Wang Jian, Qi Meng-Die, Guo Juan, Shen Ye, Lin Hui-Xin, Huang Lu-Qi
College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230038, China.
State Key Laboratory of Dao-di Herbs Breeding Base, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2017 Mar;42(5):890-895. doi: 10.19540/j.cnki.cjcmm.20170217.005.
Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis.
穿心莲在中国长期被广泛用作草药，穿心莲内酯是其主要药用成分。为了研究穿心莲内酯潜在的生物合成机制，对经茉莉酸甲酯（MeJA）处理的穿心莲叶片进行了RNA测序。在穿心莲转录组数据中，NAC转录因子家族的一个成员（ApNAC1）的表达模式与穿心莲内酯积累相匹配。通过RT-PCR克隆了ApNAC1的编码序列，GenBank登录号为KY196416。生物信息学分析表明，该基因编码一个由323个氨基酸组成的肽段，预测相对分子量为35.9 kDa，等电点为6.14。为了确定亚细胞定位，将ApNAC1-GFP在穿心莲原生质体中瞬时表达。结果表明，ApNAC1是一种定位于细胞核的蛋白质。实时定量PCR分析表明，ApNAC1基因主要在叶片中表达。与对照样品相比，经茉莉酸甲酯处理后其表达丰度急剧增加。基于其表达模式，ApNAC1基因可能参与穿心莲内酯的生物合成。ApNAC1在大肠杆菌中进行了异源表达，并用Ni-NTA琼脂糖纯化了重组蛋白。进一步的研究将有助于我们了解ApNAC1在穿心莲内酯生物合成中的功能。
