Development of transgenic Artemisia annua (Chinese wormwood) plants with an enhanced content of artemisinin, an effective anti‐malarial drug, by hairpin‐RNA‐mediated gene silencing

Abstract

Artemisinin is an effective anti-malarial drug isolated from Artemisia annua L. (Chinese wormwood), but the content of artemisinin in A. annua is low. In the present study we explored the possibility of using genetic engineering to increase the artemisinin content of A. annua by suppressing the expression of SQS (squalene synthase), a key enzyme of sterol pathway (a pathway competitive with that of artemisinin biosynthesis) by means of a hairpin-RNA-mediated RNAi (RNA interference) technique. A total of 23 independent transgenic A. annua plants were obtained through Agrobacterium tumefaciens-mediated transformation, which was confirmed by PCR and Southern-blot analyses. HPLC-evaporative light-scattering detection analysis showed that the artemisinin content of some transgenic plants was significantly increased, with the highest values reaching 31.4 mg/g dry weight, which is about 3.14-fold the content observed in untransformed control plants. Real-time reverse transcription-PCR analysis demonstrated that the expression of SQS was suppressed significantly, and GC-MS analysis showed that sterol was efficiently decreased in the transgenic plants. The present study demonstrated that genetic-engineering strategy of RNAi is an effective means of increasing artemisinin content in plants.

Platform for “Chemical Metabolic Switching” to Increase Sesquiterpene Content in Plants

“…In A. annua, artemisinin content was significantly increased in plants in which SQS was silenced, with the highest values reaching 31.4 mg g −1 dry weight, which is an approximately 3.14-fold increase in the content observed in untransformed control plants (Zhang et al 2009). Our results reveal the possibility that "chemical metabolic switching" is effective for any plant species in which transformation technology is not established and/or sesquiterpene synthase has not yet been identified.…”

“…If triterpene biosynthesis is inhibited, FPP will be used in sesquiterpene biosynthesis and the product level will increase. In A. annua, artemisinin content was significantly increased in plants by silencing squalene synthase (SQS), which biosynthesizes squalene from two molecules of FPP (Zhang et al 2009). It was recently shown that inhibition of SQS and 5-epi-aristolochene synthase by RNAi methods increased exogenous (+)-valencene accumulation in (+)-valencene synthase overexpressing tobacco (Cankar et al 2015).…”

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

“…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). Therefore, to improve artermisinin production, genetic transformation is a potentially useful strategy.…”

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).…”