Genetic Transformation of Artemisia carvifolia Buch with rol Genes Enhances Artemisinin Accumulation.

The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.01-0.8% DW). There is therefore an urgent need to design new strategies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: artemisinin (8μg/g), artesunate (2.24μg/g), dihydroartemisinin (13.6μg/g) and artemether (12.8μg/g). Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin content in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS), cytochrome P450 (CYP71AV1), and aldehyde dehydrogenase 1 (ALDH1), with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1) showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism

Sequenced DNA for identification of <i>Artemisia carvifolia</i>.

<p><i>psbA-trnH</i> sequence of <i>A</i>. <i>carvifolia</i> [NCBI: FJ418751].</p

Trichome density.

<p>Comparison of trichome density of transformed and untransformed plants of <i>A</i>. <i>carvifolia</i> (a). Graphical representation of trichomes densitiy of wild type <i>A</i>. <i>carvifolia</i> and transformants of <i>rol B</i> and <i>rol C</i> gene (b). TB1-TB4 represent <i>rol B</i> transgenics whereas TC1-TC3 represent transgenics of <i>rol C</i> gene. WT represents the control plant of <i>A</i>. <i>carvifolia</i>.</p

Vegetative propagation of <i>A</i>. <i>carvifolia</i>.

<p><i>A</i>.<i>carvifolia</i>: wild type (a), tranformant of <i>rol</i> B (b), and transformant of <i>rol</i> C gene (c).</p

Detail of vectors used for transformation of <i>A</i>. <i>carvifolia</i>.

<p>Vectors (pPCV002-CaMVBT and pPCV002-CaMVC) used in the transformation of <i>Artemisia carvifolia</i>. Fig taken from literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140266#pone.0140266.ref026" target="_blank">26</a>].</p

Southern blot analysis.

<p>Southern blot analysis of <i>rol</i> B (a) and <i>rol</i> C transgenic lines (b). TB1-TB4 represents <i>rol</i> B transgenics whereas TC1-TC3 represent <i>rol</i> C transgenics. Whereas "P” stands for the plasmid DNA run as positive control. WT indicates the wild type plant.</p

Analysis of variance for quantitative analysis of artemisinin and derivatives.

<p>Coefficient of Variation: 3.38%</p><p>Analysis of variance for quantitative analysis of artemisinin and derivatives.</p