Molecular Characterization, Expression And Phylogenetic Analysis Of Toll-like Receptor Gene Family In Goats

In order to explore molecular mechanism on disease resistance,the gene family of goat Toll-like receptors（TLRs） was chosen as candidate genes in this study.The isolation, identification and bioinformatics analyses of partial coding sequences（CDS） of 10 goat TLRs including TLR1 through TLR10 were completed and their SNPs were detected.The expression profiles of the 10 TLRs in eight tissues were examined.Correlation between performance traits and blood physiological indexes was investigated using a Boer goat population.The major results are as follows:1.Partial CDS of the 10 goat TLRs were PCR cloned and sequenced;and 13 SNPs were detected.Alignment results showed that the nucleotide sequences of goat TLRs were at least 79%identical to those of human.Also,the identity of each cloned goat TLRs to horse and bovine homologous sequences ranged from 81%to 98%.The deduced amino acid sequences of goat TLR proteins were from 69%to 97%identical to their counterparts in human,bovine and horse.Evolutionary relationship of goat TLRs with those of other mammals indicated that the goat and bovine TLRs were closely related to each other.2.Nine SNPs（TLR1₇8C＞T,TLR1₇9C＞A,TLR1₉0A＞G,TLR6₁36A＞C, TLR6₃16T＞C,TLR10₇6C＞A,TLR10₈1G＞A,TLR10₁47C＞T and TLR10₂47C＞T） were identified within the exons of TLR1,6 and 10 genes,which were mapped in the same chromosome region,in 160 individuals from 18 Chinese and Iranian indigenous goat breeds.The genotype distributions of four SNPs including three SNPs in TLR1 and the 147/C＞T in TLR10 were at Hardy-Weinberg equilibrium（P＞0.05） based on tests using THESIAS.However,the D’ values as a measure of linkage disequilibrium were 0.93 for the two SNPs of 136C＞A and 316T＞C in TLR6,1.00 for the two SNPs of 79C＞A and 90A＞G in TLR1,0.93 for the two SNPs of 76A＞C and 81A＞G and 0.85 for the two SNPs of 147C＞T and 247T＞G in TLR10,indicating their significant linkage disequilibrium.The phylogenetic tree reconstructed using the partial CDS of TLR1,6 and 10 consisted of two clusters with the Chinese Jining Grey goat different from the remaining 17 goat breeds.However,the four Iranian goat breeds were grouped into different sub-clusters:BAF was in the same sub-cluster of Arbas White Cashmere goat, GAB had a relatively close relationship with Chuandong White goat,Huai goat,Haimen goat,Matou goat and Suining goat,while the ABD and KHA were closely related to Xiang goat,Yichang White goat,Wu goat,Hainan Black goat,Nanjiang Yellow goat, Black goat and Boer goat. 3.The expression profiles of goat TLRs were examined in eight tissues including heart,liver,spleen,lung,kidney,muscle,lymph node and small intestine using reverse transcriptase polymerase chain reaction（RT-PCR）.The results showed that all of the 10 goat TLR mRNAs were expressed at reasonably high levels in the spleen as an organized immune compartment,TLR2 and 6 were expressed ubiquitously in different tissues,while the expression of the TLR10 was restricted only in spleen.4.Records of performance traits and blood samples of a Boer goat population were collected.The results showed that the EOS%was positively correlated with birth weight and body height（P＜0.05）,the MPV had a significant and positive correlation with body length and height at birth,body weight,length,height and heart girth at weaning,and the PDW had also significant and positive correlation with body weight,length,height and heart girth at weaning（P＜0.01）.However,the WBC was negatively correlated with weaning date,the LYM# was negatively correlated with body weight,height and heart girth at birth,and the RBC was also negatively correlated with body length,height and heart girth at birth and body weight at weaning（P＜0.05）;while the WBC had a significant and negative correlation with body weights,lengths,heights and heart girths at both birth and weaning（P＜0.01）.