Posted January 15, 2013 by admin in Biotechnology

Binary Vector Construction for Pyrophosphate-Dependent Phosphofructokinase (PFP-α) Sense and Antisense Gene Sequences

One of the targets in sugarcane genetic engineering is to produce high sucrose content. PFP  (pyrophosphate-dependent phosphor-fructokinase) is one of the enzyme that plays an important role in the carbon compounds metabolism. This enzyme catalyzes the phosphorylation of fructose-6-phosphate to fructose-1.6-biphosphate 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, α subunit (67 kDa) and β subunits (60 kDa). Each subunit coded by PFP α and PFP β gene. PFP enzymes occur in three forms, homodimer (β2), heterodimer (αβ) or heterotetramer (α2β2). Heterodimer or heterotetramer forms catalyze glycolytic reactions with α 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 α gene silencing may increase sugar content of sugarcane. In the absence of α 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 α gene constructs RNAi (RNA interference). This study show that RNAi PFP-α has constructed in pHannibal vector. 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 α gene fragment into pGEM7Zf, (3) the construction of RNAi sense-antisense PFP α gene in pHannibal, (4) cloning of sense-antisense constructs from pHannibal into pET-17b.

Figure 1. Restriction analysis of pJ204 vector digested by EcoRI. Lane 1, Marker ; 2, pJ204 uncut ; 3, pJ204 cut

HEAD OF RESEARCH TEAM : Dr. Sony Suhandono
TEAM MEMBERS : Dewi Murni and Tati Kristianti
OFFICIAL ADDRESS : School of Life Sciences and Technology, Institute of Technology, Bandung, Indonesia
EMAIL : sony@sith.itb.ac.id


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