Cloning and SNP screening of the TLR4 gene and the association between its polymorphism and somatic cell score in dairy cattle

Abstract

Toll-like receptor 4 (TLR4) is a cell-surface receptor that activates innate and adaptive immune responses. Recognizing a broad class of pathogen-associated molecular patterns (PAMPs), TLR4 is implicated as a receptor mediating cellular activation in response to bacterial lipopolysaccharide (LPS), a cell-wall component of gram-negative bacteria, which is the principal PAMP of TLR4. TLR4 is a candidate gene for resistance to a large number of diseases. Human models, in particular, suggest TLR4 to be a candidate gene associated with resistance to sepsis, immunodeficiencies, atherosclerosis and asthma. In this study the bovine TLR4 mRNA and genomic DNA were sequenced and their gene structures were determined through alignment of the genomic DNA sequence to the mRNA sequence. Its mRNA sequence consisted of a 2526-bp open reading frame (ORF) flanked by a 470-bp 5′-untranslated region (UTR) and a 743-bp 3′-UTR; and its genomic DNA is 11,013-bp in length and includes the three exons and two introns. Thirty-one single nucleotide polymorphisms (SNPs) were detected by sequencing polymerase chain reaction (PCR) products from 15 cattle, consisting of five randomly selected individuals from Chinese Simmental, Holstein and Sanhe cattle breeds each. Sixteen of these SNPs were in coding regions (cSNP). However, the 3′-untranslated region was comparatively conservative and no SNP was found in it. The SNP (g.8664C>T) found in the Toll IL-1 receptor (TIR) domain of the bovine TLR4 gene leads to amino acid substitution from Thr (C allele) to Ile (T allele). Three hundred and ninety seven cows from Chinese Simmental, Holstein and Sanhe cattle breeds were genotyped for SNP (g.8664C>T) using the method of created restriction site PCR together with restriction fragment length polymorphism (RFLP) by Hinf I. An association study between the SNP and somatic cell score (SCS) in the three breeds showed that the SCS of individuals with a CC genotype was significantly lower than that of the TT genotype.

Keywords: Cattle, TLR4, SNP, mastitis, SCS, somatic cell count

South African Journal of Animal Science Vol. 38 (2) 2008: pp. 101-109