| Artemisia annua L., a traditional Chinese medicinal herb of the Asteraceae well recognized for its synthesis of artemisinin. Artemisinin was the key ingredient of front-line antimalarial drugs. Artemisinin-based combination therapies(ACTs) are the most useful treatment for the cerebral and chloroquine-resistant malaria recommended by the World Health Organization. Moreover, the semi-synthetic derivatives of artemisinin have great medical value in virus, cancer and autoimmune disease. However, the content of artemisinin in the plants is very low and chemical synthesis is difficult. Therefore, it is meaningful to clone the gene on artemisinin precursor biosynthetic pathway and provide candidate genes for artemisinin metabolic engineering.The plastidial methylerythritol phosphate(MEP) pathway provides 5-carbon precursors, isopentenyl diphosphate(IPP), and its allyl isomer dimethylallyl diphosphate(DMAPP), for the biosynthesis of isoprenoid(including Artemisinin). 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase(MCT), 4-(cytidine 5’-diphospho)-2-C-methylery-thritol kinase(CMK) and 2-C-methylerythritol-2,4-cyclodiphosphate synthase(MCS) is the third, the fourth and the fifth enzyme of the MEP pathway, respectively. MCT catalyzes 2-C-methyl-D-erythritol-4-phosphate(MEP) to form 4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol(CDP-ME). CMK catalyzes CDP-ME to form 4-( cytidine-5′-diphospho)-2-C-methyl-D-erythritol-2-phosphate(CDP-ME-2P). MCS catalyzes CDP-ME-2P to form 2-C-methylerythritol-2,4-cyclodiphosphate(ME-c PP).The full-length MCT, CMK, MCS c DNA sequence(Aa MCT, Aa CMK, Aa MCS) were cloned and characterized for the first time from Artemisia annua L. The full-length c DNA of Aa MCT(Gen Bank accession No. KU365210) was 1176 bp containing a 906 bp open reading frame(ORF) encoding a polypeptide of 302 amino acids. The full-length c DNA of Aa CMK(Gen Bank accession No. KT778821) was 1462 bp containing a 1197 bp open reading frame(ORF) encoding a polypeptide of 399 amino acids. The full-length c DNA of Aa MCS(Gen Bank accession No. KT725782) was 811 bp containing a 678 bp open reading frame(ORF) encoding a polypeptide of 226 amino acids. Tissue expression pattern analysis revealed that Aa MCT, Aa CMK, Aa MCS was highly expressed in glandular secretory trichome and poorly expressed in leaf, flower, root and stem. Aa MCT, Aa CMK, Aa MCS was found to be a methyl jasmonate(Me JA)-induced genes, the expression of Aa MCT, Aa CMK, Aa MCS was significantly increased after Me JA treatment(P<0.01). The expression of Aa MCT and Aa CMK was significantly increased after SA treatment(P<0.05). Subcellular localization indicated that the GFP protein fused respectively with Aa MCT, Aa CMK, Aa MCS was targeted specifically in chloroplasts. The Arabidopsis thaliana respectively overexpressed with Aa MCT, Aa CMK, Aa MCS showed significantly increase in the content of chlorophyll a, chlorophyll b and carotenoids(P<0.01). In the three transgenic lines with Aa MCT overexpression, the content of chlorophyll a respectively increased 22.09%, 16.60%, 21.05%, the content of chlorophyll b respectively increased 23.70%, 18.00%, 17.43% and the content of carotenoids respectively increased 18.60%, 11.53%, 21.45% compared with control group. In the three transgenic lines with Aa CMK overexpression, the content of chlorophyll a respectively increased 23.83%, 22.27%, 18.26%, the content of chlorophyll b respectively increased 27.21%, 22.79%, 18.38% and the content of carotenoids respectively increased 25.24%, 16.50%, 15.53% compared with control group. In the three transgenic lines with Aa MCS overexpression, the content of chlorophyll a respectively increased 16.04%, 12.69%, 21.38%, the content of chlorophyll b respectively increased 16.18%, 16.18%, 20.59% and the content of carotenoids respectively increased 11.65%, 10.68%, 16.50% compared with control group. In all, it can be concluded that Aa MCT, Aa CMK and Aa MCS plays an influential step in isoprenoid biosynthesis. |
PDF Full Text Request