利用AtLUP1超量表达体系在拟南芥中合成羽扇豆醇
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摘要
羽扇豆醇是一种存在于多种果蔬及中草药中的五环三萜类化合物,具有抗炎症、抗氧化、抗癌等药理作用。羽扇豆醇合酶(Lupeol synthase,LUP)是羽扇豆醇合成途径中的关键酶。前期研究显示,编码该酶的基因在原核和酵母表达体系中均难以获得有生物活性的蛋白产物。本研究以拟南芥为受体,通过超量表达拟南芥羽扇豆醇合酶1(At LUP1)基因,分析利用植物细胞生产羽扇豆醇的可行性。使用气相色谱GC检测At LUP1超量表达拟南芥植株中的羽扇豆醇含量,发现48号植株中羽扇豆醇含量为0.915 5 mg·g-1,显著高于野生型。本研究证明超量表达At LUP1基因可以在拟南芥中促进羽扇豆醇的合成,为羽扇豆醇的工业化生产提供了新的思路。
Lupeol, a pentacyclic triterpenoid compound, has been found in diverse fruits, vegetables and medicinal plants. It has pharmacological effects of anti-inflammatory, anti-oxidation and anti-cancer. Lupeol synthase is a key enzyme for the biosynthesis of lupeol.Previous studies showed that it is difficult to obtain lupeol products with bioactivity either in prokaryotic or yeast expression systems. In this study, Arabidopsis was usedas a receptor for At LUP1 overexpression to analyze the feasibility of enhancing lupeol production in plant cells.The contents of lupeol in At LUP1 overexpressing strains were detected by GC, and the results showed that the lupeol content in the No.48 strain was 0.915 5 mg/g, which was significantly higher than that of the wild type. This study demonstrates that overexpression of At LUP1 could improve the biosynthesis of lupeol, thus provides a potential direction for the industrial production of lupeol.
Lupeol, a pentacyclic triterpenoid compound, has been found in diverse fruits, vegetables and medicinal plants. It has pharmacological effects of anti-inflammatory, anti-oxidation and anti-cancer. Lupeol synthase is a key enzyme for the biosynthesis of lupeol.Previous studies showed that it is difficult to obtain lupeol products with bioactivity either in prokaryotic or yeast expression systems. In this study, Arabidopsis was usedas a receptor for At LUP1 overexpression to analyze the feasibility of enhancing lupeol production in plant cells.The contents of lupeol in At LUP1 overexpressing strains were detected by GC, and the results showed that the lupeol content in the No.48 strain was 0.915 5 mg/g, which was significantly higher than that of the wild type. This study demonstrates that overexpression of At LUP1 could improve the biosynthesis of lupeol, thus provides a potential direction for the industrial production of lupeol.
引文
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[16]郭敏,刘佩,郑国华,等.p53/miR-34a通路介导羽扇豆醇的抑制人膀胱癌T24细胞增殖作用[J].中国药师,2016,19(9):1629-1632.
[17]王明,崔红霞,孙超,等.羽扇豆醇对人乳腺癌MDA-MB-231细胞侵袭转移作用及机制研究[J].药学学报,2016,51(4):558-562.
[18]郭秀春,张志娟,夏照洋,等.锦鸡儿中4种三萜类化合物的含量测定及其对糖苷酶的抑制活性[J].中国药学杂志,2017,52(6):488-493.
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[21]DELIS C,KROKIDA A,Georgiou S,et al.Role of lupeol synthase in Lotus japonicus nodule formation[J].New Phytologist,2011,189(1):335-346.
[22]陆捡花.大豆虫害响应相关基因的功能分析[D].北京:中国农业科学院,2017.
[23]金万梅,潘青华,尹淑萍,等.外源基因在转基因植物中的遗传稳定性及其转育研究进展[J].分子植物育种,2005,3(6):864-868.
[2]张扩,赵明,王美娇,等.细叶杜香叶中三萜类化学成分研究[J].中草药,2018,49(6):1250-1254.
[3]方春生,杨燕军,王如意,等.竹节蓼石油醚部位抗肿瘤活性组分研究及其成分GC-MS分析[J].今日药学,2016,28(8):538-540.
[4]马盼,莫惠雯,林坤河,等.柚花的化学成分研究[J].广西中医药,2017,40(1):77-79.
[5]王静怡,董阳阳,戚楚露,等.木棉树皮的化学成分[J].中国药科大学学报,2016,47(5):570-574.
[6]LASZCZYK M,JAGER S,SIMON-HAARHAUS B,et al.Physical,Chemical and Pharmacological Characterization of a New Oleogel-Forming Triterpene Extract from the Outer Bark of Birch(Betulae Cortex)[J].Planta Medica,2006,72(15):1389-1395.
[7]WATERMAN E,LOCKWOOD B.Active components and clinical applications of olive oil[J].Alternative Medicine Review,2007,12(4):331-342.
[8]武星彤,文亚峰.南方红豆杉分子遗传学研究进展[J].经济林研究,2017,35(2):228-232.
[9]章怀云,刘俊,毛绍名,等.天然产物活性成分提取分离研究进展[J].经济林研究,2017,35(3):244-250.
[10]PHILLIPS D R,RASBERY J M,BARTEL B,et al.Biosynthetic diversity in plant triterpene cyclization[J].Current Opinion in Plant Biology,2006,9(3):305-314.
[11]王冬.药用三萜酸细胞工厂的构建[D].天津:天津科技大学,2015.
[12]林庭庭,王冬,戴住波,等.创建酿酒酵母细胞工厂发酵生产羽扇豆醇[J].中国中药杂志,2016,41(6):1008-1015.
[13]戴鹏辉.珙桐DiMYB1基因的克隆、鉴定及功能研究[D].长沙:中南林业科技大学,2017.
[14]ZHANG X,HENRIQUES R,LIN S S,et al.Agrobacteriummediated transformation of Arabidopsis thaliana using the floral dip method[J].Nature Protocols,2006,1(2):641-646.
[15]唐泽严,杨小彤,周旭,等.羽扇豆醇抑制人肝癌Huh-7细胞增殖机制研究[J].中国药师,2017,20(9):1521-1525.
[16]郭敏,刘佩,郑国华,等.p53/miR-34a通路介导羽扇豆醇的抑制人膀胱癌T24细胞增殖作用[J].中国药师,2016,19(9):1629-1632.
[17]王明,崔红霞,孙超,等.羽扇豆醇对人乳腺癌MDA-MB-231细胞侵袭转移作用及机制研究[J].药学学报,2016,51(4):558-562.
[18]郭秀春,张志娟,夏照洋,等.锦鸡儿中4种三萜类化合物的含量测定及其对糖苷酶的抑制活性[J].中国药学杂志,2017,52(6):488-493.
[19]GUHLING O,HOBL B,YEATS T,et al.Cloning and characterization of a lupeol synthase involved in the synthesis of epicuticular wax crystals on stem and hypocotyl surfaces of Ricinus communis[J].Archives of Biochemistry&Biophysics,2006,448(1-2):60-72.
[20]FAN G,ZHAI Q,ZHAN Y.Gene Expression of Lupeol Synthase and Biosynthesis of Nitric Oxide in Cell Suspension Cultures of Betula platyphylla,in Response to a Phomopsis,Elicitor[J].Plant Molecular Biology Reporter,2013,31(2):296-302.
[21]DELIS C,KROKIDA A,Georgiou S,et al.Role of lupeol synthase in Lotus japonicus nodule formation[J].New Phytologist,2011,189(1):335-346.
[22]陆捡花.大豆虫害响应相关基因的功能分析[D].北京:中国农业科学院,2017.
[23]金万梅,潘青华,尹淑萍,等.外源基因在转基因植物中的遗传稳定性及其转育研究进展[J].分子植物育种,2005,3(6):864-868.