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. Key words: Buffalo, cloning, characterization, Glut1, citrate synthase.
Highlights
Buffalo (Bubalus bubalis) constitutes the major pillar of the dairy and meat industry of India contributing a large share to agricultural gross domestic product (GDP), maintenance and improvement of such an important domestic species requires an elaborate database
A total 90 cumulus oocyte complexes collected from 98 buffalo ovaries at 1.12/ovary recovery rate were utilized for RNA isolation (Figure 1)
The quality of cDNA was assessed by polymerase chain reaction (PCR) amplification of housekeeping gene, βactin and a solitary intact band was observed under UV transilluminator (Figure 2E)
Summary
Buffalo (Bubalus bubalis) constitutes the major pillar of the dairy and meat industry of India contributing a large share to agricultural gross domestic product (GDP), maintenance and improvement of such an important domestic species requires an elaborate database. Citrate synthase is a key regulatory metabolic enzyme that catalyzes the first step in tri-carboxylic acid (TCA) cycle, a potential regulator of aerobic energy production in highly dividing cells and is present in virtually all the cells capable of oxidative metabolism, a critical enzyme in cellular biosynthesis (Weitzman et al, 1976). Porcine cardiac tissue derived form of citrate synthase is most widely studied (Bloxham et al, 1981 and Evans et al, 1988) and it has been isolated from numerous sources. Most common form of citrate synthase consists a dimer of molecular weight 90,000 to 100,000 (Srere, 1975; Singh et al, 1970) a tetrameric form with larger subunit of molecular weight (55,000 to 60,000) was reported in some prokaryotic organisms (Weitzman, 1976). Bovine heart derived citrate synthase cDNA sequence encodes a 466 amino acids containing protein
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