Screening And Identification Of Candidate Genes Related To Antler Ossification Based On GEO Database

Antler is divided into pilose antler and hard antler.Pilose antler has low ossification degree,high content of active ingredients and high efficacy,so the medicinal value of pilose antler is higher than hard antler.In order to improve the medicinal value of antler,it is necessary to reduce the degree of ossification,so it is important to study the mechanism of ossification and its influencing factors for improving the quality of antler.Due to the limited gene expression profile data of pilose antler in GEO database,it is necessary to dig and study the genes related to ossification of pilose antler from model organisms.In this study,by mining the data set of mouse ossification related candidate genes in GEO database,the final ossification related genes were screened out through gene differential expression analysis,enrichment analysis and protein interaction network prediction.The selected TMEM119,SNAI2,DLX5,FGF7,THY1 and TAGLN genes were cloned and identified with the tip tissue of pilose antler.The experimental results are as follows:(1)By differential expression analysis,2431 up-regulated genes and 467 down-regulated genes were obtained in GSE151303 dataset,and 422 up-regulated genes and 368 downregulated genes were obtained in GSE82285 dataset.In the KEGG pathway enrichment analysis,genes in the two data sets were mainly enriched in metabolic pathway,PI3K-Akt signaling pathway,Ras signaling pathway,MAPK signaling pathway,etc.,while GO enrichment analysis showed that genes were mainly related to protein binding,metal ion binding,signal transduction,cell differentiation and other functions or processes.After protein interaction network analysis and prediction,44 and 40 ossification related genes were screened out in GSE151303 and GSE82285,respectively.(2)The c DNA sequences of TMEM119,SNAI2,DLX5,FGF7,THY1 and TAGLN genes were successfully cloned by molecular cloning technology.The sizes were 912 bp,912bp,1064 bp,585bp,529 bp and 816 bp,respectively,and all of them contained complete coding sequences.Sequence comparison showed that these genes were relatively conserved in evolution,and their m RNA sequences were more than 95% similar to the corresponding genes of cattle and sheep.(3)Real-time quantitative PCR method was used to analyze gene expression levels,and it was found that the expression levels of TMEM119 and SNAI2 genes were the highest in the middle growth stage of pilose antler,and there were significant differences between them and other stages.The expression of DLX5 gene was the highest in the late stage,which was significantly different from other stages.The expression of FGF7 gene was the highest in the middle stage and the lowest in the late stage.The expression level of THY1 gene decreased from prophase to anaphase,while that of TAGLN gene was on the contrary,and the expression levels of the two genes were not significantly different in each period.In this study,real-time fluorescence quantitative PCR was used to detect the expression of candidate genes in the tip tissue of pilose antler at different growth stages,and the important role of TMEM119,SNAI2,DLX5 and other genes in the growth and ossification of pilose was described,providing theoretical support for further research on the mechanism of ossification of pilose antler.