Transcriptome Sequencing And Bioinformatics Analysis Of Cartilage Tissues In Sika Deer Antler Growth Centers

Objective: High-throughput sequencing technology was used to screen differentially expressed genes that regulate the rapid growth and ossification of velvet antler,which provided a theoretical basis for the rapid growth and ossification mechanism of antler.Methods: The total RNA in the cartilage tissue of the antler growth center was extracted by Trizol method,and the RNA sample quality was detected by BioSpec-nano micro-UV-visible spectrophotometer and non-denaturing agarose gel electrophoresis.Sequencing library construction was performed using the TruSeq Stranded mRNA kit.Library sequencing was performed on the Illumina Hiseq 2000 platform.Data conversion was performed using Illumina HCS 1.1 software to remove low quality sequences and vector sequences.De novo assembly with Trinity software.The sequencing assembly data was compared to the nr,Swiss-Prot protein database by the BLASTX alignment program.Gene expression analysis was performed by RPKM method.Differential gene expression analysis was performed in the fast growth phase and ossified cartilage tissue using DEGseq software package,and differentially expressed genes related to cartilage growth and ossification were identified.qPCR verification was performed on a number of randomly selected factors related to bone growth and development.Results:1 Sequencing data 4.47 and 4.45 Gb were obtained by sequencing the transcriptome of the cartilage tissue of the sika deer antler growth center during the rapid growth period and ossification period.After assembly by Trinity software,the former received 48,883 contigs and,after splicing,39,377 unigenes,with an average length of 1138 nt.The latter obtained 45,668 contigs,which were spliced to obtain 37,331 unigenes with an average length of 1143 nt.2 Comparing Unigenes with the nr,Swiss-Prot protein database,a total of 20,551 Unigenes can be compared to two databases simultaneously.The GO function is classified by Unigenes on the GO database,which is classified into 61 functional categories and has the largest number of genes classified into cells and cell parts.Through the KEGG metabolic pathway analysis,a total of 26543 genes were annotated into 252 signaling pathways,with the largest number of genes annotated into Metabolic pathways and Focal adhesion.3 A total of 4422 differentially expressed genes were screened.Compared with the rapid growth phase,2493 differentially expressed genes were significantly up-regulated,and 1929 differentially expressed genes were significantly down-regulated.4 By further screening for differentially expressed genes closely related to cartilage growth and ossification,genes that are significantly up-regulated during rapid growth phase include six growth factors,Fgf7,Fgf11,Hgf,Igf1,Egfl6,and Vegfd;seven transcription factors,Atf6?,Sox12,Elf4,Elf2,Runx1,Mafk and Tcf4;three collagen components,Col8a1,Col14a1 and Col16a1.Significantly up-regulated genes during ossification include one growth factor,Vegfc;five transcription factors,Sox8,Sox9,Gtf3 a,Atf3 and Maff;five collagen components,Col9 a,Col10a,Col2a1,Col25a1 and Col27a1.5 qPCR verification of some randomly selected factors related to bone growth and development,we found that the results are basically consistent with the results of transcriptome analysis.Conclusion: The cartilage tissue of the antler growth center showed significant differences in genes such as growth factors,transcription factors,and collagen during the rapid growth phase and ossification.