转录因子PAP2对丹参酚酸类产物合成的影响
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摘要
丹参(Salvia miltiorrhiza Bunge)为唇形科鼠尾草属多年生草本植物,是我国传统名贵大宗药材之一,其主要药理活性成分为脂溶性二萜醌类和水溶性酚酸类化合物,具有活血祛瘀、养血安神、通利关脉、消除烦心等多种药理学活性,被广泛应用于心脑血管等疾病的治疗。近年来,随着丹参更多有效成分及其新的药理学作用被发现,丹参药材需求量逐年增加。目前野生丹参资源日益匮乏,栽培丹参品质逐步退化。因此利用现代生物技术手段培育优质品系具有重要的意义。长久以来,丹参水溶性成分被认为是其发挥药效的主要物质基础。目前通过丹参多倍体诱导、毛状根培养等手段能提高其酚酸性成分的含量,但收效甚微。基于分子生物学和基因工程而发展起来的药用植物次生代谢工程在提高某些药用植物特定次生代谢物或生物学特性指标方面具有广阔的应用前景。药用植物次生代谢工程所采取的策略主要有两个方面：其一是采取RNAi或过表达某些次生代谢物合成的关键酶基因进而控制其产量,而植物代谢途径是由多种酶参与的多步反应,对单个基因进行修饰有时难以奏效；其二是基于转录因子可以与植物次生代谢途径相关酶基因调控序列中相同或相似的顺式作用元件结合,达到调控整个代谢途径的特性,经分子生物学方法调节特定转录因子的表达,提高特定化合物的积累。已有的研究表明,MYB类转录因子广泛参与了植物苯丙烷类代谢途径的调节。本实验室前期研究发现丹参酚酸类活性物质合成众多相关酶基因的启动子区域均含有MYB结合位点,我们推测MYB类转录因子可能在酚酸类物质的合成及协同表达中具有重要的调控作用。为此,我们将拟南芥MYB90家族转录因子PAP2在丹参中过表达,试图筛选出某些酚酸类成分含量显著提高的株系,为日后优良丹参品系的选育提供基础。
主要研究内容及结论如下：
1.从NCBI的核酸数据库中下载拟南芥PAP2基因的全长序列,利用DNAStar软件上的ORF finder找到完整的读码框,选取拟南芥PAP2基因上750bp的片段做为目标靶片段,构建PAP2过表达的pCAMBIA1302载体,利用叶盘法介导的丹参遗传转化,将载体pCAMBIA1302/PAP2采用热激法转化入丹参,在潮霉素浓度为(Hyb5mg/1)的选择培养基上选择4-5次后,DNA水平的PCR检测获得9个阳性株系。
2.采用实时定量PCR对9个阳性株系中PAP2基因的表达水平进行检测,以及分析此转录因子基因在丹参植株中的表达模式,结果显示,获得的9个阳性株系中,有4株PAP2基因的表达水平均表现为明显的上调,且过表达的效率由高到低依次为PAP2-8、PAP2-4、PAP2-13、PAP2-7,其它获得的株系则为假阳性。在分析其器官特异性时,PAP2基因在叶和根的部位均有表达,且表达的水平有较大的差别,在茎中无表达,从表达量分析,PAP2基因在叶中的表达量最高,且远高于根中的表达量。
3.采用高效液相色谱来检测丹参不同部位的四种水溶性酚酸成分,分别为丹参素、咖啡酸、迷迭香酸以及丹酚酸B的含量。结果表明,转基因株系中丹参素、迷迭香酸以及丹酚酸B的含量均有提高,而咖啡酸含量变化不明显。
4.发现了PAP2转录因子调控丹参酚酸合成的转录激活新功能
分析了三个阳性株系中显著变化的标志性代谢产物和差异性表达基因,结果表明,PAP2转录因子通过激活苯丙烷代谢途径中PAL、C4H、4CL-1和酪氨酸代谢途径中TAT基因的表达,促进了酚酸类物质的合成积累；以及花青素代谢途径上F3’H、F3’5’H基因的表达。
5.采用Stephen Y.Lin等人的重量法来检测总木质素的含量以及酸可溶性(Acid lignin)木质素和克拉森(Klason)木质素的含量。结果表明,转基因株系无论是酸可溶性木质素还是Klason木质素含量均有所降低,从而总木质含量也有少量的降低,可能与CCR及COMT基因表达量下调相关。
6.采用Folin-Ciocalteu法、DewantoⅤ等人的氯化铝——亚硝酸钠比色法以及花青素等测定方法分别测定总酚酸、总黄酮和花青素的含量。结果表明花青素、黄酮的含量均有少量的提高。
Salvia miltiorrhiza Bunge is a well-known medicinal plant in the Labiatae family. The active constituents of S. miltiorrhiza can be divided into two groups:lipid-soluble tanshinones and water-soluble phenolic acids, its phenolic acids has been widely used for treatment of cardiovascular, cerebrovascular and heart disease. In recent years, accompanied by the growing demand of S. miltiorrhiza and a gradual reduction of its wild resources, improving the content of the active ingredients and cultivating new varieties with high quality have become the most urgent and key issues in the development of Salvia resources. For a long time, the phenolic acids from S. miltiorrhiza were considered the main components in water-decoction, which is the major form administered to patients in clinical medication of China. At present, many means have been employed for enhance the contents of phenolic acids based on hairy root cultures and changes in ploidy, but the result were not good. The development of plant metabolic engineering through specific regulation of secondary metabolism was a promising alternative strategy for generating medicinal plants with enhanced health-promoting compounds. The strategy has two aspects:first, RNAi or overexpression of some key genes in the synthesis of secondary metabolites pathway, but plant metabolic pathway was a multi-step reaction with a variety of enzymes, so a single gene modification is difficult to work. The second strategy is based on the transcription factor, which can combine with cis-acting element to control the characteristics of the metabolic pathways. Previous studies have showed that, MYB transcription factor involved in phenylpropanoid metabolism regulation widely, the molecular biology had been applied to regulate the expression of specific transcription factors to enhance the accumulation of specific compounds. Preliminary studies in our laboratory found that many genes which contain MYB binding sites were related to the synthesis of phenolic acids. In view of the above-mentioned facts, we speculate that MYB transcription factors may have an important regulatory role in the synthesis of phenolic acids. In order to provide some evidences of breeding fine strains for Salvia, so we choose the MYB90 family transcription factors PAP 2 in Arabidopsis to overexpress in S. miltiorrhiza, screening of some strains which the phenolic components were significantly improved.
The main results were as follows:
1. The 750 bp fragments, located in the 3'end of the coding region of PAP2genes, were chosen for overexpression investigation, which was introduced into S. miltiorrhiza by Agrobacterium tumefaciens-mediated gene transformation, and 9 transgenic lines were obtained by PCR screening.
2. Compared with the controls, analysis of transcription quantity by real-time PCR showed the high transcription and translation in three transgenic lines.
3. Water extracts of three transgenic lines were chosen for HPLC analysis. The PAP 2 roots and leaves, compared with control plants, shows difference. The contents of danshensu, salvianolic acid B and rosmarinci acid in transgenic lines were significantly higher than those in control,while the content of caffeic acid was not predominant.
4.PAP2 has an additional, previously unknown role as a transcriptional activator of phenolic acid biosynthesis in S. miltiorrhiza
PAP2 activated a broader spectrum of genes, such as PAL, C4H,4CLand TAT in the phenylpropanoid pathway. It is effective in enriching the formation of phenolic acids in transgenic S. miltiorrhiza.Besides, PAP2 induced F3'Hand F3'5'Hhigh transcription and enhanced the metabolic flux in anthocyanin pathway.
5. The total lignin, the Klason lignin and acid lignin in transgenic plants were detected. The results show that:Klason lignin and acid lignin were decreased in the transgenic lines, related to the decrease of CCR and COMT gene expression.
6. Total phenolic, total flavonoid and anthocyanin contents were detected. The results showed that the contents of three ingredients increased a little.
In conclusion,9 transgenic lines were obtained by PCR screening. Phenolic acids level increased diversely in leaves and roots. The total phenolic, flavonoid and anthocyanin increased slightly, while the content of lignin decreased but not significant. This research is of great significance for the further studies of the secondary metabolite regulation and molecular breeding of S. miltiorrhiza, and provides a promising strategy for genetic engineering of other medicinal plants.
主要研究内容及结论如下：
1.从NCBI的核酸数据库中下载拟南芥PAP2基因的全长序列,利用DNAStar软件上的ORF finder找到完整的读码框,选取拟南芥PAP2基因上750bp的片段做为目标靶片段,构建PAP2过表达的pCAMBIA1302载体,利用叶盘法介导的丹参遗传转化,将载体pCAMBIA1302/PAP2采用热激法转化入丹参,在潮霉素浓度为(Hyb5mg/1)的选择培养基上选择4-5次后,DNA水平的PCR检测获得9个阳性株系。
2.采用实时定量PCR对9个阳性株系中PAP2基因的表达水平进行检测,以及分析此转录因子基因在丹参植株中的表达模式,结果显示,获得的9个阳性株系中,有4株PAP2基因的表达水平均表现为明显的上调,且过表达的效率由高到低依次为PAP2-8、PAP2-4、PAP2-13、PAP2-7,其它获得的株系则为假阳性。在分析其器官特异性时,PAP2基因在叶和根的部位均有表达,且表达的水平有较大的差别,在茎中无表达,从表达量分析,PAP2基因在叶中的表达量最高,且远高于根中的表达量。
3.采用高效液相色谱来检测丹参不同部位的四种水溶性酚酸成分,分别为丹参素、咖啡酸、迷迭香酸以及丹酚酸B的含量。结果表明,转基因株系中丹参素、迷迭香酸以及丹酚酸B的含量均有提高,而咖啡酸含量变化不明显。
4.发现了PAP2转录因子调控丹参酚酸合成的转录激活新功能
分析了三个阳性株系中显著变化的标志性代谢产物和差异性表达基因,结果表明,PAP2转录因子通过激活苯丙烷代谢途径中PAL、C4H、4CL-1和酪氨酸代谢途径中TAT基因的表达,促进了酚酸类物质的合成积累；以及花青素代谢途径上F3’H、F3’5’H基因的表达。
5.采用Stephen Y.Lin等人的重量法来检测总木质素的含量以及酸可溶性(Acid lignin)木质素和克拉森(Klason)木质素的含量。结果表明,转基因株系无论是酸可溶性木质素还是Klason木质素含量均有所降低,从而总木质含量也有少量的降低,可能与CCR及COMT基因表达量下调相关。
6.采用Folin-Ciocalteu法、DewantoⅤ等人的氯化铝——亚硝酸钠比色法以及花青素等测定方法分别测定总酚酸、总黄酮和花青素的含量。结果表明花青素、黄酮的含量均有少量的提高。
Salvia miltiorrhiza Bunge is a well-known medicinal plant in the Labiatae family. The active constituents of S. miltiorrhiza can be divided into two groups:lipid-soluble tanshinones and water-soluble phenolic acids, its phenolic acids has been widely used for treatment of cardiovascular, cerebrovascular and heart disease. In recent years, accompanied by the growing demand of S. miltiorrhiza and a gradual reduction of its wild resources, improving the content of the active ingredients and cultivating new varieties with high quality have become the most urgent and key issues in the development of Salvia resources. For a long time, the phenolic acids from S. miltiorrhiza were considered the main components in water-decoction, which is the major form administered to patients in clinical medication of China. At present, many means have been employed for enhance the contents of phenolic acids based on hairy root cultures and changes in ploidy, but the result were not good. The development of plant metabolic engineering through specific regulation of secondary metabolism was a promising alternative strategy for generating medicinal plants with enhanced health-promoting compounds. The strategy has two aspects:first, RNAi or overexpression of some key genes in the synthesis of secondary metabolites pathway, but plant metabolic pathway was a multi-step reaction with a variety of enzymes, so a single gene modification is difficult to work. The second strategy is based on the transcription factor, which can combine with cis-acting element to control the characteristics of the metabolic pathways. Previous studies have showed that, MYB transcription factor involved in phenylpropanoid metabolism regulation widely, the molecular biology had been applied to regulate the expression of specific transcription factors to enhance the accumulation of specific compounds. Preliminary studies in our laboratory found that many genes which contain MYB binding sites were related to the synthesis of phenolic acids. In view of the above-mentioned facts, we speculate that MYB transcription factors may have an important regulatory role in the synthesis of phenolic acids. In order to provide some evidences of breeding fine strains for Salvia, so we choose the MYB90 family transcription factors PAP 2 in Arabidopsis to overexpress in S. miltiorrhiza, screening of some strains which the phenolic components were significantly improved.
The main results were as follows:
1. The 750 bp fragments, located in the 3'end of the coding region of PAP2genes, were chosen for overexpression investigation, which was introduced into S. miltiorrhiza by Agrobacterium tumefaciens-mediated gene transformation, and 9 transgenic lines were obtained by PCR screening.
2. Compared with the controls, analysis of transcription quantity by real-time PCR showed the high transcription and translation in three transgenic lines.
3. Water extracts of three transgenic lines were chosen for HPLC analysis. The PAP 2 roots and leaves, compared with control plants, shows difference. The contents of danshensu, salvianolic acid B and rosmarinci acid in transgenic lines were significantly higher than those in control,while the content of caffeic acid was not predominant.
4.PAP2 has an additional, previously unknown role as a transcriptional activator of phenolic acid biosynthesis in S. miltiorrhiza
PAP2 activated a broader spectrum of genes, such as PAL, C4H,4CLand TAT in the phenylpropanoid pathway. It is effective in enriching the formation of phenolic acids in transgenic S. miltiorrhiza.Besides, PAP2 induced F3'Hand F3'5'Hhigh transcription and enhanced the metabolic flux in anthocyanin pathway.
5. The total lignin, the Klason lignin and acid lignin in transgenic plants were detected. The results show that:Klason lignin and acid lignin were decreased in the transgenic lines, related to the decrease of CCR and COMT gene expression.
6. Total phenolic, total flavonoid and anthocyanin contents were detected. The results showed that the contents of three ingredients increased a little.
In conclusion,9 transgenic lines were obtained by PCR screening. Phenolic acids level increased diversely in leaves and roots. The total phenolic, flavonoid and anthocyanin increased slightly, while the content of lignin decreased but not significant. This research is of great significance for the further studies of the secondary metabolite regulation and molecular breeding of S. miltiorrhiza, and provides a promising strategy for genetic engineering of other medicinal plants.
引文
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