Molecular characterization and expression analysis of interferon γ (IFN-γ) gene in Labeo rohita (Ham.)

Abstract

Genes coding for interferon γ (IFN-γ) has been cloned and studied for functional analysis in many species. Labeo rohita commonly known as rohu, is a commercially important and widely cultured fish in South-East Asia. IFN-γ of rohu was amplified with primers based on conserved regions of other related species. 5′ and 3′ end were amplified using RACE PCR. The PCR products were cloned and sequenced. The sequence analysis of IFN-γ revealed that the genomic sequence consists of 1803 bp with 3 introns and 4 exons. An ORF of 552 nucleotides encodes for a protein of 183 amino acids. Matured IFN-γ protein has 157 amino acids with a signal peptide of 26 amino acids. Mature protein has a molecular weight of 18.7 kDa. Four RNA instability regions (ATTA) and one polyadenylation signal (AATAAA) were seen in 3′ UTR region. IFN-γ signature sequence, one glycosylation site and nuclear localisation region were also observed. Intron splicing motif (ag/gt) was observed in genomic DNA. The gene sequence shows high similarity with that of Cyprinus carpio. The expression analysis for IFN-γ in kidney, spleen, liver and gill was done by Real Time PCR. Expression of IFN-γ was more in spleen compared to gill, liver and kidney. Cultured kidney cells were induced with poly I:C and samples were collected at 2, 8, 24 and 48 h post-induction to study the effect on poly I:C of expression of IFN-γ. IFN-γ expression increased 30 fold at 2 h post- induction and increased further till 24 h.

Keywords

• Interferon γ
• Characterization
• Expression analysis