【作者】 张蕾；

【导师】 程义勇； 高志贤；

【作者基本信息】 中国人民解放军军事医学科学院 ， 营养与食品卫生学， 2009， 博士

【摘要】 丹参Salvia miltiorrhiza Bunge的根作为传统的活血化瘀药,具有祛瘀止痛、活血通经、清心除烦等功效,近来研究还发现其在治疗心血管疾病方面疗效显著。其有效化学成分主要包括:水溶性多聚酚酸类化合物和以丹参酮类化合物为代表的脂溶性化合物。由于具有天然的抗氧化作用、抗动脉粥样硬化、降低心肌耗氧量等心血管药理作用、抗菌消炎作用和明显的抗肿瘤作用,这味传统的药材受到了国内外市场的追捧。然而目前野生丹参资源由于过度采收已遭破坏,栽培品种品质退化,质量参差不齐,因此要从根本上解决当前丹参栽培中面临的问题和丹参的质量控制,直接大规模工业化生产丹参的活性成分,更好地利用这一资源,必须阐明此类物质的生物合成途径。虽然已知丹参酮类化合物为二萜生源,同植物中其它二萜类物质一样,它们也是由类异戊二烯代谢途径产生,前体物质是牻牛儿基牻牛儿基焦磷酸(geranylgeranyl pyrophosphate,GGPP),但关于GGPP以后的具体过程却仍不清楚。GGPP是由GGPP合酶催化异戊烯基焦磷酸( isoprenyl pyrophosphate, IPP)和倍半萜前体法呢基焦磷酸( farnesyl pyrophosphate,FPP,C15)缩合形成的,它是二萜化合物的通用前体,为多种二萜、类胡萝卜素、叶绿素、醌和维生素E侧链等的提供构建骨架。由于GGPP是多种初生和次生代谢物的共同前体,因此,GGPP合酶能够起到调节碳流的作用,从而成为初生和次生代谢途径中的关键酶之一。GGPP合酶普遍存在于植物、动物和细菌中,具有很高的保守性,在进化关系上具有明显的种族特异性,动物、植物、细菌、真菌和古细菌来源的GGPS蛋白序列在进化上属于不同的分支。对模式植物拟南芥GGPS的研究表明,在植物体内存在多个GGPS基因,不同酶型的GGPS在细胞中的定位不同,它们的表达亦具有时空特异性,其在代谢途径中的作用亦不同。已经知道的以GGPP为前体的具有药用价值的化合物有紫杉醇、银杏酚等,且已证明GGPS是决定红豆杉植物合成紫杉醇类物质的关键酶之一。利用GGPS中两个富含天冬氨酸的保守区域FARM(the first aspartate-rich motif,II区)和SARM(the second aspartate-rich motif,V区)设计简并引物,通过同源扩增的方法获得400bp左右的与已报道其它植物中的GGPP合酶序列相似的核心序列,再用cDNA末端快速扩增技术(RACE)得到3’端和5’端序列,拼接三段序列,最终获得两条长度分别为1 298bp和1 177bp的丹参GGPScDNA序列,将其命名为SmGGPS1和SmGGPS2。生物信息学分析表明,它们分别具有1 095bp和1035bp的ORF框,与已报道的多个植物GGPS基因具有60%以上的一致性;推断其编码364个和344个氨基酸序列,分子质量分别为38 986.6和37 373.4,等电点分别为6.29和6.78,均具有多聚异戊二烯基合成酶的特异序列;TargetP 1.1在线分析它们的N端可能分别具有52个氨基酸和19个氨基酸的信号肽序列,引导这两个蛋白依次定位于质体和线粒体。RT-PCR半定量分析表明,这两个基因在所分析的各组织中均有表达,但SmGGPS1在成熟期(花期)叶片中的表达最强,而SmGGPS2则在成熟期的根和成花中表达最强;基因结构分析表明SmGGPS1基因有一397bp的内含子,而SmGGPS2则不带内含子。通过Gateway技术构建酵母表达载体,将其转化至GGPS缺陷的低温敏感型酵母菌株SFNY368,利用功能互补的原理,确证了SmGGPS1编码蛋白的确具有GGPP合酶;而SmGGPS2却不能很好的回复SFNY368的低温生长能力,分析可能是其信号肽序列的定位功能影响到该基因在酵母中的表达,需要进一步去除信号肽序列再确证其功能。针对经过生化功能确定的SmGGPS1基因,分别构建了RNAi载体、过表达载体及两个相关的阴性对照空载体。利用SmGGPS1-RNAi载体,首次建立了发根农杆菌ACCC10060介导的外源基因转化丹参的体系,所得转基因毛状根具有抗生素和GFP双筛选标记,平均诱导率和转化率均可达50%以上。用同样的转化方法分别获得SmGGPS1过表达载体及其空载体对照转基因毛状根,分别选择20株过表达株系和17株空载体对照株系进行液体培养,45天后用代谢组学提取方法提取化合物,通过高效液相色谱(HPLC)检测分析,结果表明,过表达株系和空载体对照株系中含有丹参酮IIA的株系分别占90%和100%,含有丹参酮I的均为35%,而含有隐丹参酮的株系则分别占20%和71%;三种丹参酮类化合物的总量在空载体对照株系中基本呈正态分布,而过表达株系中除了15#特别多外,其余样本含量均不显著;这说明SmGGPS1的过表达导致了隐丹参酮含量的显著降低,进而引起了丹参酮类化合物总量的减少;选取19个由HPLC检测得到的化合物,对其进行主成分分析,结果显示过表达株系和空载体对照株系完全可以从化合物的角度被分开,说明过表达组的化合物检测结果并不是随机事件,而是SmGGPS1基因表达水平改变后的整体特征;同时找到了5个可作为代谢标识物的物质;聚类分析将这19个化合物分成了三类,其中两类可能与丹参酮类化合物的合成相关。利用根癌农杆菌介导转化分别得到了32株SmGGPS1-RNAi2、10株SmGGPS1-RNAi3和41株pDONR(-)转基因植株,其中有8株RNAi2植株和1株RNAi3植株肉眼可见的显著表型为根缩短、叶发黄,这提示赤霉素含量的升高及叶绿素和/或类胡萝卜素含量的降低。结论:首次获得了两个丹参GGPP合酶基因,并通过酵母互补实验确证了SmGGPS1的生化功能;首次建立了发根农杆菌介导外源载体转化丹参的体系;通过分析过表达转基因毛状根中化合物的变化和RNAi转基因植株的表型,将这两个结果相联系,可以推测SmGGPS1在丹参中的主要作用之一是合成叶绿素和/或类胡萝卜素,而不参与赤霉素和丹参酮类物质的合成;但同时结果亦指示,在GGPP之后,赤霉素和丹参酮类物质在生物合成途径中可能有一段共同的合成步骤,而后才进入各自的特异分支途径中。更多还原

【Abstract】 The root of Salvia miltiorrhiza Bunge (Danshen) is the traditional medicine with the function of invigorating the circulation of blood and removing blood stasis, and recently was found to remarkably affect in the treatment of cardiovascular and cerebrovascular diseases. It contains two kinds of biologically active compounds, water-soluble poly-phenolic compounds and fat-soluble compounds , including tanshinones. Because of having the functions of natural anti-oxidation, anti-atherosis, antibacterial and anti-tumor, the Chinese herb is more popular all of the world. However, the wild resources of Danshen have been disrupted by excess picking, and the quality of cultivars degenerates and intermingles, so in order to radically solve these problems, to industrializedly product the active compenents of Danshen, and to well exploit the herb, the biosynthesis pathway must be elucidated. It has been known that tanshinones are the diterpenoid derivatives, like other diterpenoids, they come from the isoprenoid metabolic pathway, and the precursor is geranylgeranyl pyrophosphate (GGPP), but the detail process since GGPP is still unclear. GGPP is the product of the condensation of isoprenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP, C15), catalyzed by GGPP synthase. It is the universal precursor of diterpenoids, providing the framework of other diterpenoids, carotenoid, chlorophyll, quinine and the side of vitamin E. Because GGPP is the common precursor of primary and secondary metabolites, GGPP synthase can regulate the flux of carbon, and then become one of the key enzymes among the primary and secondary metabolism pathways.GGPS is ubiquitous in plants, animals and bacteria. It has the well conservative sequences, and the phylogenetic analysis shows the proteins have the evident phyletic specific, and the GGPS of animals, plants, bacteria, fungi and archaea come from the different branchs. The studies of GGPS in the model plant, Arabidopsis thaliana, show that there are several GGPS genes in plants, transporting to the different organelles, expressing temporally and spacially, so the functions in metabolic pathways are distinct. It has been known GGPP is also the precursor of the taxol and ginkgolide which are officinal chemicals, and it has been proved that GGPS is one of the key enzymes in the synthesis pathway of taxols in yew.The degenerate primers were designed based on the conservative regions, the first aspartae-rich motif and the second aspartate-rich motif, of GGPS protein sequences from public databases. The core target fragment of about 400bp was obtained from Danshen root by use of homologous cDNA amplification, and then the sides of the core fragment were complemented with the RACE technology, finally the 1 298bp and 1 177bp cDNA were obtained, respectively named SmGGPS1 and SmGGPS2. Bioinformatics analysis showed that the two sequences have respectively 1 095bp and 1035bp ORF, more than 60% identity to the reported plant GGPS genes; they were deduced to encode 364 and 344 amino acids, the molecular weight of the two proteins are respectively 38 986.6 and 37 373.4, pI are 6.29 and 6.78, and the polyprenyl synthetase signature is both found. By TargetP 1.1, it is predicted that SmGGPS1 has a plastid targeting signal peptide of about 52 amino acid at the N-terminal end, and SmGGPS2 has a mitochondrion targeting signal peptide of about 19 amino acid at the N-terminal end. RT-PCR semi-quantitative analysis showed that the two genes expresse in the all tested tissues, and SmGGPS1 with much higher level of expression in the leaves in the flowering stage, SmGGPS2 with much higher level of expression in the roots and flowers in the flowering stage. SmGGPS1 has a 397 bp intron, and SmGGPS2 has no intron.By the Gateway technology, the vectors expressed in yeast was constructed, and transformed to SFNY368 that is sensitive to lower temperature because of the absent GGPS. According to the functional complement, it was validated that SmGGPS1 encodes the functional GGPP synthase, while SmGGPS2 cannot well complement to the function of SFNY368.It was explained that the signal peptide of SmGGPS2 influenced its expression in yeast, and it must be validated further.For SmGGPS1, RNAi vectors, overexpression vector and the two control vectors were constructed. With RNAi vectors, for the first time, Agrobacterium rhizogenes-mediated transformation of Salvia miltiorrhiza Bunge was performed, brought the transgenetic hairy root with the two screening markers of antibiotic and GFP, and the induction rate and the average transformation rate were both more than 50%. Taking use of the transforming method, the SmGGPS1 overexpression and the control transgenetic hairy root were obtained, then 20 lines overexpression transgenetic hairy root and 17 lines control transgenetic hairy root were cultured in liquid culture medium. After 45 days, the compounds were extracted by the metabolism methods. The results of analysis by HPLC showed the ratios of the lines of overexpression transgenetic hairy root and control transgenetic hairy root containing tanshanone IIA are respectively 90% and 100%, containing tanshanone I all 35%, containing cryptotanshinone respectively 20% and 71%. On the distribution of all tanshanones, in the control transgenetic hairy roots, it showed basically normal distribution, while in overexpression transgenetic hairy roots, it didn’t show distinct, except NO.15. The result showed that the over expression of SmGGPS1 induced the sharp decrease of cryptotanshinone content, and then the lower of all tanshanones content. 19 compounds detected by HPLC were selected and analysized by PCA, the result showed the components of overexpression lines and control lines can be apart, proving that over-expression group of compounds tested is not a random event, but the overall characteristics by cause of the expression level of SmGGPS1 gene change; and at the same time, five metabolic markers were found. The cluster result of chemicals showed the nineteen components of chosen can be classed to three sort, and two of them might be correlative with the synthesis of tanshinones. By Agrobacterium tumefaciens-mediated method, 32 lines of RNAi2, 10 lines of RNAi3, and 41 lines of pDONR(-) transgenetic plants were obtained, and 8 lines of RNAi2 and 1 lines of RNAi3 ones exhibited macroscopicly the reduced roots and yellow leafs, which hinted the increased content of gibberellin and the decreased content of chlorophyll and (or) carotenoid.Conclusion: For the first time, the two GGPS genes of Salvia miltiorrhiza Bunge were obtained, and then the function of SmGGPS1 was confirmed with yeast complement test. And firstly, the Agrobacterium rhizogene-transformed transgenetic method to Danshen was established. From the type relationship of overexpreesion transgenetic hairy roots and RNAi transgenetic plants, it can be concluded that one of the function of SmGGPS1 is to synthesize chlorophyll and (or) carotenoid, but not gibberellin and tanshinones. At the same time, it can be presumed that the biosynthesis pathway of gibberellin and tanshinones shares several steps.更多还原