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

https://doi.org/10.22146/ijbiotech.27956

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

Abstract


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.

Keywords


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

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DOI: https://doi.org/10.22146/ijbiotech.27956

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