Molecular cloning, characterization, and promoter analysis of the isochorismate synthase （AalCS1） gene from Artemisia annua  

青蒿异分支酸合酶基因的克隆及功能分析（英文）

作　　者：Lu-yao WANG Ying ZHANG Xue-qing FU Ting-ting ZHANG Jia-wei MA Li-da ZHANG Hong-mei QIAN Ke-xuan TANG Shan LI Jing-ya ZHAO 

机构地区：[1]Plant Bioteehnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China [2]School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China

出　　处：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2017年第8期662-673,共12页浙江大学学报（英文版）B辑（生物医学与生物技术）

基　　金：supported by the National High-Tech R&D Program(863)of China(No.22011AA100605)

摘　　要：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.Isochorismate synthase （ICS） is a crucial enzyme in the salicylic acid （SA） synthesis pathway. Thefull-length complementary DNA （cDNA） sequence of the ICS gene was isolated from Artemisia annua L. The gene,named AalCS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinfor-matics and comparative study revealed that the polypeptide protein of AalCS1 had high homology with ICSs from otherplant species. Southern blot analysis suggested that AalCS1 might be a single-copy gene. Analysis of the 1470-bppromoter of AalCS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site（MBS）, and TCA-elements. An analysis of AalCS1 transcript levels in multifarious tissues of A. annua using quantita-tive real-time polymerase chain reaction （qRT-PCR） showed that old leaves had the highest transcription levels.AalCS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by thepresence of the predicted cis-acting elements in the promoter region of AalCSI. Overexpressing transgenic plants andRNA interference transgenic lines of AalCSl were generated and their expression was compared. High-performanceliquid chromatography （HPLC） results from leaf tissue of transgenic A. annua showed an increase in artemisinincontent in the overexpressing plants. These results confirm that AalCS1 is involved in the isochorismate pathway.

关 键 词：Salicylic acid ARTEMISIA annua L. Quantitative real-time POLYMERASE CHAIN reaction (qRT-PCR) Isochorismate SYNTHASE 

分 类 号：Q943.2[生物学—植物学]