Increasing Artemisinin Content In Artemisia Annua L. Through Genetic Engineering

Artemisinin, a sesquiterpene lactone endoperoxide derived from plant Artemisia annua L., is the most effective anti-malarial drug. Since the synthesis of artemisinin is very difficult, artemisinin is mainly extracted from the aerial part of A. annua plants. However, the low content of artemisinin in the plants hampers its commercialization. So, increasing artemisinin production is of great significance.In this study, to increase artemisinin production from two aspects was investigated. Firstly, using genetic engineering method, several key genes involved in the artemisinin biosynthetic pathway were overexpressed in transgenic A. annua plants. Secondly, in order to increase artemisinin content in A. annua, phytohormone ABA was used to treat A. annua plants. Besides, in order to study the expression regulation of cyp71av1 gene, its promoter was isolated and analysized. The results are as following.(1) Several plant expression vectors containing the key genes involved in the artemisinin biosynthesis were constructed.(2) A series of transgenic A. annua were obtained using agrobacterium-mediated transformation. Twenty-five, 43, 10, 38 and 20 independent transgenic plants containing ads alone, fps alone, ads and fps together, fps and hmgr together, cyp71av1 and cpr together were obtained.(3) Some transgenic plants were analyzed by Southern blot. Results showed that foreign DNA fragments were randomly inserted into A. annua genome.(4) Artemisinin contents were determined by HPLC-ELSD, and high artemisinin-yielding plants were screened out from transgenic plants that were transformed with different genes. The highest artemisinin content was 1.8-fold of that in control among the transgenic plants transformed with ads gene, 2.2-fold among those transformed with fps gene, 2-fold among those transformed with fps and ads genes, 1.8-fold among those transformed with fps and hmgr genes, 2.4-fold among those transformed with cyp71av1 and cpr genes.(5) A. annua plants were treated with ABA at three different concentrations (1μM,10μM,100μM). Results showed that ABA treatment could significantly increase the artemisinin content in A. annua plants. Especially, when the A. annua plants were treated by 10μM ABA, the artemisinin content was increased by 65%.(6) Both A. annua plants and cell suspension cultures were treated by 10μM ABA, and then the expression of genes involved in artemisinin biosynthesis was analyzed using semi-quantitive RT-PCR. Results showed that the expression of hmgr, fps, cyp71av1 and cpr was induced by ABA.(7) The promoter of cyp71av1 gene was isolated using TAIL-PCR and genomic DNA walking. Sequence analysis showed that TATA box was located at -25 to -30 nt and CAAT box was located at -53 to -57 nt.(8) The transcription start site of cyp71av1 gene was determined by 5'-RACE. Result showed that the transcription start site was A, 18 bp upstream of the ATG codon.(9) A series of 5'-end deletions of cyp71av1 promoter were fused to gus report gene. These vectors could be used to analyze the cis-elements in the promoter.