Characterization Of The Promoter Of Artemisia Annua Amorpha-4,11-diene Synthase(ADS) Gene Using Homologous And Heterologous Expression As Well As Deletion Analysis

Artemisinin, a sesquiterpene lactone endoperoxide isolated fromArtemisia annua L.(A. annua) of Asteraceae family, is a widely used anti-malarial compound.Nowadays, artemisinin is in great demand on international markets;however, A. annua plants, the main source of artemisinin, are of low contentof this vital compound (0.1%-0.8%). Therefore, it is significantly importantto enhance artemisinin yield in A. annua. Artemisinin is specificallybiosynthesized and stored in A. annua glandular trichomes, making trichome-specific promoters a great value to artemisinin trichome engineering.The Amorpha-4,11-diene Synthase (ADS) from A. annua is a keyenzyme in artemisinin biosynthetic pathway, and the ADS gene is specificallyexpressed in A. annua glandular trichomes (T-shaped trichomes not included).Therefore, it is attractive to know whether the ADS promoter is trichome-specific as well.In this study, we cloned the ADS promoter (2935bp) from A. annuagenome. Based on the GUS reporter system, the ADS promoter is studiedthrough homologous expression in A. annua, and heterologous expression aswell as deletion analysis in Arabidopsis thaliana (A. thaliana). Results andconclusions of this study are as follows:1. The ADS promoter is trichome-specific in both A. annua and A.thaliana.Considerable differences lie in both structures and sizes of trichomes inA. annua and A. thaliana: The Artemisia trichomes are multicellular andglandular, while the Arabidopsis ones are unicellular and non-glandular.Nevertheless, GUS staining of A. annua leaves transformed with the ADS promoter and of A. annua leaves transformed with the same promoter wereboth well confined to trichomes, and could not be observed in other tissues ofthe leaves. These results correspond with the phenomenon that the ADS geneis specifically expressed in A. annua trichomes, and indicate that thetrichome specificity of the ADS promoter is of cross-species feature.2. Key elements are probably located in the region of-350to-300bp upstream of the transcription start site of the ADS promoter that maywell explain the trichome specificity of the promoter.When deleted to as short as merely-350bp upstream of the transcriptionstart site of the ADS promoter (2935bp in full length), the resulting deletedfragment was still sufficient for the trichome-specific expression in A.thaliana. However, when subsequently deleted to-300bp, the specificitydisappeared; instead, expressions of the downstream reporter gene wereobserved across the whole leaves. Therefore, it could be conjectured that the50-bp region from-350to-300bp probably contain key cis-acting elementsthat are responsible for the trichome-specific feature of the ADS promoter.3. The frequencies of transgenic A. thaliana plants displayingtrichome-specific expressions varied between different lines, and all thelines with deleted fragments of the ADS promoter showed lowerfrequencies than the line with the full-length ADS promoter.In this study, for the full-length ADS promoter or a certain deleted ADSpromoter fragment, its trichome specificity percentage refers to the ratiobetween the number of its transgenic Arabidopsis plants displaying trichomespecificity and the number of transgenic plants in total. Compared to thetrichome specificity percentage of the full-length ADS promoter, percentagesof all the deleted ones decreased sharply. The percentage of the full-lengthADS promoter was around70%, however that of Δ-800or Δ-550was onlyaround1/5, and that of Δ-400, with the highest percentage among all thedeleted ADS promoter fragments, was only45.83%. These results indicatethat unknown complex mechanisms are hidden behind the trichome-specificphenomenon of the ADS promoter, awaiting further studies.4. Activities of all the deleted ADS promoter fragments wereuniversally higher than that of the full-length ADS promoter.Based on the quantitative GUS assay results of transgenic A. thaliana, Δ-350was the highest, with3.27times that of the full-length promoter; andthat of Δ-400was the lowest, with2.09times. In addition, the activity of Δ-350was significantly higher than that of Δ-400as well. These resultsindicate that there are probably regions in charge of negative regulationsunstream of-800bp or between-400to-350bp.In this study, a series of deleted ADS promoter fragments were obtainedthrough deletion analysis, among which the shortest one is of only417bp(Δ-350). Activities of those fragments were all higher than that of the full-length ADS promoter, while still retaining the trichome specificity. Thosefragments will be of great value to studies on molecular mechanisms oftrichome specificity, constructions of super trichome-specific promoters, aswell as metabolic engineering in glandular trichome.