【作者】 金宏滨；

【导师】 唐克轩；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 植物次生代谢产物中许多成分具有极高的药用价值,如红豆杉产生的紫杉醇等次生代谢产物在临床治疗中、晚期癌症中疗效明显,长春花产生的长春碱和长春新碱等多种次生代谢产物具有抗肿瘤等作用,而银杏产生的黄酮类化合物和银杏内酯等次生代谢产物也因其越来越多的药用价值被发现而引起人们的广泛关注。然而,天然植株中许多目标次生代谢产物含量极低,而且由于人们对药用植物的盲目采集,许多植物趋于濒危,而引种栽培、化学合成及植物组织培养等由于成本高、产量低,阻碍了次生代谢产物的商业化生产。人们发现开展植物次生代谢工程和代谢调控研究是提高植物次生代谢产物含量的有效方法之一。系统深入研究药用次生代谢产物生物合成途径及其累积的分子调控机理是利用基因工程技术提高药用次生代谢产物含量的前提和关键。其中,ABC(ATP-Binding Cassette)转运蛋白由于可将植物细胞产生的有毒次生代谢产物从细胞内转运到细胞外或胞内细胞器以减少对细胞自身的毒性,已成为植物代谢工程和代谢调控研究的重要方向之一。红豆杉(Taxus)、长春花(Catharanthus roseus)和银杏(Ginkgo biloba)是可产生重要次生代谢产物的药用植物,是植物次生代谢研究的重要材料。为了深入了解这三种药用植物中重要次生代谢产物生物合成与累积的分子机制,本文从ABC转运蛋白及其相关基因的克隆和表达分析等方面开展了一系列的工作,取得了以下结果:1.首次成功地从东北红豆杉中克隆了一个ABC转运蛋白基因Tcmdr1(GenBank登录号:DQ660357)。该基因属于MDR亚家族,全长cDNA为4485 bp,含有一个长度为3951 bp的开放阅读框,编码一个具有1316个氨基酸残基的蛋白。序列比对和结构预测发现,TcMDR1蛋白与其它MDR蛋白尤其是植物MDR蛋白具有较高的同源性, TcMDR1蛋白有两个跨膜域TMD和两个核苷酸结合域NBD按照正向的“TMD1-NBD1-TMD2-NBD2”顺序排列,这与典型的有功能的MDR类ABC转运蛋白是一致的,而且在TcMDR1蛋白序列中也发现了所有ABC转运蛋白共同的高度保守基序:“Walker A”、“Walker B”和C基序,这表明TcMDR1可能与其他MDR类ABC转运蛋白一样都是通过结合和水解ATP为特定的底物转运提供能量。组织特异性表达分析结果表明,Tcmdr1在根、茎和叶中的表达都很高。Southern blot结果显示,东北红豆杉中Tcmdr1基因属于一个小基因家族。此外,还成功构建了含Tcmdr1的酵母表达载体pDR196-Tcmdr1并转化了酵母突变菌株AD12345678,得到了酵母转化菌株。进一步通过TcMDR1蛋白表达的检测和底物分析确定TcMDR1蛋白的转运功能将有助于深入研究TcMDR1在提高特定药用次生代谢产物中的潜在作用。2.首次成功地从长春花中克隆了一个ABC转运蛋白基因Crmdr1(GenBank登录号:DQ660356)。该基因属于MDR亚家族,全长cDNA为4395 bp,含有一个3801 bp的开放阅读框,编码一个具有1266个氨基酸残基的蛋白。序列比对和结构预测发现,CrMDR1蛋白与其它MDR蛋白具有较高的同源性,而且预测的CrMDR1蛋白与TcMDR1一样具有典型的MDR类ABC转运蛋白的结构特点,这表明CrMDR1可能与其他MDR类ABC转运蛋白一样能通过结合和水解ATP为特定底物的转运提供能量。组织特异性表达分析结果表明,Crmdr1基因在叶片中表达较弱,而在根和茎等部位表达量较高。Southern blot结果显示,长春花中Crmdr1基因属于一个小基因家族。3.首次成功地从银杏中克隆了一个ABC转运蛋白基因Gbmdr1(GenBank登录号:DQ779968)。该基因属于MDR亚家族,全长cDNA为4275 bp,含有一个3840 bp的开放阅读框,编码一个具有1279个氨基酸残基的蛋白。序列比对和结构预测发现,GbMDR1与其它MDR蛋白具有较高的同源性,预测的GbMDR1蛋白具有典型的MDR类ABC转运蛋白的结构特点,这表明GbMDR1可能与其他MDR类ABC转运蛋白具有同样的功能,即通过结合和水解ATP为特定底物的转运提供能量。组织特异性表达分析结果表明,Gbmdr1基因在根中表达水平极低,而在茎和叶等部位表达量较高。Southern blot结果显示银杏中Gbmdr1基因属于一个小基因家族。4.首次对药用植物MDR类ABC转运蛋白基因进行了比较研究。序列比较结果显示,长春花CrMDR1、黄芪CjMDR和东北红豆杉TcMDR1蛋白同源性较高(超过60%),而银杏GbMDR1同源性则相对较低,仅为40%左右,这可能与银杏这一物种进化上较为古老有关。研究还发现,GbMDR1和TcMDR1蛋白结构中都具有AtPGP4和CjMDR等内向摄取蛋白共有的N末端突出卷曲序列,推测GbMDR1和TcMDR1可能也具有内向摄取功能,进一步的酵母和植物转化及底物分析研究将有助于对这三个ABC转运蛋白进行确切的功能鉴定。进化树分析显示MDR蛋白总体分为三簇,其中药用植物MDR蛋白CrMDR1、CjMDR和TcMDR1都位于第I簇,与内向转运蛋白AtMDR4同在一簇中。通过组织特异性表达分析比较发现,这四个基因的组织表达谱不相同,对其进行功能分析将有助于阐明药用植物中MDR类ABC转运蛋白的主要作用机制及这些机制与药用次生代谢产物累积的关系,为进一步通过基因工程方法提高目的次生代谢产物含量提供依据。5.对紫杉醇累积相关ABC转运蛋白基因进行研究需要详细了解紫杉醇的生物合成途径。本研究首次成功地从曼地亚红豆杉中克隆了一个紫杉醇生物合成相关基因Tmmecs(GenBank登录号:DQ286391),该基因编码2-C-甲基-D-赤藓醇-2,4-环焦磷酸合成酶,全长cDNA为1081 bp,含有一个741 bp的开放阅读框,编码一个具有247个氨基酸残基的蛋白。序列比对和进化分析结果显示, TmMECS蛋白与其它植物MECS蛋白具有较高的同源性。同源三维结构建模分析显示,TmMECS与E.coli来源的MECS蛋白具有相似的三维空间结构,而且在TmMECS结构中也发现了MECS蛋白家族中高度保守的氨基酸残基Asp98、His100和His132,提示TmMECS与其他MECS可能具有相似的生物学功能;组织特异性表达分析表明,Tmmecs是一个组成型表达的基因,在植物根、茎和叶中均呈高水平表达。Southern blot杂交结果显示Tmmecs基因属于一个小基因家族。通过将该基因在大肠杆菌中的超量表达实验证实该酶能够推动大肠杆菌中β-胡萝卜素合成的代谢流向下游流动导致β-胡萝卜素的累积增加,从而证明了TmMECS蛋白具有催化功能。本文从东北红豆杉、长春花和银杏三种药用植物中分别克隆了一个MDR类ABC转运蛋白基因并对其进行了结构及生物信息学分析,为今后对植物ABC转运蛋白中MDR这类最大的全分子亚家族进行克隆及功能研究提供了重要的实验依据;对四个药用植物MDR类ABC转运蛋白基因的结构及组织表达进行了比较分析,对其进行功能分析将有助于阐明药用植物中MDR类ABC转运蛋白的主要作用机制及这些机制与药用次生代谢产物累积的关系,为进一步通过基因工程方法提高目的次生代谢产物含量提供依据;从曼地亚红豆杉中克隆了紫杉醇生物合成相关基因Tmmecs并验证了功能,丰富了紫杉醇生物合成途径研究的基因资源,也为进一步研究红豆杉中与紫杉醇累积相关的ABC转运蛋白基因提供了前提和基础。更多还原

【Abstract】 Plants produce a large number of secondary metabolites of important medicinal values, such as vinblastine and vincristine in Catharanthus roseus, taxol in Taxus, flavonoids and ginkgolides in Ginkgo biloba. Vinblastine, vincristine and taxol are highly effective anti-tumor medicines, while flavonoids and ginkgolides are well-known for their preventive and therapeutic functions against cardiovascular and cerebrovascular diseases. But most of these secondary metabolites are produced at very low levels in the natural plants and many of the natural plants are threatened with extinction due to overharvest. Other methods such as plant cultivation, chemical synthesis, plant cell and tissue culture are not suitable for commercialization of the secondary metabolites because of the high cost and low yield. As one of the most promising approaches, metabolic engineering has been developed to improve the product composition and increase the product yield in plants and cultured cell systems. However, the biosynthetic pathways of the medicinal secondary metabolites and the detailed molecular regulation of their accumulation are yet to be fully elucidated. ABC transporters are among the important research areas for plant metabolic engineering because of their capabilities of transporting the toxic secondary metabolites produced by plant cells either out of the cells or into the organelles so as to reduce the toxicity to the cells themselves.Taxus,Catharanthus roseus and Ginkgo biloba are important medicinal plants which can produce valuable secondary metabolites and they are important materials for the study of plant secondary metabolism. In order to further understand the biosynthetic pathways and the accumulating molecular basis of the valuable secondary metabolites in the medicinal plants, gene cloning, expression profiles and a series of work of ABC transporter genes were performed and reported in this study as follows:1. A novel full-length cDNA encoding an ABC transporter protein (named as Tcmdr1; GenBank accession number: DQ660357) was cloned from Taxus cuspidata by rapid amplification of cDNA ends (RACE) for the first time. This gene,a member of MDR subfamily,had a total length of 4385 bp with an open reading frame (ORF) of 3951 bp encoding a predicted polypeptide of 1316 amino acids. Sequence analysis showed that TcMDR1 had high similarity with other plant MDRs. The domains analysis showed that TcMDR1 possessed two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) arranged in“TMD1-NBD1-TMD2-NBD2”direction, consistent with MDR-type ABC transporters. Within NBDs three characteristic motifs common to all ABC transporters,“Walker A”,“Walker B”and C motif, were found. Expression patterns analysis revealed that Tcmdr1 expressed at high levels in the root, stem and leaf. Southern blot analysis showed that Tcmdr1 belonged to a low-copy gene family. These results indicated that TcMDR1 was a MDR-type ABC transporter protein that might be involved in the transport and accumulation of secondary metabolites in T. cuspidata. The expression construct pDR196-Tcmdr1 containing Tcmdr1 was constructed and transformed into mutant yeast strain AD12345678. The detection of TcMDR1 protein expression and further substrate analysis will disclose the exact function of TcMDR1, which may help us to increase the yield of certain medicinal secondary metabolites through genetic engineering.2. A novel full-length cDNA encoding an ABC transporter protein (named as Crmdr1; GenBank accession number: DQ660356) was cloned from Catharanthus roseus by RACE for the first time. This gene, belonging to MDR subfamily, had a total length of 4395 bp with an ORF of 3801 bp encoding a predicted polypeptide of 1266 amino acids. The CrMDR1 shared high identity with other plant MDRs. The domains analysis showed that CrMDR1 possessed the common structural characteristics of all functional MDR-type ABC transporter proteins. Expression pattern analysis revealed that Crmdr1 was enriched in the root and stem, but low in the leaf. Southern blot analysis showed that Crmdr1 belonged to a low-copy gene family. These results indicated that CrMDR1 was an MDR-type ABC transporter protein that might be involved in the transport and accumulation of secondary metabolites in C. roseus.3. A new full-length cDNA encoding an ABC transporter protein (named as Gbmdr1; GenBank accession number: DQ779968) was cloned from Ginkgo biloba by RACE for the first time. This gene, which was included in MDR-type ABC transporters, had a total length of 4275 bp with an open reading frame of 3840 bp encoding a predicted polypeptide of 1279 amino acids. The GbMDR1 possessed high homology with other MDRs. Amino acid sequence analysis showed that GbMDR1 had the common structural characteristics of all functional MDR-type ABC transporter proteins. Expression pattern analysis revealed that Gbmdr1 was enriched in the stem and leaf, but very low in the root. Southern blot analysis showed that Gbmdr1 belonged to a low-copy gene family. These results indicated that GbMDR1 was an MDR-type ABC transporter protein that might be involved in the transport and accumulation of secondary metabolites in G. biloba.4. MDR-type ABC transporter proteins from medicinal plants were compared for the first time. The sequence comparison showed that CrMDR1 from C. roseus, CjMDR from C. japonica and TcMDR1 from T. cuspidata shared higher homology among each other (60%), while GbMDR1 from G. biloba shared lower homology with the other three (about 40%). In addition,it was found that GbMDR1 and TcMDR1 possessed the coiled-coil structure on N-terminus which was the common structural characteristics of the inward-uptaking AtPGP4 and CjMDR. Therefore, it was supposed that GbMDR1 and TcMDR1 might also possess the function of inward uptake. Phylogenetic analysis showed that plant MDR proteins were grouped generally into three classes. Together with inward-uptaking AtPGP4, CrMDR1,CjMDR and TcMDR from medicinal plants were classified into Class I. Expression patterns analyses revealed great differences among the four MDR genes from medicinal plants. The function determination of these MDR-type ABC transporters is needed to illustrate the main action styles of MDR-type ABC transporter proteins from medicinal plants and the relationship between the MDR proteins and the accumulation of medicinal secondary metabolites.5. The study of ABC transporter genes involved in the accumulation of Taxol requires the detailed acquaintance of the biosynthetic pathway of Taxol. In this study a new full-length cDNA encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (designated as Tmmecs, GenBank accession number: DQ286391), the fifth enzyme of the nonmevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis and further Taxol biosynthesis, was isolated from T. media by RACE for the first time. The full-length cDNA of Tmmecs was 1081 bp containing a 741 bp ORF encoding a deduced protein of 247 amino acid residues. Comparative and bioinformatic analyses revealed that TmMECS had extensive homology with MECSs from other plant species. Phylogenetic analysis indicated that TmMECS was more ancient than other plant MECSs. The homology-based 3D structural modeling of TmMECS was analyzed, which showed TmMECS had a similar 3D structure with the E.coli MECS. Besides, the three highly conserved amino acid residues of MECS family were also found in TmMECS including Asp98, His100 and His132, correspondingly, suggesting that TmMECS had a similar biological function with other MECSs. Southern blot analysis revealed that Tmmecs belonged to a small gene family. Tissue expression pattern analysis indicated that Tmmecs expressed constitutively in all tested tissues including roots, stems and leaves. Furthermore, overexpression of Tmmecs in the E.coli pushed forward the metabolic flux ofβ-carotin synthesis and led to the increase ofβ-carotin accumulation, confirming that TmMECS had enzymic activity.In this paper three MDR-type ABC transporter genes were cloned from medicinal plant T. cuspidata,C. roseus and G. biloba and, meanwhile,their structural and bioinformatic analyses were carried out, which helped to establish a significant experimental basis for the isolation and functional research of plant ABC transporters of MDR subfamily,the largest full-molecule subfamily. The structures and tissue expression of four MDR-type ABC transporter genes from four medicinal plants were compared and further functional analysis would be helpful to illustrate the main action styles of MDR-type ABC transporter proteins from medicinal plants and the relationship between the action styles of MDR proteins and the accumulation of medicinal secondary metabolites, which would be beneficial to the increase of target secondary metabolites by genetic engineering.Tmmecs, a gene involved in Taxol biosynthesis, was isolated from T. media and its function was confirmed, which enriched the gene data of Taxol biosynthesis and helped further study of ABC transporter genes related with the accumulation of Taxol in Taxus.更多还原