红花钓钟柳中松果菊苷的分离纯化、动态变化及松果菊苷生物合成途径中部分酶的基因工程研究
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摘要
松果菊苷既是一种咖啡酸类衍生物,也是一种苯乙醇苷类化合物,具有多种药理学活性,如抗氧化、抗炎、抗肿瘤、神经保护作用、保肝作用以及对免疫系统产生刺激作用。松果菊苷最早发现于狭叶松果菊,其含量约为0.2%,后亦在克氏钓钟柳中发现,含量约为0.09%。目前,松果菊苷主要从一种寄生于梭梭的管花肉苁蓉中提取。在2005版中华人民共和国药典中,管花肉苁蓉的唯一质量控制标准是,松果菊苷的含量不低于1.0%。管花肉苁蓉只分布于沙漠和戈壁一带,生长缓慢,且难于栽种。然而,近年来随着松果菊苷商业价值的不断提高,管花肉苁蓉的市场需求也在不断增长,管花肉苁蓉被过度开发,已经成为濒危植物。因此,寻找管花肉苁蓉的替代资源显得尤为紧迫。
本研究通过高效液相色谱筛选到富含松果菊苷的药源植物红花钓钟柳(Penstemon barbatus (Can.) Roth),利用高速逆流色谱快速分离制备松果菊苷纯品用于波谱学鉴定。为了提高次生代谢产物松果菊苷的含量,利用分子生物学方法,在其生物合成的基因工程领域开展了探索性研究。获得的主要结果如下：
1.首次发现红花钓钟柳中含有松果菊苷,并测定了根和叶中含量的年变化。
通过筛选首次发现红花钓钟柳含有松果菊苷,其根和叶中松果菊苷的含量随季节变化,在一年中,最高含量分别在十一月和五月达到7.25±0.36 mg/g和9.09±0.32 mg/g。结果表明,红花钓钟柳中松果菊苷的含量十分接近管花肉苁蓉中松果菊苷的含量水平,其有可能替代管花肉苁蓉,而用于规模化生产松果菊苷。
2.利用循环高速逆流色谱等技术从红花钓钟柳根中得到含量为96.3%的松果菊苷纯品42.0 mg,并确定其结构。
从红花钓钟柳中分离得到松果菊苷纯品。利用50%甲醇超声提取20 g红花钓钟柳根的粉末,所得到的甲醇提取物经AB-8树脂预纯化后,以正丁醇：甲醇(1：1,v/v)为溶剂系统,通过经典高速逆流色谱和循环高速逆流色谱纯化。最终获得了纯度为96.3%的松果菊苷42.0mg。循环HSCCC的回收率为91.0%。获得的松果菊苷纯品由IR,~1H NMR and ~(13)CNMR进行了结构确认。
3.完成了狭叶松果菊中3-脱氢奎尼酸合成酶基因aroB的克隆及其组织表达特征研究。
以狭叶松果菊(Echinacea.angustifolia DC)组培苗为材料,用Trizol法提取总RNA,根据已有的3-脱氢奎尼酸合成酶基因aroB保守氨基酸序列设计简并引物,通过RT-PCR扩增得到1条289bp的aroB基因同源片段。利用该已知中间序列,通过快速扩增cDNA末端技术(RACE)及序列拼接,最终得到该基因cDNA序列,命名为EanaroB, Genbank登录号为EU293857。分析结果表明,EanaroB cDNA长1424bp,包含一个1326bp的开放读码框,氨基酸序列同源分析结果发现,EanaroB编码的氨基酸序列与番茄、葡萄、拟南芥、山毛榉的aroB同源性都在80%左右,表明aroB氨基酸序列具有较强的保守性。半定量RT-PCR结果表明,狭叶松果菊EanaroB基因在狭叶松果菊的根、茎、叶、花中均有表达,但花和叶中的表达量较高,在根和茎中较少。
4.构建了pCAMBIA2301G-EaC4H1高效植物表达载体并获得基因工程农杆菌
对含有源于狭叶松果菊的EaC4H1 (Genbank登录号为EU676019)的pMD18-T和中间表达载体pCAMBIA2301G分别进行SacⅠ和XbaⅠ双酶切。由于EaC4Hl基因内含有SacⅠ酶切位点,经不完全酶切产生4条电泳条带,回收1602bp的目的基因条带和约12kb的pCAMBIA2301G载体骨架条带。将回收的pCAMBIA2301G载体骨架与酶切后产生的EaC4H1目的基因片段连接,连接产物转化大肠杆菌DH5α。抽提质粒后转化农杆菌。经PCR验证,获得与目的基因大小一致的条带。结果表明工程农杆菌LBA4404-pCAMBIA2301G-EaC4H1已构建成功。上述工程菌可用于遗传转化狭叶松果菊或红花钓钟柳,对在分子遗传学水平上探讨C4H在咖啡酸衍生物生物合成中的作用机理,并为进一步获得富含松果菊苷的转基因狭叶松果菊和红花钓钟柳奠定了基础。
Echinacoside was known not only as a caffeic acid derivative, but also as a phenylethanoid glycoside. It possesses a spectrum of beneficial activities, such as antioxidation, anti-inflammatory, antineoplastic, neuroprotective and hepatoprotective activities. Moreover, this compound can boost the immunoprotective efficacy. Echinacoside was firstly reported to be extracted from Echinacea angustifolia DC with a content around 0.2% and subsequently prepared from the aerial part of Penstemon crandallii A. Nels with a content of 0.09%. Currently, echinacoside is largely extracted from Cistanche tubulosa, a perennial parasitical plant on the roots of Haloxylox ammodendron and H. persicum, with the content of no less than 1.0%. The content of echinacoside is the only quality standard for the herbal medicine of C. tubulosa in Pharmacopoeia of the People's Republic of China. C. tubulosa is a slow-growing plant endemic to arid lands and deserts and recalcitrant to domestication. Increasingly demands for the soaring medication value of echinacoside further endangered C. tubulosa. New alternative source other than C. tubulosa for echinacoside is urgent both for commercial demands and environmental protection.
Taking the adventage of high performance liquid chromatography, this research was focusing on the screening of the alternative source that was abound in echinacoside. High-speed counter-current chromatography was imployed for the rapid separation and purification of echinacoside. The structure of the purified echinacoside was confirmed by IR,~1H NMR and ~(13)C NMR. In order to enhance the content of echinacoside, the secondary metabolism of the biosynthetic pathway of echinacoside was studied by means of molecular biology as well. The main results are listed as follows:
1. The annual dynamics of echinacoside in the leaves and the roots of P. barbatus
By screening, echinacoside was firstly reported to be isolated from P. barbatus in this study. The content of echinacoside varied within one year and the peak contents of it were 9.09±0.32 mg/g in the leaves and 7.25±0.36 mg/g in the roots annually. The result indicated that the content of echinacoside in P. barbatus was almost the same as that in C. tubulosa. Potentially, it might be an ideal source for preparation of large scale of echinacoside.
2.42.0 mg echinacoside with a purity of 96.3% was isolated from the root of P. barbatus by recycling high-speed counter-current chromatography
Echinacoside was isolated from P. barbatus by HSCCC for the first time. The methanolic extracts from 20 g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography and recycling high-speed counter-current chromatography with the solvent system n-butanol-water (1:1, v/v). Totally 42.0 mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR,~1H NMR and ~(13)C NMR is identical to the standard sample. The result demonstrated that echinacoside could be rapidly and efficiently isolated from P. barbatus by conventional high-speed counter-current chromatography and recycling high-speed counter-current chromatography.
3. Cloning and expression characterization of 3-dehydroquinate synthase cDNA from E. Angustifolia
Total RNA was extracted from tissue cultures of E. Angustifolia with Trizol method. A cDNA fragment of 289 bp was amplified by RT-PCR with a pair of degenerated primer designed according to the reported conserved amino acid sequence of 3-dehydroquinate synthase (aroB). The cDNA of aroB, named EanaroB (GenBank No. EU293857), of E. Angustifolia was obtained by Rapid amplification of cDNA ends and sequence spliced based on the known cDNA fragment. Sequence analysis indicated that EanaroB is 1424 bp and contains a 1326 bp open reading frame encoding a 442 predicated amino acid residues which shared arround 80% identity with other plant aroB, such as Lycopersicon esculentum, Vitis vinifera, Fagus sylvatica and Arabidopsis thaliana. Analysis of semi-quantitative RT-PCR indicated that aroB gene was transcribed in leaf and flower, less in root and stem.
4. Construction of plant high efficient expression vector pCAMBIA2301 G-EaC4Hl and the obtainment of bioengineering agrobacterium
pMD18-T--EaC4H1 and intermediate vector pCAMBIA2301G were digested by Sac I and Xba I. Since there was a restriction endonuclease site within the coding region of EaC4Hl,4 bands of pMD18-T-EaC4H1 were obtained by incomplete degistion. The target gene EaC4Hl of 1612 bp and the large fragment of pCAMBIA2301G of 12 kb were recovered and ligated. The recombinant plasmid was transformed into Agrobacterium tumefaciens strain LBA4404. This work was approved by PCR and DNA sequencing. The pCAMBIA2301G-EaC4H1-carrying LBA4404 could be then used for plant transformation. This work underlay the first step for studing the biosynthetic pathway of caffeic acid derivatives and improving the content of echinacoside in E. angustifolia and P. barbatus.
本研究通过高效液相色谱筛选到富含松果菊苷的药源植物红花钓钟柳(Penstemon barbatus (Can.) Roth),利用高速逆流色谱快速分离制备松果菊苷纯品用于波谱学鉴定。为了提高次生代谢产物松果菊苷的含量,利用分子生物学方法,在其生物合成的基因工程领域开展了探索性研究。获得的主要结果如下：
1.首次发现红花钓钟柳中含有松果菊苷,并测定了根和叶中含量的年变化。
通过筛选首次发现红花钓钟柳含有松果菊苷,其根和叶中松果菊苷的含量随季节变化,在一年中,最高含量分别在十一月和五月达到7.25±0.36 mg/g和9.09±0.32 mg/g。结果表明,红花钓钟柳中松果菊苷的含量十分接近管花肉苁蓉中松果菊苷的含量水平,其有可能替代管花肉苁蓉,而用于规模化生产松果菊苷。
2.利用循环高速逆流色谱等技术从红花钓钟柳根中得到含量为96.3%的松果菊苷纯品42.0 mg,并确定其结构。
从红花钓钟柳中分离得到松果菊苷纯品。利用50%甲醇超声提取20 g红花钓钟柳根的粉末,所得到的甲醇提取物经AB-8树脂预纯化后,以正丁醇：甲醇(1：1,v/v)为溶剂系统,通过经典高速逆流色谱和循环高速逆流色谱纯化。最终获得了纯度为96.3%的松果菊苷42.0mg。循环HSCCC的回收率为91.0%。获得的松果菊苷纯品由IR,~1H NMR and ~(13)CNMR进行了结构确认。
3.完成了狭叶松果菊中3-脱氢奎尼酸合成酶基因aroB的克隆及其组织表达特征研究。
以狭叶松果菊(Echinacea.angustifolia DC)组培苗为材料,用Trizol法提取总RNA,根据已有的3-脱氢奎尼酸合成酶基因aroB保守氨基酸序列设计简并引物,通过RT-PCR扩增得到1条289bp的aroB基因同源片段。利用该已知中间序列,通过快速扩增cDNA末端技术(RACE)及序列拼接,最终得到该基因cDNA序列,命名为EanaroB, Genbank登录号为EU293857。分析结果表明,EanaroB cDNA长1424bp,包含一个1326bp的开放读码框,氨基酸序列同源分析结果发现,EanaroB编码的氨基酸序列与番茄、葡萄、拟南芥、山毛榉的aroB同源性都在80%左右,表明aroB氨基酸序列具有较强的保守性。半定量RT-PCR结果表明,狭叶松果菊EanaroB基因在狭叶松果菊的根、茎、叶、花中均有表达,但花和叶中的表达量较高,在根和茎中较少。
4.构建了pCAMBIA2301G-EaC4H1高效植物表达载体并获得基因工程农杆菌
对含有源于狭叶松果菊的EaC4H1 (Genbank登录号为EU676019)的pMD18-T和中间表达载体pCAMBIA2301G分别进行SacⅠ和XbaⅠ双酶切。由于EaC4Hl基因内含有SacⅠ酶切位点,经不完全酶切产生4条电泳条带,回收1602bp的目的基因条带和约12kb的pCAMBIA2301G载体骨架条带。将回收的pCAMBIA2301G载体骨架与酶切后产生的EaC4H1目的基因片段连接,连接产物转化大肠杆菌DH5α。抽提质粒后转化农杆菌。经PCR验证,获得与目的基因大小一致的条带。结果表明工程农杆菌LBA4404-pCAMBIA2301G-EaC4H1已构建成功。上述工程菌可用于遗传转化狭叶松果菊或红花钓钟柳,对在分子遗传学水平上探讨C4H在咖啡酸衍生物生物合成中的作用机理,并为进一步获得富含松果菊苷的转基因狭叶松果菊和红花钓钟柳奠定了基础。
Echinacoside was known not only as a caffeic acid derivative, but also as a phenylethanoid glycoside. It possesses a spectrum of beneficial activities, such as antioxidation, anti-inflammatory, antineoplastic, neuroprotective and hepatoprotective activities. Moreover, this compound can boost the immunoprotective efficacy. Echinacoside was firstly reported to be extracted from Echinacea angustifolia DC with a content around 0.2% and subsequently prepared from the aerial part of Penstemon crandallii A. Nels with a content of 0.09%. Currently, echinacoside is largely extracted from Cistanche tubulosa, a perennial parasitical plant on the roots of Haloxylox ammodendron and H. persicum, with the content of no less than 1.0%. The content of echinacoside is the only quality standard for the herbal medicine of C. tubulosa in Pharmacopoeia of the People's Republic of China. C. tubulosa is a slow-growing plant endemic to arid lands and deserts and recalcitrant to domestication. Increasingly demands for the soaring medication value of echinacoside further endangered C. tubulosa. New alternative source other than C. tubulosa for echinacoside is urgent both for commercial demands and environmental protection.
Taking the adventage of high performance liquid chromatography, this research was focusing on the screening of the alternative source that was abound in echinacoside. High-speed counter-current chromatography was imployed for the rapid separation and purification of echinacoside. The structure of the purified echinacoside was confirmed by IR,~1H NMR and ~(13)C NMR. In order to enhance the content of echinacoside, the secondary metabolism of the biosynthetic pathway of echinacoside was studied by means of molecular biology as well. The main results are listed as follows:
1. The annual dynamics of echinacoside in the leaves and the roots of P. barbatus
By screening, echinacoside was firstly reported to be isolated from P. barbatus in this study. The content of echinacoside varied within one year and the peak contents of it were 9.09±0.32 mg/g in the leaves and 7.25±0.36 mg/g in the roots annually. The result indicated that the content of echinacoside in P. barbatus was almost the same as that in C. tubulosa. Potentially, it might be an ideal source for preparation of large scale of echinacoside.
2.42.0 mg echinacoside with a purity of 96.3% was isolated from the root of P. barbatus by recycling high-speed counter-current chromatography
Echinacoside was isolated from P. barbatus by HSCCC for the first time. The methanolic extracts from 20 g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography and recycling high-speed counter-current chromatography with the solvent system n-butanol-water (1:1, v/v). Totally 42.0 mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR,~1H NMR and ~(13)C NMR is identical to the standard sample. The result demonstrated that echinacoside could be rapidly and efficiently isolated from P. barbatus by conventional high-speed counter-current chromatography and recycling high-speed counter-current chromatography.
3. Cloning and expression characterization of 3-dehydroquinate synthase cDNA from E. Angustifolia
Total RNA was extracted from tissue cultures of E. Angustifolia with Trizol method. A cDNA fragment of 289 bp was amplified by RT-PCR with a pair of degenerated primer designed according to the reported conserved amino acid sequence of 3-dehydroquinate synthase (aroB). The cDNA of aroB, named EanaroB (GenBank No. EU293857), of E. Angustifolia was obtained by Rapid amplification of cDNA ends and sequence spliced based on the known cDNA fragment. Sequence analysis indicated that EanaroB is 1424 bp and contains a 1326 bp open reading frame encoding a 442 predicated amino acid residues which shared arround 80% identity with other plant aroB, such as Lycopersicon esculentum, Vitis vinifera, Fagus sylvatica and Arabidopsis thaliana. Analysis of semi-quantitative RT-PCR indicated that aroB gene was transcribed in leaf and flower, less in root and stem.
4. Construction of plant high efficient expression vector pCAMBIA2301 G-EaC4Hl and the obtainment of bioengineering agrobacterium
pMD18-T--EaC4H1 and intermediate vector pCAMBIA2301G were digested by Sac I and Xba I. Since there was a restriction endonuclease site within the coding region of EaC4Hl,4 bands of pMD18-T-EaC4H1 were obtained by incomplete degistion. The target gene EaC4Hl of 1612 bp and the large fragment of pCAMBIA2301G of 12 kb were recovered and ligated. The recombinant plasmid was transformed into Agrobacterium tumefaciens strain LBA4404. This work was approved by PCR and DNA sequencing. The pCAMBIA2301G-EaC4H1-carrying LBA4404 could be then used for plant transformation. This work underlay the first step for studing the biosynthetic pathway of caffeic acid derivatives and improving the content of echinacoside in E. angustifolia and P. barbatus.
引文
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