Transcriptome Analysis And Differentially Expressed Genes Screening Of Antler Tips At Different Growth Stages

Deer antlers are the unique appendages of cervidae capable of repeated rounds ofcasting and regeneration. Antlers are derived from a region of the periosteumoverlying the frontal bone that is known as antlerogenic periosteum (AP). Pedicleinitiation is caused by direct stimulation of androgen hormones on the stem cellslocated in the AP, subsequently, the first antlers develop by a combination ofintramembranous and endochondral ossification from the tip of the fully formedpedicle, followed by rapid growth, and the fastest growth rate can reach up to2cmper day. In autumn, the antler attains its full size and becomes totally calcified,resulting in the shedding of the velvet skin, and is not cast until the following spring,which triggers another round of antler regeneration, ossification and casting. Themechanisms regulating antler growth have always been the spotlight of the biologicalresearch, and its unique growth process has fascinated many biologists for centuries.The transcriptome sequencing technology based on the Illumina/Solexahigh-throughput sequencing platforms can detect the overall transcription of anyspecies at the single-nucleotide level, and analyze the structure of transcriptions andgene expression levels. Meanwhile, this technology can also detect rare and noveltranscripts, therefor providing the most comprehensive transcriptome information. Incontrast to microarrays, transcriptome sequencing does not require specificpre-designed probes based on prior knowledge of gene sequences, and is particularlyattractive for non-model organisms with genomic sequences that are yet to bedetermined (De novo), with the characteristics of high-throughput, fast, accurate andlow-cost. Transcriptome studies can reveal gene structure and gene function from theoverall level, and elucidate the molecular mechanisms in specific biological processesand disease process.In the present study, transcriptomes of antler tips (growth centre) at the stages ofrapid growth and ossification were sequenced using Illumina/Solexa sequencing technology, respectively. Transcriptome de novo assembly was carried out with shortreads assembling program–SOAPdenovo, thus,transcriptome databases at differentgrowth stages were established; Sequence alignment, functional annotation andpathway analysis against protein databases including nr, Swiss-Prot, KEGG and COGwere performed; By means of differential screening, differentially expressed genes(DEGs) related to rapid growth and ossification between different stages wereanalyzed, therefor, to explore the mechanisms regulating antler growth.1. Based on these studies, we generated over80million short reads, each readhad a90-bp length, the data sets were deposited in the NCBI ArrayExpress repository,the accession number were E-MTAB-802and E-MTAB-853. These short reads wereassembled, resulting in116,504Unigenes with a mean size of522bp and an N50of682bp. The majority of the Unigenes showed gap lengths that were less than5%ofthe total length, which accounted for87%of total Unigenes numbers. Although the3'ends of the assembled Unigenes contained relatively fewer numbers of reads, theother positions of the assembled Unigenes showed a greater and more evendistribution. Comparisons with the Sika deer EST database using BLASTN revealedthat our transcriptome dataset had both a high degree of consistency (78.57%) withprevious EST data and an approximate42times increase in coverage.2. Among the Unigenes,39,047genes (33.52%of All-Unigenes) get a CDSregion as well as a sequence direction. The rest genes were analyzed using thesoftware named ESTScan to predict the coding regions as well as sequence direction,the results indicated that2,120genes may represent new protein coding sequences.GO functional classification showed that antler growth closely related to extracellularmatrix synthesis, ligand/receptor interactions and enzyme catalysis reactions. COGfunctional classification showed that protein synthesis and DNA damage and repairplayed key roles in regulating antler growth. KEGG pathway analysis showed thatcell metabolism, cytoskeleton connection and extracellular matrix receptor interactionwere the main regulatory pathways during antler growth. There were also22types ofgrowth factors,39types of transcription factors and25types of extracellular matrixrelated to antler growth. 3. According to differential screening,16,905DEGs were identified (︱log2Ratio︱≥1and FDR≤0.001). We also performed Gene Ontology (GO) functionalenrichment and pathway enrichment analysis of the gene expression patterns. Wefound that most of the significantly changed genes were correlated to cell metabolism,protein synthesis and translation, extracellular matrix receptor interaction. There were53types of DEGs related to protein synthesis (consisted of32types of ribosomalproteins,18types of translation initiation factors and3types of elongation factors),and19types of DEGs related to cartilage growth and ossification. These genes playedimportant roles in protein synthesis, cell proliferation and apoptosis, and ossificationduring antler growth.4. We selected20types of genes related to protein synthesis (incluing ribosomalproteins, translation initiation factors and elongation factors) and19types of genesinvolved in rapid growth and ossification (including growth factors, transcriptionfactors and extracellular matrix proteins) for qPCR validation. The results showedconsistent expression patterns with the observed findings in transcriptome analysis.The significant agreement between the data sets indicated that the abundance of theIllumina sequences from the Sika deer transcriptome closely mirrors the actualexpression level.In this study, the transcriptome database of sika deer antler tips were successfullyconstructed using the next-generation high-throughput sequencing technology, andsequence alignment, functional annotation and pathway analysis against proteindatabases were performed. Based on these results, by means of differential screening,differentially expressed genes (DEGs) related to rapid growth and ossificationbetween different stages were analyzed, therefor, this study provides a basis forfurther research on gene structure and function of deer antlers.