| Velvet is the only known mammalian organ wich had the capability of complete regeneration after cast, and any mammalian tissues & organs can not compare with its fast growing rate. Over the years, many scholars from various fields, using various scientific methods had studied its internal mechanism of this special, unique biological phenomenon, and there still not any study could clear the velvet growing mechanism fundamentally.A cDNA library using velvet tip tissue of Sika Deer (Cervus nippon Tem minck) was constructed, and it was subjected to 5'EST sequencing and analysis, and bioinformatics method and real Time PCR technique used to analyze the fractional growth and ossification-related genes. The potential results are as follows:1. Totally 1012 clones were randomly selected from cDNA library of velvet tip tissue and sequenced,906 readable sequences that are more than 100 by were produced by analysis with CAP3 software. And the average length of the 487 ESTs was 390.48 bp, the GC content is 44.56%. Using the Phrap software, the 906 effective ESTs has been cluster analyzed and assembleed into 701 UniGene including 86 contigs and 615 contigs were composed of only one EST which was called singlet. The GenBank accession numbers are GH546162-GH546861 and GH571843.2. Analysising by using the BLASTx and BLASTn programs,580 known or putative functional gene,74 unknown gene,47 no hits (maybe new genes). The mostly homological proteins were from Bos taurus, accounted for 81.3%.The genes have been annotated functions according to GO three classification levels (the molecular function, biological process and cell component). Made clear at all levels of gene expression in the overall situation, to construct Gene Expression Profiling of Velvet tip tissue in Northeast sika deer and, based on the results of BLAST annotations, by further clustering analysis, a total of 42 with Velvet Tip Tissue development-related functional genes were obtained.3. The seven genes related to development at velvet tip tissue were selected for expression analysis using real time revers transxription-polymerase chain reaction (RT-PCR), and five of them after re-sequencing and measuring communication that have full-length cDNA of the gene of bioinformatics analysis.The results show that:①The full-length cDNA of the bone sialoproteinⅡfrom velvet tip tissue of Cervus nippon hortulorum was 1576 bp, contains a long open reading frame of 936 and encoded 311a mino acid,with a calculated molecular mass of 34.1 kD and a theoretical isoelectric point of 4.05. BSP-Ⅱprotein is hydropathicity protein, contain BSP-Ⅱdomain,signal peptide and two transmembrane domain were found, it might be secretory protein. The BSP-Ⅱprotein has been localized in extracellular. Secondary structure with a-helix and random coil-based. Real-time PCR results showed that:in the skin layer and the mesenchymal layer is almost not expressed, the precartilage layer and the cartilage layer were expressed, and the cartilage layer of expression intensity was significantly higher than the precartilage layer, its expression with velvet mineralization process of the tissue showed a significant positive correlation suggesting that the gene as the major structural protein of bone matrix to participate in velvet tissue calcification.②The full-length cDNA of the osteopontin from velvet tip tissue of Cervus nippon hortulorum was1406 bp, contains a long open reading frame of 840 and encoded 279 a mino acid, with a calculated molecular mass of 30.99 kD and a theoretical isoelectric point of 4.40. OPN protein is hydropathicity protein, contain Osteopontin domain, signal peptide and two transmembrane domain were found, it might be secretory protein. The OPN protein has been localized in extracellular. Secondary structure with random coil-based. Real-time PCR results showed that:the expression from a velvet precartilage layer of the skin layer to a gradual increase, to the cartilage layer down regulation. Speculated that the gene through the osteoclast-mediated adhesion of cells and mineralized tissues involved in velvet tissue calcification.③The full-length cDNA of the decorin from velvet tip tissue of Cervus nippon hortulorum was 1831 bp, contains a long open reading frame of 1083 and encoded 360 a mino acid, with a calculated molecular mass of 39.9 kD and a theoretical isoelectric point of 8.8. DCN protein is hydropathicity protein, contain two LRR-RI domain, signal peptide and three transmembrane domain were found, it might be secretory protein. The DCN protein has been localized in cytoplasmic and extracellular. Secondary structure with a-helix and random coil-based. Real-time PCR results showed that:the expression of mesenchymal layer was significantly higher than the other three tissues, speculated that the antifibrotic activity of the gene may be the maintenance rapid growth of mesenchymal layer of velvet important regulator factors.④The full-length cDNA of the calmodulin from velvet tip tissue of Cervus nippon hortulorum was 1527 bp,contains a long open reading frame of 462 and encoded 149 a mino acid, with a calculated molecular mass of 16.9 kD and a theoretical isoelectric point of 4.09. CaM protein is hydropathicity protein, contain two EFh domain, no signal peptide and transmembrane domain were found, it might be non-secretory protein. The CaM protein has been localized in nuclear and mitochondrial. Secondary structure with a-helix-based. Real-time PCR results showed that:the expression of precartilage from the skin layer to a gradual down regulation, to the cartilage layer step-up, and the skin layer were significantly higher than the other three tissues, speculated that high expression of the gene can be seen to promote skin tissue growth of velvet has a positive role. ⑤The full-length cDNA of the annexin A2 from velvet tip tissue of Cervus nippon hortulorum was 1487 bp, contains a long open reading frame of 1050 and encoded 349 a mino acid, with a calculated molecular mass of 39.5 kD and a theoretical isoelectric point of 6.92.ANXA2 protein is hydropathicity protein,contain tetrad ANXA2 domain,no signal peptide and transmembrane domain were found,it might be non-secretory protein. The ANXA2 protein has been localized in nuclear. Secondary structure with a-helix-based. Real-time PCR results showed that:in the velvet skin layer and the mesenchymal layer expression is low,whereas in the precartilage layer to the cartilage layer but maintains high expression,suggesting that the gene through participation in velvet Ca2+ ion channel formation, and thus to participate in velvet tissue calcification.⑥Real-time PCR of fibronectin 1 gene results showed that:the expression of cartilage from the skin layer to gradually increased, and expression of the precartilage layer and the cartilage layer was significantly higher than the skin layer and the mesenchymal layer, speculated that the gene can be directly stimulate osteoblast growth and differentiation and proliferation, to participate in a the process of bone formation.⑦Real-time PCR of osteonection gene results showed that:in the velvet tip of the gene expression levels of different tissues with similar osteopontin, suggesting that the gene start the mineralization process, to promote mineral deposition in collagen plays an important role.The analysis shows that bone sialoproteinⅡ, osteopontin, membrane associated protein, fibronectin binding factor 1 and bone tissues in the antler tip is similar expressions, that velvet skin layer and the expression of mesenchymal layer of low The first layer to the cartilage layer of cartilage has maintained a high expression level, they are probably an important factor in the ossification to promote the antler bone formation process.The calmodulin and decorin may be a important regulatory factor of the rapid growth of velvet skin layer and mesenchymal layer. The above experimental results from the gene level for study development mechanism of antler provides the basis for use of test data.
|
PDF Full Text Request