TRANSFORMASI TRANSIEN GEN PENGKODE FARNESYL PYROPHOSPHATE SYNTHASE (FPS) KE DALAM DAUN ARTEMISIA ANNUA L. (WILD TYPE)

• Main Author: Reni Sijabat, Veronika
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45082
• Institution: Institut Teknologi Bandung

Artemisinin is a secondary metabolite of Artemisia annua L. that used as an antimalarial. Low levels of artemisinin in A. annua makes artemisinin-based malaria treatment being relatively expensive. An strategy that used to increase artemisinin levels is through genetic engineering involving biosynthetic pathway of artemisinin. This study aimed to construct pCAMBIA-fps plasmid and observe fps gene expression in A. annua L. leaves (wild-type). Farnesyl pyrophosphate synthase (FPS) is an important enzyme in artemisinin biosynthesis. The gene encoding fps enzyme is transformed transiently to A. annua L. leaves to increase artemisinin levels. To transforms fps gene in to A. annua, this gene should be inserted to binary vector pCAMBIA 1303. FPS gene used in this study is a synthetic gene that has been in the pUC57cloning vector. pUC57-fps plasmid and pCAMBIA 1303 plasmid were cut using restriction enzymes SpeI and NcoI. Construction was conducted by ligating two DNA fragments that have been truncated and subsequently transformed in Escherichia coli strain DH5?. pCAMBIA-fps plasmid was transformed into A. annua L. leaves through Agrobacterium tumefaciens AGL1. Transient transformation is performed by vacuum infiltration method. The result showed that fps gene was succesfully constructed and confirmed by analysis of migration, analysis of PCR products with CaMV forward primer and GUS reverse primer that gave product on the size of 1655 bp, restriction analysis with SacI and NheI restriction enzyme that gave 3 bands on the size of 3442, 4369, and 5577 bp, and determination of nucleotide sequence tthat give 100% identity conformitywith GenBank data. Fps gene from pCAMBIA-fps plasmid has successfully transformed into A. annua L. leaves through vacuum infiltration method and has confirmed by GUS histochemical test and determination of PCR products using CaMV forward primer and GUS reverse primer and give product on the size of 1655 bp.