| Artemisinin-based combinationtherapies (ACTs) is an effective medicine for the treatment of malaria. At present the commercial source of artemisinin relies mainly on medicinal plant Artemisia annua L.(A. annua). Glandular trichomes (AaGSTs) are sites for artemisinin biosynthesis, secretion and accumulation. The normal development of AaGSTs is directly related to production of artemisinin. This study cloned a gene, named TAR1 (TRICHOME AND ARTEMISININ REGULATOR 1) from A. annua, which participated in the development of trchomes (including AaGSTs and T-shaped non-glandular trichomes) and artemisinin biosynthesis. Through bioinformatics analysis and a series of gene function research, the study finally proved that the gene is involved in development of trichomes and directly regulate artemisinin biosynthesis by bingding to promoters of ADS and CYP71AV1. In addition, a metabolic engeneering strategy was carried out to improve artemisinin production by overexpressing TAR1.The evolutionary tree and gene sequence analysis showed TAR1 was a transcription factor of AP2/ERF family and its homologous genes was associated with wax synthesis. TAR1-GFP fusion protein results confirmed that the gene was nuclei location. Electrophoretic mobility shift assay (EMSA) experiment confirmed that TAR1 genes could combined with GCC-box which was the basic characteristic of ERF sub-family. These results showed that TAR1 was indeed an AP2/ERF transcription factors.The spatial and temporalanalysis results by quantitative RT-PCR (qRT-PCR) showed that TAR1 was mainly expressed in young leaves, buds and gradually reduced as grew. To further analyzed the express patterns of TAR1, the research cloned the promoter of TAR1 and construct histocellular and in vivo protein localization vector. By generation of TAR1pro-GUS, it proved TAR1 was expressed in leaf meristem, young buds and trchomes (including AaGSTs and T-shaped non-glandular trichomes). In addition, the fluorescence of TAR1-GFP fusion protein in transgenic A. annua plant was localized in nuclei of young leaves by experiment of TAR1pro:TAR1-GFP. These results testified TAR1 expressed and functioned in young tissues and organs.To further study biological function of TAR1, we successfully obtained TAR1 silenced (TAR1-RNAi) plant and explored the its function from the aspects of genes, phenotype and compounds. We also studied the molecular regulatory mechanism of TAR1 on artemisinin biosynthesis.· We observed trichomes in leaf surface of transgenic TAR1-RNAi plants since TAR1 was involved in development of trichomes in A. annua. When transcript levels of TAR1 were decreased, trichomes were appeared abnormal development. Compared to the same period of wild type plants, most of AaGSTs were abnormal inflated top and the T-shaped non-glandular trichomes were extended, distorted and form reticular structure.· Since its homologous were involved in wax biosynthesis, we also analyzed the wax constituents and content in TAR1-RNAi transgenic plant. The scanning electron microscopy (SEM) results showed abnormal accumulation of wax on leaf surface of TAR1-RNAi plant. When treated with 75% alcohol, the chlorophyll in TAR1-RNAi transgenic plant could be taken off quickly compared with wild type, which indicated the change of permeability of leaf surface. The GC-MS were used to determine the wax content. The results further confirmed that the wax content decreased. These results inferred that TAR1 might influence the development of trichomes by regulating wax biosynthesis.· To study the function of TAR1 in artemisinin biosynthesis, we used HPLC-MS/MS to determine the content of artemisinin, dihydroartemisinic acid and artemisinic acid in TAR1-RNAi transgenic plant. In different TAR1-RNAi strains, their content was significantly lower. The result indicated TAR1 was involved in artemisinin biosynthesis. Furthermore, we analyzed expression levels of nine genes which related to artemisinin biosynthesis in TAR1-RNAi plants. The result supported that TAR1 could affect transcript levels of many genes in artemisinin biosynthetic pathway, especially, key genes in downstream of artemisinin synthesis, ADS and CYP71AV1.· ADS and CYP71AV1 transcripts changed significantly in TAR1-RNAi strains. The promoter regions of ADS and CYP71AV1 contained CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs which were the binding sites of AP2 transcription factors. The result of EMSA and yeast single hybriad indicated that TAR1 could control ADS and CYP71AV1 directly. Therefore, TAR1 could control artemisinin biosynthesis by regulating ADS and CYP71AV1.Taken together, we have a completely analysis of TAR1 in biological function. As the important role of TAR1 in trichome development and artemisinin biosynthesis, we constructed TAR1 overexpressed (TAR1-OX) transgenic plants. The results showed a increased content of artemisinin, dihydroartemisinic acid and artemisinic acid in TAR1-OX transgenic plants. Besides, the genes involved in artemisnin biosynthesis also altered in TAR1-OX strains. It is to say we can increase artemisinin content by overexpressing TAR1.In conclusion, TAR1 could regulate development of trichomes and affect wax biosynthesis.In addition, TAR1 could also impact artemisinin biosynthesis by directly regulating ADS and CYP71AV1, and finally influence the content of artemisinin, artemisinic acid and dihydroartemisinic acid. This study firstly reported a developmental gene involved in trichomes in A. Annua, which could influence the formation of trichomes, the synthesis of wax and the regulation of artemisinin biosynthesis in A. annua. Because of the defense function of trichomes in plant, the TAR1 may be a potential target gene to improve resistance and artemisinin content in A. annua. In this paper, a strategy, from the perspective of trichome involved gene (TAR1), was carried out to increase the content of artemisinin. This is a novel and promising metabolic engineering strategy and is useful to cultivate good quality and high yield strain of A. Annua in theoretical and practical. |
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