【作者】 王敬；

【导师】 开国银；

【作者基本信息】 上海师范大学 ， 遗传学， 2009， 硕士

【摘要】 丹参始载于《神农本草经》,为我国传统名贵中药材,具有祛瘀止痛、活血调经、养心除烦等功效。丹参主要成分包括脂溶性的二萜醌类化合物(丹参酮类成分)和水溶性的酚酸类化合物,二者均具有较强的药理活性,其中以丹参酮IIA为代表的二萜醌类化合物在抑菌、消炎、抗氧化及抗癌等方面有着广泛的应用,临床需求巨大。但是,由于丹参生长周期长(2年以上),在传统的栽培模式下,面临着品质严重退化、生产成本相对过高等诸多弊端;通过化学合成的方法,其过程十分的繁琐,成本极高且易造成环境污染;在离体条件下的细胞培养方法获得的细胞其活性成分积累量很低而且稳定性很差。因此,利用现代基因工程技术将丹参药用活性成分生物合成途径中的关键酶基因导入到丹参中,获得转基因的发根、细胞系或再生植株,并进行大规模的培养,是提高丹参药用活性成分的含量和解决丹参药源问题的最佳途径。而有关丹参次生代谢产物生物合成途径方面的研究报道较少,因此本实验以丹参为材料,以cDNA末端快速扩增(Rapid amplification of cDNA ends, RACE)为技术平台,以同源克隆为基本思路,首次克隆得到了三个与丹参酮类成分次生代谢相关的基因:(1-deoxy-d-xylulose 5-phosphate synthase I)1-脱氧木酮糖-5-磷酸合成酶I,(1-deoxy-d-xylulose 5-phosphate synthase II)1-脱氧木酮糖-5-磷酸合成酶II和(3-hydroxy-3-methylglutaryl-CoA synthase)3-羟基-3-甲基戊二酰辅酶A合成酶的全长cDNA序列,并从其序列特征分析、表达分析、诱导调控等方面开展了一系列的工作,并取得了以下成果:1、成功地从丹参中克隆出了SmDXSI基因,该基因全长2519 bp,包含一个完整的2145 bp的开放阅读框(Open reading frame, ORF),编码714个氨基酸残基。根据蛋白比对的结果,SmDXSI基因编码的氨基酸序列与其它物种中报道的已知DXSI蛋白序列具有高度的同源性。序列分析发现该蛋白含有一个硫胺素焦磷酸盐绑定区域(thiamin pyrophosphate binding domain),一个保守的嘧啶绑定区域(DRAG domain),和一个保守的组氨酸残基。组织特异性表达分析结果表明,SmDXSI基因在丹参中为组成型表达,且叶中的表达比茎和根高,根里表达很弱。丹参毛状根诱导处理结果表明,诱导子处理对SmDXSI基因的表达基本上没有影响,为非诱导应答型基因。2、成功地从丹参中克隆出了SmDXSII基因,该基因全长2522 bp,包含一个完整的2175 bp的开放阅读框,编码724个氨基酸残基。根据蛋白比对的结果,SmDXSII基因编码的氨基酸序列与其它物种中报道的已知DXSII蛋白序列具有高度的同源性。序列分析发现该蛋白含有一个硫胺素焦磷酸盐绑定区域(thiamin pyrophosphate binding domain),一个保守的嘧啶绑定区域(DRAG domain),和一个保守的组氨酸残基。组织特异表达分析结果表明,SmDXSII基因不是一个组成型表达的基因,SmDXSII基因在根里表达量最高,在茎和叶里几乎检测不到表达。丹参毛状根诱导处理结果表明,SmDXSII基因至少在转录水平上受到诱导调控,是一个诱导应答型的基因,MJ、Ag+均能非常有效地诱导SmDXSII基因表达水平的上调。其中MJ的诱导效应相对显著。3、成功地从丹参中克隆出了SmHMGS基因,该基因全长1655 bp,包含一个完整的1383 bp的ORF,编码460个氨基酸残基。根据蛋白比对的结果,SmHMGS基因编码的氨基酸序列与其它物种中报道的已知HMGS蛋白序列具有高度的同源性。组织特异性表达分析结果表明,该基因在丹参中为组成性表达,在叶片中的表达最为强烈,在茎部的表达次之,在根里的表达最弱。丹参毛状根诱导处理结果表明,SmHMGS基因至少在转录水平上受到诱导调控,也是一个诱导应答型的基因,MJ、YE均能诱导SmHMGS基因表达水平的上调。本文系统地从丹参中克隆到了三个与丹参酮生物合成相关的基因,为今后利用基因工程技术改良丹参、提高丹参酮类次生代谢产物含量打下了坚实的基础;分析了丹参酮生物合成途径中的三个相关酶基因在丹参不同部位的组织特异表达特征,为深入了解丹参酮类成分在不同部位的含量差异的遗传基础提供了初步的分子证据;比较分析了MJ、YE和Ag+三种诱导子对丹参酮生物合成途径的调控效应,从基因表达水平上探讨了这些诱导子对丹参酮生物合成途径可能的分子调控机制,具有十分重要的理论和现实意义。更多还原

【Abstract】 Danshen, first recorded in the Shen long’s herbal classic, was a famous traditional Chinese medicinal herb, which has effects such as dissolving stasis and odynolysis, blood-activating and regulating of the menstrual function, nourishing the heart and relieving restlessness. The active ingredients of S.miltiorrhiza were fat-soluble diterpene quinine compounds and water-soluble phenolic acid ones, both of which had strong pharmacological activity. The diterpene quinine compounds, such as Tanshinone IIA, were widely applied in bacteriostasis, diminishing inflammation, oxidation resistance and anticancer et al, so the clinical requirement is tremendous.However, because of the long growth cycle(more than two years), there were many defects in the S.miltiorrhiza’s traditional culture, such as the degeneration of quality, and the relative large cost of production;there were also many defects in the chemosynthesis, such as the complicated course, the huge cost, the environment pollution; the active ingredients production were very low and unstable in cell culture. The modern genetic engineering technologies were used to put the key-enzyme of the metabolic pathway into the S.miltiorrhiza’s genome and then the transgenetic hairy root, transgenetic cell line and the regeneration plant were gained and to be cultivated on a large scale. These methods can provide us a new way to produce tanshinones and this way can be considered as an optimal path to resolve the problem such as the low production of tanshinones and the the shortage of the S.miltiorrhiza’s resources.Until now, there are few researches and reports of the molecular biology about the biosynthesis of S.miltiorrhiza’s secondary metabolites. So the S.miltiorrhiza was used as experimental material, RACE was used as technology platform and the homologous clone used as experimental considerations in this experiment. Three genes, involved the tanshinones’biosynthesis, were first cloned and characterized, including: 1-deoxy-d-xylulose 5-phosphate synthaseI, 1-deoxy-d-xylulose 5-phosphate synthaseII, 3-hydroxy-3-methylglutaryl-coenzyme A. The Tissue expression profile analysis and the expression profile analysis treated with the elicitors were also tested in this experiment. The results obtained were showed as below.1. A new full-length cDNA (2519bp) encoding 1-deoxy-d-xylulose 5-phosphate synthase I (designated as SmDXS I) was successfully isolated from S.miltiorrhiza by rapid amplification of cDNA ends (RACE). The full-length cDNA containd a 2145bp ORF (Open Reading Frame), encoding a deduced protein of 714 amino acid residues. Amino acid sequence comparison analysis showed that SmDXS I had high similarity with DXSI from other reported plants. Squence analysis showed that this protein contained a TPP banding domain(thiamin pyrophosphate binding domain), a conserved (DRAG domain)and a conserved His. Tissue expression profile analysis showed that SmDXS1 was detected in all tested tissues but at different levels with the highest expression in leaves, followed by in stems and roots, so it was considered to be a constitutively expressing gene. The Profile of SmDXSI under induction of elicitors showed that the elicitors treatments had few effect on the expression of SmDXSI which was not an elicitors-responsive gene.2. A new full-length cDNA (2522bp) encoding 1-deoxy-d-xylulose 5-phosphate synthase I (designated as SmDXSII) was successfully isolated from S.miltiorrhiza by rapid amplification of cDNA ends (RACE). The full-length cDNA containd a 2175bp ORF (Open Reading Frame), encoding a deduced protein of 724 amino acid residues. Amino acid sequence comparison analysis showed that SmDXSII had high similarity with DXSII from other reported plants. Squence analysis showed that this protein contained a TPP banding domain(thiamin pyrophosphate binding domain), a conserved (DRAG domain)and a conserved His. Tissue expression profile analysis showed that SmDXSII expression was weakly detected in roots and barely in stems and leaves, so it was considered to a non-constitutively expressing gene. The Profile of SmDXSII under induction of elicitors showed that SmDXSII was regulated in the transcription level at least; it was an elicitors-responsive gene, methyl jasmonate (MJ) and silver ion (Ag+) both can effectively up-regulated the expression of SmDXSII, but MJ’s effects was better than Ag+.3. A new full-length cDNA (1655bp) encoding 3-hydroxy-3-methylglutaryl-CoA synthase (designated as SmHMGS) was successfully isolated from S.miltiorrhiza by rapid amplification of cDNA ends (RACE). The full-length cDNA containd a 1383bp ORF (Open Reading Frame), encoding a deduced protein of 460 amino acid residues. Amino acid sequence comparison analysis showed that SmHMGS had high similarity with HMGS from other reported plants. Tissue expression profile analysis showed that SmHMGS expression was detected in all tested tissues but at different levels with the highest expression in leaves, followed by in stems and roots, so it was considered to be a constitutively expressing gene. The profile of SmHMGS under induction of elicitors showed that SmHMGS was regulated in the transcription level at least; it was an elicitors-responsive gene, methyl jasmonate (MJ) and Yeast Extract (YE) both can effectively regulated the expression of SmHMGS.In this experiment, three genes involved in the tanshinones biosynthesis were isolated from S.miltiorrhiza, which can be very helpful for inhancing the tanshinones’production and S.miltiorrhiza’s genetic improvement; Tissue expression pattern analyses of these cloned genes provides primary molecular evidence for further understanding of the genetic basis of tanshinones content diversity in different parts; expression profile analyses treated with the elicitors were carried out to provide useful information to further understand induction expression and molecular regulation mechanism of genes encoding related enzymes involved in tanshinones’biosynthesis.更多还原