闫静 向贵生 冯垒 杨梅 郭敏 张广辉

摘  要：草酰輔酶A合成酶参与了三七主要止血活性成分三七素的生物合成，研究三七草酰辅酶A合成酶基因（PnAAE41）的分子特征及表达特征，从而完善三七素生物合成途径以及成分积累调控机制等方面的研究。以三七为实验材料，在前期转录组研究基础上，设计特异性引物，通过RT-PCR扩增获得1个三七AAE基因的cDNA全长。采用生物信息学方法分析其分子特性，通过qRT-PCR技术分析PnAAE41基因的时空表达特异性。通过异源表达探索进行PnAAE41最佳蛋白最佳的诱导条件的探索，随后以颜色反应作为定性研究的基础。序列分析结果表明，PnAAE41的开放阅读框长度为1572 bp，编码523个氨基酸，登录号为MW242343。系统进化分析显示，PnAAE41蛋白与拟南芥AtAAE蛋白亲缘关系最近。预测PnAAE41蛋白主要定位于细胞质膜上，不含信号肽序列，二级结构中α-螺旋和无规则卷曲占大部分比例。qRT-PCR分析表明，PnAAE41基因主要在根和茎中优势表达。异源表达最佳诱导条件为0.5 mmol/L异丙基-β-D-硫代半乳糖苷（IPTG），16 ℃诱导9 h，该蛋白对草酸具有催化作用。本研究结果为进一步深入研究AAE在三七素生物合成途径中的功能提供理论基础。

关键词：三七;草酰辅酶A合成酶;生物信息学分析;时空表达;原核表达

中图分类号：S567.23+6      文献标识码：A

Clone and Prokaryotic Expression Analysis of Acyl-activating Enzyme Gene from Panax notoginseng

YAN Jing1，2，3， XIANG Guisheng1，2， FENG Lei1，2，3， YANG Mei3， GUO Min3， ZHANG Guanghui1，2*

1. National & Local Joint Engineering Research Center on Germplasms Utilization & Innovation of Chinese Materials in Southwest/ ， Yunnan Agricultural University， Kunming， Yunnan 650201， China; 2. Yunnan Provincial Key Laboratory of Medicinal Plant Bio?logy， Yunnan Agricultural University， Kunming， Yunnan 650201， China; 3. College of Agriculture and Biotechnology， Yunnan Agricultural University， Kunming， Yunnan 650201， China

Abstract： Acyl-activating enzyme （PnAAE41） of Panax notoginseng is involved in the biosynthesis of dencichine，the main hemostatic active ingredient. The molecular characteristics and expression characteristics of Acyl-activating enzyme was explored to provide a basic theory for further research on the biosynthesis pathway of dencichine and the regulation mechanism of component accumulation. The full length of PnAAE41 gene was obtained by RT-PCR. The molecular properties was analyzed by bioinformatics methods. The spatio-temporal expression specificity of PnAAE41 gene was determined by real-time quantitative PCR. The heterologous expression was used to explore the optimal induction condition. The color reaction was used as the basis for qualitative research. The sequence analysis results showed that PnAAE41 was 1572 bp， encoding 523 amino acids， and the login number was MW242343. Phylogenetic analysis showed that PnAAE41 was most closely related to Arabidopsis AtAAE. PnAAE41 was predicted to be mainly located on the plasma membrane， which did not contain signal peptide sequence， and its secondary structure was mainly composed of alpha helix and random coil. The optimal induction condition was 0.5 mmol/L IPTG and 16 ℃ induction for 9 h. The protein had a catalytic effect on oxalic acid. The study would provide a theoretical basis for the further research on the role of AAE in the biosynthesis of dencichine.

Keywords： Panax notoginseng; acyl-activating enzyme; bioinformatics analysis; spatio-temporal expression; prokaryotic expression

DOI： 10.3969/j.issn.1000-2561.2021.07.008

三七[Panax notoginseng （Burk） F. H. Chen]为五加科人参属多年生草本植物，以根及根茎入药，是我国重要的中药材大品种。三七具有散瘀止血、消肿定痛、防止脑缺血、止血、降脂和保护肝脏等功效，与人参共享“北参南七”的美誉。三七总皂苷广泛用于治疗冠心病、心绞痛、脑血管后遗症和高血压等。……