TRANSFORMASI TRANSIEN GEN ARTEMISINIC ALDEHYDE ? ???????? (????????) DOUBLE BOND REDUCTASE (DBR2) KE DALAM ARTEMISIA ANNUA L.

• Main Author: Ditya Maulana Lubis, Andre
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45053
• Institution: Institut Teknologi Bandung

Increasing global demand of artemisinin has not been proportional with production capacity from existing resources. Genetic engineering-based strategy for A. annua L. source plant has needed to overproduce of key enzymes in artemisinin biosynthetic pathway. One of the key enzymes was artemisinic aldehyde ? 11 (13) double bond reductase (dbr2). In this research, transient transformation of recombinant A. tumefaciens has done so dbr2 gene transfered into the existing tissue on the leaves of A. annua. pCAMBIA-dbr2 construction has used restriction method through kanamycin selection of E. coli DH5?. The confirmation result of plasmid showed slower migration profile than pCAMBIA1303, and a different restriction fragment size of pCAMBIA-dbr2 (5687 bp, 4550 bp, 3481 bp) instead of pCAMBIA1303 (5638 bp , 3455 bp) with NheI and SacI. PCR product size and nucleotide sequence alignment with GeneBank were also positive to contain the gene. Confirmed pCAMBIA-dbr2 has been introduced into A. tumefaciens AGL1 and confirmed again by colony PCR. Leaves of A. annua were infected by positive A. tumefaciens recombinant (OD600 ? 1) in liquid MS suplemented with acetosyringone, and Silwet S-408. Sample in vacuum within minutes in dark, and co-cultivated on MS co-cultivation for 3 days. The leaves have washed in cefotaxime and divided for evaluation purpose. Transient transformation have done in triplicate. In GUS histochemical test, showed positive blue spot result of pCAMBIA1303 and pCAMBIA-dbr2 with CV less than 5%. Analysis of PCR product from genomic DNA showed a positive result of inserted dbr2 recombinant. As a conclusion, pCAMBIA-dbr2 construct has been successfull, but the expression result of dbr2 gene has not been preconcerted.