Enhanced artemisinin yield by expression of rol genes in Artemisia annua.
BACKGROUND
Despite 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.


METHODS
In 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.


RESULTS
Transgenics of rol B 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.


CONCLUSIONS
Thus 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.
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. erum.dilshad@gmail.com.
Erum
E
Dilshad
Laboratorio de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Barcelona, Barcelona, Spain. rcusido@ub.edu.
Rosa Maria
RM
Cusido
Laboratorio de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Barcelona, Barcelona, Spain. javierpalazon@ub.edu.
Javier
J
Palazon
Laboratorio de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Barcelona, Barcelona, Spain. kems3estrada@gmail.com.
Karla Ramirez
KR
Estrada
Laboratorio de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Barcelona, Barcelona, Spain. mbonfill@ub.edu.
Mercedes
M
Bonfill
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. bushramirza@qau.edu.pk.
Bushra
B
Mirza
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000962:Antimalarials / Q000378:metabolism*; D031172:Artemisia annua / Q000235:genetics* / Q000378:metabolism*; D037621:Artemisinins / Q000378:metabolism*; D002853:Chromatography, Liquid; D015870:Gene Expression; D017343:Genes, Plant; D013058:Mass Spectrometry; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014170:Transformation, Genetic
D000962:Antimalarials; D037621:Artemisinins; C031327:artemisinin