Molecular cloning and characterization of glucose transporter 1 (glut1) and citrate synthase cDNA in buffalo (Bubalus bubalis)

Abstract

Glucose transporter type-1 (glut1) and citrate synthase plays crucial role in glucose transport and regulation of tricarboxylic acid cycle (TCA) cycle in mammalian energy metabolism. The present study was aimed to clone and characterize glut1 and citrate synthase cDNA in water buffalo (Bubalus bubalis). Total of 90 cumulus oocyte complexes (COCs) were used for mRNA isolation and reverse transcribed to cDNA, which was further used in polymerase chain reaction (PCR) amplification of glut1 and citrate synthase. PCR products of glut1 and citrate synthase were cloned by T/A cloning using pGEM-T easy vector and further  sequenced. Gene sequence analysis of glut1 and citrate synthase revealed that they have open reading frame of 1479 (encoding 492 aa) and 1401 bp (encoding 466 aa), respectively. Further phylogenic analysis of gene and deduced amino acid sequences suggests that bubaline glut1 shares ∼ 89 to 98% and ∼ 97 to 99% similarity at nucleotide and amino acid level respectively  whereas citrate synthase shared ∼ 89 to 99% at nucleotide and ∼ 96 to 99% at amino acid level respectively with other domestic species and human. Predicted protein structures of buffalo glut1 protein accentuate the presence of crucial amino acids involved in glucose transport moreover the essential catalytic residues are highly conserved in buffalo citrate synthase.

Keywords: Buffalo, cloning, characterization, Glut1, citrate synthase.