Transient transformation of artemisinic aldehyde ∆ 11 (13) double bond reductase (dbr2) gene into Artemisia annua L.

Elfahmi Elfahmi(1*), Fany Mutia Cahyani(2), Andre Ditya Maulana Lubis(3), Tati Kristanti(4), Sony Suhandono(5)

(1) School of Pharmacy, Bandung Institute of Technology, Jl. Ganesha 10 Bandung 40132
(2) School of Pharmacy, Bandung Institute of Technology, Jl. Ganesha 10 Bandung 40132
(3) School of Pharmacy, Bandung Institute of Technology, Jl. Ganesha 10 Bandung 40132
(4) School of Life Sciences and Technology, Bandung Institute of Technology, Jl. Ganesha 10, Bandung 40132
(5) School of Life Sciences and Technology, Bandung Institute of Technology, Jl. Ganesha 10, Bandung 40132
(*) Corresponding Author


Global demand of antimalarial drug artemisinin has a gap with production capacity from existing sources since the low content of this compound from Artemia annua L. Genetic engineering-based strategy for A. annua plant on key enzymes in artemisinin biosynthetic pathway is needed. Artemisinic aldehyde ∆ 11 (13)  double bond reductase (dbr2) is one of the key enzyme on artemisinin biosynthesis which was studied in this research. Agrobacterium tumefaciens-mediated transformation of A. annua using dbr2 was carried out. Synthetic dbr2 was ligated into pCAMBIA1303 and transformed into Escherichia coli DH5α. pCAMBIA1303-dbr2 plasmid was transformed to A. tumefaciens AGL1. Leaves of  A. annua were infected by positive transformant of recombinant A. tumefaciens (OD600 ≈ 1) supplemented with acetosyringone 50 ppm, and Silwet S-408 0.02%. Samples were incubated in desiccators connected with vacuum pump, this method is called infiltration vacuum. Leaves were covered in dark for 45 min, and co-cultivated on MS co-cultivation media for 3 days. All leaves were washed in 300 ppm cefotaxime and divided into 2 parts; 3 leaves for GUS histochemical assay and 300 mg of leaves for HPLC analysis. Transient transformation was done in triplicate. In GUS histochemical assay, pCAMBIA1303 and pCAMBIA-dbr2 showed positive blue spot where coefficient of variance was less than 5%. PCR analysis for genomic DNA of transformed  A. annua showed a positive result of inserted dbr2 recombinant indicated by migration profile and direct sequencing analysis. It could be concluded that pCAMBIA-dbr2 construct and transformation into  A. annua have been successfully performed.


Artemisia annua L.; artemisinin; construction; malaria; pCAMBIA-dbr2; transient

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Brown, G.D. 2010. The biosynthesis of artemisinin (Qinghaosu) and the phytochemistry of Artemisia annua L. Molecules, 15, 7603-7698,

Cahyadi, A. (2011) Transformasi genetik Artemisia annua L. menggunakan Agrobacterium dalam rangka produksi artemisinin. Master's thesis, School of Pharmacy ITB, Bandung.

Cahyani, F.M. (2016) Transformasi gen Amorpha-4,11-Dien Synthase (ADS) dan Protein 19 (P19) pada tumbuhan Artemisia annua L. serta pengaruhnya terhadap kandungan artemisinin sebagai antimalaria. Master's thesis, School of Pharmacy ITB, Bandung.

Chamidah, N. (2013) Transformasi gen Amorfa-4,11-Dien Sintase (ADS) ke dalam kultur in vitro Artemisia annua L. menggunakan Agrobacterium tumefaciens AGL-1 untuk meningkatkan kadar artemisinin. Bachelor thesis, School of Pharmacy ITB, Bandung.

Chen, H., Nelson, R.S., and Sherwood, J.L. 1994. Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. BioTechniques, 16(4), 664–670.

Covello, P.S., Teoh, K.H., Polichuk, D.R., Reed, D.W., and Nowak, G. 2007. Functional genomics and the biosynthesis of artemisinin. Phytochemistry, 68, 1864–1871.

Depkes RI. (2008) Infodatin: situasi malaria di Indonesia, Depkes RI, Jakarta.

Dewick, P.M. (2009) Medicinal natural products: a biosynthetic approach. West Sussex: John Wiley & Sons Ltd. pp. 197–199.

Elfahmi, Suhandono, S., Cahyadi, A. 2014. Optimization of genetic transformation of Artemisia annua L. using Agrobacterium for artemisinin production. Pharmacogn. Mag. 10 (Suppl 1), S176–S180.

Jefferson, R.A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system, Plant Mol. Biol. Rep., 5(4), 387–405.

Komari, T., Takakura, Y., Ueki, J., Kato, N., Ishida, Y., and Hiei, Y. (2006) Binary vectors and Super-binary vectors, In: Wang K (ed), Agrobacterium protocols 2nd ed., vol. 1. New Jersey: Humana Press. pp. 15-42.

Koo, J., Kim, Y., Kim, J., Yeom, M., Lee, I.C., and Nam, H.G. 2007. A GUS/luciferase fusion reporter for plant gene trapping and for assay of promoter activity with luciferin-dependent control of the reporter protein stability. Plant Cell Physiol., 48(8), 1121–1131.

Lee, L.Y., and Gelvin, S.B. 2008. T-DNA binary vectors and systems. Plant Physiol., 146, 325–332.

Leuzinger, K., Dent, M., Hurtado, J., Stahnke, J., Lai, H., Zhou, X., and Chen, Q. 2013. Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. J. Visualized Exp., 77, 1–9.

Martin, T., Wöhner, R.V., Hummel, S., Willmitzer, L., and Frommer, W.B. (1992) The GUS reporter system as a tool to study plant gene expression, In: Gallagher SR (ed), GUS protocols: using the GUS gene as a reporter of gene expression. San Diego: Academic Press Inc. pp. 23–39.

Michelis, C.B., Hooykaas, P.J.J., Hondel, C.A.M.J.J., and Ram, A.F.J. 2008. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori, Nat. Protoc., 3(10), 1671–1678.

Nguyen, K.T., and Arsenault, P.R. 2011. Trichomes + roots + ROS = Artemisinin: regulating artemisinin biosynthesis in Artemisia annua L. in vitro cell. Dev. Biol. – Plant, 47, 329–338.

Olofsson, L., Engström, A., Lundgren, A., and Brodelius, P.E. 2011. Relative expression of genes of terpene metabolism in different tissues of Artemisia annua L.. BMC Plant Biol., 11, 45.

Rao, A.Q., Bakhsh, A., Kiani, S., Shahzad, K., Shahid, A.A., Husnain, T., and Riazuddin, S. 2009. The myth of plant transformation, Biotechnol. Adv., 27, 753–763.

Rivera, A.L., Lim, M.G., Fernández, F., and Loske, A.M. 2012. Physical methods for genetic plant transformation. Phys. Life Rev., 9, 308–345.

Ruijter, N.C.Ad., Verhees, J., Leuween, Wv., and Krol, Arvd. 2003. Evaluation and comparison of the GUS, LUC and GFP reporter system for gene expression studies in plants. Plant Biol., 5, 105–113.

Sambrook, J., Russell, D.W. (2001) Molecular Cloning: A laboratory manual, 3rd ed., New York: Cold Spring Harbor Laboratory Press.

Simmons, C.W., Gheynst, J.Svd., and Upadhyaya, S.K. 2009. A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression. Biotechnol. Bioeng. 102(3), 965–970.

Tang, K., Shen, Q., Yan, T., and Fu, X. 2014. Transgenic approach to increase artemisinin content in Artemisia annua L. Plant Cell Rep., 33(4), 605–615.

WHO (2015) World malaria report 2015. Geneva: World Health Organization.

Yuan, Y., Liu, W., Zhang, Q., Xiang, L., Liu, X., Chen, M., Lin, Z., Wang, Q., and Liao, Z. 2015. Overexpression of artemisinic aldehyde Δ11 (13) reductase gene–enhanced artemisinin and its relative metabolite biosynthesis in transgenic Artemisia annua L. Biotechnol. Appl. Biochem., 62(1), 17–23.

Zhang, Y.S., Teoh, K.H., Reed, D.W., Maes, L., Goossens, A., Olson, D.J., Ross, A.R., and Covello, P.S. 2008. The molecular cloning of artemisinic aldehyde delta11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J. Biol. Chem., 283(31), 21501–21508.


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