| Jiangkou Luobo Pig is an excellent local livestock and poultry breed in Guizhou Province.It has the characteristics of delicate meat and rich flavor.Therefore,the screening of effective molecular markers is of great significance for the selection and breeding of Jiangkou Luobo pigs.STAT3 is an important transcriptional activator in the JAK-STAT pathway and plays an important role in regulating lipid metabolism.UCP2 and UCP3,as members of the uncoupling protein family,have a significant impact on the energy metabolism patterns of animal organisms,so they are all As an important candidate gene for improving production performance and meat quality of livestock and poultry.In this study,Jiangkou Luobo pigs were used as experimental materials to screen SNPs by direct sequencing of mixed DNA pools,andbioinformatics analysis,population genetic parameter analysis and linkage disequilibrium analysis were performed.Direct sequencing was used to classify individual genotypes of SNPs,and Genotypes and correlation analysis between each genotype and meat quality traits was conducted.At the same time,At the same time,qPCR was used to detect the expression of different tissues and different months of age in each gene.The correlation between the expression of these three genes in the longissimus dorsi muscle and meat quality traits was also analyzed.The main results are as follows:1.A polymorphic site(STAT3-C56621T)was screened in the STAT3 gene,located in the 16 exon,which is a synonymous mutation.Bioinformatics analysis showed that the mutation site had a great influence on the conformation of secondary structure of RNA.The minimum free energy increased,and the structural stability decreased.Only mutant TT and heterozygous TC were found in the C56621 T site of the STAT3 gene in the sample population,where T is the dominant allele with a gene frequency of 0.7430.The correlation between genotype and meat quality analysis found that the TT type of Jiangkou Luobo pigs marble and cooked meat rate was significantly higher than TC type,TT type shear force and water loss rate was significantly lower than TC type pig,overall TT type pork quality is good For TC type pigs.The expression analysis of STAT3 gene in different tissues and different ages of Jiangkou Luobo pigs showed: Compared with different tissues,the expression levels in lung,duodenum and spleen were significantly higher than those in other tissues.The expression of STAT3 gene in the longissimus dorsi of Jiangkou Luobo pig was positively correlated with intramuscular fat content,marbling score and cooked meat rate,and negatively correlated with shear force.The expression of STAT3 gene in the longissimus dorsi muscle of mutant TT individuals was significant higher than heterozygous CT.At different ages,the expression levels of STAT3 gene in the longissimus dorsi,psoas muscle,lung and duodenum were not related to the age of the kidney,stomach,liver and heart.The growth of STAT3 gene in the longissimusdorsi of Jiangkou was positively correlated with intramuscular fat content,marble stain score and cooked meat rate,and negatively correlated with shear force.The expression of STAT3 gene in the dorsal long muscle of mutant TT individuals was significant.Higher than heterozygous CT.The expression of STAT3 gene in kidney,stomach,liver and heart was not related to the age of month.The expression of STAT3 gene in longissimus dorsi,psoas major,lung and duodenum increased with the age of month.2.Six SNPs were screened on the UCP2 gene,three of which were located in the promoter and the other three were located in the 3’UTR region.A 17 bp insertion sequence was found in the UCP2 gene promoter sequence of Jiangkou Luobo pig compared with the Genbank reference sequence,bioinformatics analysis indicated that three polymorphic loci and 17 bp inserts on the promoter may result in increased binding efficiency of UCP2 gene transcription factors.The three in the 3’ UTR regions affect the mRNA secondary structure stability to varying degrees.Linkage disequilibrium analysis of UCP2 gene in sample population showed that there was strong linkage disequilibrium between Promoter-T-366 A,Promoter-G-161 C,UCP2-T5728 G,UCP2-T5774 G,UCP2-C5878 T except Promoter-T-366 A and UCP2-C5878 T sites The genotype and meat quality correlation analysis showed that the Promoter-T-366 A site TA type,Promoter-G-161 C site GC type,UCP2-C5878 T site CT type,the shear force difference was significantly higher than other genotypes.The UCP2-T5728 G and UCP2-T5774 G sites are completely linked,and both TT-type marbled patterns are significantly higher than the TG-type.The expression of UCP2 gene in different tissues and different ages of Jiangkou Luobo pigs showed that the expression of UCP2 was highest in lung,liver and spleen,and the expression in kidney and heart was at medium level.The expression of UCP2 gene in the psoas muscle,duodenum and stomach is low.There was no significant difference between the duodenum and the stomach at different ages,and the rest of the tissues showed a trend of decreasing with the increase of the age.Correlation analysis between UCP2 gene expression and meat quality index in the longissimus dorsi of Jiangkou Luobo showed that UCP2 gene expression was significantlynegatively correlated with marbling score and intramuscular fat content,and was significantly positively correlated with shear force.The expression of heterozygous TA in the promoter-T-366 A site of UCP2 gene was significantly higher than that in wild-type TT,Promoter-G-161 C site heterozygous GC expression was significantly higher than mutant CC and wild type GG.The expression levels of heterozygous individuals at UCP2-T5728 G,UCP2-T5774 G,and UCP2-C5878 T were significantly higher than those of mutant and wild-type individuals.2.Six SNP sites were screened on the UCP3 gene,three in the 5’ UTR region and three in the 3’ UTR region.The UCP3-A3127 G and UCP3-C10439 T sites did not affect the secondary structure stability of mRNA.The A>T mutations in UCP3-C10441 A and UCP3-A10479G/T sites reduced the stability of mRNA secondary structure.UCP3-T3205 C and UCP3-A10479G/T sites A>G mutations enhance mRNA secondary structure stability.Among the UCP3-A10479G/T loci,there are two types of mutations: A>G-type conversion and A>T-type transversion,and 4 genotypes were detected,including wild-type AA,mutant GG and TT,and Heterozygous AG,the remaining sites showed three genotypes.The linkage disequilibrium analysis of UCP3 gene revealed that the UCP3-A3127 G and UCP3-T3205 C sites,UCP3-C10439 T and UCP3-G10466 A sites were completely linked unbalanced.Analysis of genotype and meat quality correlation showed that the marbling scores of different genotypes of UCP3-A10479G/T sites were significantly different.Marbling scores and intramuscular fat content mutants were significantly higher than wild type and heterozygous.There was no significant difference between other loci genotype and meat quality index.The expression of UCP3 gene in different tissues and different ages of Jiangkou Luobo pigs showed that the UCP3 gene in Jiangkou Luobo pig has the highest expression in the longissimus dorsi and psoas muscle,followed by the heart.The expression in the duodenum,lung,liver,spleen,kidney and stomach is extremely low.In the different ages of Jiangkou Luobo pigs,the expression of the longissimus dorsi and psoas muscles increased significantly with the increase of the age of the month,and the difference was significant;the expression levels in the duodenum,lung,liver,spleen,kidney and stomach were all low in each month.The expression level of UCP3 gene was significantly positively correlated with intramuscular fat content,and was significantly negatively correlated with shear force,and a strong positive correlation with the marbling score.In the UCP3-A10479G/T site,which had a significant effect on the marbling score,the expression levels of mutant GG and TT were significantly higher than those of heterozygous and wild type. |
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