| The local pigs in our country have distinct germplasm characteristics.The excellent stress resistance is one of the important germplasm characteristics of local pigs.However,due to the complexity of its phenotype,it is difficult to determine the traits and genetic composition.In order to reveal the potential relationship between the gene expression pattern of pig peripheral blood and different stress tolerance phenotypes in different genetic backgrounds,and to identify SNP loci with species characteristics.In the early stage,based on the analysis of the stress resistance phenotype,blood routine and serum immune index of Changbai Mountain with Min pig(shorted as crossbred wild boars),Min pig and Large White pig.Six individuals were used to compare the transcriptomics of peripheral blood with high-throughput sequencing method.The accuracy of the analysis was verified by qRT-PCR method.The genetic mechanism of stress-resistant phenotype was extracted from the perspective of system biology.The high-resolution melting curve method(HRM)was used to analyze the polymorphism of TNF-a gene promoter region in crossbred wild boars,Min pig and Large White pig and the correlation between the polymorphism and serum TNF-a content.Furthermore,the effect of polymorphic site of TNF-a gene promoter region on gene transcription was revealed By constructing dual luciferase vector,cell transfection and luciferase assay,it was confirmed that SNP in promoter region had an effect on TNF-a gene expression.The results show:1.Comparative transcriptomic analysis of peripheral blood showed that the Q30 values of all samples were above 87%.MDS analysis showed that the similarity of samples in each group was higher,and the differences between samples were larger.155 differentialy expressed genes were obtained by comparison between crossbred wild boars and Large White pigs 213 differentially expressed genes were obtained by comparison between Min pigs and Large White pigs.229 differentially expressed genes were obtained by comparison between crossbred wild boars and Min pigs.The GO annotation and KEGG pathway analysis showed that the differentially expressed genes in the crossbred wild boars population were mainly enriched in the signaling pathways related to the immune regulation mechanism.The qRT-PCR results showed that the differentially expressed genes obtained by RNA-Seq analysis in each group were relative.The trend of expression was consistent with the trend of expression of each gene measured by qRT-PCR.2.There are 7 single nucleotide polymorphic loci(SNPs)in the promoter region of TNF-a gene in pig populations with different genetic backgrounds.The G-1256 and G-1239 locus regions were selected by HRM analysis to show that there are 4 genotypes in the promoter region of TNF-a gene in three pig populations,among which CC genotype is the dominant genotype,and the genotype frequency is in the crossbred wild boars population.Up to 73.0%,followed by 52.0%for pigs and 31.0%for Large White pigs.The significant difference analysis showed that the serum TNF-a content of CC genotypes in crossbred wild boars and Large White pig was significantly higher than that of other genotype individuals(p<0.05).All pigs were regarded as a unified population,and the significant difference analysis also showed that the serum TNF-a content of CC genotype individuals was also significantly higher than other genotype individuals(p<0.05).Transcription factor binding site prediction revealed that the CC genotype G-1239 site A constitutes a potential transcriptional binding site for the NF-?B subunit RelB.However,the determination of different haplotypes by constructing the dual luciferase vector had no significant effect on TNF-a gene expression.In summary,this experiment compared the transcriptomics of peripheral blood of pigs with different genetic backgrounds and analyzed the single nucleotide polymorphisms of the TNF-a gene promoter region in different genetic background pig populations.The experiment laid the foundation for further study of the regulatory pathways affecting the stress traits of pigs,explaining the molecular mechanism of the formation of stress resistance traits and explaining the genetic mechanism of local pigs' high resistance to stress. |
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