Cloning and sequencing of hfq (host factor required for synthesis of bacteriophage Q beta RNA) gene of Salmonella Typhimurium isolated from poultry

Research (Published online: 14-05-2015)

9. Cloning and sequencing of hfq (host factor required for synthesis of bacteriophage Q beta RNA) gene of Salmonella Typhimurium isolated from poultry - Parthasarathi Behera, Muhammed Kutty, Bhaskar Sharma, Ajay Kumar and Meeta Saxena

Veterinary World, 8(5): 610-614

   doi: 10.14202/vetworld.2015.610-614

Abstract

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Aim: The aim was to clone and sequence hfq gene of Salmonella Typhimurium strain PM-45 and compare its sequence with hfq gene of other serovar of Salmonella.

Materials and Methods: Salmonella Typhimurium strain PM-45 was procured from the G. B. Pant University of Agriculture and Technology, Pantnagar, India. The genomic DNA was isolated from Salmonella Typhimurium. Hfq gene was polymerase chain reaction (PCR) amplified from the DNA using specific primers, which was subsequently cloned into pET32a vector and transformed intoEscherichia coli BL21 pLys cells. The recombinant plasmid was isolated and subjected to restriction enzyme digestion as well as PCR. The clone was then sequenced. The sequence was analyzed and submitted in GenBank.

Results: PCR produced an amplicon of 309 bp. Restriction digestion of the recombinant plasmid released the desired insert. The hfqsequence shows 100% homology with similar sequences from other Salmonella Typhimurium isolates. Both nucleotide and amino acid sequences are highly conserved. The submitted sequence is having Genbank accession no KM998764.

Conclusion: Hfq, the hexameric RNA binding protein is one of the most important post-transcriptional regulator of bacteria. The sequence of hfq gene of Salmonella Typhimurium is highly conserved within and between Salmonella enterica serovars. This gene sequence is probably under heavy selection pressure to maintain the conformational integrity of its product in spite of its being not a survival gene.

Keywords: cloning, hfq, RNA binding protein, sequencing, Salmonella Typhimurium.