Characterization of pyrophosphate-dependent phosphofructokinase α-subunit gene from sugarcane showing gene without intron

Abstract

Abstract Pyrophosphate-dependent Phosphofructokinase or PFP is an enzyme that regulate sucrose metabolism. It consists of α- and β-subunits, which encoded by PFPα and PFPβ genes, respectively. Sugarcane PFPa has a strong function in glycolysis and has the potency to be engineered to increase sugarcane yield. Hence, the purpose of this work was to isolate, clone and characterize the sugarcane PFPα gene. Total RNA was isolated from leafrolls of TD 91 sugarcane variety. cDNA synthesis followed by DNA amplification of PFPα gene were performed using degenerate primers. cDNA and DNA fragments were ligated into pGEM-T Easy vector, which were subsequently introduced to E. coli competent cells. EcoRI were used to cut the plasmids for sequencing. Finally, homology searching was conducted using BLASTn, and then the nucleotide sequence was translated to a protein sequence using Bioedit. The results showed that PFPα cDNA fragment was 900 bp in length. The translated PFPa protein showed binding sites for fructose-6-phosphate and fructose-1,6-biphosphate, which are conserved in all family members of PFP. In silico analysis of the DNA fragment showed gene without intron. In conclusion, the PFPα gene from sugarcane has been successfully isolated, cloned and characterized.