中国中药杂志

2018, v.43(02) 309-315

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穿心莲中1个新NADPH-细胞色素P450还原酶的克隆及功能鉴定
Cloning and functional identification of a new NADPH-cytochrome P450 reductase in Andrographis paniculata

齐梦蝶;王健;马晓晶;张权;王方方;康莹;林慧馨;刘勇;
QI Meng-die;WANG Jian;MA Xiao-jing;ZHANG Quan;WANG Fang-fang;KANG Ying;LIN Hui-xin;LIU Yong;Beijing University of Chinese Medicine;State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

摘要(Abstract):

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.

关键词(KeyWords): 穿心莲内酯;生物合成途径;细胞色素P450还原酶;原核表达;功能鉴定
andrographolide;biosynthetic pathway;cytochrome P450 reductase;prokaryotic expression;functional identification

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(ZZ2016026)

作者(Author): 齐梦蝶;王健;马晓晶;张权;王方方;康莹;林慧馨;刘勇;
QI Meng-die;WANG Jian;MA Xiao-jing;ZHANG Quan;WANG Fang-fang;KANG Ying;LIN Hui-xin;LIU Yong;Beijing University of Chinese Medicine;State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;

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DOI: 10.19540/j.cnki.cjcmm.20171030.011

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