KONSTRUKSI PLASMID pCAMBIA YANG MENGANDUNG GEN ALDEHYDE DEHYDROGENASE (ALDH1) UNTUK TUJUAN TRANSFORMASI KE TANAMAN ARTEMISIA ANNUA L

• Main Author: Liawan Sanjaya, Hans
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/44364
• Institution: Institut Teknologi Bandung

The demand of artemisinin for ACT (artemisinin-based combination therapies) increases every year because the earlier drug, especially Cinchona sp. compounds, already resisted by malarian caused parasite, Plasmodium sp. This circumstance cause the need of solution to increase the production of artemisinin, one of the strategy that oftenly used is genetic engineering. Agroinfiltration is used on Artemisia annua to overproduce the enzyme that responsible for the increasing of artemisinin content. There are 5 enzyme responsible in artemisinin biosynthesis pathway; farnesyil diphospate synthase (FPS), amorpha-4,11-diene synthase (ADS), cytochrome-dependent amorpha-4,11-diene 12- monooxygenase (CYP71AV1), aldehyde artemisinic ?11(13) double-bound reductase (DBR2), dan aldehyde dehydrogenase (ALDH1). Aldehyde dehydrogenase oxidize the aldehyde functional group in artemisinic/dihydroartemisinic aldehyde into carboxylic acid, producing the artemisinic/dihydroartemisinic acid. This research aimed to contruct a plasmid pCAMBIA1303 contained ALDH1 gene. The ALDH1 gene was constructed into vector pCAMBIA1303 using cut and paste method. SpeI and PmlI were used for the restriction process in plasmid. The constructed vector was confirmed by migration analysis, restriction analysis, PCR, and sequencing. The constructed plasmid restriction analysis did not give any band in 2150 bp compared to the empty pCAMBIA as control. PCR amplification with F-ALDH and R-ALDH primer gave 1512 bp band in the electroforegram. The sequence analysis with BLASTN showed >92% query coverage and 100% identity score. Based on the data analysis, it could be concluded that ALDH1 has been successfully inserted into pCAMBIA1303 that could be used for transformation into A. annua.