The Tissue Localization Of TFAR1 Promoter And Enhancement Of Artemisinin Biosynthesis By Overexpressing TFAR1 In Artemisia Annua L

Artemisinin, a sesquiterpene lactone extracted from the plant of Artemisia annua L., is considered to be the best drug against chloroquine-resistant and cerebral malarial. The Word Health Organization (WHO) recommended the Artemisinin-based combination therapies (ACTs) as the best treatment of malaria. But in the natural Artemisia annua L., the artemisinin content of the dry weight only accounted for 0.01% to 0.6%, can not meet the demands of the market. In recent years, along with the research of artemisinin biosynthetic pathway and the study of key enzyme genes, genetic technology reconstruction project of artemisinin biosynthetic pathway is considered to be one of most effective means to improve the content of artemisinin.The gene TFAR1 encoded a member of the fatty acyl-coenzyme A reductase family. TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. In this study, according to the TFAR1 gene sequence reported by NCBI (accession number:GU733320), we have cloned TFAR1 gene from Artemisia annua L. by PCR, and the bioinformatics and phylogenetic analysis revealed that TFARl belongs to the FAR (fatty acyl-CoA reductase) superfamily. Quantitative PCR showed that expression level of the TFAR1 were significantly increased (p<0.01) after MeJA and ABA treatment. We have constructed the plant expression vector of pCAMBIA2301+-TFAR1 and transferred into leaves of Artemisia annua L.. When the regenerated shoots grew, the primers were designed to detect whether the regenerated plants were authentic transgenic plants of A. annua L.. After PCR detection we have identificated seven independent transformed plants with overexpression of TFAR1 gene and were named as T1, T2, T3, T4, T5, T6, T7. Using EF1α as reference gene, the relative expression of TFAR1 gene in transgenic plants were detected by fluorescence quantitative PCR. The results of relative expression of TFAR1 gene in transgenic plants showed that compared with the control, transgenic lines T3, T4, T5, T6, T7 expression showed significantly increased(p<0.01). Using high performance liquid chromatography (HPLC) detection the content of artemisinin, dihydroartemisinic acid, and artemisia acid. The detection reslts of artemisinin content by HPLC indicated that the content of artemisinin of all transgenic lines compared with the control were increased at different degrees, which the artemisinin content of T2, T4, T5, T6, T7 were significantly increased (p<0.01) compared with the control. T5 transgenic line, with a highest content of 20.19±1.29mg·g-1, is about 1.25 to 1.3 times compared with the artemisinin content of control plants. The detection reslts of dihydroartemisinic acid by HPLC indicated that all transgenic lines compared with the control were increased at different degrees. The content of dihydroartemisinic acid in transgenic lines T1, T3, T5, T6 were significantly increased (p<0.01) compared with the control. T6 transgenic line, with a highest dihydroartemisinic acid content of 8.74±2.06 mg·g-1, is about 2.55 to 3.3 times compared with the control plants. The artemisinic acid content of transgenic plants compared with the control were significantly increased (p<0.01) except of T2 transgenic line (p<0.05), the highest artemisinic acid content of T6 transgenic line is 3.5-7.5 times higher than the control. The above results show that, it can promote the synthesis of artemisinin and its metabolic products by over-expression of TFAR1 gene in Artemisia annua L.In addition to confirm the function of TFAR1 gene in transgenic level, we still have cloned TFAR1 promoter by the PCR technology according to the genomic sequence(unpublished) of Artemisia annua L. And its cis-acting element analysis showed that the promoter of TFAR1 is 2243bp, containing MeJA response element, and MYC binding element, WRKY binding element, MYB binding element. Then we construct the promoter functional verification vector containing TFAR1 promoter fragment named pCAMBIA1305-TFAR1, and transferred into Arabidopsis. Transformed seeds were screened on (MS+Hygr (50mg·L-1)+Cef (200mg·L-1) to get the Hygromycin-resistant plantlets. The expressions of pTFAR1::GUS were detected at seedlings, stems, leaves, flowers, pTFARl::GUS was highly specific expressed in glandular tichome of leaves. From the above results, it can be concluded that TFAR1 promoter shown glandular trichomes-specific expression patterns.