Cloning Of TLR4 Gene And It's Signal Transduction Gene, SNPs Detection And Association Analysis Of TLR4 Gene With Mastitis In Dairy Cattle

Mastitis is one of most prevalent and costly diseases in dairy cattle housing, and the incidence rate is very high(25%-60%), which is mainly caused by a broad spectrum of bacterial and pathogens. The toll-like receptors (TLRs) is a family of cell-surface signalling molecules that play a fundamental role in the immune response to recognize pathogens, which bind to specific pathogen-associated molecular patterns (PAMPs). TLR4 is a member of TLRs, and recognizes a class of PAMPs presented by lipopolysaccharides and lipoteichoic acid, a cell-wall component of Gram-negative bacteria, and which induces the over expression of inflammatory factors by signal transduction, then confer disease resistance in body. In order to explore molecular mechanism on mastitis resistance, the bovine TLR4 gene was researched as a candidate gene of mastitis resistance for Chinese Simmental, Sanhe cattle and Chinese Holstein in this study. The isolation, identification and bioinformatics analysis of cDNA sequence for bovine TLR4 gene and its signal transduction genes (IRAK1, IRAK2, IRAK-M, IRAK4, TRAF6, TOLLIP and TIRAP) and amino acids sequence deduced by the cDNA were performed by molecular biology and bioinformatics approaches. The genomic DNA of bovine TLR4 gene was isolated and the SNPs were detected. Afterwards, the association between four SNPs of TLR4 gene and mastitis was analyzed with general linear model (GLM) procedure using SAS software. The main results were as follows:1. The complete cDNA sequences of TLR4, IRAK2, IRAK-M and IRAK4 gene were cloned and identified by combining the e-PCR, RT-PCR and RACE methods,. Many alternative splices were discovered, of which, two for IRAK2, three for IRAK-M and two for IRAK4.2. The main cDNA sequences and partial UTR sequences of IRAK1, TRAF6, TOLLIP and TIRAP, of which contained complete CDS, were cloned and identified.3. The tissues expression of TLR4, IRAK1, IRAK2, IRAK-M, IRAK4, TRAF6 and TOLLIP gene were detected in 11 tissues such as mammary gland, liver, muscle, duodenum, adipose, uterus, kidney, heart, lung, pancreas and ovary by RT-PCR, the results showed that the expression of TLR4, IRAK1 and IRAK4 gene were detected in all 11 tissues, but the expressions of IRAK2 gene in muscle tissue, IRAK-M in adipose tissue, TOLLIP in muscle, duodenum, uterus and lung tissues were not detected.4. The amino acids sequence deduced by ORF of TLR4 cDNA was analyzed and the character and protein domain were predicted by bio-informatics, the results showed bovine TLR4 protein was consisted of 841 amino acids, in which 1-23 amino acids were signal peptide, 635-657 amino acids were transmembrane helices structure, that was to say 1-634 amino acids were outside of membrane, and 658-841 amino acids were inside of membrane; the TLR4 protein contained LRR, LRR-TYP, LRRCT and TIR domain, and the function was inferred that bovine TLR4 protein might induce immune response and conferred to disease resistance of pathogens infection by signal transduction pathway.5. The character and function prediction analysis of amino acids sequences deduced by cDNA of IRAK1, IRAK2, IRAK-M, IRAK4, TRAF6, TOLLIP and TIRAP indicated that they were the main proteins in TLR4 signal transduction pathways. IRAK2a and IRAK2b were the alternative splices of IRAK2, the former contained death domain, but the latter don't, so we inferred that the function of IRAK2a and IRAK2b might be different for TLR4 signal transduction according to function of death domain.6. The complete DNA of bovine TLR4 gene was isolated, then the SNPs were detected by sequencing, the results indicated that there are total of 31 SNPs in TLR4 DNA, of which 2 in 5′UTR, 4 in exon1, 11 in intron 1, 1 in exon2, 2 in intron2 and 11 in exon3.7. The population genetic polymorphisms of the sites for 5′UTR(G/C mutation), exon2 -24(G/C mutation), T4CRBR2(T/C mutation) and TIR2AA(C/T mutation) in Chinese Simmental, Sanhe cattle and Chinese Holstein were detected by RFLP, SSCP and CRS-PCR. The result showed that the mutations of all sites detected in all populations were moderate polymorphism except that Chinese Simmental was a lowest polymorphism in TIR2AA site.8. Meanwhile, the effect of polymorphism of TLR4 gene on somatic cell score (SCS) indicated that the cattle with AA genotype in exon2-24 and TIR2AA showed lower somatic cell score than that of other genotypes (P<0.05). In short, the allele A might play an important role in mastitis resistance in cattle. But somatic cell score of cattle with different genotypes in 5′UTR and T4CRBR2 showed no significant difference (P>0.05).