Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua

Abstract

Isochorismate synthase (ICS) is a crucial enzyme in the salicylic acid (SA) synthesis pathway. The full-length complementary DNA (cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site (MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction (qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography (HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.

摘要

目 的

研究青蒿异分支酸合酶的表达模式, 评价其对青蒿素含量的影响。

创新点

该研究首次克隆了青蒿异分支酸合酶基因(AaICS1), 并发现AaICS1 影响青蒿素的合成, 为更有效地开发利用青蒿提供了新思路。

方 法

根据青蒿转录组数据, 利用聚合酶链式反应(PCR)克隆AaICS1 基因和启动子, 并进行多重序列分析和启动子作用元件预测。通过实时定量PCR( qRT-PCR ) 对AaICS1 进行表达分析, 用Southern 杂交分析AaICS1 的拷贝数。构建AaICS1过表达载体和干扰表达载体, 转化青蒿获得转基因植株, 用高效液相色谱法(HPLC)分析青蒿素含量。

结 论

AaICS1 含一个总长为1710 bp 的完整阅读框, 编码570 个氨基酸, 与其它植物的ICS 基因具有较高的相似性。Southern 杂交结果表明AaICS1 为单拷贝(图4), qRT-PCR 结果显示该基因能够响应伤害、干旱、盐胁迫和水杨酸的处理, 处理后基因表达量提高(图6), 和启动子作用元件预测相符。qRT-PCR 结果显示过表达转基因青蒿中AaICS1 表达量提高, 干扰转基因青蒿中该基因表达量降低(图7)。HPLC 显示过表达AaICS1 转基因植株中青蒿素含量提升, 最高可达对照的1.9倍(图8)。