Overexpression Of Artemisinic Aldehyde Δ11(13) Reductase Gene Enhanced Artemisinin And Its Relative Metabolite Biosynthesis In Transgenic Artemisia Annua L.

Artemisinin, a sesquiterpene lactone extracted from the traditional Chinese medicinal herb of Artemisia annua L., is universally considered to be the most effective drug against malaria,especially for chloroquine-resistant and cerebral malarial. Artemisinin-based combination therapies (ACTs) have been recommended as the preferred treatment method of drug-resistant malaria by the Word Health Organization (WHO) for its high efficacy, fast action, and no serious side effects. However, the artemisinin content in the plants of Artemisia annua L. is very low, ranging from0.01to1%of dry weight and this has been strongly limited the artemisinin supply. As a result, the production of artemisinin can not meet the increasing demands. Additionally, artemisinin as a complex molecule can be chemically synthesized, but the cost of chemical synthesis was too high to afford, and also toxiferous with very limited production. Therefore, the plants of A. annua are the main source of artemisinin extraction. It is urgent to develop A. annua with higher content of artemisinin.The gene DBR2encoded a member of the enoate reductase family with similarity to plant12-oxophytodienoate reductases which was found to be reductase activity of artemisinic aldehyde△11(13) double bond to generate dihyroartemisinic aldehyde (precursor of dihydrartemisinic acid) and directed artemisinic aldehyde to artemisinin biosynthetic pathway. With a more in-depth study of artemisinin biosynthesis, it has been confirmed that enhancing the artemisinin content through transgenic technology is one of effective methods. Presently, the checkpoint genes involved in artemisinin biosynthetic pathway have been used to genetically modify artemisinin biosynthetic pathway in transgenic A. annua plants. However, there is no genetically modified evidence to confirm the role of DBR2in the artemisinin biosynthesis pathway. Therefor, in the present study, DBR2gene was used to genetically modify the biosynthetic pathway of artemisinin by the gene-overexpression strategy and attempted to describe effects of artemisinin and its derivatives by overexpression of DBR2gene.Artemisinic aldehydc△11(13) reductase gene (DBR2) from A. annua was cloned by PCR and used as target gene to construct the plant expression vector named as pCAMBIA1305-DBR2. Then, the plant expression vector was obtained by Agrohacterium tunefaciens strain LBA4404to generatc engineered bacteria which was used for genetic transformation of A. annua. Transformed explants were screened by8mg-L-1Hygromycin to get the hygromycin-resistant plantlets. Hygromycin-resistant regenerated plantlets, being to2-3cm long, were cut and transferred into rooting MS medium with0.05mg-L-1NAA,200mg-L-1cefotaxime to get hygromycin-resistant plants. Genomic PCR was used to confirm the authentic transgenic plants of A. annua through detecting the hygromycin-resistant gene(Hygr) and DBR2gene. Besides, virDl gene was also detected through the PCR analysis to avoid the interference of Agrobacterium to PCR results. As a result, the fragments of Hygr and DBR2gene were amplified from transgenic A. annua with overexpression of DBR2, but the fragments of virDl gene did not occur in the transgenic A. annua with overexpression of DBR2. Finally, Seven independent transgenic plants with overexpression of DBR2gene were obtained and named as D1、D2、D3、D4、D5、D6、D7, respectively. Simultaneously, qPCR was used to analyze the expression of the DBR2gene in plants of A. annua at the transcriptional level and RPII gene was used as the reference gene. The result showed that the gene expression level of DBR2was dramatically upregulated in all the transgenic plants. The HPLC analysis of target product indicated that artemisinin contents were improved in all transgenic lines, which were1.92±0.69mg·g-1、1.50±0.20mg-g-1、1.73±0.08mg·g-1、91+0.21mg·g-1、1.72±0.30mg·g-1、1.95±0.24mg·g-1、2.14±0.20mg·g-1,respectively. D7transgenic line, with a highest content of2.14±0.20mg·g-1,is about2.26times compared with the content of non-transgenic plants (0.94±0.35mg·g-1, DW). Dihydroartemisinic acid as the direct precursor for artemisinin was also detected. The HPLC analysis results demonstrated that all the transgenic lines with DBR2overexpression had higher contents of dihydroartemisinic acid, which were1.23±0.20mg·g-1、1.46±0.36mg·g-1、1.39±0.39mg·g-1、1.09±0.27mg·g-1、1.51±0.38mg·g-1、50±0.16mg·g-1、1.93±0.19mg·g-1, respectively. The highest content of1.93±0.19mg·g-1is about2.22folds of nontransgenic control(0.87±0.03mg·g-1, DW). Interestingly, it was also found that the contents of arteannuin B and its direct precursor artemisinic acid, in the branch pathway competing against artemisinin biosynthesis, were also enhanced in all the seven DBR2-overexpressed transgenic lines. Especially, the contents of artemisinic acid were dramatically increased, varying from5.48to9.06times compared with control. In all, it can be concluded that DBR2was a useful structural gene to develop genetically modified A. annua plants with the higher yield of artemisinin and its relative metabolite, which can be provided as the first-hand material for the industrial production of artemisimn.