Screening And Functional Analysis Of Tarim Red Deer (Cervus Elaphus Yarkandensis) Hair Color Differentially Expressed Genes

Red deer(Cervus elaphus)is a representative large ungulate animal in the forest and desert forest environment of Xinjiang,and plays an important ecological role in the arid and semi-arid forest ecosystem of Xinjiang.There are 22 subspecies of red deer in the world according to the difference of their individual size,hair color and horn type.Hair color is one of the most important indicators for the classification of red deer subspecies.However,the study on genes related to the control of hair color at the molecular level has not been reported.In this study,the genes that related to control the hair color of red deer were obtained through the high-throughput transcriptome sequencing analysis of Tarim red deer and Tianshan red deer by using RNA-Seq technology,and real-time fluorescence quantitative PCR analysis of differentially expressed candidate genes.This would lay the foundation for elucidating the molecular mechanism of the morphological characteristics of Tarim red deer.The results are as follows:1.A total of 47.51 Gb Clean Data were obtained from transcriptome sequencing of skin tissues of Tarim red deer and Tianshan red deer,and the Clean Data of each sample reached 7.29 Gb.The percentage of Q30 base was 85.01% or more,and86,277 Unigenes were obtained after assembly,of which 27,802 Unigenes were longer than 1 kb.A total of 25,038 Unigenes with annotated information was obtained by comparing Unigenes with NR,Swiss-Prot,KEGG,COG,KOG,GO and Pofam databases.Through differential expression analysis,the number of 922 differentially expressed genes was obtained,among them 495 genes were up-regulated and 427 genes were down-regulated.Preliminary analysis showed that,the up-regulated genes such as KRT75,Ggt1 and POMC and the down-regulated gene MITF were most likely relate to hair color formation directly.2.BLAST was used to compare differentially expressed genes with NR,Swiss-Prot,Pfam and eggNOG databases.The results showed that 736,585,644 and698 genes could match the known sequences in these databases.The results of directhomology classification of gene products that comes from the comparison of differentially expressed genes with COG databases showed that,186 differentially expressed genes were classified into 26 gene families and the largest number of annotated genes were belong to General function predictaion only(R).3.The results of GO enrichment analysis showed that,568 differentially expressed genes were enriched in GO term,which were divided into three categories namely: biological processes,cell components and molecular functions.Tricarboxylic acid cycle is the most concentrated in biological processes,fibrillar collagen trimer is the most concentrated in cell composition,and FK506 binding is the most concentrated in molecular function.4.A total of 480 genes was involved in 240 pathways through KEGG analysis of differentially expressed genes.Eight signaling pathways with significant differences were obtained,among them ECM-receptor interaction pathway was found for the most significant difference,followed by protein digestion and absorption as well as PI3K-Akt signaling pathway.5.Seven differentially expressed genes including MITF,Ggt1,VDR,PTPRF,CIITA,ARPC5 L and POMC were analyzed by qRT-PCR to verify the reliability of transcriptome sequencing.The results showed that,the up-regulation or down-regulation trend of qRT-PCR of these seven genes was consistent with that of RNA-Seq,indicating that the sequencing results of transcriptome were reliable.6.VDR gene was selected from differentially expressed genes and the protein structure was predicted by bioinformatics analysis.VDR protein is a hydrophilic protein and has strong liposolubility.It is relatively conservative in species evolution,the protein encoded by VDR has certain stability and most likely located in the endoplasmic reticulum.At the same time,VDR protein is a structural protein without transmembrane region and signal peptide.However,the VDR protein is rich in phosphorylation sites,and this post-translational modification of the protein has important implications for a range of protein functions.This study would provide basic information for future studies on the molecular mechanism of the morphological characteristics of Tarim red deer,and also providesscientific and effective data information for further exploration and searching of new genes and related functional genes structure of Tarim red deer.