【作者】 廖志华；

【导师】 唐克轩；

【作者基本信息】 复旦大学 ， 遗传学， 2004， 博士

【摘要】 紫杉醇的生物合成来源于经典的MVA途径和新近发现的DXP途径。为研究紫杉醇前体生物合成途径的分子生物学，本文首先建立了从成熟的曼地亚红豆杉等裸子植物的各种成熟组织中提取高质量RNA的方法；在此基础上，采用RACE方法从曼地亚红豆杉中首次克隆了MVA途径上三个重要基因的全长cDNA，包括tmhmgr(曼地亚红豆杉3-羟基-3-甲基戊二酰CoA合成酶基因)，tmfps(曼地亚红豆杉法呢基焦磷酸合成酶基因)，tmipi(曼地亚红豆杉异戊烯焦磷酸异构酶基因)；同时克隆了DXP途径上的两个限速酶tmdxs(曼地亚红豆杉5-磷酸脱氧木酮糖合成酶基因)和tmdxr(曼地亚红豆杉5-磷酸脱氧木酮糖还原异构酶基因)的全长cDNA；克隆了为紫杉醇提供二萜骨架(20碳原子)的tmggpps(曼地亚红豆杉香叶基香叶基焦磷酸合成酶基因)的基因组序列，并获得全长cDNA；由于这些基因都是首次从裸子植物中获得，故对这些基因及其编码的酶进行了详尽的生物信息学分析；采用遗传功能互补的方法验证了tmhmgr、tmfps、tmggpps基因的功能，结果显示，tmhmgr能够弥补酵母JRY2394缺失的HMGR功能，tmfps能够弥补酵母CC25缺失的FPS功能，tmggpps能够弥补酵母SFNY368缺失的GGPPS功能，证明它们编码相应的功能蛋白；在大肠杆菌中过量表达tmipi推动DXP途径代谢流向下游流动，促进β-胡萝卜素的生物合成而验证了tmipi的功能；Southern杂交结果表明，tmhmgr、tmfps、tmipi、tmdxs在曼地亚红豆杉中都是其相应基因家族的成员；Northern杂交结果表明，tmhmgr在曼地亚红豆杉根、茎和叶中都有表达，但表达量有差异，tmhmgr在地上部分的表达高于地下部分，这与紫杉醇的药用部位相一致；tmfps在根、茎、叶中都有表达，在根和树干中的表达量没有明显差异，在针叶中的表达量高于根和树干。首次发现tmggpps是一个没有内含子的基因，有本底表达水平，在用甲基茉莉酸处理曼地亚红豆杉细权只k李讨阮七学位专七火。一~l~~胞后，其表达量大为提高且维持较长时间，有利于紫杉醇的积累;获得了切茗邵砂，上游调控序列，通过生物信息学分析，发现了与长春花str基因MeJA反应元件G一box一样的顺式作用元件CACGTC，该元件可能是MeJA诱导加g盯，，表达上调的分子基础;分别采用紫外线和甲基茉莉酸处理曼地亚红豆杉细胞，结果显示，tme众‘在处理后细胞中的表达水平提高，这表明，紫外线和甲基茉莉酸是促使该基因表达上调的两个信号;作为紫杉醇前体生物合成途径中的关键基因，切啥邵少，和tm加都受到MeJA的影响而上调表达，这个现象的发现为MeJA诱导紫杉醇超量积累的分子机制的阐明提供了事实依据。本文关于这些基因的研究为深入阐明紫杉醇生物合成的分子生物学、分子遗传学和生物化学机理莫定了重要基础，为紫杉醇的代谢工程提供了更多可能的调控靶点。 喜树碱、经基喜树碱、长春花碱和长春新碱都是高效的抗癌天然药物，属于菇类叫垛生物碱(Terpenoid hidole Alkalnids，TIAs)，但在植物中的含量都很低。本文对这些抗癌叫噪生物碱的代谢工程进行了研究。从长春花中克隆了TIAs生物合成途径上的关键酶基因tdc、str和g10h，以及调控长春花T认s生物合成途径上多个基因的转录因子。rc“了，构建了双元植物表达载体:p1304++比沦勺tr、pl304++g]oh、pl304++orca了、pl3o4++g 20人+orca了，导入了根癌农杆菌LB从404并获得了工程菌株，同时导入卸甲型根癌农杆菌C58CI(P形A4)并获得了工程菌株;用LBA4404+P 1 304+十r改十‘tr遗传转化喜树，用15。留ml潮霉素筛选，获得多个独立转化的抗性愈伤组织，在多次继代除菌培养后，用PCR在继代培养抗性愈伤组织的基因组DNA中检测到转入的潮霉素抗性基因、外源目的基因tdc、s介:同时用Rl’- PCR检测到了tdc和str的表达;HPLC分析结果表明，转基因喜树愈伤组织中轻基喜树碱和喜树碱含量比普通愈伤组织中的含量显著提高，转基因喜树愈伤组织中经基喜树碱最高含量达到1.87mg/g干重，喜树碱最高含量达到6.52m砂g干重，证明长春花TIAs生物合成上游途径上的基因可以用于具有共通途径的喜树TIAs的代谢工程，提高喜树中喜树碱和经基喜树碱的含量。用c58CI(p形A4)，C58CI印形A4)+P 1304++g 10h，C58CI(p瓦A4)+pl3o4++orca了，C58CI印形A4)+p1304++g 10h+O rca3分别转化了长春花，获得了多个毛状根，在多次继代除菌培养后，用PCR在继代培养的毛状根(C 58CI印形A4)转化获得的长春花毛状根)的基因组DNA中检测到导致并维持毛状根表型的rolB和rolC基因，并用RpPCR检测到rolB和rolC基因的表达;在其他三种携带目的基因的工程菌转化获得的长春花毛状根基因组DNA中检测到潮霉素抗性基因，rolB和rolC基因，同时用 Rl’- PCR检测到潮霉素抗性基因、rolB和roIC基因的表达。这些结果表明我们获得了长春花毛状根、转gI0h的长春花毛状根、转口rc心的长春花毛状根、共转化g10h和。rca了的长春花毛状根，为进一步研究转基因对长春掇只k李例卜七学州叶七火、.咳洲奉生用“幻做的，曰阳七传月.~.一花TIAs代谢组的影响提供了重要的材料，同时为利用转基因毛状根开展长春花抗癌菇类叫噪生物碱代谢工程奠定了坚实的基础。更多还原

【Abstract】 Taxol, one of the most potent and efficient anti-tumor agents, is originated from the classic MVA pathway and recently unveiled DXP pathway, In order to map the biosynthesis of taxol precursors at the level of molecular genetics, an easy and efficient protocol was developed for isolating good-quality total RNA from various mature tissues including fruits, leaves, stems and roots of Taxus plant. On the basis of this protocol, the full-length cDNAs of three key genes involved in the MVA pathway were cloned through RACE method, characterized and functionally identified, which was respectively tmhmgr(the gene encoding 3-Hydroxy-3-methylglutaryl-CoA reductase from Taxus media), tmfps (the gene encoding Farnesyl diphosphate synthase from Taxus media) and tmipi (the gene encoding Isopentenyl diphosphate isomerase from Taxus media). Two key genes encoding the committed-step enzymes involved in the DXP pathway, tmdxs (the gene encoding 1-Deoxy-D-xylulose 5-phosphate synthase from Taxus media) and tmdxr (the gene encoding 1-Deoxy-D-xylulose 5-phosphate reductoisomerase from Taxus media), and the geranylgeranyl diphosphate synthase gene from Taxus media and its correlative genomic sequence were also cloned and characterized. The different mutant yeast strains were applied to respectively identify the function of tmhmgr, tmfps and tmggpps by functionally genetic complementation; the function of tmipi was identified by over expressing in E. coli strain XL1-Blue with the carotenoid-producing plasmid pAC-BETA, whichresulted in pushing forward the metabolic flux to the downstream of the DXP pathway. Southern blot analysis revealed that tmhmgr, tmjps, tmipi or tmdxs was not a single-gene but belonged to its gene family in Taxus media. Northern blot analysis showed that tmhmgr and tmjps expressed in the roots, stems and needles of Taxus media, but with higher level of expression for tmhmgr in the needles and stems, that was coincident with the fact that stems and needles were used for extracting taxol and its derivatives, tmjps expressed constitutively in roots, stems and needles, tmggpps was an intron-free gene with the low level of constitutive expression; when Taxus media cells were treated with MeJA, the expression of tmggpps increased greatly and maintained high expression level in a relative long time that was helpful to the accumulation of taxol. A G-box was found in the upstream sequence of tmggpps, which was the same as the MeJA-responsive G-box of the strictosidine synthase gene in C. roseus, and this might explain that tmggpps was up-regulated by MeJA. When Taxus media cells were treated with UV and MeJA, RT-PCR of tmdxs revealed that UV and MeJA could up regulate its expression. The responses of tmggpps and tmdxs to MeJA might give reasons that MeJA induced over production of taxol. It was important to clone and characterize the genes described before, which was very helpful to map the taxol biosynthetic pathways at the level of molecular genetics and biochemistry and also provided more potential targets for metabolic engineering of taxol.Camptothecin, vinblastine and vincristine were potent anti-tumor agents, all belonging to TIAs but with very low contents in planta. Therefore, metabolic engineering of these TIAs would be a good alternative way to solve the shortage problem of these agents. The three key genes, including tdc (the gene encoding tryptophan decarboxylase from Catharanthus roseus), str (the gene encoding strictosidine synthase from C. roseus) and g10h (the gene encoding geraniol 10-hydroxylase from C. roseus), were isolated by RT-PCR and used for constructing plant expression vectors. ORCA3, a transcription regulator involved in the TIAs biosynthetic pathway in C. roseus, was also cloned and used for constructing plant expression vector. After a series of molecular operation, four plant expression vectors were obtained: p1304++tdc+str, p1304++g10h, p1304++orca3, p1304++g10h+orca3, and the resulting four vectors were introduced into Agrobacterium tumejaciens strain LBA4404 and disarmed strain更多还原