Identification And Functional Of Genes Related To Intramuscular Fat Deposition In Guizhou Yellow Chicken

Intramuscular fat(IMF)content was an important factor affecting the quality of meat,which was closely related to flavor,tenderness,moisture content of meat.Different species had variations in IMF content,and even within the same species,there were differences in IMF content between individuals.However,Intramuscular fat content had been difficult to measure in vivo and could only be measured after slaughter,which was costly and time-consuming,so the search for genes that affect intramuscular fat deposition was always a hot topic for scientists.The Guizhou yellow chicken was one of the typical representatives of yellow-feathered broilers in China,which had early sexual maturity and easy fattening characteristics.Therefore,in this study,using individuals with different intramuscular fat content in Guizhou yellow chickens as the research object,with the aim of analyzing the molecular regulatory mechanisms and signaling pathways affecting intramuscular fat deposition in Guizhou yellow chickens at the transcriptome level,searching for candidate genes affecting intramuscular fat deposition in Guizhou yellow chickens,as well as validating them at the cytological level,and providing a theoretical basis for the selection and breeding of intramuscular fat content in yellow-finned broiler chickens.(1)It was used to determine the slaughter performance and meat quality,fatty acid composition and muscle fibre characteristics of 30 Guizhou yellow chickens under the same nutritional conditions at 120 days of age;according to the level of IMF content the group was divided into high intramuscular fat group(IMFH group)and low intramuscular content group(IMFL group).The results showed that there were very significant differences between the high intramuscular fat content group and the low intramuscular fat content group(P<0.01);for slaughter performance the differences between the two groups were not significant.Highly significant difference in pectoral shear force between IMFH group and IMFL group(P<0.01).The highest saturated fatty acid content of both groups with palmitic acid;linoleic acid was significantly higher than in the IMFH group(P<0.01),and arachidonic acid significantly higher than in the IMFL group(P<0.05).Muscle fibres density were significantly higher among the IMFH group than the IMFL group.Compared to the IMFL group,the IMFH group had better meat quality than the IMFL group.(2)Selecting the 4 highest(H group,n=4)and lowest(L group,n=4)pectoral muscle tissue samples from each of the high-IMF group and the low-IMF group to be transcriptome sequenced.The sequencing results showed that 259 differentially expressed genes were screened between the two groups,including 195 up regulated genes and 64 down-regulated genes,with GO and KEGG enrichment analysis performed of the screened differentially expressed genes,there were 5 pathways including focal adhesion,extracellular matrix(ECM)-receptor interaction,cell adhesion molecules,regulation of actin cytoskeleton,and PPAR signaling pathways that might be involved in IMF deposition;among them,differentially expressed genes were highly enriched in focal adhesion and ECM-receptor interaction pathway,some of which could be key genes affecting intramuscular fat deposition.There were 6randomly selected up-regulated genes(CAPN2,PLTP,ABHD6)and down-regulated genes(LPIN1,ANKRD2,MCEE)differentially expressed genes validated by fluorescent quantitative PCR.qRT-PCR expression levels were consistent with RNASeq sequencing results.(3)Using fluorescence quantitative PCR to identify the mRNA expression levels of interfering COL1A1 gene with fat deposition-related genes COL1A1,PLTP,ABHD6 and LPIN1 in chicken intramuscular adipocytes.The effect of interfering with the COL1A1 gene on intramuscular fat deposition was examined by establishing an RNA interference lentiviral expression vector for the COL1A1 gene,cultivating chicken intramuscular adipocytes,lipogenesis-induced differentiation,and oil-red O staining techniques.The results showed that interference to COL1A1 gene reduce the proliferation of adipocytes at 48 h,72h and 96 h points of time.There were two time points of 4d and 8d after lipogenesis-induced differentiation observed by Oil Red O staining,which resulted in a significant decrease in adipocyte differentiation and lipid droplet deposition capacity.qPCR results showed that the relative expression of fat deposition-related genes LAMA3,ABHD6 and PTGS2 were significantly decreased at4d(P<0.05),and ABHD6,LPIN1 and PTGS2 were highly significantly decreased at 8d(P<0.01);triglyceride and cholesterol contents were significantly decreased after transfection with interfering COL1A1 at 4d and 8d(P< 0.01).In summary,this paper compares the different intramuscular fat contents of Guizhou yellow chickens,the higher intramuscular fat content,the more saturated fatty acid content,and also have denser muscle fibres,the meat with better nutrition and taste.Based on transcriptome sequencing analysis of pectoral muscle tissues from high and low intramuscular adipose groups,it was screened for 259 differentially expressed genes,which were highly enriched in focal adhesion and ECM-receptor interaction pathways,in which genes from these two pathways(CAPN2,COL1A1,COL1A2,COL6A1,COL6A2,COL6A3)may play important regulatory roles in intramuscular fat deposition.The COL1A1 gene enhances the proliferative differentiation and lipid deposition ability of intramuscular adipocytes in chicken.In chicken preadipocytes,COL1A1 gene promoted the proliferation,differentiation and lipid deposition of adipocytes.COL1A1 gene promotes the accumulation of triglyceride and cholesterol content in chicken preadipocytes and has regulatory effects on fat deposition-related genes LAMA3,ABHD6,LPIN1 and PTGS2.