京大戟三萜成分及生物合成途径中关键酶研究
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摘要
京大戟是药用植物大戟的干燥根,是一种民间常用药材,由于其对水肿和各种原因引起的腹水有独特的疗效而广为应用。目前因为环境破坏,可用作药材来源的野生大戟已经越来越少,同时大戟又是多年生的宿根植物,大戟栽培需要很多年才能形成可供药材的根。因此,如何解决京大戟药材资源日渐匮乏问题已成为科研工作者所面临的一项严峻挑战,是中药现代化领域重要课题之一,具有十分重要的意义。京大戟中的药理成分主要是三萜类化合物。植物中的萜类化合物代谢途径研究得已很透彻,利用代谢途径中的关键酶基因调控萜类化合物的生物合成也取得了一定的进展。因此解决京大戟药源短缺问题,可从不同的角度进行研究,如改变药材泡制方法提高三萜类活性成分的萃取率,分析三萜类成分进而通过生物工程技术有目的地提高活性成分的含量等。本论文针对这一问题主要进行了以下几方面的研究。
1)为阐明京大戟中的三萜类组分及三萜类是否有活性,本文采用GC/MS对京大戟氯仿提取的弱极性成分进行了分析,结果检测出23种化合物,主要包含三萜类、甾类和酯类等物质。其中三萜类化合物含量最高。体外抗肿瘤实验结果表明,京大戟弱极性成分对人非小细胞肺癌NCI-H460有一定的抑制作用,当氯仿萃取物浓度达到20μg·mL-1时,抑制率达到为99.51%。清除DPPH自由基能力测试结果表明,京大戟弱极性成分具有一定的清除DPPH自由基的能力。在氯仿萃取物浓度在4 mg/mL时,清除率最高,达96.68%。
2)为研究如何较大幅度地提高京大戟药材中的三萜类化合物的萃取率,本文运用响应面法优化了京大戟三萜类化合物的超声提取工艺,通过回归分析和验证实验得到的最优提取工艺条件为:乙醇浓度为95%,液料比为20 mL/g,超声提取温度为70℃,超声时间为32 min,超声波功率390 W,在此优化条件下的京大戟总三萜的萃取率达到5.46%。
3)为研究三萜类生物合成途径中的关键酶,本文通过RACE技术从药用植物大戟中成功克隆了3-羟基-3甲基戊二酰辅酶A还原酶(HMGR; EC1.1.1.34,GenBank收录号为EF062569)基因的全长cDNA序列,序列全长为2200 bp,含有一个1752 bp大小的开放阅读框,编码583个氨基酸序列。通过在线和非在线的分子软件分析表明预测的药用植物大戟的HMGR的氨基酸序列和其他植物的HMGR同源性很高,有两个跨膜区和一个催化区。预测的3D模型是一个典型的HMGR的三维结构模型。Southern杂交分析结果显示药用植物大戟叶基因组中最多以两个拷贝形式存在,RT-PCR分析结果表明药用植物大戟HMGR基因在根、茎和叶中都有表达,在根中的表达量高于茎,茎中的表达量高于叶。功能测验实验表明药用植物大戟中的HMGR能增加大肠杆菌中胡萝卜素的积累,从而证明了该基因编码一个功能蛋白。
4)通过RACE技术从药用植物大戟中成功克隆了三萜类生物合成途径中的关键酶法呢基焦磷酸合成酶(FPS,GenBank收录号为FJ755465)基因的全长cDNA序列,该序列全长为1431 bp,含有一个长为1029 bp的开放阅读框,编码342个氨基酸序列。通过在线和非在线的分子软件分析表明预测的药用植物大戟的FPS的氨基酸序列和其他植物的FPS同源性很高,有5个保守区序列。通过氨基酸序列构建的进化树表明药用植物大戟的FPS属于植物列群。Southern杂交分析结果表明药用植物大戟叶基因组中有一个FPS基因家族。RT-PCR分析结果表明FPS在根、茎和叶中都有表达,在根中的表达量高于茎,茎中的表达量高于叶。功能互补实验表明药用植物大戟的FPS基因能够弥补酵母CC25缺失的FPS功能,证明所克隆的基因能够编码相应的具有FPS功能的蛋白。
5)通过甲基茉莉酸和水杨酸两个诱导子诱导药用植物大戟愈伤组织分析两个功能酶对外界环境的响应情况,结果表明两个诱导子都能诱导两个功能酶基因的表达。甲基茉莉酸处理的愈伤组织,EpHMGR基因的表达量在第12 h达到最高,随后缓慢减低。EpFPS基因的表达量在第48 h达到最高,之后缓慢减低。水杨酸处理的愈伤组织,EpHMGR基因的表达量在48 h内缓慢增加,在第72 h达到最高。EpFPS基因的表达量在24 h达到最高。推测三萜类化合物的含量可随外界环境刺激而相应增加。实验结果为通过一定的化学手段提高京大戟中的活性成分含量提供了理论基础,同时为利用生物工程技术提高京大戟中的三萜类含量提供了候选基因。
“Jing Daji”, the root of Euphorbia pekinensis Rupr, is a common medicine in the folk, and widely used for treatment of edema and ascites caused by various reasons. Wild resources of Euphorbia pekinensis has become more scarce at present because of environmental disruption. At the same time Euphorbia pekinensis is a perennial plant with persistent roots and need many years cultivation, therefore it is a major challenge for researchers and is an important issue for the field of traditional Chinese medicine modernization to solve the decreasing resources. The main medicine composition of Euphorbia pekinensis are triterpenoids. The research on the metabolic pathway of triterpenoids is more clearly and key genes in metabolic pathway regulated triterpenoids biosynthesis have made some progress. Some works can be done from different directions to solve resource shortage of medicine plant, such as changing extracting method to improve the rate of extraction of active ingredients, or analyzing the active components and using biological engineering technology to improve the content of the active ingredient. The following aspects had been done in this study:
1) The chemical components of non-polar factions exctracted from the roots of Euphorbia pekinensis were analyzed by GC/MS. Twenty-three compounds were separated and identified. Triterpenoids, steroids and esters were the major components, and triterpenoids were the more. The chemical components of non-polar factions could inhibit the growth of cell line NCI-H460 cell with an inhibiting rate of 99.51% at 20μg/mL and the DPPH radical scavenging rate was 96.68% when the concentration was 4 mg/mL.
2) Response surface methodology was used to optimize the extraction technique for total triterpenoids from roots of Euphorbia pekinensis by supersonic wave. By regression analysis and verification experiment, the results showed that the optimum conditions of alcohol concentration, solid liquid ratio, extraction temperature, ultrasonic extraction time and ultrasonic power were 95%, 20 mL/g, 70℃, 32 min and 390 W respectively.
3) A full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569 ) was 2200 bp containing a 1752 bp ORF encoding 583 amino acids. Bioinformatic analyses revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that at most two copies of EpHMGR gene existed in Euphorbia Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis.
4) A cDNA encoding farnesyl diphosphate synthase (FPS) from E.pekinensis was cloned and characterized. The full-length cDNA named EpFPS (Genbank Accession Number FJ755465) contained 1431 bp with an open reading frame of 1029 bp encoding a polypeptie of 342 amino acids. The deduced amino acid sequence of the EpFPS named EpFPS exhibited a high homology with other plant FPS, and contained five conserved domains. Phylogenetic analysis showed that EpFPS belonged to the plant FPS group. Southern blot analysis revealed a small FPS gene family in E.pekinensis. Tissue expression pattern analysis revealed that EpFPS expressed strongly in root, weak in leaf and stem. Functional complementation of EpFPS in an ERG20-disrupted yeast strain indicated that the cloned cDNA encoded a farnesyl diphosphate synthase.
5) Salicylic acid (SA) and methyl jasmonate (MeJA) were used to study the response of two functional gene to two elicitors. To MeJA treated callus, the content of EpHMGR expression was the highest after 12 h, and then was slowly lower, while the content of EpFPS expression was the highest after 48 h, then slower lower. To SA treated callus, the content of EpHMGR expression was the highest after 12 h, and then was slowly lower, while the content of EpFPS expression was the highest after 48 h, then slower lower. The results provides a theoretical basis for the adoption of certain chemical means and provides candidate genes to use biological engineering technology to increase active ingredients in Euphorbia pekinensis.
1)为阐明京大戟中的三萜类组分及三萜类是否有活性,本文采用GC/MS对京大戟氯仿提取的弱极性成分进行了分析,结果检测出23种化合物,主要包含三萜类、甾类和酯类等物质。其中三萜类化合物含量最高。体外抗肿瘤实验结果表明,京大戟弱极性成分对人非小细胞肺癌NCI-H460有一定的抑制作用,当氯仿萃取物浓度达到20μg·mL-1时,抑制率达到为99.51%。清除DPPH自由基能力测试结果表明,京大戟弱极性成分具有一定的清除DPPH自由基的能力。在氯仿萃取物浓度在4 mg/mL时,清除率最高,达96.68%。
2)为研究如何较大幅度地提高京大戟药材中的三萜类化合物的萃取率,本文运用响应面法优化了京大戟三萜类化合物的超声提取工艺,通过回归分析和验证实验得到的最优提取工艺条件为:乙醇浓度为95%,液料比为20 mL/g,超声提取温度为70℃,超声时间为32 min,超声波功率390 W,在此优化条件下的京大戟总三萜的萃取率达到5.46%。
3)为研究三萜类生物合成途径中的关键酶,本文通过RACE技术从药用植物大戟中成功克隆了3-羟基-3甲基戊二酰辅酶A还原酶(HMGR; EC1.1.1.34,GenBank收录号为EF062569)基因的全长cDNA序列,序列全长为2200 bp,含有一个1752 bp大小的开放阅读框,编码583个氨基酸序列。通过在线和非在线的分子软件分析表明预测的药用植物大戟的HMGR的氨基酸序列和其他植物的HMGR同源性很高,有两个跨膜区和一个催化区。预测的3D模型是一个典型的HMGR的三维结构模型。Southern杂交分析结果显示药用植物大戟叶基因组中最多以两个拷贝形式存在,RT-PCR分析结果表明药用植物大戟HMGR基因在根、茎和叶中都有表达,在根中的表达量高于茎,茎中的表达量高于叶。功能测验实验表明药用植物大戟中的HMGR能增加大肠杆菌中胡萝卜素的积累,从而证明了该基因编码一个功能蛋白。
4)通过RACE技术从药用植物大戟中成功克隆了三萜类生物合成途径中的关键酶法呢基焦磷酸合成酶(FPS,GenBank收录号为FJ755465)基因的全长cDNA序列,该序列全长为1431 bp,含有一个长为1029 bp的开放阅读框,编码342个氨基酸序列。通过在线和非在线的分子软件分析表明预测的药用植物大戟的FPS的氨基酸序列和其他植物的FPS同源性很高,有5个保守区序列。通过氨基酸序列构建的进化树表明药用植物大戟的FPS属于植物列群。Southern杂交分析结果表明药用植物大戟叶基因组中有一个FPS基因家族。RT-PCR分析结果表明FPS在根、茎和叶中都有表达,在根中的表达量高于茎,茎中的表达量高于叶。功能互补实验表明药用植物大戟的FPS基因能够弥补酵母CC25缺失的FPS功能,证明所克隆的基因能够编码相应的具有FPS功能的蛋白。
5)通过甲基茉莉酸和水杨酸两个诱导子诱导药用植物大戟愈伤组织分析两个功能酶对外界环境的响应情况,结果表明两个诱导子都能诱导两个功能酶基因的表达。甲基茉莉酸处理的愈伤组织,EpHMGR基因的表达量在第12 h达到最高,随后缓慢减低。EpFPS基因的表达量在第48 h达到最高,之后缓慢减低。水杨酸处理的愈伤组织,EpHMGR基因的表达量在48 h内缓慢增加,在第72 h达到最高。EpFPS基因的表达量在24 h达到最高。推测三萜类化合物的含量可随外界环境刺激而相应增加。实验结果为通过一定的化学手段提高京大戟中的活性成分含量提供了理论基础,同时为利用生物工程技术提高京大戟中的三萜类含量提供了候选基因。
“Jing Daji”, the root of Euphorbia pekinensis Rupr, is a common medicine in the folk, and widely used for treatment of edema and ascites caused by various reasons. Wild resources of Euphorbia pekinensis has become more scarce at present because of environmental disruption. At the same time Euphorbia pekinensis is a perennial plant with persistent roots and need many years cultivation, therefore it is a major challenge for researchers and is an important issue for the field of traditional Chinese medicine modernization to solve the decreasing resources. The main medicine composition of Euphorbia pekinensis are triterpenoids. The research on the metabolic pathway of triterpenoids is more clearly and key genes in metabolic pathway regulated triterpenoids biosynthesis have made some progress. Some works can be done from different directions to solve resource shortage of medicine plant, such as changing extracting method to improve the rate of extraction of active ingredients, or analyzing the active components and using biological engineering technology to improve the content of the active ingredient. The following aspects had been done in this study:
1) The chemical components of non-polar factions exctracted from the roots of Euphorbia pekinensis were analyzed by GC/MS. Twenty-three compounds were separated and identified. Triterpenoids, steroids and esters were the major components, and triterpenoids were the more. The chemical components of non-polar factions could inhibit the growth of cell line NCI-H460 cell with an inhibiting rate of 99.51% at 20μg/mL and the DPPH radical scavenging rate was 96.68% when the concentration was 4 mg/mL.
2) Response surface methodology was used to optimize the extraction technique for total triterpenoids from roots of Euphorbia pekinensis by supersonic wave. By regression analysis and verification experiment, the results showed that the optimum conditions of alcohol concentration, solid liquid ratio, extraction temperature, ultrasonic extraction time and ultrasonic power were 95%, 20 mL/g, 70℃, 32 min and 390 W respectively.
3) A full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569 ) was 2200 bp containing a 1752 bp ORF encoding 583 amino acids. Bioinformatic analyses revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that at most two copies of EpHMGR gene existed in Euphorbia Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis.
4) A cDNA encoding farnesyl diphosphate synthase (FPS) from E.pekinensis was cloned and characterized. The full-length cDNA named EpFPS (Genbank Accession Number FJ755465) contained 1431 bp with an open reading frame of 1029 bp encoding a polypeptie of 342 amino acids. The deduced amino acid sequence of the EpFPS named EpFPS exhibited a high homology with other plant FPS, and contained five conserved domains. Phylogenetic analysis showed that EpFPS belonged to the plant FPS group. Southern blot analysis revealed a small FPS gene family in E.pekinensis. Tissue expression pattern analysis revealed that EpFPS expressed strongly in root, weak in leaf and stem. Functional complementation of EpFPS in an ERG20-disrupted yeast strain indicated that the cloned cDNA encoded a farnesyl diphosphate synthase.
5) Salicylic acid (SA) and methyl jasmonate (MeJA) were used to study the response of two functional gene to two elicitors. To MeJA treated callus, the content of EpHMGR expression was the highest after 12 h, and then was slowly lower, while the content of EpFPS expression was the highest after 48 h, then slower lower. To SA treated callus, the content of EpHMGR expression was the highest after 12 h, and then was slowly lower, while the content of EpFPS expression was the highest after 48 h, then slower lower. The results provides a theoretical basis for the adoption of certain chemical means and provides candidate genes to use biological engineering technology to increase active ingredients in Euphorbia pekinensis.
引文
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