珠子参中法尼基焦磷酸合酶(FPS)对皂苷生物合成的影响研究

doi:10.7525/j.issn.1673-5102.2018.04.017

摘要/Abstract

摘要： 珠子参作为名贵中草药已有上千年应用历史，珠子参皂苷是珠子参的主要活性成分，由达玛烷型和齐墩果烷型三萜皂苷组成。目前，对珠子参皂苷的生物合成了解甚少，有必要开展与皂苷合成相关的基础研究工作。已有报道表明，法尼基焦磷酸合酶（FPS）可能是植物中皂苷合成的关键酶。本研究克隆了珠子参FPS基因（PjFPS）的cDNA序列，并对其进行了生物信息学分析。基于PjFPS序列构建了珠子参过表达载体pCAMBIA2300s-PjFPS，将其转化到珠子参细胞中，成功获得阳性转基因细胞；测定了阳性细胞系中PjFPS基因的相对表达量、FPS酶活、皂苷以及植物甾醇含量的变化。结果表明，与野生型珠子参细胞相比，PjFPS转基因珠子参细胞系中，PjFPS基因的相对表达量、FPS酶活以及皂苷含量均有不同程度的提高。而且，由于皂苷合成代谢流的增加，也促进了相关重要关键酶基因的表达。在效果最好的阳性细胞中，PjFPS基因的相对表达量、FPS酶活、皂苷含量分别为对照的12、4和3倍；同时，转基因细胞系的植物甾醇含量也有所升高。本研究通过对皂苷合成途径关键酶基因的调控实现了对皂苷生物合成的调节，为获得高效、稳定的珠子参皂苷合成调控技术提供理论参考和依据。

关键词: 珠子参, 人参属, 人参皂苷, 三萜, 过表达

Abstract: Panax japonicus recognized as a valuable Chinese herb has been used for thousands of years. P.japonicus saponins are the main bioactive ingredients composed by dammarane-type and oleanane-type triterpenoid saponins. So far, because of the lack of study about the biosynthesis of P.japonicus saponins, it's necessary to carry out the basic research on saponins biosynthesis. Farnesyl pyrophosphate synthase(FPS) may be a key enzyme in the synthetic pathway of saponins in plants. The cDNA sequence of PjFPS was cloned and bioinformatics analysis was conducted. Overexpression vector pCAMBIA2300s-PjFPS was constructed, and then transferred into the P.japonicus cells to obtain transgenic cell lines successfully. The relative expression levels of PjFPS, the enzyme activities of PjFPS, the contents of PJS and phytosterols in transgenic cell lines were determined. All of them in PjFPS transgenic cells achieved enhancements compared with the control. Furthermore, the expressions of key enzyme genes were promoted because the metabolic flux of saponins was increased. Especially, the expression level of PjFPS, the enzyme activity and total P.japonicus saponins content of the best-performing positive cell line were 12, 4 and 3 times of those in control, respectively. Meanwhile, the content of phytosterol in transgenic cell lines was also enhanced. This study realized the biosynthetic regulation of saponins by controlling the expression of a key enzyme gene, and thus to provide theoretical reference and basis for acquiring an efficient and stable technique of regulating the P.japonicus saponins biosynthesis.

Key words: Panax japonicus, Panax, ginsenoside, triterpenoid, overexpression

中图分类号:

Q942.5

刘美佳, 于怡琳, 姜森, 张翔, 刘迪秋, 葛锋. 珠子参中法尼基焦磷酸合酶(FPS)对皂苷生物合成的影响研究[J]. 植物研究, 2018, 38(4): 611-618.

LIU Mei-Jia, YU Yi-Lin, JIANG Sen, ZHANG Xiang, LIU Di-Qiu, GE Feng. Effect of Farnesyl-pyrophosphate Synthase(FPS) on the Biosynthesis of Saponins in Panax japonicus[J]. Bulletin of Botanical Research, 2018, 38(4): 611-618.

参考文献

1. Francis G,Kerem Z,Makkar H P,et al. The biological action of saponins in animal systems:a review[J]. The British Journal of Nutrition,2002,88(6):587-605.
2. Han L K,Zheng Y N,Yoshikawa M,et al. Anti-obesity effects of chikusetsusaponins isolated from Panax japonicus rhizomes[J]. BMC Complementary and Alternative Medicine,2005,5:9.
3. Matsuda H,Morikawa T,Ueda H,et al. Medicinal foodstuffs. XXVⅡ. Saponin constituents of Gotu Kola(2):structures of New Ursane-and Oleanane-type triterpene,oligoglycosides,centellasaponins B,C and D,from Centella asiatica cultivated in Sri Lanka[J]. Chemical & Pharmaceutical Bulletin,2001,49(10):1368-1371.
4. Yoshizaki K,Devkota H P,Fujino H,et al. Saponins composition of rhizomes,taproots,and lateral roots of Satsuma-ninjin(Panax japonicus)[J]. Chemical & Pharmaceutical Bulletin,2013,61(3):344-350.
5. Qi L W,Wang C Z,Yuan C S. Ginsenosides from American ginseng:chemical and pharmacological diversity[J]. Phytochemistry,2011,72(8):689-699.
6. Zhang H,Wang H F,Liu Y,et al. The haematopoietic effect of Panax japonicus on blood deficiency model mice[J]. Journal of Ethnopharmacology,2014,154(3):818-824.
7. 左锐,袁丁. 竹节参化学成分和药理活性研究进展[J]. 时珍国医国药,2015,16(9):838-839,841.Zuo R,Yuan D. Research on chemical constituents and biological activities of Rhizoma of Panax japonicus[J]. Lishizhen Medicine and Materia Medica Research,2005,16(9):838-389,841.
8. Luo H M,Sun C,Sun Y Z,et al. Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers[J]. BMC Genomics,2011,12(S5):S5.
9. Huang Z P,Zhang L N. Chemical structures of water-soluble polysaccharides from Rhizoma Panacis japonici[J]. Carbohydrate Research,2009,344(9):1136-1140.
10. Yamahara J,Kubomura Y,Miki K,et al. Anti-ulcer action of Panax japonicus rhizome[J]. Journal of Ethnopharmacology,1987,19(1):95-101.
11. Matsuda H,Samukawa K,Fukuda S,et al. Studies of Panax japonicus fibrinolysis[J]. Planta Medica,1989,55(1):18-21.
12. Kim Y K,Kim Y B,Kim J K,et al. Molecular cloning and characterization of mevalonic acid(MVA) pathway genes and triterpene accumulation in Panax ginseng[J]. Journal of the Korean Society for Applied Biological Chemistry,2014,57(3):289-295.
13. 黄壮嘉,刘迪秋,葛锋,等. 植物三萜皂苷生物合成中关键后修饰酶研究进展[J]. 西北植物学报,2014,34(10):2137-2144.Huang Z J,Liu D Q,Ge F,et al. Advances in studies on key post-modification enzymes in triterpenoid saponins biosynthesis[J]. Acta Botanica Boreali-occidentalia Sinica,2014,34(10):2137-2144.
14. Chappell J. Biochemistry and molecular biology of the isoprenoid biosynthetic pathway in plants[J]. Annual Review of Plant Physiology and Plant Molecular Biology,1995,46(1):521-547.
15. Closa M,Vranová E,Bortolotti C,et al. The Arabidopsis thaliana FPP synthase isozymes have overlapping and specific functions in isoprenoid biosynthesis,and complete loss of FPP synthase activity causes early developmental arrest[J]. The Plant Journal,2010,63(3):512-525.
16. Yang Y,Ge F,Sun Y,et al. Strengthening triterpene saponins biosynthesis by over-expression of farnesyl pyrophosphate synthase gene and RNA interference of cycloartenol synthase gene in Panax notoginseng cells[J]. Molecules,2017,22(4):581.
17. Tansakul P,Shibuya M,Kushiro T,et al. Dammarenediol-Ⅱ synthase,the first dedicated enzyme for ginsenoside biosynthesis,in Panax ginseng[J]. FEBS Letters,2006,580(22):5143-5149.
18. Rai A,Yamazaki M,Takahashi H,et al. RNA-seq transcriptome analysis of Panax japonicus,and its comparison with other Panax species to identify potential genes involved in the saponins biosynthesis[J]. Frontiers in Plant Science,2016,7:481.
19. Huang Z W,Lin J C,Cheng Z X,et al. Production of oleanane-type sapogenin in transgenic rice via expression of β-amyrin synthase gene from Panax japonicus C. A. Mey[J]. BMC Biotechnology,2015,15:45.
20. Zhang L,Huang Y S. Molecular cloning and expression of SS gene from Panax japonicus[J]. Nanomedicine:Nanotechnology,Biology,and Medicine,2016,12(2):524.
21. Kim O T,Kim S H,Ohyama K,et al. Upregulation of phytosterol and triterpene biosynthesis in Centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate synthase[J]. Plant Cell Reports,2010,29(4):403-411.
22. 阮琴妹,曹雄军,孙化鹏,等. 洋常春藤法呢基焦磷酸合酶基因的克隆与序列分析[J]. 湖南农业大学学报:自然科学版,2016,42(2):136-141.Ruan Q M,Cao X J,Sun H P,et al. Cloning and sequence analyzing of farnesyl pyrophosphate synthase from Hedera helix[J]. Journal of Hunan Agricultural University:Natural Sciences,2016,42(2):136-141.
23. 吴耀生,陈莉,蓝秀万,等. 三七与其它植物FPS序列的比较分析[J]. 西北植物学报,2007,27(8):1563-1570.Wu Y S,Chen L,Lan X W,et al. Comparison analysis of FPS from Panax notoginseng with other plant FPS registered in GenBank[J]. Acta Botanica Boreali-Occidentalia Sinica,2007,27(8):1563-1570.
24. 刘佳,全雪丽,姜明亮,等. 低温胁迫对人参皂苷生物合成途径基因家族表达特性的影响研究[J]. 中草药,2016,47(11):1956-1961.Liu J,Quan X L,Jiang M L,et al. Effect of cold stress on expression characteristic of gene families of ginsenoside biosynthesis pathway[J]. Chinese Traditional and Herbal Drugs,2016,47(11):1956-1961.
25. 王淋,杜红岩,乌云塔娜. 杜仲MVA和MEP途径相关基因的亚细胞定位与表达分析[J]. 植物研究,2017,37(1):52-62.Wang L,Du H Y,Wuyun T N. Subcellular localization and expression analysis of genes from Eucommia ulmoides involved in MVA and MEP pathway[J]. Bulletin of Botanical Research,2017,37(1):52-62.
26. Kim Y K,Kim Y B,Uddin M P,et al. Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase[J]. ACS Synthetic Biology,2014,3(10):773-779.
27. 王冰莹,杨永霞,闰鼎,等. fps基因过量表达对烟叶类胡萝卜素生物合成的影响[J]. 中国烟草学报,2012,18(2):44-48.Wang B Y,Yang Y X,Yan D,et al. Effect of over-expression of farnesyl pyrophosphate synthase(fps) gene on biosynthesis of Carotenoids in tobacco[J]. Acta Tabacaria Sinica,2012,18(2):44-48.
28. 崔红,慕平利,李雪君,等. 烟草fps转基因发状根的离体培养及其抗菌性[J]. 湖南农业大学学报:自然科学版,2008,34(4):422-425.Cui H,Mu P L,Li X J,et al. fps transgenic hairy roots culture in vitro and its resistance to Phytophthoro parasitica[J]. Journal of Hunan Agricultural University:Natural Sciences,2008,34(4):422-425.
29. Deng B,Zhang P,GE F,et al. Enhancement of triterpenoid saponins biosynthesis in Panax notoginseng cells by co-overexpressions of 3-hydroxy-3-methylglutaryl CoA reductase and squalene synthase genes[J]. Biochemical Engineering Journal,2017,122:38-46.

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