Sequence Characterization and Phylogenetic analysis of TLR4 Gene in Vechur Cattle

Vechur cattle, an indigenous breed of Kerala are highly disease resistant. Toll-like receptors (TLRs) perform a vital role in disease resistance through their recognition of pathogen associated molecular patterns (PAMPs). In this study, the mRNA of Toll-like receptor 4 (TLR4) gene of Vechur cattle was sequenced, characterized and compared with other species. Total RNA from milk somatic cells of Vechur cattle was isolated and converted into cDNA using reverse transcriptase kit. The cDNA of TLR4 gene was amplified with the designed primers and the product was sequenced by primer walking technique. The mRNA sequence of TLR4 spans 2766 bp with an open reading frame (ORF) of 2526 bp coding for 841 amino acids. TLR4 sequence of Vechur cattle with Bos taurus sequence showed 99 per cent homology and exposed 7 nucleotide variations (4 non-synonymous and 3 synonymous substitution). The ectodomain of Vechur TLR4 displayed 13 LRRs and change in amino acid at LRR5 domain was observed when compared to Bos taurus. Leucine (16.05 per cent) and serine (8.08 per cent) were found to be major amino acids contributing to primary structure of TLR4 protein. Secondary structure displayed portions of amino acid sequence contributing to formation of alpha helix (50.18 per cent), beta turn (17.60 per cent) and random coil (32.22 per cent). Tertiary structure of TLR4 protein revealed distinctive horseshoe fold, composed of repeated strand-turn-alpha helix structure. Phylogenetic tree of TLR4 gene showed that Vechur cattle closely related to Bos taurus. The observed structural variation in the mRNA sequence of TLR4 gene in Vechur cattle breed will provide an important basis for further study on the relationship between polymorphisms and host disease resistance.