Molecular Cloning Of Transcription Factor AaWD40 And RNA Interference Of DXR Gene In Artemisia Annua L.

Artemisinin, an endoperoxide sesquiterpene lactone produced by the traditional Chinese medicinal herb Artemisia annua L., is an effective drug against cerebral malaria and chloroquine-resistant strains of Plasmodium falciparum. The biosynthesis of artemisinin is mainly located in the glandular trichomes of A. annua. There is an enormous demand for artemisinin supply all over the world. However, its low content in A. annua limits the commercialization of artemisinin production greatly. Transcription factor WD40 is involved in the development of glandular trichome and 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene(DXR) is a key gene of artemisinin biosynthesis pathway. To better understand and regulate the biosynthesis of artemisinin, it is meaningful to further explore those related genes. In the present study, a new WD40 family transcription factor Aa WD40 was cloned and we studied on role of DXR gene on growth and artemisinin biosynthesis in A. annua using RNA interference technology.Firstly, a full length c DNA encoding a WD40 transcription factor(named as Aa WD40) was cloned and characterized from A. annua for the first time. The full length of Aa WD40 was 1498 bp containing 209 bp 5’ untranslated region, 183 bp 3’ untransled region and a 1106 bp open reading frame encoding a polypeptide with 368 amino acids, with a putative isoelectric point 4.64 and calculated molecular weight about 41.29 k Da. The results of sequence alignment and BLASTP search in Genbank database showed Aa WD40 had high homology with many other plant WD40 genes. Conserved domain analysis of Aa WD40 protein revealed that it contained four WD-repeat motifs. In three-dimensional structure predicted by Swiss-Modeling software, Aa WD40 protein had similar domain with other plant WD40. A phylogenetic tree of different WD40 s from other species was constructed. The result revealed that plant WD40 s originated from a common ancestor and had the closest relationship with Dp WDR and Gh TTG4.To construct the RNA interference(RNAi) vector targeting DXR gene, we chose an interference fragment(523 bp) with the analysis of BLAST alignment. DXR-RNAi vector was constructed using Gateway clone technology with entry vector p DONR221 and purpose vector p SGRNAi. After confirmation by PCR, sequencing and restriction enzyme analysis, the recombinant plasmid was successfully transferred into Agrobacterium strain LBA4404. Then transgenic plants of A. annua were developed following Agrobacterium-mediated transformation of leaf explants. Thirty-six plants were screened out with kanamycin-resistance, acclimatized and transplanted.The screened transgenic A. annua plants were further identified by PCR. The expression levels of DXR gene in five positive transgenic plants were determined by real-time PCR. Results showed that DXR-RNAi significantly suppressed the expression of DXR in A. annua, average 68% lower than that in control. It was observed that the transgenic plants had longer roots and stems, smaller leaves, lower density but larger area of glandular trichome than that in non-transgenic group. And the contents of artemisinin and artemisinic acid were down-regulated 39% and 58% after DXR-RNAi, respectively.In conclusion, this is the first study on molecular clone of WD40 transcription factor in A. annua. The c DNA full length information of Aa WD40 will be useful to explore the functions of WD40 transcription factor, especially on the development of glandular trichome in A. annua. Moreover, the RNA inference of DXR gene was carried out to investigate on the roles of this gene. And results revealed that DXR gene was relevant with glandular trichome development for the first time in A. annua.