摘要
从穿心莲叶片中克隆了1个NAC家族的转录因子,命名为ApNAC1,Gen Bank注册号为KY196416。生物信息学分析表明该基因的完整编码区包含972 bp,编码1个323个氨基酸的多肽,预测蛋白相对分子质量为35.9 k Da,等电点为6.14。原生质体瞬时表达研究证实了ApNAC1-GFP融合蛋白特异定位在穿心莲叶肉细胞的细胞核中,是一个核定位蛋白。实时定量PCR结果表明ApNAC1基因表达与穿心莲内酯的积累模式一致:主要在穿心莲的叶片里表达,而且茉莉酸甲酯处理能急剧上调其表达水平。ApNAC1基因在大肠杆菌中进行异源表达,并且被成功纯化。该研究为进一步探索ApNAC1蛋白参与穿心莲内酯生物合成的研究奠定了基础。
Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medic- inal 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 acces- sion number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predic- ted relative molecular weight of 35.9 kDa and isoelectric point of 6. 14. To confirm the subcellular localization, ApNAC1-GFP was tran- siently 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 an- drographolide 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.
出处
《中国中药杂志》
CAS
CSCD
北大核心
2017年第5期890-895,共6页
China Journal of Chinese Materia Medica
基金
国家杰出青年科学基金项目(81325023)
中央本级重大增减支项目(20160302)
关键词
穿心莲
NAC转录因子
亚细胞定位
表达模式
原核表达
Andrographis paniculata
NAC transcription factor
subcellular localization
expression pattern
heterologous expression
