| Toll-like receptors(TLRs)are a group of highly conserved,pattern-recognition receptors commonly found in animals and through specific recognition of pathogen-associated molecular patterns(PAMPs)activate downstream signaling pathways to induce the expression of anti-microbial,chemotactic,cytokine,and co-stimulatory molecules to participate in the body’s immune response.To get the polymorphism of TLRs in main sheep breeds in Guizhou Province,TLRs genes of Guizhou Black Goat,Guizhou White Goat,Qianbei Ma Goat,Boer Goat and Hu Sheep were taken as the research object and the sequence information of TLR1~TLR10 genes of different sheep breeds were obtained by cloning and sequencing.Single nucleotide polymorphism(SNP)of TLRs gene in different sheep breeds were analyzed by software of bioinformatics,protein secondary structure analysis,protein tertiary structure analysis and Protein functional domain analysis.Then the expression of TLRs genes in the blood samples and lungs samples of different sheep breeds were detected by Taq Man real-time fluorescence quantitative PCR method and the expression quantities of TLRs were analyzed.The results of this research will provide basic data for the correlation between the TLRs polymorphism and disease susceptibility.1.Study on TLRs gene polymorphism of different sheep breeds:The technologies of gene amplification,cloning,sequencing and sequence analysis were used to get the information of TLRs genes and genes polymorphism from blood samples of Guizhou Black Goat,Guizhou White Goat,Qianbei Ma Goat,Boer Goat and Hu Sheep.TLRs gene nucleotide homology analysis of different sheep breeds showed that TLR1~TLR10 genes homology of 4 goat breeds were above 98.7% and the homology of TLR1~TLR10 genes between 4 goat breeds and Hu Sheep were 97.7%~99.8%.Phylogenetic tree analysis showed that TLR1~TLR10genes of the 4 goat breeds and Hu Sheep were in different branches.Sequence analysis showed that 184 missense mutations were found in TLR1~TLR10 genes of 5 sheep breeds,including 86 missense mutations.A total of 28 SNPs were found in goat intraspecific,including 9 missense mutations,distribution in TLR2,6,7,8,9 genes.78 SNPs were found in Hu sheep,including 44 missense mutations and 78 SNPs were found in 4 goat interspecific,Containing 33 missense mutations and distribution in TLR1~6 genes.The changes of protein secondary structure,protein tertiary structure and Protein functional domain before and after TLRs missense mutations were analyzed by online server.The results showed that the protein secondary structure of TLR1~TLR10 between goat breeds and Hu sheep were all different and 80 missense mutations caused changes in the secondary structure of TLRs.The analysis of the TLRs genes TIR domain protein tertiary structure revealed that there were difference in the TLR1,2 and 6 between goats and Hu sheep.Protein tertiary structure changes were found at the goats TLR2 gene missense mutation site A1922G(Tyr→Cys),the goats TLR5 missense mutation site A2198G(Asn → Ser)and the Hu sheep TLR7 missense mutation site A3056G(Glu → Gly).TLRs genes Protein functional domain analysis showed there were difference in the TLR1,4,5 and 10 genes between goats and Hu sheep and 13 missense mutations caused changes in the molecular structure of TLRs.2.Study on the TLRs expression polymorphism of Different breeds:Based on the sequence of sheep TLR1~TLR10 genes,specific primers and Taq Man probes were designed to construct real-time fluorescence quantitative PCR for detecting TLRs m RNA.The expression of TLRs m RNA in blood samples and lung samples from 5 sheep breeds were detected using those established methods and the expression polymorphism of TLRs m RNA in different sheep was analyzed.The results showed that those established real-time fluorescence quantitative PCR methods could be used for the quantitative detection of sheep TLRs m RNA expression,all of them have good stability.The detection results of Taq Man real-time fluorescence quantitative PCR methods for the expression of sheep TLR1~TLR10m RNA showed the TLR1~TLR10 m RNA were expressed in 50 blood samples and 50 lung samples from 5 sheep breeds.The expression of TLR1 m RNA in Hu Sheep blood samples was significantly higher than those of the other 4 goat breeds(p<0.01)and the expression of TLR1 m RNA in lung samples of Guizhou White Goat was significantly higher than those of the other 4 sheep breeds(p<0.01).The TLR2 m RNA expression in blood samples of Guizhou White Goat,Qianbei Ma Goat and Hu Sheep was significantly higher than those of the other 2goat breeds(p<0.01)and the expression of TLR2 m RNA in lung samples of 4 goat breeds was significantly higher than that of the Hu Sheep(p<0.01).The TLR3 m RNA expression in Hu Sheep blood samples was significantly higher than those of the other 4 sheep breeds(p<0.01)and the expression of TLR3 m RNA in lung samples of 4 goat breeds was significantly higher than that of the Hu Sheep(p<0.01).The TLR4 m RNA expression in blood samples of Guizhou White Goat was significantly higher than those of the other 4 sheep breeds(p<0.01)and the TLR4 m RNA expression in the lung samples of 4 goat breeds was significantly higher than that of the Hu Sheep(p<0.01).The expression of TLR5 m RNA in blood samples from Guizhou White Goat and Qianbei Ma Goat was significantly higher than those of the other 3sheep breeds(p<0.01)and the expression of TLR5 m RNA in lung samples from Qianbei Ma Goat was significantly higher than those of the other 4 sheep breeds(p<0.01).The expression of TLR6 m RNA in the blood samples of Guizhou White Goat and Boer Goat was significantly higher than those of the other 3 sheep breeds(p<0.01)and the expression of TLR6 m RNA in the lung samples of Guizhou Black Goat,Qianbei Ma Goat and Boer Goat was significantly higher than those of the other 2 sheep breeds(p<0.01).The expression of TLR7 m RNA in blood samples of Guizhou White Goat was significantly higher than those of the other 4 sheep breeds(p<0.01)and the expression of TLR7 m RNA in lung samples of Boer Goat was significantly higher than those of the other 4 sheep breeds(p<0.01).The expression of TLR8 m RNA in the blood samples of Guizhou White Goat was significantly higher than those of the other 4 sheep breeds(p<0.01)and the expression of TLR8 m RNA in the lung samples of the Qianbei Ma Goat was significantly higher than those of the other 4 sheep breeds(p<0.01).The expression of TLR9 m RNA in the lung samples of Qianbei Ma Goat was higher than in the Boer Goat(p<0.05)and significantly higher than those of the other 3 sheep breeds(p<0.01).The expression of TLR10 m RNA in blood samples and lung samples of Boer Goat were significantly higher than those of the other 4 sheep breeds(p<0.01).Overall,TLR2,4,7,8 m RNA expression in blood samples were higher than the remaining TLRs,and TLR2,3,4,5,8 m RNA expression levels in lung samples were higher than the remaining TLRs.In summary,The TLRs genes of sheep have abundant polymorphisms,among which 106 SNPs are found in the intraspecific and interspecific of goat breeds.There were 42 were missense mutations and 16 missense mutations caused the Toll-like receptor proteins secondary structure,tertiary structure and molecular structure polymorphism;TLRs m RNA were expressed in both blood samples and lung samples and the expression quantities were polymorphic.The TLR2,4,7 and 8 m RNA had higher expression in the blood samples and the TLR2,3,4,5 and 8 m RNA had higher expression in the lung samples.The expression of TLR2、4、5、7、8 m RNA in Boer Goat blood samples was lower than those of the other 4 sheep breeds and the expression of TLR4,8 and 9 m RNA in Guizhou White Goat blood samples was significantly higher than those of the other 4 sheep breeds.The TLR5 and TLR8 m RNA expressions of lung samples in the Qianbei Ma Goat are higher than those of the other 4 sheep breeds.the expression of TLR2,3,4,5,6,8,9 m RNA in Hu Sheep lung samples were lower than those of the other 4 goat breeds. |
