Screening And Bioinformatics Analyzing The Development Related Genes Of Sika Deer Antler (Cervus Nippon)

Deer antlers are bony organs which are cast and fully regenerate each year. Antlers are the only mammalian appendages capable of regeneration, No any other mammal can naturally regenerate their lost organ, and thus provide a unique model for investigating the mechanisms that underlie mammalian organ regeneration. However, the underlying moleculer mechanism remains poorly understood. Antler development is a uniquely intense and complex process, which involves endochondral ossification and intramembranous ossification. A complex'blastema-like'structure or'antler-bud'then forms, however, unlike the regenerative process in the newt, most evidence (albeit indirect) suggests that this does not involve reversal of the differentiated state but is stem cell (periosteal cells) based. In the microenvironment, the division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals.Cell differentiation,proliferation and apoptosis in the developing antler of Sika deer (Cervus Nippon), was regulated under gene expression. the mesenchymal layer at the very tip of the velvet antler plays an important role in apical morphogenesis,as to deer antler development.The aim of the study was to screen transcripts differentially expressed during sika deer antler development and identify the antler growing–related genes through profiling the differential gene expression between two different stages of the mesenchymal layer at the very tip of the velvet antler. Here we apply the complementary DNA amplified fragment length polymorphism (cDNA-AFLP) technique, a highly reproducible differential display method based on restriction enzyme digests and selective amplification under high stringency conditions, Then, using RT-PCR, to identify the differentially expressed genes in the tissues . Using 64 different primer combinations via cDNA-AFLP technique and silver staining, many transcript tags were found differentially expressed in two antler developmental stages, and 9 transcripts were selected and subject to clone and sequence. Subsequently, the information of these expression genes were analyzed by bioinformatics based on 9 selected genes representing different patterns. The first to pre-process the ESTs, then cluster ESTs into contig sequences, and the second to get further information of combine contig and unique ESTs, from non-redundant sequences and EST database that can be systematically compared and GO that can be annotated.Our results showed that 2 ESTs were derived from one gene, assembling the cluster into a contig that is AF78. We blasted this contig and singlets and annotated. Through the analysis, we obtained the related gene information. The 2 upregulated genes were AF1 and AF2. The former was unknown gene, the latter had homology with Homo sapiens transient receptor potential cation channel, subfamily V, member 4 (TRPV4). TRPV4 channel is transient receptor potentiall vanilloid that plays vital role in chondrogenesis, and is the non-selective cation channel that has the moderate permeability to the calcium ion. The 6 downregulated genes were AF3-AF6, AF78 (contig) and AF9. The function of AF6 gene was unknown; The AF3 had homologous sequence with homo sapiens'FAS, which is a pro-apoptosis gene, a member of the tumor necrosis factor (TNF) receptor superfamily, as an important mediator of apoptotic cell death; The AF4 had homologous sequence with homo sapiens Wingless-type MMTV integration site family, member 3(WNT3), is a member of the WNT gene family, The WNT gene family encodes secreted signaling proteins.These proteins have implicated in cell proliferation and differentiation and in several developmental processes; The AF5 had homologous sequence with homo sapiens transforming growth factor beta 1(TGF-β1). TGF-β1 is a member of the transforming growth factor beta superfamily of cytokines. It is a secreted protein that performs many cellular functions, including the control of cell growth,proliferation,differentiation and apoptosis; The AF78 had homologous sequence with Macaca mulatta Cyclin-dependent kinase 2 (CDK2), CDK2 is a member of a large family of protein kinases that initiate the principal transitions of the eukaryotic cell cycle; The AF9 had homologous sequence with homo sapiens apoptotic protease activating factor 1(APAF1), Apaf-1 is a cytosolic protein involved in cell death or apoptosis. The genes with known functions were involved in signal transduction, such as TGF/TGFR1 signals, Wnt/β-action signal pathway, cell division, proliferation, differentiation and apoptosis, playing indispensable roles in the development of deer antler. Several genes are novel or identified first time in deer. Then identifying the target genes expression in the tissues by RT- PCR. Our studies have laid the foundation for further investigation on antlerogenesis and organogenesis.