Overexpression of farnesyl pyrophosphate synthase (FPS) gene affected artemisinin content and growth of Artemisia annua L.

Abstract

Transgenic plants of Artemisia annua L., a medicinal plant that produces the compound artemisinin which has an anti-malarial activity, were developed following Agrobacterium tumefaciens-mediated transformation of leaf explants. A. tumefaciens strain EHA105 carrying either pCAMBIA1301 or pCAMBIAFPS was used. Both plasmids harbored the hygromycin phosphotransferase II (hptII) gene as a selectable gene, but the latter plasmid also harbored the gene encoding for farnesyl pyrophosphate synthase (FPS), a key enzyme for artemisinin biosynthesis. Shoot regeneration was observed either directly from leaf sections or via intervening callus when explants were incubated on solidified Murashige and Skoog (MS) (1962) medium containing 0.1 mg l -1 a-naphthaleneacetic acid (NAA), 1 mg l -1 N 6 -benzyladenine (BA), 30 mg l -1 meropenem and 10 mg l -1 hygromycin. Applying vacuum infiltration dramatically increased transformation efficiency up to 7.3 and 19.7% when plasmids with and without FPS gene were used, respectively. All putative transgenic regenerants showed positive bands of hptII gene following Southern blot analysis. Expression of FPS was observed in all transgenic lines, and FPS over-expressed lines exhibited higher artemisinin content and yield, of 2.5-and 3.6-fold, respectively, than that detected in wild-type plants. A relatively high correlation (R 2 = 0.78) was observed between level of expression of FPS and artemisinin content. However, gene silencing was detected in some transgenic lines, especially for those lines containing two copies of the FPS transgene, and with some lines exhibiting reduced growth.

Overexpression of the cytochrome P450 monooxygenase (cyp71av1) and cytochrome P450 reductase (cpr) genes increased artemisinin content in Artemisia annua (Asteraceae)

“…It has been reported that multiple copies of CaMV 35S promoters may cause cosuppression (Napoli et al, 1990;McNellis et al, 1994;Ohashi et al, 2002), so that could be the reason why some of the transgenic plants displayed a lower expression of gene cpr compared to the controls. Likewise, other researchers have also reported a similar phenomenon, so cosuppression phenomenon occurs in overexpression transgenic plant regularity (Yang et al, 2008;Banyai et al, 2010).…”

“…Transferring the hmgr gene from Catharanthus roseus L. into A. annua resulted in a significantly higher HMGR activity compared with wild-type controls in leaf tissue and an increase of 22.5% artemisinin content compared with wild-type A. annua (Aquil et al, 2009). Overexpression of the farnesyl pyrophosphate synthase gene (fps) exhibited significant enhancement of artemisinin yields of about 2.5 times in comparison to the wild-type plants (Banyai et al, 2010). Suppressing the expression of squalene synthase, a key enzyme of the sterol pathway, which is competitive with the amorpha-4,11-diene accumulation pathway, produced a 3.14-fold increase in artemisinin content in transformed plants compared with untransformed control plants (Zhang et al, 2009).…”

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua

“…7). Researchers have reported that a co-suppression phenomenon can often occur in overexpressing transgenic plants (Yang et al, 2008;Banyai et al, 2010).…”

“…In the SA biosynthetic pathway of the transgenic A. annua, overexpression of the key enzyme genes could be an effective way to increase the artemisinin content (Han et al, 2006;Aquil et al, 2009;Banyai et al, 2010;Nafis et al, 2011). As SA treatment can improve the artemisinin content, the overexpression of a key gene in the SA biosynthesis pathway might increase endogenous SA, leading to improved artemisinin content (Fig.…”

An ecological implication of glandular trichome-sequestered artemisinin: as a sink of biotic/abiotic stress-triggered singlet oxygen

“…During last two decades, a lot of initiatives to engineer artemisinin biosynthesis genes towards enhanced artemisinin production has been lunched (Farhi et al 2013). The transgenic endeavors in A. annua include 'pulling carbon flux' to artemisinin via the introduction of a single or multiple artemisinin biosynthesis genes Aquil et al 2009;Banyai et al 2010;Elfahmi and Chahyadi 2014;Nafis et al 2011;Shen et al 2012;Yuan et al 2014) or 'shutting carbon flux' from steroids or other isoprenoids to artemisinin through anti-sense/RNA interference (Chen et al 2011;Feng et al 2009;Yang et al 2008;Zhang et al 2009). In the former situation, artemisinin biosynthesis is enhanced due to the increase of the copy numbers of artemisinin biosynthesis genes; in the latter one, other isoprenoid biogenesis is suppressed due to the knockdown of one or more genes (Lange and Ahkami 2013).…”