GUS expression controlled by the <i>FS</i> promoter in transgenic plants of <i>Artemisia annua</i>.

<p>A: young leaf; B: close-up of young leaf; C: leaf; D: old leaf; E: root.</p

Plant-produced recombinant full-length hemagglutinin (rHA0) analysis using SDS-PAGE and western blot.

<p>A: Probe with anti-His antibodies, Lane 1, molecular weight marker; lane 2, purified rHA0 (5 µg) from total soluble protein (TSP) at 6 days post infiltration (dpi) (SDS-PAGE); lane 3, TSP (40 µg) from untreated <i>Nicotiana benthamiana</i> leaves (negative control; 0 dpi); lanes 4 to 8, TSP (40 µg protein per lane) from the agro-infiltrated leaves of <i>N. benthamiana</i> at 3, 6, 9, 12 and 15 dpi; lane 9, purified rHA0 (5 µg) at 6 dpi; lane 10, purified rHA0 (5 µg) at 6 dpi after PNGase F digestion. The shift to a lower molecular mass upon treatment with PNGase F is indicative of N-linked glycosylation on rHA0; B: Probe with anti-HA antibody, Lane 1, molecular weight marker, lane 2, purified rHA0 (5 µg) at 6 dpi.</p

Studies on the Expression of Sesquiterpene Synthases Using Promoter-β-Glucuronidase Fusions in Transgenic <i>Artemisia annua</i> L

<div><p>In order to better understand the influence of sesquiterpene synthases on artemisinin yield in <i>Artemisia annua</i>, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), <i>epi</i>-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of <i>cis</i>-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic <i>A. annua</i> plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of <i>A. annua</i> to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the <i>GUS</i> gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the <i>CPS</i>, <i>ECS</i> or <i>GAS</i> gene may not improve artemsinin yield. However, blocking the expression of <i>FS</i> may have effects on artemisinin production.</p></div

Relative expression of the wild-type <i>pADS</i> (A), <i>pCPS</i> (B), <i>pECS</i> (C), and <i>pFS</i> (D) in leaves after wounding.

<p>The β-actin promoter activity was set to 1.0.</p

Enzymes in <i>Artemisia annua</i> utilizing farnesyl diphosphate as substrate.

<p>ADS: amorpha-4,11-diene synthase; CPS: β-caryophyllene synthase; ECS: <i>epi</i>-cedrol synthase; FS: β-farnesene synthase; GAS: germacrene A synthase; GDS: germacrene D synthase; SQS: squalene synthase; PPO: diphosphate moiety</p

Wounding of leaves of transgenic <i>Artemisia annua</i> carrying the <i>pECS::GUS</i> fusion.

<p>A: unwounded; B: immediately after wounding; C: 1h; D: 2h; E: 4h; F: 8h; G: 12h; H: 24h; I: 48h.</p

Phylogenetic relationships between the A/mallard/Sweden/7206/2004(H7N7) isolate and representative H7 sequences available in public databases.

<p>Bayesian phylogeny of HA amino acid sequences (denoted by GenBank accession number and isolated description) was inferred using the MrBayes 3.1.2 program and the Jones model of substation with gamma-distributed rate variation across sites. Nodes with posterior probability support ≥95% are indicated. The Swedish H7 sequence isolated is highlighted in bold face. Scale bar indicates the number of amino acid changes per branch length.</p

Relative expression of plant-produced recombinant hemagglutinin was measured by quantitative real-time PCR.

<p>The expression levels was measured at 3, 6, 9, 12 and 15 dpi (days post infiltration) and are normalized to expression of control genes, the ubiquitin (<i>ubi3</i>) and elongation factor-1 (<i>EF-1</i>). The results represent two separate experiments, each time performed including three technical replicates.</p

Hemagglutination assay.

<p>Triplicates of purified plant-produced hemagglutinin (rHA0) were two-fold serially diluted beginning with a concentration of 10 µg/mL from the stock of 400 µg/mL and mixed with chicken erythrocytes. Wells in the bottom row contains bovine serum albumin (BSA) as a negative control starting with a concentration of 10 µg/mL from 400 µg/mL stock. The lowest concentration (µg/mL) of the well showing complete agglutinating activity of erythrocytes was considered as hemagglutination titers (HT).</p

Some features of the cloned sesquiterpene promoters.

*<p>The position of the TSS (+1) is given as bases upstream of the ATG start codon.</p>**<p>The position of the TATA- and CAAT-boxes are given relative to the TSS.</p