Enhanced artemisinin yield by expression of rol genes in Artemisia annua

Abstract

BackgroundDespite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug.MethodsIn this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC–MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined.ResultsTransgenics of rolB gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4–12-fold increase in artesunate and 1.2–3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the rol B gene had higher expression of these genes than rol C transformants. TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant.ConclusionThus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.

Cannabis sativa: The Plant of the Thousand and One Molecules

“…It has been recently shown that the transformation of A. annua with the rolB and rolC genes of Agrobacterium rhizogenes led to plants with an increased content of artemisinin ( Dilshad et al, 2015 ). The rol genes are known for their stimulatory action on plant secondary metabolism ( Bulgakov, 2008 ).…”

Rol genes enhance the biosynthesis of antioxidants in Artemisia carvifolia Buch

“…Protein of the rol A gene binds to DNA and stimulates growth, whereas the rol B gene is involved in the regulation of the auxin signal transduction pathway [ 15 ] and is a potent inducer of plant secondary metabolism, increasing the resveratrol production in Vitis amurensis [ 16 ] and anthraquinones in Rubia cardifolia [ 17 ]. The rol C gene encodes cytokinin glucosidase and stimulates the production of many secondary compounds in various plants [ 18 – 22 ].…”

Cloning and characterization of sesquiterpene synthase genes from the <italic>Ganoderma sinense </italic>genome

“…随着越来越多有价值的倍半萜化合物如抗疟疾的 青蒿素、显著抗癌活性的土荆芥内酯等 [22,23] 先后被发 现, 倍半萜的生物合成途径的研究日渐深入. 倍半萜合 酶在倍半萜的生物合成过程中起着至关重要的作用, 有实验证明, 通过过量表达倍半萜合酶基因可以提高 倍半萜化合物含量 [13,16] .…”