毛冬青三萜C-28位氧化酶基因的克隆与功能研究
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摘要
目的对毛冬青三萜C-28位氧化酶基因进行挖掘与功能鉴定。方法在前期获得的毛冬青转录组数据中,筛选出1个可能参与毛冬青三萜C-28位氧化的CYP基因(IpAO1),将IpAO1与毛冬青三萜合酶IpAS1共转化酿酒酵母,验证IpAO1的功能。结果 Western Blot结果表明,重组酵母能够表达与预期大小相符的IpAS1蛋白和IpAO1蛋白。GC-MS分析结果显示,仅表达IpAS1基因的重组酵母产生了α-香树脂醇和β-香树脂醇,而共表达IpAS1和IpAO1基因的重组酵母合成了熊果酸和齐墩果酸。结论来源于毛冬青的IpAO1是编码一个α-香树脂醇/β-香树脂醇的C-28位氧化酶。
引文
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[2] 冯华芬. 毛冬青叶化学成分研究[D].广西师范大学硕士学位论文, 2012.
[3] Cao D, Wang Q, Jin J, et al. Simultaneous Qualitative and Quantitative Analyses of Triterpenoids in Ilex pubescens by Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry[J].Phytochem Anal, 2018,29(2):168.
[4] 张双伟, 梁宏宇, 陈洁, 等. 中药毛冬青对慢性心力衰竭大鼠炎症因子IL-1β和NF-κB表达的影响[J].新中医, 2013,45(8):180.
[5] 郑关毅, 石旺清, 陈晓东, 等. 毛冬青甲素对大鼠脑缺血再灌注后bFGF、GAP-43的表达及神经元再生的影响[J].药学学报, 2011,46(9):1065.
[6] Zhou K, Qiao K, Edgar S, et al. Distributing a metabolic pathway among a microbial consortium enhances production of natural products[J].Nat Biotechnol, 2015,33(4):377.
[7] Petersen M, Seitz HU. Reconstitution of cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove[J].Biochem J, 1988,252(2):537.
[8] Hayashi H, Hanaoka S, Tanaka S, et al. Glycyrrhetinic acid 24-hydroxylase activity in microsomes of cultured licorice cells[J].Phytochemistry, 1993,34(5):1303.
[9] 汪思远, 蒋世翠, 王康宇, 等. 植物细胞色素P450的研究进展[J].吉林蔬菜, 2014, (4):41.
[10] Moses T, Pollier J, Almagro L, et al. Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum[J].PNAS, 2014,111(4):1634.
[11] Kunii M, Kitahama Y, Fukushima EO, et al. β-Amyrin oxidation by oat CYP51H10 expressed heterologously in yeast cells: the first example of CYP51-dependent metabolism other than the 14-demethylation of sterol precursors[J].Biol Pharm Bull,2012,35(5):801.
[12] Wen L, Yun X, Zheng X, et al. Transcriptomic Comparison Reveals Candidate Genes for Triterpenoid Biosynthesis in Two Closely Related Ilex Species[J].Front Plant Sci, 2017,8:634.
[13] Urban P, Mignotte C, Kazmaier M, et al. Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5[J].J Biol Chem,1997,272(31):19176.
[14] Tamura K, Dudley J, Nei M, et al. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[J].Mol Biol Evol, 2007,24(8):1596.
[15] Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap[J].Evolution, 1985,39(4):783.
[16] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[J].Nucleic Acids Res, 1994,22(22):4673.
[17] Carelli M, Biazzi E, Panara F, et al.Medicago truncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins[J].Plant Cell, 2011,23(8):3070.
[18] Chapple C. Molecular-genetic analysis of plant cytochrome p450-dependent monooxygenases[J].Annu Rev Plant Physiol Plant Mol Biol, 1998,49(1):311.
[19] Rupasinghe S, Schuler MA. Homology modeling of plant cytochrome P450s[J].Phytochem Rev,2006,5(2-3):473.
[20] 贺丽虹, 赵淑娟, 胡之璧. 植物细胞色素P450基因与功能研究进展[J].药物生物技术, 2008,15(2):142.
[21] Nelson DR, Schuler MA, Paquette SM, et al. Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot[J].Plant Physiol, 2004,135(2):756.
[22] Sakai JI, Ando M. A Taxatetraene from Microbial Transformation of Sinenxan A[J].Chin Chem Lett,2005,16(6):22.
[23] Han JY, Hwang HS, Choi SW, et al. Cytochrome P450 CYP716A53v2 catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in Panax ginseng[J].Plant Cell Physiol, 2012,53(9):1535.