Study On The Regulation Of CaN Gene On CFL2 Gene Expression And Meat Quality In Jiangkou Luobo Pigs

CFL2 gene is the main coding gene of cofilin,a member of actin depolymerization factor（ADF/cofilin）family,in skeletal muscle.Cofilin has the function of regulating actin assembly and plays an important role in muscle development and muscle regeneration.Therefore,CFL2 gene may have a direct impact on meat quality of livestock and poultry.Calcium regulated phosphatase（Ca N）is a kind of protein phosphatase regulated by Ca²⁺,which is an important upstream regulatory molecule of cofilin and a heterodimer composed of catalytic subunit（Cn A）and regulatory subunit（Cn B）.In muscle tissue,Cn A and Cn B are mainly encoded by PPP3CA and PPP3R1 genes.As an effector,Ca N is involved in the regulation of various cell functions,and has been proved to have an important influence on the type transformation of muscle fibers,and has a regulatory effect on the meat quality of livestock and poultry.In this study,Jiangkou radish pig,a good local breed in Guizhou Province,was used as the experimental object.The SNPs of PPP3R1,PPP3CA and CFL2 genes were detected by direct sequencing and bioinformatics analysis was carried out.Genotyping of each site.The relative expression of three genes in the longissimus dorsi muscle and 11 meat quality indexes were measured to research the correlation among SNPs,meat quality indexes and gene expression and the relationship between PPP3R1,PPP3CA and CFL2 gene expression.In addition,the gene expression in 9 main tissues of Jiangkou radish pigs of different months were measured to study the expression of PPP3R1,PPP3CA and CFL2 genes on the time and space.The main results are as follows:1.Two SNPs were detected in CFL2 gene,CFL2-A-397G and CFL2-G163C,which were located in promoter and 5’UTR respectively.CFL2-A-397G increased the number of transcription factor binding sites.CFL2-G163C can enhance m RNA secondary structure stability.The meat color of wild type（AA）at CFL2-A-397G locus was significantly higher than that of heterozygous type（AG）,and the quality of wild type pork was better than that of heterozygous type.The expression of CFL2gene in longissimus dorsi was significantly negatively correlated with meat color and significantly positively correlated with shear stress.The variation of CFL2-A-397G locus will result in the increase of CFL2 gene expression and the decrease of PPP3R1gene expression.The expression of CFL2 gene was the highest in the heart,followed by the longissimus dorsi,psoas major,duodenum and stomach,and lower in the lung,spleen and kidney.The expression of CFL2 gene in the longissimus dorsi,duodenum,stomach,heart and psoas muscle increased wide with age,and in the liver increased slightly with age.2.Three SNPs were detected in PPP3R1 gene,PPP3R1-G-1119T,PPP3R1-A-1134C and PPP3R1-C-1310T,all located in the promoter.PPP3R1-A-1134C is located in a predicted high core promoter region,and in a promoter marker TATA frame.The meat color of wild type（AA）at PPP3R1-A-1134C was significantly higher than that of heterozygous type（AC）and mutant type（CC）,and the quality of wild type pork was better than that of other two genotypes.The mutation of PPP3R1-A-1134C resulted in the down-regulation of gene expression,and the expression of mutant（CC）was significantly lower than that of wild type（AA）.The expression of PPP3R1 gene in skeletal muscle was the highest,significantly higher than that in other tissues;the expression of PPP3R1 gene in heart was the lowest,significantly lower than that in other tissues;there was no significant difference in the expression of PPP3R1 gene in other tissues.It can be seen in the longissimus dorsi,duodenum,stomach and psoas major muscle that the trend of increasing with the age of months.In duodenum,there was significant difference in the amount of expression in different months.3.Three SNPs were detected in PPP3CA gene,PPP3CA-A-918C is located in the promoter,PPP3CA-G154767A and PPP3CA-G316451A are located in the exon,which are synonymous mutations.PPP3CA-A-918C site predicted to be located near the core promoter region,PPP3CA-G316451A site had no effect on the secondary structure of m RNA,PPP3CA-G154767A site would enhance the stable type of secondary structure of m RNA.The marbling scores of wild type（GG）and heterozygous type（GA）at PPP3CA-G316451A site were significantly higher than those of mutant type（AA）.The expression of PPP3CA gene in the longissimus dorsi was positively correlated with p H₂₄.The expression of PPP3CA gene in skeletal muscle and spleen was higher than that in other tissues,followed by spleen.Except for the relatively low level of kidney,there was no significant difference in other tissues.The expression in the longissimus dorsi,the heart and the psoas maximus increased significantly with the age of the month,while that in the spleen increased slightly with the age of the month,but the increase in the age of the month became smaller and smaller.4.There was a significant negative correlation between the expression of CFL2and PPP3R1 in the longissimus dorsi muscle.Moreover,when the expression of CFL2 gene is significantly increased,the expression of PPP3R1 gene is significantly decreased,but when the expression of PPP3R1 gene is significantly decreased,there is no significant difference in the expression of CFL2 gene,indicating that the change of CFL2 gene expression has a certain impact on the expression of PPP3R1 gene,while the change of PPP3R1 gene expression has no impact on the expression of CFL2 gene.