Screening Of Key Genes Related To Fat Deposition Of Jinghai Yellow Chicken

Intramuscular fat?IMF?content is associated with multiple meat quality characteristics,such as tenderness,juiciness,flavor level and water holding capacity.With the improvement of living standard,people pay greater attention to the meat equality.Now,increasing the IMF content has been one of the breeding aims of the breeder.However,the quality and flavor of chicken have decreased in recent decades as a result of genetic selection for faster growth and increased feed conversion efficiency.Moreover,selection for faster growth is accompanied by excessive accumulation of abdominal and visceral fat.Therefore,to increase IMF while reducing the deposition of abdominal fat has become a major goal for the improvement of chicken.In the present study,RNA-seq technology was used to characterize the transcriptome of abdominal and intramuscular preadipocytes during adipogenic differentiation as well as identify IncRNAs and analyze the structure of IncRNAs.Then,the differentially expressed genes were identified in abdominal and intramuscular preadipocytes during differentiation.Candidate factors and pathways involved in differentiation of abdominal and intramuscular preadipocytes were identified using bioinformatics analysis.Finally,by comparing the gene expression level,differences between transcriptome of abdominal and intramuscular during differentiation were analyzed.The main results are as follows:1.Screening of key genes related to abdominal fat depositionChicken primary abdominal preadipocytes were isolated and cultured from abdominal fat tissue of chicken,and induced to differentiation using differentiation medium.Oil O staining results showed that we isolated abdominal preadipocytes successfully.27,023 IncRNAs were identified in chicken abdominal preadipocytes during differentiation.Structural analysis indicated that lncRNAs in abdominal preadipocytes have characteristics of short sequence,short open reading frame,and few exon number.Function prediction suggested that IncRNAs were mainly enriched in protein modification process,cellular protein modification process,and regulation of signaling.4,232 differentially expressed IncRNAs and 1,656 differentially expressed mRNAs were screened during the differentiation of abdominal preadipocytes differentiation.Function annotation showed that differentially expressed genes were mainly enriched in cell cycle,mesenchymal differentiation,and cell cycle process.Pathway analysis indicated that differentially expressed genes were mainly enriched in cell cycle,DNA replication,and PPAR signaling pathway.Six stage-specific modules were identified using WGCNA program.Dozens of highly connected genes were identified in six stage-specific modules by visualizing them in Cytoscape,including XLOC₀52948,gga-mir-30c,SCD and KLF15 gene.Those highly connected genes might be important candidate factors regulating chicken abdominal preadipocytes differentiation.2.Screening of key genes related to intramuscular fat depositionChicken primary intramuscular preadipocytes were isolated and cultured from muscle tissue of chicken,and induced to differentiation using differentiation medium.Oil O staining results showed that we isolated intramuscular preadipocytes successfully.26,172 lncRNAs were identified in chicken intramuscular preadipocytes during differentiation.Structural analysis indicated that IncRNAs in abdominal preadipocytes have characteristics of short sequence,short open reading frame,and few exon number.Function prediction suggested that IncRNAs were mainly enriched in gene expression,cellular macromolecule biosynthetic process and RNA metabolic process.4,694 differentially expressed lncRNAs and 2,169 differentially expressed mRNAs were screened during the differentiation of abdominal preadipocytes differentiation.Function annotation showed that differentially expressed genes were mainly enriched in regulation of signaling,regulation cell communication,and regulation signal transduction.Pathway analysis indicated that differentially expressed genes were mainly enriched in focal adhesion,MAPK,and PPAR signaling pathway.Six stage-specific modules were identified using WGCNA program.Dozens of highly connected genes were identified in six stage-specific modules by visualizing them in Cytoscape,including XLOC₀13577,gga-mir-146b,BMP3 and MYOD1 gene.Those highly connected genes might be important candidate factors regulating chicken intramuscular preadipocytes differentiation.3.Mechanism comparison between abdominal and intramuscular fat depositionStructure comparative analysis showed that IncRNAs in abdominal and intramuscular preadipocytes both have features of short sequence,short open reading frame,and few exon number.The expression level of IncRNAs was much lower than that of mRNAs in both preadipocytes.The number of differentially expressed gene between abdominal and intramuscular preadipocytes decreased with the differentiation time went on.2,942?1,877 mRNAs and 1,065 lncRNAs?,3,788?1,964 mRNAs and 1,824 lncRNAs?,2,872?1,194 mRNAs and 1,678 lncRNAs?,and 2,214?1,206 mRNAs and 1,008 lncRNAs?genes were differentially expressed at day 0,2,4,and 6 of differentiation.GO annotation indicated that differentially expressed genes at day 0 were significantly enriched in response to growth factor,cellular response to growth factor stimulus,and regulation of locomotion.While they were muscle structure development,muscle organ development,and cellular response to growth factor stimulus at day 2,mesenchymal cell differentiation,regulation of cell migration,and cell adhesion at day 4,and enzyme linked receptor protein signaling pathway,locomotion,and localization of cell at day 6 of differentiation.Pathway analysis showed that differentially expressed genes were enriched in many pathways involved in preadipocyte differentiation,such as PPAR and glyceride metabolism pathway.Finally,we compared the differentially expressed genes between two preadipocytes during differentiation.The result showed that only 253 IncRNAs were differentially expressed both in abdominal and intramuscular preadipocytes,while it was 911 when it comes to mRNAs.Common genes were significantly enriched in more that 200 biological processes,including cell cycle,cell cycle process,and chromosome segregation.Pathway analysis showed that the common genes were mainly significantly enriched in dozens of pathways,including the cell cycle,ECM-receptor interaction,and focal adhesion.Several pathways involved in adipogenesis were also found,such as PPAR,p53l Foxo,and TGF-beta signaling pathway.4.Validation of target interaction between gga-mir-30c-2 and XLOC 060155 and SIX4The high-throughput sequencing and gene co-expression results showed that XLOC₀60155,gga-mir-30c,and SIX4 are important candidate genes regulating the differentiation of chicken preadipocyte.High correlation was found between the expression pattern of gga-mir-30c and XLOC₀60155 as well as gga-mir-30c and SIX4 gene.RT-qPCR results showed that the expression pattern of XLOC₀60155 is significantly negative correlated with that of gga-mir-30c?r=-0.97,P<0.01?;The expression pattern of gga-mir-30c is significantly negative correlated with that of SLX4?r=-0.684,P<0.05?.Target prediction indicates that binding sites of gga-mir-30c-5p were found on the sequences of XLOC₀60155 and SIX4 genes.Dual luciferase test results showed that gga-mir-30c-5p could significantly reduce the luciferase expression activity by binding with XLOC₀60155.And gga-mir-30c-5p could significantly reduce the luciferase expression activity by binding with the 3'UTR region of SIX4 gene.Moreover,the dual-luciferase expression activity was significantly increased after mutant treatment of the binding sites.The results confirmed that XLOC₀60155 and SIX4 gene is targets of gga-mir-30c?...