Abstract:

This study aimed to detect the SNPs of CDKN2A gene in Wenshang Barred chicken,and perform the analysis of its bioinformatics and expressions in skin tissues.Based on the high quality chromosome-level genome of Wenshang Barred chicken assembled by the research group of Resource Conservation and Evaluation in Jiangsu Institute of Poultry Science,the sequences were blasted to chicken reference genome (GRCg7b) and the SNPs were acquired.These SNPs were detected and genotyped by MassARRAY nucleic acid mass spectrometry in the chicken population.Bioinformatics software was used to predict the regulatory sequence of CDKN2A gene.The physicochemical properties and structural characteristics of the encoded protein were analyzed.The expressions of this gene in the skin tissues of Wenshang Barred chicken at different periods were detected by transcriptome and Real-Time Fluorescence Quantitative PCR.The results showed that the total length of CDKN2A and its CDS were respectively 9.854 kb and 183 bp,encoding 60 amino acids.Forty-one SNPs were found in this gene and its upstream and downstream 1 kb,of which four SNPs were newly discovered loci.These four new SNPs were located in the downstream and the intron,respectively.The first exon had two SNPs,both of which were missense mutations:V9D and C10R.The detection rates of 34 SNPs among the 41 SNPs were all over 91% in the Wenshang Barred chicken population.These SNPs were almost homozygous mutants without polymorphism.Eight promoters and five CpG islands were predicted in the 2 kb upstream of CDKN2A in Wenshang Barred chicken.The promoter region contained a total of 12 transcription factor binding sites,including Sp1,MEB-1,YY1,C/EBPα,AP-2α.The protein encoded by this gene was a positively charged,unstable hydrophilic protein with a molecular mass of 7.30 ku and a theoretical isoelectric point of 12.82.The predictive subcellular localization of the protein was mainly in mitochondria.The protein had no signal peptide regions and signal peptide shear sites.It had eight potential phosphorylation sites and no glycosylation sites.The protein contained a typical TRP_2 (transient receptor potential ion channelⅡ) conserved domain and its secondary and tertiary structures were mainly α-helices and random coil.In addition,it had interactions with MDM2,NPM1,USP36 proteins.The expressions of CDKN2A gene in the back skins increased from 1 to 35 days old in Wenshang Barred chicken.

Key words: Wenshang Barred chicken, CDKN2A, SNP variations, Bioinformatics analysis, Tissue expression