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， Beijing 100102， China.
State Key Laboratory Breeding Base of Dao-di Herbs， National Resource Center for Chinese Materia Medica， China Academy of Chinese Medical Sciences， Beijing 100700， China.
Zhongguo Zhong Yao Za Zhi. 2018 Jan;43(2):309-315. doi: 10.19540/j.cnki.cjcmm.20171030.011.
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 . The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 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 (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.
穿心莲内酯是中药穿心莲中的主要活性成分，具有多种药理活性，广泛应用于临床实践。然而，其生物合成途径尚未明确。细胞色素P450还原酶为CYP450提供电子，在CYP450催化过程中起重要作用。本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。通过原核表达获得了ApCPR4蛋白。经过分离纯化后，对其酶活性进行了鉴定。结果表明，ApCPR4能够以NADPH依赖的方式还原细胞色素c和铁氰化物。为了验证其功能，将ApCPR4和CYP76AH1共转化到酵母工程菌中。结果表明，ApCPR4能够帮助CYP76AH1在酵母中催化锈醇的形成。实时定量PCR结果表明，在茉莉酸甲酯（MeJA）处理的叶片中，ApCPR4的表达逐渐增加。该表达模式与MeJA诱导和积累穿心莲内酯的趋势一致，表明ApCPR4与穿心莲内酯的生物合成有关。
