Screening Ang Functional Verification Of Genes Related To Abdominal Fat Deposition In Nandan-Yao Chicken

One problem caused by excessively increasing growth rate and feed conversion efficiency for local chicken breeding is excessive fat deposition.Excessive fat deposition will not only cause waste of feed and lower slaughter rate,but also cause environmental pollution and economic losses to the broiler industry.Fat deposition is a complex process regulated by multiple factors,and the molecular mechanism of its regulation is still unclear.Nandan-Yao chicken is an excellent local breed in Guangxi.It is characterized by thin skin and crispy meat with less subcutaneous fat.In order to explore the genes and regulatory pathways related to abdominal fat deposition in Nandan-Yao chickens,this experiment took Nandan-Yao chickens as the research object and analyzed the differentially expressed genes of high and low abdominal fat birds using RNA-seq.We selected several candidate genes for further analysis.The results are as follows:1.RNA-seq analysis and qPCR verification.The total RNA of abdominal fat in the Nandan-Yao chickens aged 120-day-old were extracted with Trizol,and RNA-seq transcriptome sequencing was performed.Transcriptome analysis detected 1222 differentially expressed genes.GO enrichment and KEGG pathway analysis were performed on these differentially expressed genes.The significantly enriched pathways are mainly involved in the developmental growth and the synthesis of pantothenic acid and coenzymes related to fat metabolism(P <0.05).In order to verify the accuracy of the transcriptome,q PCR was used to detect the expression levels of 5 candidate genes in the high and low abdominal fat groups,including DGKD(diacylglycerol kinase delta),PLA2G4A(phospholipase A2 group IVA),SREBF1(sterol regulatory element binding transcription factor 1),DDX5(DEAD-box helicase 5)and RFC2(replication factor C subunit 2).The results showed that the trend of q PCR is consistent with that of RNA-seq,indicating that the results of the transcriptome are credible.The expression levels of DDX5 and RFC2 genes in abdominal fat in the high abdominal fat group were significantly lower than those in the low abdominal fat group(P <0.01).2.Cloning,bioinformatics and tissue expression profiling of DDX5 and RFC2 genes.Through cloning and sequence analysis of the DDX5 and RFC2 gene,we found that the full length of the CDS region of the DDX5 gene and RFC2 gene was 1788 bp and 1080 bp,encoding 595 and 359 amino acids,respectively;there were two synonymous mutations(c.504C> T and c.507A> G)and one missense mutation(c.1060T> C,the 354 th amino acid mutated from tryptophan to arginine acid)in the coding region of DDX5 gene.There is one synonymous mutation in the coding region of RFC2 gene(c.336C> T).Using q PCR to detect the expression level of DDX5 and RFC2 gene in different tissues such as sebum,abdominal fat,duodenum,liver,kidney,testis and ovary.It was found that the expression levels of DDX5 and RFC2 genes in abdominal fat of females were significantly higher than those in males(P <0.01),while the expression level in duodenum of females were significantly lower than that in males(P <0.01),the expression level of RFC2 in testis was significantly higher than that in ovary(P <0.01).3.Construction and verification of DDX5 and RFC2 genes eukaryotic expression vectors.The DDX5 and RFC2 cloning vectors were used as templates for PCR amplification of DDX5 and RFC2,which were connected to the p EGFP-N1 eukaryotic expression vector,and the accuracy of the vector was verified by PCR,double digestion and sequencing.The p EGFP-N1-DDX5 and p EGFP-N1-RFC2 recombinant plasmids were transferred into chicken DF1 cell line.After 24 hours,green fluorescence was observed under a fluorescence microscope,indicating that this study successfully constructed p EGFP-N1-DDX5 and p EGFP-N1-RFC2 eukaryotic expression vectors.In conclusion,through RNA-seq transcriptome analysis,gene cloning sequence analysis,tissue expression profiling analysis and quantitative analysis,it was confirmed that DDX5 and RFC2 may be important candidate genes for fat deposition in Nandan-Yao chicken.The eukaryotic expression vector of the two genes was constructed and the function of the vector was verified at the cellular level,which laid the foundation for the further determination of the functions of the two genes.