Molecular Cloning And Functional Characterization Of Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate Reductase Gene From Artemisia Annua L

Artemisinin, a sesquiterpene endoperoxide lactone extracted from the traditional Chinese medicinal plant of Artemisia annua L., is the first choice for malaria treatment, especially for chloroquine-resistant and cerebral malarial. Artemisinin has advantages of good solubility in the blood, high efficacy and no serious side effects. Artemisinin-based combination therapies(ACTs) have been recommended as the best treatment method of malaria by the Word Health Organization(WHO). However, the content of artemisinin is very low in the natural Artemisia annua L., ranging from 0.01 to 0.8% of the dry weight. Additionally, the chemical synthesis of artemisinin could not be put into production due to complex chemical synthesis route, high cost, low production and strong toxicity. Therefore, the yield of artemisinin, reduce the artemisinin drugs cost, market demand is very necessary. Therefore, it is necessary to improve the production of artemisinin and reduce the cost of artemisinin drugs for alleviating the market demand. Recently, the research of artemisinin biosynthetic pathway and the key enzyme genes, genetic engineering of artemisinin biosynthetic pathway is considered to be one of most effective ways to improve the artemisinin content.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. IPP and DMAPP is the precursor of the terpenoid biosynthesis. HDR affect artemisinin biosynthesis by the formation of IPP and DMAPP.In this study, we have cloned Aa HDR2 gene from Artemisia annua L. and the bioinformatics analysis revealed that Aa HDR2 amino acid sequence with others have high similarity: the consistency with the HDR1 of Artemisia annua L. is 81.8%, the consistency with the HDR of Tanacetum parthenium is 84.6%, the consistency with the HDR of Vitis vinifera is 78.5%. The results above indicated that Aa HDR2 belonged to the HDR family. And the functional complementation assay showed that Aa HDR2 did have the enzymatic function of HDR, using E. coli mutant MG1655ara<>HDR as host cell. According to gene expression analysis based on q PCR, it was found that Aa HDR2 had much higher expression level in glandular secretory trichomes(GST) than that in roots, stems, leaves and flowers. Further, the plant hormones such as Me JA, ABA and GA3 respectively up-regulated the expression level of Aa HDR2 significantly. The subcellular localization assay showed that Aa HDR2 fused with GFP at its N-terminal specifically targeted in chloroplasts. We have constructed the plant expression vector of p HB- Aa HDR2 and transferred into Arabidopsis thaliana. We collected of the seeds Arabidopsis thaliana and germinated into seedlings on MS medium with 250 mg.l-1 cefotaxime and 50 mg.l-1 hygromycin at 25℃ in the cycle of 16 h light and 8 h dark, growing 2 weeks. Genomic PCR was used to confirm the authentic transgenic plants of Arabidopsis thaliana through detecting the hygromycin-resistant gene(Hygr) and Aa HDR2 gene. To detect the content of chlorophyll a, chlorophyll b and carotenoids in transgenic Arabidopsis thaliana. The result indicated that the content of chlorophyll a in control was 0.500±0.010mg/g, in the transgenic lines were 0.567±0.006mg/g 、0.613±0.024mg/g、0.607±0.002mg/g respectively and compared with the control were increased at 13.42%、22.58%、21.29%, the content of chlorophyll b in control was 0.207±0.003mg/g, in the transgenic lines were 0.222±0.007mg/g、0.239±0.002mg/g、0.235±0.004mg/g respectively and compared with the control were increased at 7.10%、15.16%、13.50%, the content of carotenoids was 0.095±0.002mg/g, in the transgenic lines were 0.109±0.002mg/g 、 0.116±0.002mg/g 、 0.120±0.001mg/g respectively, and compared with the control were increased at 14.92%、22.75%、27.42%. The content of chlorophyll a, chlorophyll b and carotenoids in transgenic lines were significantly increased(p<0.01) compared with the control, that because of the Aa HDR2-overexpressing improve the biosynthesis of isoprenoids including chlorophyll a, chlorophyll b and carotenoids. The result indicated that Aa HDR2 have an important role in terpenoid biosynthesis, and the result can provide experimental basis for Aa HDR2-overexpressing in Artemisia annua L.. In summary, molecular cloning and functional characterization of Aa HDR2 in favour of illuminating the molecular regulation mechanism of artemisinin biosynthesis and providing a possible candidate gene for improving artemisinin content by genetic engineering in Artemisia annua L..