Identification And Functional Validation Of Key Genes Regulating Polyunsaturated Fatty Acid Ratio In Muscle Of Huang Shan Black Chicken

The content,proportion and types of fatty acids play an important role in improving the quality and flavor of chicken.Identifying genes associated with fatty acid composition could provide valuable insights into the complex genetic networks of genes underling variation in fatty acids synthesis.Paired-end RNA sequencing(RNA-Seq)was conducted to explore the chicken transcriptome from the thigh muscle tissue of 6 Huang shan Black Chickens with 3 extremely high and 3 low phenotypic values for polyunsaturated fatty acid percentage.Integrated interpretation of differential gene expression and formerly reported quantitative trait loci indicated the most promising candidate genes affecting fatty acid concentration.Fatty Acid Desaturase 2(FADS2)gene is used as a key candidate gene for functional verification.Construct a lentiviral vector delivering the FADS2 gene-targeted(si RNA)and determine infection efficiency and expression of the vector in the chcken adipocytes cell,and ultimately verify interference efficiency on FADS2 gene.At the same time,the effect of FADS2 gene on the expression of 19 key functional genes of fatty acid metabolism and the regulation of fatty acid metabolism were investigated by using RNA interference technique.(1)Detection of fatty acid content:By detecting the fatty acid composition of 20 samples,the thigh muscle tissue of 6 Huang shan Black Chickens with 3 extremely high(0.324?0.329?0.324)and 3 low(0.285?0.261?0.265)(P<0.01)phenotypic values for polyunsaturated fatty acid percentage were selected as experimental materials.(2)Identification of key genes by RNA-seq:In total,we revealed 274 differentially expressed genes(DEGs)Gene ontology and pathway analysis demonstrated that the DEGs were enriched in particular biological processes affecting biosynthesis of unsaturated fatty acids,fatty acid metabolism and cell junction-related pathways and so on.Integrated interpretation of differential gene expression and formerly reported quantitative trait loci indicated that FADS2,DCN,FRZB,OGN,PRKAG3,LHFP,CHCHD10,CYTL1,FBLN5 and ADGRD1 are the most promising candidate genes affecting polyunsaturated fatty acid percentage.(3)Functional validation of key genes:Three si RNA sequences targeting the coding region of gallus FADS2 m RNA were designed.Additionally,q RT-PCR revealed that the FADS2 m RNA expression was obviously down-regulated in the interference group compared with the negative control group,and FADS2-si RNA F1 was the most efficient sequence with interference efficiency 60%.The recombinant lentiviral vector FADS2-si RNA and its gene-silenced model in the chcken adipocyte cell were both constructed successfully.The decrease in expression observed after knockdown of FADS2 relative to the control group averaged 14.91%,24.59%,54.56%,29.59%,39.57%,11.55%,45.94%,67.56%,195.49%,67.55% and 31.62% for ELOVL5,FADS1,ACACA,ACSL1,LPL,PPARG,LPIN1,ADFP,XDH,PLIN2,ACOX1;12.63%,27.33%,14.19%,23.42%,19.22%,16.59%,14.88%,34.14% for ELOVL6,ACLY,CD36,ACSS2,SREBF1,INSIG1,GPAM,PNPLA2.FADS2 gene was identified as the key gene affecting polyunsaturated fatty acid percentage in Huang Shan black chicken,which provided a theoretical basis for later identification of the regulation pathway of fatty acids.