穿心莲中1个新NADPH-细胞色素P450还原酶的克隆及功能鉴定
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摘要
穿心莲内酯为传统中药穿心莲中的主要有效成分,具有多种药理活性,广泛运用于临床,但其生物合成途径尚未被解析。细胞色素P450还原酶为CYP450提供电子,在CYP450的催化过程中起重要作用。该研究通过同源比对筛选并克隆了穿心莲CPR的编码序列,命名为ApCPR4。原核表达ApCPR4蛋白,分离纯化后进行体外酶活鉴定,发现ApCPR4能够以NADPH依赖性方式还原细胞色素c和铁氰化物。为了验证其体内功能,将ApCPR4与CYP76 AH1共转化入酵母工程菌,结果表明,ApCPR4在酵母体内能够协助CYP76AH1催化生成铁锈醇。实时定量PCR结果显示,在茉莉酸甲酯(Me JA)处理的叶片中,ApCPR4的表达量逐渐增加。该表达模式与穿心莲内酯受Me JA诱导积累的趋势一致,推测ApCPR4与穿心莲内酯的生物合成相关。
Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata,with a variety of pharmacological activity,widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study,the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment,named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification,the enzyme activity was identified in vitro. The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its in vivo function,ApCPR4 and CYP76 AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76 AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate( Me JA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by Me JA,suggesting that ApCPR4 was associated with biosynthesis of andrographolide.
Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata,with a variety of pharmacological activity,widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study,the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment,named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification,the enzyme activity was identified in vitro. The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its in vivo function,ApCPR4 and CYP76 AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76 AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate( Me JA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by Me JA,suggesting that ApCPR4 was associated with biosynthesis of andrographolide.
引文
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[18]苏炜华,黄珑,黄宁,等.甘蔗细胞色素P450还原酶基因的RT-PCR扩增与表达分析[J].应用与环境生物学报,2016,22(2):173.
[19]刘玉忠,申业,荣齐仙,等.丹参转录因子Sm WRKY1蛋白的表达和纯化条件优化研究[J].中国中药杂志,2014,39(7):1214.
[20]Lin H,Wang J,Qi M,et al.Molecular cloning and functional characterization of multiple NADPH-cytochrome P450 reductases from Andrographis parriculata[J].Int J Biol Macromol,2017,102:208.
[21]阮仁余,孔建强,郑晓东,等.中国红豆杉细胞色素P450还原酶的基因克隆、表达与活性分析[J].遗传,2010,32(11):1187.
[22]Liu D,Zhou X,Li M,et al.Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm,Helicoverpa armigera[J].Gene,2014,545(2):262.
[23]Zhou Y J,Gao W,Rong Q,et al.Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production[J].J Am Chem Soc,2012,134(6):3234.
[24]Sharma S N,Jha Z,Sinha R K,et al.Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata[J].Physiol Plant,2015,153(2):221.
[2]靳鑫,时圣明,张东方,等.穿心莲化学成分的研究(Ⅱ)[J].中草药,2014,45(2):164.
[3]吴亦晴,虞劲祥,程志红.二萜类化合物微生物转化研究进展[J].中国生化药物杂志,2016,36(3):9.
[4]褚晨亮.穿心莲药材的化学成分和质量控制研究[D].广州:广东药学院,2013.
[5]陈娟,谷巍,段金廒,等.不同生长期穿心莲活性成分及关键酶基因差异表达研究[J].中草药,2014,45(21):3149.
[6]陈咏梅,张韻慧,张莉华,等.穿心莲抗肿瘤转移化学成分研究[J].湖北中医药大学学报,2016,18(6):46.
[7]Misra R C,Garg A,Roy S,et al.Involvement of an ent-copalyldiphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata[J].Plant Sci,2015,240:50.
[8]Shen Q,Li Li,Jiang Y,et al.Functional characterization of entcopalyldiphosphate synthase from Andrographis paniculata with putative involvement in andrographolides biosynthesis[J].Biotechnol Lett,2016,38(1):131.
[9]Srivastava N,Akhila A.Biosynthesis of andrographolide in Andrographis paniculata[J].Phytochemistry,2010,71(11):1298.
[10]Guengerich F P,Martin M V,Sohl C D,et al.Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase[J].Nat Protoc,2009,4(9):1245.
[11]程丽,李宜奎,李晓丹,等.植物CPR基因密码子偏好性及聚类分析[J].分子植物育种,2017,15(5):1672.
[12]Wang M,Roberts D L,Paschke R,et al.Three-dimensional structure of NADPH-cytochrome P450 reductase:prototype for FMNand FAD-containing enzymes[J].Proc Natl Acad Sci USA,1997,94(16):8411.
[13]Jensen K,Moller B L.Plant NADPH-cytochrome P450 oxidoreductases[J].Phytochemistry,2010,71(2/3):132.
[14]Elmore C L,Porter T D.Modification of the nucleotide cofactorbinding site of cytochrome P-450 reductase to enhance turnover with NADH in vivo[J].J Biol Chem,2002,277(50):48960.
[15]Zhao C,Tang T,Feng X,et al.Cloning and characterisation of NADPH-dependent cytochrome P450 reductase gene in the cotton bollworm,Helicoverpa armigera[J].Pest Manage Sci,2014,70(1):130.
[16]何海,郭继云,舒少华,等.茯苓细胞色素P450还原酶基因的克隆与生物信息学分析[J].中草药,2016,47(16):2909.
[17]凌淑萍,张会敏,苏闪闪,等.洋甘菊细胞色素P450还原酶基因(CPR)的分子克隆及表达分析[J].农业生物技术学报,2014,22(5):580.
[18]苏炜华,黄珑,黄宁,等.甘蔗细胞色素P450还原酶基因的RT-PCR扩增与表达分析[J].应用与环境生物学报,2016,22(2):173.
[19]刘玉忠,申业,荣齐仙,等.丹参转录因子Sm WRKY1蛋白的表达和纯化条件优化研究[J].中国中药杂志,2014,39(7):1214.
[20]Lin H,Wang J,Qi M,et al.Molecular cloning and functional characterization of multiple NADPH-cytochrome P450 reductases from Andrographis parriculata[J].Int J Biol Macromol,2017,102:208.
[21]阮仁余,孔建强,郑晓东,等.中国红豆杉细胞色素P450还原酶的基因克隆、表达与活性分析[J].遗传,2010,32(11):1187.
[22]Liu D,Zhou X,Li M,et al.Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm,Helicoverpa armigera[J].Gene,2014,545(2):262.
[23]Zhou Y J,Gao W,Rong Q,et al.Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production[J].J Am Chem Soc,2012,134(6):3234.
[24]Sharma S N,Jha Z,Sinha R K,et al.Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata[J].Physiol Plant,2015,153(2):221.