Study On The Characteristics Of Collagen Of Guizhou Local Pig And Its Effect On Meat Quality

The local pork in Guizhou is of excellent quality,good taste and flavor,thick skin,and fragrant glutinous meat,which is deeply loved by consumers.In this experiment,Guizhou local pig Kele pig and Zongdihua pig(Guanling pig type)were used as the research objects,and the ternary hybrid pigs were used as controls to explore the effect of collagen content and structural characteristics of Guizhou local pig on meat quality,and to study FAS,FABP1,COL3A1,LOX genes and other related genes' tissue expression;at the same time,studies have shown that fat deposition affects the formation of mature cross-links between collagen molecules,but the specific regulatory mechanism is still unclear.Therefore,by cloning the CDS region of FAS and FABP1 genes,this study constructed a eukaryotic expression vector to explore the effect of fat deposition on collagen and its cross-linking at the cellular level,aiming to explore the material basis and possible regulatory mechanism of local pork quality characteristics,Provide a reference basis for the selection of meat quality breeding.The main findings are as follows:1.The detection of collagen content in different tissues(subcutaneous fat,dorsal longest muscle)of 3 varieties;research results show that there are differences in collagen content in different tissues,all three varieties have subcutaneous fat collagen content higher than the longest dorsal muscle.Among them,the subcutaneous fat of Zongdihua pig and the collagen content in the longest muscle of the back were significantly higher than that of Kele pig and ternary cross-breed pig(P<0.01),with the best glutinous meat and viscoelasticity;Kele pig subcutaneous The content of fat and collagen in the longest muscle of the back is centered,which is significantly lower than that of Zongdihua pig(P<0.01),and significantly higher than that of the external ternary hybrid pig(P<0.05).2.There is no correlation between the content of pyridine cross-linking(HP and LP)between breeds and within the breed.Hydroxylysylpyridinium(HP)is covalently cross-linked in the longest dorsal muscle tissue,with Zongdihua pig being the highest.Among the subcutaneous fat tissues,the Zongdihua pig was the lowest,which was very significantly different from the Kele pig and the ternary cross-breeding pig(P<0.01);the covalent crosslinking of lysylpyridinium(LP)was the most subcutaneous fat and back The content of long muscles is the lowest in Zongdihua pigs,and the content in the longest muscle of the back is very significantly different from that of Kele pigs and external ternary cross-breeding pigs(P<0.01).The ternary cross-breeding pigs showed a very significant difference(P<0.01),which was not significantly different from the Kele pigs.The low cross-linking content of Zongdihua pig indicates that the muscle tenderness is better.3.COL3A1,LOX,FAS,FABP1 gene m RNA are expressed in the subcutaneous fat and longest muscle of Zongdihua pig,Kele pig and ternary crossbred pig,but the expression levels are different.The expression level of COL3A1 gene m RNA was highest in the subcutaneous fat and longest muscle of Zongdihua pig,and the expression level of subcutaneous fat m RNA was significantly higher than that of longest muscle(P<0.01).It indicates that the tissues with high collagen content have higher expression level of COL3A1 gene m RNA;while the LOX gene m RNA expression law is consistent with HP and LP covalent cross-linking content,and the m RNA expression level in subcutaneous fat of Zongdihua pig is extremely low In the outer ternary cross-bred pigs,the expression levels in the subcutaneous fat and longest muscle of the Kele pig were significantly lower than that of the outer ternary cross-breed pig(P<0.01),slightly lower than the Kele pig,but the difference was not significant.The expression of LOX gene m RNA was positively correlated with HP and LP cross-linking;the m RNA expression of fat deposition related genes FAS and FABP1 gene was consistent in the three pig breeds,all of which were higher in tissues with higher fat content,FAS and FABP1 gene m RNA expression in subcutaneous fat are significantly higher than the longest muscle.In tissues with higher m RNA expression of FAS gene and FABP1 gene,COL3A1 gene expression is higher,while LOX gene expression is lower.It shows that in the tissues with more fat deposits,the collagen content is higher,and the cross-linking of HP and LP is lower.4.Successfully cloned the FAS gene and FABP1 gene CDS regions,constructedeukaryotic expression vectors p EGFP-C1-FAS,p EGFP-C1-FABP1,and compared the sequencing results with the original sequence of pig FAS and FABP1 genes in NCBI genebank,the results showed success Construct the overexpression vectors p EGFP-C1-FAS and p EGFP-C1-FABP1.5.The subcutaneous fat cells of Zongdihua pig were successfully isolated and cultured,and the recombinant plasmids p EGFP-C1-FAS and p EGFP-C1-FABP1 were transfected into fat cells.The expression of COL3A1 and LOX genes were detected by q RT-PCR method.When expressing FAS and FABP1 genes,the expression level of COL3A1 gene was significantly or extremely significantly increased compared with the blank control group,but the LOX gene expression level was significantly or extremely significantly lower than that of the blank control group.It shows that fat deposition has positive regulation on COL3A1 gene and negative regulation on LOX gene.After 36 hours of transfection,the cell supernatant was taken for lysyl oxidase(LOX)activity detection.The results showed that the concentration of LOX enzyme activity was significantly lower than that of the blank control group.This indicates that fat deposition has an inhibitory effect on the activity of lysyl oxidase(LOX).The results show that fat deposition has a positive regulation effect on collagen synthesis,and by inhibiting the activity of lysyl oxidase(LOX)to inhibit the production of pyridine crosslinks,the content of mature crosslinks between collagen molecules is reduced and the tenderness of pork is improved.The research results provide a theoretical basis for maintaining the excellent meat quality characteristics and breeding of Guizhou local pig breeds.