Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Publication year range
1.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-690502

ABSTRACT

Artemisinin is a preferred medicine in the treatment of malaria. In this study, AaCMK, a key gene involved in the upstream pathway of artemisinin biosynthesis, was cloned and characterized from Artemisia annua for the first time. The full-length cDNA of AaCMK was 1 462 bp and contained an ORF of 1 197 bp that encoded a 399-anomo-acid polypeptide. Tissue expression pattern analysis showed that AaCMK was expressed in leaves, flowers, roots and stems, but with higher expression level in glandular secretory trichomes. In addition, the expression of AaCMK was markedly increased after MeJA treatment. Subcellular localization showed that the protein encoded by AaCMK was localized in chloroplast. Overexpression of AaCMK in Arabidopsis increased the contents of chlorophyll a, chlorophyll b and carotenoids. These results suggest that AaCMK plays an important role in the biosynthesis of terpenoids in A. annua and this research provids a candidate gene that could be used for engineering the artemisinin biosynthesis.

2.
Acta Pharmaceutica Sinica ; (12): 1791-2016.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-779373

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 MG1655araHDR 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.

3.
Acta Pharmaceutica Sinica ; (12): 1334-2016.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-779316

ABSTRACT

The plastidial methylerythritol phosphate (MEP) pathway provides 5-carbon precursors to the biosynthesis of isoprenoid (including artemisinin). 2-C-Methyl-D-erythritol-4-phosphate cytidylyltransferase (MCT) is the third enzyme of the MEP pathway, which catalyzes 2-C-methyl-D-erythritol-4-phosphate to form 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol. The full-length MCT cDNA sequence (AaMCT) was cloned and characterized for the first time from Artemisia annua L. Analysis of tissue expression pattern revealed that AaMCT was highly expressed in glandular secretory trichome and poorly expressed in leaf, flower, root and stem. AaMCT was found to be a methyl jasmonate (MeJA)-induced genes, the expression of AaMCT was significantly increased after MeJA treatment. Subcellular localization indicated that the GFP protein fused with AaMCT was targeted specifically in chloroplasts. The transgenic plants of Arabidopsis thaliana with AaMCT overexpression exhibited a significantly increase in the content of chlorophyll a, chlorophyll b and carotenoids, demonstrating that AaMCT kinase plays an influential role in isoprenoid biosynthesis.

4.
Acta Pharmaceutica Sinica ; (12): 1346-1352, 2014.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-299128

ABSTRACT

Artemisnin is a novel sesquiterpene lactone with an internal peroxide bridge structure, which is extracted from traditional Chinese herb Artemisia annua L. (Qinghao). Recommended by World Health Organization, artemisinin is the first-line drug in the treatment of encephalic and chloroquine-resistant malaria. In the present study, transgenic A. annua plants were developed by overexpressing the key enzymes involved in the biosynthetic pathway of artemisinin. Based on Agrobacterium-mediated transformation methods, transgenic plants of A. annua with overexpression of both HDR and ADS were obtained through hygromycin screening. The genomic PCR analysis confirmed six transgenic lines in which both HDR and ADS were integrated into genome. The gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had higher expression levels of HDR and ADS than the non-transgenic control (except ah3 in which the expression level of ADS showed no significant difference compared with control); and the HPLC analysis of artemisinin demonstrated that transgenic A. annua plants produced artemisinin at significantly higher level than non-transgenic plants. Especially, the highest content of artemisinin was found in transgenic line ah70, in which the artemisinin content was 3.48 times compared with that in non-transgenic lines. In summary, overexpression of HDR and ADS facilitated artemisinin biosynthesis and this method could be applied to develop transgenic plants of A. annua with higher yield of artemisinin.


Subject(s)
Artemisia annua , Genetics , Metabolism , Artemisinins , Metabolism , Biosynthetic Pathways , Drugs, Chinese Herbal , Mixed Function Oxygenases , Genetics , Oxidoreductases , Genetics , Plant Proteins , Genetics , Plants, Genetically Modified , Genetics , Metabolism , Plants, Medicinal , Genetics , Metabolism
5.
Mol Phylogenet Evol ; 49(3): 702-14, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18849001

ABSTRACT

The genus Croomia (Stemonaceae) comprises three herbaceous perennial species that are distributed in temperate-deciduous forests in Southeastern North America (C.pauciflora) and East Asia (C. japonica, C. heterosepala). The two Asian species have abutting ranges in South Japan, but C. japonica also occurs disjunctively on the adjacent Asiatic mainland in East China. In our phylogenetic analysis of Croomia, based on chloroplast (cp) DNA sequence variation of the trnL-F region, and rooted with Stemona spp., the two Asian species are identified as sister that likely diverged in the Mid-to-Late Pleistocene (0.84-0.13 mya), whereas the divergence of C. pauciflora dates back to the Late Plio-/Pleistocene (<2.6 mya). Phylogeographical analysis of the two East Asian species detected seven cpDNA (trnL-F) haplotypes across 16 populations surveyed, and all of those were fixed for a particular cpDNA haplotype (H(E)=0.0, G(ST)=1). A survey of inter-simple sequence repeats (ISSRs) markers also detected remarkably low levels of within-population diversity (C. japonica: H(E)=0.085; C. heterosepala: H(E)=0.125), and high levels of inter-population differentiation (C. japonica: Phi(ST)=0.736; C. heterosepala: Phi(ST)=0.550), at least partly due to pronounced regional genetic substructure within both species. Non-overlapping distributions of cpDNA haplotypes and strong genetic (cpDNA/ISSR) differentiation among populations and/or regions accord with findings of a nested clade analysis, which inferred allopatric fragmentation as the major process influencing the spatial haplotype distribution of the two species. Based on mismatch distribution analysis and neutrality tests, we do not find evidence of population expansion in both species. Overall, we conclude that components of temperate-deciduous forest types in South Japan and East China are particularly sensitive to range fragmentation, isolation, and enhanced (incipient) species formation through climate-induced expansions of other forest types over glacial and interglacial periods of the (Late) Quaternary.


Subject(s)
DNA Fingerprinting , DNA, Chloroplast/genetics , Evolution, Molecular , Phylogeny , Stemonaceae/genetics , China , DNA, Plant/genetics , Genetic Markers , Genetics, Population , Geography , Haplotypes , Japan , Likelihood Functions , Models, Genetic , Polymorphism, Genetic , Repetitive Sequences, Nucleic Acid , Stemonaceae/classification
SELECTION OF CITATIONS
SEARCH DETAIL
...