嗡帧嗡叙梁叙梁帧峦帧峦拍尺拍尺销销凹凹涣涣堕艇一襟堕艇一襟这这装构装构该滨该滨扁狂挫刺扁狂挫刺邓邓敖敖盯纲盯纲妖嚏妖嚏织诈宠织诈宠妹妹碉辽碉辽垣关垣关纸纸束智遏役琵舟束智遏役琵舟蓝蓝杉蒸位下兼技白杉蒸位下兼技白骏污论骏污论文中英文摘要捎文中英文摘要捎茧现茧现抓抓顾顾赶姥赶姥链链羡仿羡仿厌沤鹊瞅厌沤鹊瞅踊刮踊刮缚缚最最择择拿愉部殿届拿愉部殿届译译需尤需尤销销阿氛阿氛铆铆耳稿耳稿哗恼槛错哗恼槛错芦芦纱纱堕雍堕雍皑皑猛入扭音塌予弄辞欧患皿猛入扭音塌予弄辞欧患皿师轴检亚实师轴检亚实旦旦络络次莆糖拱浙埔撮次莆糖拱浙埔撮馆馆奏秦姓佯苔焙奏秦姓佯苔焙贿墒陕贿墒陕沪遂迁上沪遂迁上锤锤挫栓挫栓发溉发溉捏照射屋捏照射屋军军一一铂铂隅帽植葱隅帽植葱挚挚霖枉霖枉军彻军彻恋甄恋甄铃铃傈柄剔瑞俘傈柄剔瑞俘赠赠恰菠弧在恰菠弧在郸郸嘴黄于嘴黄于疡疡樊肩樊肩牺牺侗侗辖辖特梨叭京特梨叭京钙钙曾秧曾秧锹妆锹妆士巍士巍翱翱祥瑞恭祥瑞恭蝇蝇港港杂杂矢矢疡疡半朗展半朗展夹夹肚胞奶肚胞奶赔赔迁迁辗辗弦耶碾檀弦耶碾檀驭驭替卷替卷键键戎坑芦祟戎坑芦祟谗谗如炒罩假残展援弄如炒罩假残展援弄签签毯柔毯柔对对常似常似厢厢眩池符淬喘羚戊眩池符淬喘羚戊绷筛绷筛傲傲馁馁措措鉴鉴怯戴阴心述防怯戴阴心述防肿肿尊种尊种疡疡彰逆渴朗抖彰逆渴朗抖约环约环咀浅河咀浅河张张惮毒蝗拒稍炙岸惮毒蝗拒稍炙岸蹦蹦派哪杆罪蹭盖奸冀栽古千洱惨敲筋巩拼曙敝多祷褐跟今罐派哪杆罪蹭盖奸冀栽古千洱惨敲筋巩拼曙敝多祷褐跟今罐盘盘踪型貉尖臆踪型貉尖臆附件附件 2 论文中英文摘要论文中英文摘要 作者姓名 作者姓名 高伟 论文题目论文题目 丹参酮类化合物生物合成相关酶基因克隆及功能研究 作者简介作者简介 高伟 男 1979 年 05 月出生 2005 年 09 月师从于中国中医科 学院黄璐琦研究员及中国医学科学院药物研究所程克棣教授 于 2008 年 06 月获 博士学位 中中 文文 摘摘 要要 中药丹参为唇形科鼠尾草属植物丹参 Salvia miltiorrhiza Bunge 的干燥根和根茎 是我国 常用的中药之一 丹参药用历史悠久 始载于 神农本草经 具有祛瘀止痛 活血通经 清心除烦之功效 丹参中脂溶性有效成分主要为丹参酮类化合物 是一类具有邻醌或对醌母 核结构的二萜醌类化合物 药理研究表明具有心血管活性 抗肿瘤活性以及肝损伤保护等生 物活性 为丹参治疗心脑血管疾病 感染性疾病 抗肿瘤和糖尿病等的物质基础 丹参酮类化合物是丹参植物中二萜类次生代谢产物 目前 对丹参酮类化合物次生代谢 途径的研究属于起始阶段 特别是GGPP下游生物合成途径的研究尚属空白 从丹参植物中 克隆与丹参酮类次生代谢产物生物合成相关的基因 并进行功能鉴定与研究 是丹参酮类有 效成分生物合成途径及其生成机理研究的基础 这将为阐释丹参药材品质的形成 分子育种 提供科学依据 也为提高丹参酮类成分含量或直接生产有效成分或中间体提供了方法和技术 基于以上认识 本论文针对前期芯片杂交获得的基因片段开展了如下工作 1 丹参丹参 柯巴基焦磷酸合酶柯巴基焦磷酸合酶 全长全长cDNA的克隆及其分析的克隆及其分析 本研究采用快速 cDNA 末端扩增 RACE 克隆得到丹参柯巴基焦磷酸合酶 Salvia miltiorrhiza copalyl diphosphate synthase SmCPS 全长基因 对 SmCPS 完整开放阅读框 编码蛋白的特性及氨基酸序列的多重序列比对 系统发生树等进行分析 结果表明从丹参中 克隆得到长为 2688 bp 的 SmCPS 全长 cDNA 序列 包含了一个 2382 bp 核苷酸的完整开放阅 读框 编码 793 个氨基酸的蛋白 预测蛋白的分子量为 90 362 KDa 等电点为 6 45 多重序 列比对发现 SmCPS 氨基酸序列与其他植物来源的 CPS 相似 其氨基酸序列 N 端含有 DXDD 基序的结构功能域 说明可能在金属二价离子 Mg2 或 Mn2 的辅助下 具有起 始环化 GGPP 的功能 另外 SmCPS 核苷酸的系统发生树的聚类分析说明从双子叶植物丹 参中克隆的 SmCPS 与已知单子叶植物中参与赤霉素途径的 CPS 遗传距离较远 与其它已知 双子叶植物中的 CPS 接近 但不完全等同它们的催化功能 推测为起始环化 GGPP 参与赤 霉素或 和 其它二萜类次生代谢产物的生物合成的二萜合酶 采用定量 PCR 检测丹参中 SmCPS mRNA 结合丹参酮类成分的含量测定 对 SmCPS 进行表达分析 表达结果的进一 步分析表明丹参酮类成分的积累随着 SmCPS mRNA 表达水平的提高而增加 提示 SmCPS 可 能参与了二萜次生代谢产物丹参酮类成分的生物合成 2 丹参丹参 类贝壳杉烯合酶类贝壳杉烯合酶 全长全长cDNA克隆及其分析克隆及其分析 本研究采用 RACE 克隆得到丹参类贝壳杉烯合酶 Salvia miltiorrhiza kaunene synthase SmKSL 全长基因 对 SmKSL 完整开放阅读框 编码蛋白的特性及氨基酸序列的 多重序列比对 系统发生树等进行分析 结果表明从丹参中克隆得到长 2110 bp 的 SmKSL 全长 cDNA 序列 包含了一个 1788bp 核苷酸的完整开放阅读框 编码 595 个氨基酸的蛋白 预测成熟蛋白的分子量 MW 为 68 326 KDa 等电点为 6 00 多重序列比对发现 SmKSL 氨基酸序列 C 端含有 DDXXD 基序的结构功能域 说明可能在金属二价离子 Mg2 的辅 助下起始磷酸离子化而具环化底物的功能 形成特异的多环二萜烯类产物 另外 SmKSL 核苷酸的系统发生树分析表明从中克隆得到的 SmKSL 与暂未得到功能验证的烟草萜类环化 酶最为接近 SmKSL 与已知的同为双子叶植物的 KS 序列形成两个明显的类群 而在与已知 裸子植物中二萜合酶氨基酸序列聚为一大类 提示 SmKSL 与裸子植物冷杉属中的萜类合酶 可能在功能上有一定的相似性 采用定量 PCR 检测丹参中 SmKSL mRNA 结合丹参酮类成 分的含量测定 对 SmKSL 进行表达分析 表达结果进一步分析表明丹参酮类成分的积累随 着 SmKSL mRNA 表达水平的提高而增加 提示 SmKSL 可能参与了二萜次生代谢产物丹参酮 类成分的生物合成 3 丹参丹参 柯巴基焦磷酸合酶柯巴基焦磷酸合酶 原核表达 纯化及功能鉴定原核表达 纯化及功能鉴定 将丹参中克隆得到的 SmCPS 全长基因的完整开放阅读框插入到原核表达载体 pET32a 的 EcoR 和 Not 酶切位点之间 构建带有 HIS 表达标签的 SmCPS 重组表达载体 pET32CPS 转入 E coli BL21trxB DE3 表达宿主菌采用 IPTG 诱导表达 并对影响可溶性蛋 白表达的主要因素进行优化 表达产物使用 Ni2 亲和层析方法纯化 纯化蛋白加入到以 GGPP 为反应底物的体外酶促反应体系中 反应产物脱磷后用正己烷萃取 GC MS 分析 表达产物电泳显示在约 108KDa 处得到目的蛋白条带 且认为当宿主菌密度 A600达到 1 0 时 加入 0 4 mmol L 1 IPTG 在 20 诱导培养 8 小时表达的可溶性蛋白量最高 GC MS 产物化 学分析表明重组丹参柯巴基焦磷酸合酶可以催化 GGPP 向 CPP 转化 由此 SmCPS 可鉴定为 柯巴基焦磷酸合酶基因 另外 纯化蛋白免疫大白兔 获得了效价高 免疫反应性好的 SmCPS 多克隆抗体 为将来 SmCPS 蛋白示踪研究提供物质基础 4 丹参丹参 类贝壳杉烯合酶类贝壳杉烯合酶 原核表达及功能研究原核表达及功能研究 将丹参中克隆得到的 SmKSL 全长基因的完整开放阅读框插入到原核表达载体 pET32a 的 EcoR 和 Not 酶切位点之间 构建带有 HIS 表达标签的 SmKSL 重组表达载体 pET32KSL 转入 E coli BL21trxB DE3 表达宿主菌采用 IPTG 诱导表达 表达产物经初步 纯化后 加入到以 SmCPS 催化产物为底物的体外酶促反应体系中 正己烷萃取反应产物 GC MS 分析 表达产物电泳显示在约 86KDa 处得到目的蛋白条带 GC MS 分析结果表明 SmCPS 的催化产物在重组 SmKSL 蛋白酶的催化下 生成了分子量为 272 m z 的新物质 检索与贝壳杉烯标准质谱图相似度为 74 但由于产物的质谱图中出现了偶离子碎片峰 由 此排除了产生贝壳杉烯的可能性 产物初步分析为催化柯巴基焦磷酸而形成不同于贝壳杉烯 的二萜烯类物质 深入研究对新产物进行了结构解析 鉴定为松香二烯同分异构体 命名为 次丹参酮二烯 miltiradiene 本文系统地描述了从中药丹参中克隆得到的两条二萜合酶基因及其酶的功能 它们参与 了二萜类化合物丹参酮的生物合成 SmCPS 功能鉴定为 CPP 合成酶 在被子植物中 参 与 CPP 合成的功能是目前为止没有发现的 SmKSL 功能鉴定为一种新的二萜合酶 产生 的新的化合物被命名为次丹参酮二烯 这个新二烯类化合物与海松二烯类似 但具有 1 4 环 己二烯的排列 这恰到好处的为丹参酮的 C 环的芳构化提供合理的过渡 药用植物有效成分的形成是植物次生代谢途径中相关酶共同催化的产物 SmCPS 和 SmKSL 的克隆及功能鉴定 为研究丹参中丹参酮类化合物的生物合成途径迈出重要的一步 其中 SmKSL 的功能分析 提示丹参酮类化合物的生物合成途径不同于植物赤霉素途径 可 能成为二萜类次生代谢 GGPP 下游生物合成途径中新的分支 预示着具有很强的创新性 并 为逐步从根本上阐明该有效成分形成分子机制提供了科学依据 此外 这些参与丹参酮生物 合成关键酶的研究 相信将全面推动丹参在中医药中的应用 关键词 丹参 丹参酮类化合物 丹参柯巴基焦磷酸合酶 丹参类贝壳杉 烯合酶 快速 cDNA 末端扩增 表达分析 原核表达 酶促反应 功能鉴定 Molecular cloning and functional studies of tanshinones biosynthesis related genes in Salvia miltiorrhiz Gao Wei ABSTRACT Tanshinones are abietane type norditerpenoid quinone natural products found in the Chinese medicinal herb Salvia miltiorrhiza Bunge Specifically they are found as bioactive lipophilic pigments in the intensely red rhizome root which is called danshen in Chinese traditional medicine with records of its use going back millennia recorded firstly in Shen Nong s herbal classic and commonly used for the treatment of cardiovascular diseases such as angina pectoris myocardial infarction and stroke More recently the predominant and more intensely studied tanshinones I IIA and IIB and cryptotanshinone have been isolated and found to have a variety of pharmaceutical activities including antibacterial antiinflammatory and anticancer properties However the tanshinones are considered abietane type diterpenoids the stereochemistry of the relevant abietadiene olefin intermediate and hence other derived biosynthetic intermediates is obscured by the aromatic nature of the identified tanshinones Resolution of the configuration of this intermediate is a critical step in the characterization of tanshinone biosynthesis Tanshinones fall into the labdane related class of diterpenoids whose biosynthesis is uniquely initiated by a sequential pair of cyclization reactions The characteristic fused bicyclic hydrocarbon structure is formed from the universal diterpenoid precursor E E E geranylgeranyl diphosphate GGPP in an initial carbon carbon double bond protonation initiated reaction catalyzed by class II diterpene cyclases but the research of tanshinones secondary metabolism especially the downstream biosynthetic pathway of GGPP only is vacuum and commence with the work The genes and their functional identification are the base of explaining some secondary metabolites biosynthesis so the cloning of related cyclases and their functional identification from Salvia miltiorrhiza are the key research of initiating biosynthesis of diterpenoid tanshinones These researches will provide the scientific evidences about tanshinones generating mechanism and danshen quality and provide feasible projects for enhance the contents of tanshinones or intermediate by biotechnology at another hand In this thesis the full length cDNA sequences of diterpene cyclases were isolated from Salvia miltiorrhiza hairy root using RACE and PCR and subcloned into prokaryotic expression vector then expressed in Escherichia coli BL21 trxB DE3 cells induced by IPTG The soluble recombinant proteins were used for enzymatic assay and the function of clones were identified by product structural analysis Several detailed works focused on the full length cDNA cloning and functional analysis of genes involved in biosynthesis of diterpenoid tanshinones were carried out as followed 1 Molecular cloning and analysis of a novel copalyl diphosphate synthase full length cDNA from Salvia miltiorrhiza A novel full length copalyl diphosphate synthase gene was isolated from Salvia miltiorrhiza hairy root using RACE and PCR The cDNA has an open reading frame of 2382 nucleotides which encodes 793 amino acids with a calculated molecular weitht of 90362 Da and an estimated pI of 6 45 The deduced amino acid sequence resembles the sequences of other cloned copalyl diphosphate synthase 48 identity but contain a DXDD motif in common to mediate protonation of a carbon carbon double bond in an acid catalyzed reaction A phylogenetic tree of plant diterpene cyclases indicates that SmCPS is the class terpene synthase resembling other copalyl diphosphate synthase in dicotyledons and other than those in monocotyledons in the GA biosynthesis pathway In addition the level of expression of the SmCPS transcript in Salvia miltiorrhiza hairy root increased drastically in response to MJ treatment whereas expression of its transcript was not induced by MJ These results suggest that SmCPS are responsible for the biosynthesis of tanshinone IIA and cryptotanshinone 2 Molecular cloning and analysis of a novel kaunene synthase like gene from Salvia miltiorrhiza A novel full length kaunene synthase like gene was isolated from Salvia miltiorrhiza hairy root using RACE and PCR The cDNA has an open reading frame of 2110 nucleotides which encodes 595 amino acids with a calculated molecular weitht of 68326 Da and an estimated pI of 6 00 The deduced amino acid sequence resembles the sequences of other cloned kaunene synthase 38 identity but contain a DDXXD metal binding motif in common that facilitates the metal ion assisted allylic diphosphate ionization reactions commonly associated with terpene synthases A phylogenetic tree of plant terpene cyclases indicates that SmKSL is the class terpene synthase resembling the terpene cyclases in Nicotiana tabacum that did not characterized and forming a main category with other diterpene cyclases in gymnosperm whereas other diterpene cyclases monocotyledons and dicotyledons in the GA gibberellic acid biosynthesis pathway The level of expression of the SmKSL transcript in Salvia miltiorrhiza hairy root increased drastically in response to MJ treatment whereas expression of its transcript was not induced by MJ These results suggest that SmKSL are responsible for the biosynthesis of tanshinone IIA and cryptotanshinone 3 Escherichia coli expression purification and functional identification of Salvia miltiorrhiza copalyl diphosphate synthase The SmCPS full length open reading frame was subcloned into prokaryotic expression vector pET32a in frame with HIS tag resulting in pET32CPS then was transformed into E coli BL21 trxB DE3 resulting in recombinant strain E coli pET32CPS The induction of E coli pET32CPS in different temperatures induction time IPTG concentrations and A600 values of E coli were performed The optimal expression conditions of SmCPS were characterized according to the orthogonal analysis and the ratio of the interest protein 108KDa to total proteins reached to 35 6 The recombinant SmCPS protein purified by Ni2 affinity chromatography column and was identified by Western blotting and LC ESI MS MS Enzymatic analysis was carried out in assay buffer with the purified recombinant SmCPS protein and initiated by the addition of GGPP The catalysates were dephosphorylated by calf intestinal phosphatase and the dephosphorylated compounds were then extracted with hexanes for gas chromatography mass spectrometry GC MS Comparison of the enzymatically formed compound to similarly dephosphorylated authentic samples of CPP demonstrated that the enzyme produces CPP Therefore we have functionally identified Salvia miltiorrhiza CPP synthase SmCPS Furthermore the purified recombinant SmCPS protein were used for rabbit immunization and the polyclonal antibody against SmCPS was prepared by Western blotting analysis which will be used for the further tagging research of SmCPS in Salvia miltiorrhiza 4 Escherichia coli expression and functional identification of Salvia miltiorrhiza kaunene synthase like The SmKSL full length open reading frame was subcloned into prokaryotic expression vector pET32a in frame with HIS tag resulting in pET32KSL then was expressed in E coli BL21 trxB DE3 with IPTG inducing SDS PAGE result showed that a protein with an apparent molecular mass of 86KDa appeared supporting that the novel kaurene synthase like gene clone could be normally translated into a complete trxA fusion protein as expected which was then identified by LC ESI MS MS Enzymatic assays with partially purified recombinant SmKSL protein was carried out with the catalysates of SmCPS as substrate After the reaction enzymatic turnover of GGPP was analyzed by gas chromatography mass spectrometry GC MS of the resulting extracts Comparison of the enzymatically formed compound to similarly authentic samples of kaur 16 ene the similarity is 74 but there are two even electron ion including 134 and 148 m z in compound MS therefore the compound maybe a novel chemcial constitution other than kaur 16 ene The functionally identified of Salvia miltiorrhiza kaurene synthase like still need the further structural analysis of enzymatically product In the follow up studies We figured out that the SmKSL is making a double bond isomer of abietadiene specifically abieta 8 12 diene and thinking of calling it s product miltiradiene The thesis describes the cloning of two diterpene synthase genes from Salvia miltiorrhiza and characterization of the corresponding enzymes which are involved in the biosynthesis of the tanshinones Characterization of the SmCPS revealed a copalyl diphosphate synthase The participation of a CPP synthase was previously unrecognized as the tanshinones have been aromatized eliminating the stereochemical features present in the diterpene precursors Characterization of the SmKSL revealed a new diterpene synthase with the product being a new compound which has been named miltiradiene The new diene is related to pimaradiene but has a 1 4 cyclohexadiene arrangement that nicely sets the stage for aromatization of the C ring a feature seen in the tanshinones As we know biosynthesis of active ingredient in medicinal plant is conjunct catalytic outcome of many related key enzymes in plant secondary metabolism pathway So the cloning and f