Molecular Mechanism Of TSHR (G558R) Mutation Regulating Heat Stress Response In Chickens

The purpose of this experiment is to screen the candidate genes that regulate the heat stress response in chickens,and to study the molecular mechanism of the G558 R mutation in the TSHR gene regulating the heat stress response in chickens.First download tropical and temperate local chickens from the database(5 tropical chickens totaling 30,3 temperate chickens totaling 26),using comparative population genomics(Fst and Pi)to detect positive selection genes in tropical chickens,and screen out TSHR Gene,this gene has a G/A mutation at 41020238,causing the 558 th glycine to be mutated to arginine.In order to further verify the role of TSHR gene G558 R mutation in regulating chicken heat stress response,TSHR gene mutant,wild-type and empty vector expression vectors were constructed and transfected into fibroblasts and cultured at 28°C,37°C and 43°C,respectively 5h.The CCK8 method was used to determine the cell viability at different culture temperatures,and at the same time,the RNA of the cells at a culture temperature of 37°C was extracted to build a library for RNA-seq,and the differentially expressed genes of different genotype vectors were analyzed.Finally,it was verified in vivo that the G558 R mutation of TSHR gene regulates the heat stress response of chickens.9 and 81 male and female males and females of Huainan Ephedra chickens with heterozygous individuals at TSHR gene mutation sites were selected and bred according to the male-female ratio of 1:9.About 500 breeding eggs were collected and pedigree hatched.Divide the test population into mutant group(n=64),wild-type group(n=54)and heterozygous group(n=128),record body weight and body size.At the age of 20 weeks,30 males were randomly selected from each of the mutant group and the wild type group,and they were divided into a mutant heat stress group,a mutant control group,a wild type heat stress group,and a wild type control group.15 animals,the heat stress condition was 33±1℃(20:00-8:00 the next day),and the slaughter performance and meat quality were determined.At the same time,the blood of mutant and wild-type chickens before and after heat stress was collected,and the blood biochemical indexes were measured.To study the differences in heat stress response of different genotypes of chickens.The results showed that there were 34 genes subject to positive selection in tropical chickens,and missense mutations occurred in the TSHR gene.In the cell-level verification test,it was found that although the cell viability of the mutant group was significantly lower than that of the wild-type group(P<0.05)at the treatment temperature of 28°C and 37°C,the cell viability of the mutant group was significantly lower at the treatment temperature of43°C.The cell viability was significantly higher than that of the wild-type group and the empty vector group(P<0.05).A total of 5 differentially expressed genes were screened for the wild-type group and the empty vector group,of which 2 genes were up-regulated and 3genes were down-regulated in the wild-type group;16 differentially expressed genes were screened for the wild-type group and the mutant group.All up-regulated in the mutant group.These genes are mainly enriched in the pathways of body temperature regulation,inflammation and lipid metabolism.In the live level verification test,the two groups of chickens had no significant differences in body weight,body size and slaughter performance(P>0.05).The a* of the wild type heat stress group was 21.0% lower than that of the mutant heat stress group(P<0.05).The drip loss and cooking loss of the wild type heat stress group and the mutant heat stress group were significantly higher than their control Group(P<0.05).The ALT,LDH and CK of the mutant heat stress group were significantly lower than those of the wild type heat stress group(P<0.05),and other blood biochemical indexes were not significantly different(P>0.05).In summary,TSHR gene G558 R mutation can be used as a candidate gene for regulating heat stress response in chickens.It may regulate heat stress response in chickens by upregulating cellular inflammatory response and heat shock protein-related gene expression.