| Pork is the main source of protein for humans and it is rich in protein and trace elements.Intramuscular fat(IMF)content is one of the most important factors influencing meat quality.IMF directly affects the colour,tenderness and flavour of the meat.The androgen,Testosterone(T)and Dihydrotestosterone(DHT)are among the main factors influencing fat deposition,which can regulate fat production by affecting the activity of adipogenic enzymes.Androgens are also a major contributor to the fishy smell of pork due to the presence of T,which gives pork an unpleasant odour.CYP3A29 is a pig-specific cytochrome enzyme that can metabolize androgens due to its 6β-hydroxylation activity.Structural variation(SVs)is closely related to the phenotype,and the Deletion is the main form of the structural variation.The presence of short interspersed nuclear elements(SINE)is the main way of causing the Deletion.The meat of Xiang pigs has the higher IMF,the better tenderness and juiciness.Previous laboratory studies have identified a sequence Insertion/Deletion structural variation at the 3’UTR of the CYP3A29 gene in Xiang pigs.The aim of this study is to investigate the relationship between the SV of CYP3A29 and the formation of the characteristic muscle tissue in Xiang pigs,and to provide auxiliary molecular markers and theoretical references for the study of the formation in the characteristic muscle tissue of Xiang pigs and the breeding and selection of pigs.The results of this study are as follows:1.Validation of the site at the SV and the population genetics by PCR and chi-square test.The results found that the SV of CYP3A29-3’UTR did exist and showed abundant polymorphism in both Xiang pigs and Large White pigs.And the II,ID and DD genotypes were detected in two pigs.The D allele frequency being significantly higher than the I allele in Xiang pigs,and the I allele frequency being significantly higher than the D allele in Large White pigs;The results ofX~2-test indicate that the SV in CYP3A29-3’UTR was satisfying the law of the Hardy-Weinberg(P>0.05)in the Xiang pigs,but not in the large white pigs(P<0.05).2.The effect of the SV on the m RNA expression of CYP3A29 gene was examined by q-PCR.And the results showed that the expression of m RNA in ID genotype was highly significant higher than that of II and DD genotype(P<0.01),Although there was no statistical difference between DD genotype and II genotype,DD was slightly higher than II.Using ELISA to detect the protein amount of the gene in different genotypes,the results showed that the ID genotype was highly significant higher than II and DD(P<0.01),and DD was slightly higher than type II,but there is no statistically different.3.The androgen(T,DHT)content of different genotypes in CYP3A29 was detected by ELISA,and the T and DHT content was correlated with the expression of CYP3A29 gene.The results of T concentration showed that ID genotype was significantly higher than DD and II(P<0.05),and The results of DHT concentration showed that DD genotype was highly significant than ID and type II(P<0.01);the T concentration was significantly correlated with genotype(P<0.01),but the DHT concentration was not(P>0.01).And there was no correlation between T and DHT.4.The analysis of bioinformatics in the SV using UCSC,mi RBase,PITA,and RBPsuite software.The results showed that the SV contained a SINE(241 bp)which belongs to the Pre0＿SSt RNA family;snd the SV may bind 33 mi RNAs and 13 RBP.5.By observing the fluorescence intensity of transfected cells,and the protein expression were detected by WB.The results showed that the fluorescence intensity and protein expression of EGFP in the p-EGFP-SINE+group were extremely significantly lower than those in the p-EGFP-C1 and p-EGFP-SINE-groups,but no significant difference.In summary,we finally demonstrated that the presence of the SINE in the CYP3A29-3’UTR structural variant region would lead to the down-regulation of the expression of this gene,the down-regulation of this gene can regulate the content of T,which is one of the factors of fat,and it reduces the concentration of T.It may be one of the important factors in the formation of the characteristic muscle tissue of the Xiang pigs. |
