RNAi Construction of Pirophosphat–dependent Phosphofructokinase (PFP)-? gene of sugarcane (Saccharum officinarum L.) in expression vector pET- 17b (E.coli)

• Main Author: Murni, Dewi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34902
• Institution: Institut Teknologi Bandung

Increasing of sugar (sucrose) content of sugarcane influence by the allocation of carbon compound that resulted through photosynthesis in carbohydrate metabolism. The carbon compounds could be used for respiration (glycolysis) or sucrose biosynthesis (gluconeogenesis). One of the enzyme that plays an important role in the carbon compounds metabolism is a pyrophosphate-dependent phosphor-fructokinase (PFP). This enzyme catalyzes the phosphorylation of fructose-6-phosphate to fructose-1.6biphosphate on the sucrose metabolic pathway. Enzyme activity was negatively correlated with sucrose content in sugarcane internodes, the lower the enzyme activity higher sucrose content. In plant, PFP enzymes consisted of two pairs of different subunits, ? subunit (67 kDa) and ? subunits (60 kDa). Each subunit coded by PFP ? and PFP ? gene. PFP enzymes occur in three forms, homodimer (?2), heterodimer (??) or heterotetramer (?2?2). Heterodimer or heterotetramer forms catalyze glycolytic reactions with ? subunits that played a role in the regulation of this enzyme. Homodimeric forms play a role in the process of gluconeogenesis which increase the levels of sucrose in plants. PFP ? gene silencing may increase sugar content of sugarcane. In the absence of ? subunit, the enzyme will form homodimeric structure which will favour catalyze gluconeogenesis reaction. Silencing of the PFP ? gene expression could be done using double-strand RNA PFP ? gene constructs RNAi (RNA interference). This study aims to construct sense-antisense of the PFP ? gene with expression vector pET-17b. This construction was made in four stages, (1) designing RNAi using BLOCK-IT ™ RNAi Designer PFP Invitrogen and ordering a synthetic gene, (2) insertion of PFP ? gene fragment into pGEM7Zf, (3) the construction of RNAi sense-antisense PFP ? gene in pHannibal, (4) cloning of sense-antisense constructs from pHannibal into pET-17b. The results shows that the most favourable gene silencing target is sequence number 1 to 300 bp of PFP ? gene. PFP ? gene successfully ligated to pGEM7Zf through EcoRI restriction side. The sequences showed 100% similarity with PFP ? sequences that had been published by Suhandono and Alamsyah in GenBank (2007). Then from pGEM7Zf, the PFP ? gene has also been successfully constructed at the KpnI-XhoI (sense) and BamHIXbaI (antisense) restriction site of the Hannibal to produced fragments of sense-antisense PFP ? sized 1452 bp. The success of ligation was confirmed by PCR crude and restriction with XhoI that showed the band around 1500 bp. Sequencing of pHannibal + PFP ? sense showed 99% homology with pHannibal (Acc.no. AJ311872.1) and 100% with sugarcane PFP ? (Acc.no. AB270695.1). PFP ? sense-antisense successfully transferred from pHannibal to pET-17b in the XhoI restriction site that was confirmed by crude PCR and obtained 1500 bp sized