【作者】 于湘莉；

【导师】 元英进；

【作者基本信息】 天津大学 ， 生物化工， 2004， 硕士

【摘要】 紫杉醇生物合成上游途径是指紫杉醇生物合成途径中GGPP之前的部分,包括MVA途径、DXP途径和从IPP缩合形成GGPP的代谢途径。为从曼地红豆杉中克隆紫杉醇生物合成上游途径中的重要酶基因,首先建立了一种从曼地亚红豆杉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克隆了六个重要酶的基因的全长cDNA:MVA途径上的3-羟基-3-甲基戊二酰CoA合成酶基因(tmhmgr)和异戊烯焦磷酸异构酶基因(tmipi)、DXP途径上的5-磷酸脱氧木酮糖合成酶基因(tmdxs)和5-磷酸脱氧木酮糖还原异构酶基因(tmdxr)、从IPP缩合形成GGPP途径上的曼法呢基焦磷酸合成酶基因(tmfps)和香叶基香叶基焦磷酸合成酶基因(tmggpps)。除tmggpps外其他五个基因都是首次从裸子植物中获得。酵母遗传功能互补的方法证实了tmhmgr、tmfps、tmggpps基因都编码相应的功能蛋白,而tmipi也能通过其在大肠杆菌中过量表达促进β-胡萝卜素的生物合成,说明它是一个编码有功能的IPI蛋白的基因。Southern杂交表明,在曼地亚红豆杉中tmhmgr、tmfps、tmipi、tmdxs都是其相应基因家族的成员。Northern杂交表明,这些基因在根、茎、叶中都有表达,但一般来说针叶中的表达量高于根和树干。通过MeJA诱导实验,发现tmggpps和tmdxs在对照实验有本底表达,但MeJA处理东北红豆杉细胞导致其长时间超量表达。通过克隆tmggpps基因的基因组序列,发现tmggpps是一个没有内含子的基因。利用染色体步移技术,获得了tmggpps上游启动子调控序列,在其中发现了与长春花str基因MeJA反应元件G-box一样的顺式作用元件CACGTC,该元件可能是MeJA诱导tmggpps表达上调的分子基础,有助于阐明MeJA诱导紫杉醇超量积累的分子机制。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机制奠定了重要基础,为紫杉醇的代谢工程提供了更多可能的调控靶点。更多还原

【Abstract】 The upstream biosynthesis pathway of Taxol is metabolic pathway before GGPP. As the common biosynthesis pathway of biterpenoids, it includs MVA pathway, DXP pathway and the part from IPP to GGPP. In order to clone genes coding the important enzymes in this upstream biosynthesis pathway of taxol, 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 six key genes were cloned through RACE method, characterized, which were respectively tmhmgr(the gene encoding 3-Hydroxy-3-methylglutaryl-CoA reductase from Taxus media), tmipi (the gene encoding Isopentenyl diphosphate isomerase from Taxus media) involved in the MVA 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) involved in DXP pathway, tmfps (the gene encoding Farnesyl diphosphate synthase from Taxus media), tmggpps (the geranylgeranyl diphosphate synthase gene from Taxus media) from the biosynthesis pathway from IPP to GGPP. 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, which resulted in pushing forward the metabolic flux to the downstream of the DXP pathway. Southern blot analysis revealed that tmhmgr, tmfps, tmipi or tmdxs was not a single-gene but belonged to its gene family in Taxus media. Northern blot analysis showed that tmhmgr and tmfps 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. tmfps expressed constitutively in roots, stems and needles. when Taxus cuspidata 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.The genomic sequence of tmggpps was also cloned and characterized. it was <WP=5>found that tmggpps was an intron-free gene with the low level of constitutive expression. 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. 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.更多还原