| Artemisinin, a new and very potent antimalarial drug, is extracted from annual herb Artemisia annua L. Due to its sesquiterpene lactone structure with an endoperoxide bridge, it is deemed as an active drug against both chloroquine-resistant and chloroquinine-sensitive forms by Plasmodium falciparum, as well as cerebral malaria. Artemisinin Combination Therapies (ACTs) were the recommended treatment of malaria by World Health Organization (WHO). In addition to antimalarial, artemisinin also has anti-progesterone, anti-fibrotic, anti-schistosomiasis, Toxoplasma gondii, anti-arrhythmic and tumor cytotoxicitysuch. Artemisinin and its derivatives are efficient, fast, low toxicity, and new type of anti-malarial drugs. However, the supply of artemisinin is not far from enough in the international market. Although artemisinin could be synthesized chemically, it could not be put into production due to high cost and strong toxicity. In recent years, with the development of molecular biology research of artemisinin biosynthetic pathway, great progress of metabolic regulation of artemisinin biosynthesis has been made at molecular level. Several important functional genes involved in the biosynthetic pathway of artemisinin have been identified that can be used to genetically modify the artemisinin pathway.Hydroxymethylbutenyl4-diphosphate reductase is an essential enzyme of the MEP pathway, which catalyzes the conversion of hydroxymethylbutenyl4-diphosphate (HMBPP) into isopentenyl pyrophosphate and dimethylallyl diphosphate (DMAPP) in a ratio of5:1. The expression of hdr gene has a closely relative with artemisinin biosynthesis, which could provide enough substrate for downstream pathway. Amorpha-4,11-diene synthase is the first essential enzyme of the artemisinin downstream pathway, which catalyzes the conversion of farnesyl diphosphate (FDP) into amorpha-4,11-diene. The expression of ads gene can make more upstream substrate transform into objective product.In order to investigate the molecular mechanism of artemisinin biosynthesis and provide targets for artemisinin metabolic engineering, hydro xymethylbutenyl4-diphosphate reductase (HDR) and amorpha-4,11-diene synthase (ADS) gene from A. annua are used target genes. This study cloned ads and hdr gene from A. annua. Plant expression vector pCAMBIA1304+-ads-hdr was constructed and introduced into Agrobacterium tumefaciens strain LBA4404to generate engineered strain LBA4404-pCAMBIA1304+-ads-hdr. Through leaf-disc transformation method, recombinant vector harboring pCAMBIA1304+-ads-hdr was introduced into A. annua followed by screening with hygromycin to obtain transgenic plantlets of A. annua. After2-3cm hygromycin-resistant shoots were obtained, they were separated and sub-cultured in MS+0.05mg·L-1NAA+250mg·L-1Cab, which is suitable for rooting of shoots. Genomic PCR was applied to detect the vir gene, hygr gene, and target genes ads and hdr. Finally,6independent transgenic lines were confirmed by PCR analysis in all the hygromycin-resistant plant materials, the number were ah3, ahll, ah20, ah27, ah28and ah70.The transgenic A. annua lines were micropropagated in MS+1mg·L-16-BA+0.05mg·L-1NAA to produce plenty of shoots followed by separated and cultured in MS medium to get more plants to hardening and transplanting in experimental field. In middle of August, transgenic and non-transgenic A. annua were collected and subjected to artemisinin content and gene expression levels analysis. After detection with HPLC-ELSD, it was confirmed that artemisinin content varied with different lines. Artemisinin contents in ah3, ahll, ah20, ah27, ah28and ah70transgenic lines were0.53mg·g-1DW,0.51mg·g-1DW,0.27mg·g-1DW,0.46mg·g-1DW,0.39mg·g-1DW,0.47mg·g-1DW respectively. Transgenic line ah70showed the highest artemisinin production, which was enhanced by2.48-time (0.53mg·g-1DW) compared to that in non-transgenic A. annua (0.15mg·g-1DW). Field experimental found that ah3, ah20and ah70lines had different traits, which manifested as undersized, easy to topple over and prematureness.The genes expression is basically consistent with the level of artemisinin content. Due to the different synthesis ability between A. annua plants, the genes expression not absolutely consistent with artemisinin content. The highest gene expression of ads and hdr of ah27relatively had low artemisinin content, while the highest artemisinin content of ah70had not the highest gene expression. Overall, these two genes ads and hdr have important role in artemisinin biosynthetic pathway. This study proves that two-gene strategy can break the rate-limited step in A. annua artemisinin biosynthetic pathway, and improve artemisinin synthesis ability to produce more artemisinin. This study also proves that overexpression A. annua endogenous genes ads and hdr can enhance artemisinin content. |
PDF Full Text Request