【作者】 刘万宏；

【导师】 廖志华； 陈敏；

【作者基本信息】 西南大学 ， 遗传学， 2008， 硕士

【摘要】 紫杉醇是从药用植物红豆杉中分离出的具有抗癌作用的天然产物,是最好的天然抗癌药物之一。由于红豆杉生长缓慢且紫杉醇含量非常低,所以完全不能满足患者的需求。开展红豆杉的基因工程为解决药源缺乏的问题提供了崭新的思路,因此开展紫杉醇生物合成途径的分子生物学研究是开展红豆杉基因工程的首要工作。紫杉醇前体生物合成源于新近发现的MEP途径。为研究紫杉醇前体生物合成途径的分子生物学,本文采用cDNA末端快速扩增技术(Rapid amplification of cDNA ends,RACE)方法从曼地亚红豆杉(Taxus media)中首次克隆了MEP途径上两个关键酶基因的全长cDNA,包括曼地亚红豆杉2-C-甲基-D-赤藓醇-4-磷酸胱氨酰转移酶基因(2-C-Methyl-D-erythritol4-phosphate cytidyltransferase,MECT)和曼地亚红豆杉2-C-甲基赤藓醇-2,4-环焦磷酸合成酶基因(TmMECPS),并对这些基因进行了详尽的分析和功能验证。2-C-甲基-D-赤藓醇-4-磷酸胱氨酰转移酶是紫杉醇前体生物合成MEP途径上的第三个酶。本试验采用RACE方法,第一次从曼地亚红豆杉中获得了MECT基因(命名为TmMECT)。TmMECT全长1301 bp,包括5’和3’非翻译区、polyA尾巴和一个编码939 bp的开放阅读框(Open Reading Frame,ORF),它编码一条312个氨基酸残基的MECT蛋白质,其分子量预测为34.33kDa,等电点为8.96;组织差异表达研究显示,TmMECT在红豆杉根、叶、树皮、茎中均有表达,在叶和树皮中表达量较高。颜色互补试验表明TmMECT可以加速β-胡萝卜素的积累。对TmMECT基因的克隆和功能分析将会有助于在分子水平上更好地了解MECT在紫杉醇生物合成中的作用。2-C-甲基赤藓醇-2,4-环焦磷酸合成酶(2C-methyl-D-erythritol 2,4-cyclodiphosphatesynthase,MECPS)是紫杉醇前体生物合成MEP途径中第五步反应酶。利用RACE技术首次从曼地亚红豆杉中获得MECPS基因(命名为TmMECPS)。根据生物信息学分析TmMECPS基因cDNA全长为899 bp,含有一个726 bp的开放阅读框(ORF),编码一个由241个氨基酸残基组成的蛋白质;预测的分子量约为26.1 kDa,等电点(pI)为8.22。同源比对与生物信息学分析表明,TmMECPS蛋白与其它物种的MECPS蛋白具有较高的同源性,并且具有MECPS蛋白家族的保守区域。系统发生树分析表明,TmMECPS蛋白在进化上比其它绝大多数植物的MECPS蛋白更为古老。组织表达分析表明,TmMECPS基因在根、茎、皮、叶中均有表达,但表达水平存在差异,叶与皮中表达量较根与茎要高。功能验证试验表明,TmMECPS蛋白能够促进β-类胡萝卜素的产生与积累。TmMECPS基因的克隆与研究为进一步了解TmMECPS在紫杉醇合成途径中的作用打下了基础。更多还原

【Abstract】 Taxol,isolated from Taxus species,is one of the best anti-tumor drugs.However,Taxus species is nationwide-protected with slow growth rate and low taxol content.To solve the problem of limited taxol availability,genetic engineering technology offers a brand new way.Therefore,The molecular biology research of taxol biosynthesis pathways is a prerequisite work for it’s engineering.Taxol is originated from recently unveiled MEP pathway.In order to map the biosynthesis of taxol precursors at the level of molecular genetics,The full-length cDNAs of two key genes involved in the MEP pathway were cloned through RACE method,characterized and functionally identified,which was respectively TmMECT(the gene encoding 2-C-Methyl-D-erythritol 4-phosphate cytidyltransferase from Taxus media),TmMECPS(the gene encoding 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase from Taxus media).2-C-Methyl-D-erythritol 4-phosphate cytidyltransferase(MECT,EC:2.7.7.60)is the third enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate(MEP)Taxol biosynthesis.The full-length MECT cDNA sequence(designated TmMECT)was cloned and characterized for the first time from Taxus media,using Rapid Amplification of cDNA Ends(RACE)technique.The full-length cDNA of TmMECT was 1301-bp containing 5’and 3’ untranslated regions,poly A tail and a 939 bp open reading frame(ORF)encoding a peptide of 312 amino acids with a calculated molecular mass of 34.33 kDa and an isoelectric point of 8.96.Tissue expression pattern analysis indicated that TmMECT expressed constitutively in all tissues including leaves,roots,barks and stems,with higher expression in leaves and barks.The color complemetion assay demonstrated that TmMECT could accelerate the accumulation ofβ-carotene.The cloning and characterization of TmMECT will be helpful to understand more about the role of MECT involved in the Taxol biosynthesis at the molecular level. 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase(MECPS,EC:4.6.1.12)is the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and is involved in the methylerythritol phosphate(MEP)pathway of Taxol biosynthesis.The full-length cDNA sequence(designated as TmMECPS)was cloned and characterized for the first time from Taxus media,using RACE technique.According to the bioinformations analysis,the full-length cDNA of TmMECPS was 899 bp containing a 726-bp ORF encoding a peptide of 241 amino acids with a calculated molecular mass of 26.1 kDa and an isoelectric point of 8.22.Comparative and bioinformatic analyses revealed that TmMECPS had extensive homology with MECPSs from other species and contained conserved residues owned by the MECPS protein family.Phylogenetic analysis indicated that TmMECPS was more ancient than other plant MECPSs.Tissue expression pattern analysis indicated that TmMECPS expressed in all tested tissues including roots,stems, barks and leaves but at different levels,with higher expression levels in tender barks and leaves than roots and stems.The color complementation assay demonstrated that TmMECPS could promoted the accumulation ofβ-carotene.The cloning and characterization of TmMECPS will be helpful to understand more about the role of MECPS involved in the Taxol biosynthesis at the molecular level.更多还原