Molecular cloning and functional characterization of the gene encoding hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase gene from Artemisia annua L.
10.16438/j.0513-4870.2016-0246
- VernacularTitle:黄花蒿羟甲基丁烯基-4-磷酸还原酶基因克隆与功能研究
- Author:
Fang CAO
1
;
Jing XIA
1
;
Yu-pei CHEN
1
;
Man ZHANG
1
;
Li-en XIANG
1
;
Jun-lan ZENG
1
;
Min CHEN
2
;
Xiao-zhong LAN
3
;
Zhi-hua LIAO
1
Author Information
1. Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Life Sciences, Southwest University, Chongqing 400715, China
2. College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
3. Medicinal Plant Research Centre, Tibet Agricultural and Animal Husbandry College, Nyingchi 860000, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
Artemisia annua L.;
hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase;
functional complement;
gene expression;
subcellular localization;
overexpression
- From:
Acta Pharmaceutica Sinica
2016;51(11):1791-
- CountryChina
- Language:Chinese
-
Abstract:
Artemisinin is the first choice for malaria treatment. The plastidial MEP pathway provides 5-carbon precursors (IPP and its isomer DMAPP) for the biosynthesis of isoprenoid (including artemisinin). Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) is the last enzyme involved in the MEP pathway, which catalyzes HMBPP to form IPP and DMAPP. In this study, we isolated the full-length cDNA of HDR from Artemisia annua L. (AaHDR2) and performed functional analysis. According to gene expression analysis of AaHDR2 (GenBank:KX058541) and AaHDR1 reported ever (GenBank:ADC84348.1) by qPCR, we found that AaHDR1 and AaHDR2 had much higher expression level in trichomes than that in roots, stems, leaves and flowers. AaHDR2 had much higher expression level in flowers than that in leaves. Further, the plant hormones such as MeJA and ABA respectively up-regulated the expression level of AaHDR1 and AaHDR2 significantly, but GA3 up-regulated the expression level of AaHDR2 only. The gene expression analysis of AaHDR1 and AaHDR2 showed that AaHDR2 had a greater contribution than AaHDR1 to isoprenoid biosynthesis (including artemisinin). We used AaHDR2 for the following experiments. Bioinformatic analysis indicated that AaHDR2 belonged to the HDR family and the functional complementation assay showed that AaHDR2 did have the enzymatic function of HDR, using E. coli mutant MG1655ara<>HDR as host cell. The subcellular localization assay showed that AaHDR2 fused with GFP at its N-terminal specifically targeted in chloroplasts. Finally, AaHDR2 was overexpressed in Arabidopsis thaliana. The AaHDR2-overexpressing plants produced the isoprenoids including chlorophyll a, chlorophyll b and carotenoids at significantly higher levels than the wild-type Arabidopsis plants. In summary, AaHDR2 might be a candidate gene for genetic improvement of the isoprenoid biosynthesis.
