Regulatory Function Of Classical Signal Transduction System In Antler Growth Process

Purpose: Using transcriptome sequencing technology discussing the distribution ofdifferentially expressed genes in Wnt, IHH, and mTOR signaling pathway, study on therelationship between Wnt, IHH, mTOR signal pathway and antler growth as well as thecandidate gene in antler growth process. Discussion of these signal transduction system in themolecular mechanism of antler growth in drug, reveal its clinical significance.Method: Initial stage and final phase of ossification of antler tips total RNA was isolatedusing TRIzol reagent and RNA quality was tested using agarose gel electrophoresis, BioSpecnano uv vis spectrophotometer and Agilent Technologies2100Bioanalyzer analyzer.We getunigene sequence datas based on sequencing original data after the assembly splicing usedTrinity software, using Illumina/Solexa high-throughput sequencing technology for antlertranscriptome depth sequencing. Those Unigenes were used for KEGG metabolism pathwayanalysis to screen pathway related differentially expressed genes. Verify the accuracy oftranscriptome sequencing using qPCR. Using geNorm and NormFinder software selectreference genes.Result:1. Initial stage and final phase of ossification of antler tips total RNA were used forthe non degeneration agarose electrophoresis and28s and18s band is clear, BioSpec-nanotrace uv-vis spectrophotometer testing and sample density were1384.23ng/μl and2420.85ng/μl, Agilent Technologies2100Bioanalyzer detection and RIN value were7.6and7.9.2. Based on high throughput sequencing and sequence assembly, we generated almost80million short reads; construct a more comprehensive transcriptome database.3.Through analysis of the deer antler transcriptomic gene expression level and KEGGmetabolic pathways annotation information, a total of20kinds of signaling molecules was inmTOR signaling pathways,15kinds of signaling molecules was in IHH signaling pathways,17kinds of signaling molecules was in Wnt signaling pathway.4.According to the results of the analysis of differentially expressed genes, including sevenkinds of difference distribution of signaling molecules (︱log2Ratio︱1and FDR acuitieswere0.001or higher) targeting mTOR signaling system, the expression of the seven kinds of signaling molecules were in the late early than chopped raw ossification, and downstreamfactors’ S6’in the most significant; IHH signaling pathways leading members of secretoryglycoprotein ligand Hh type signal, transmembrane protein receptor Ptch and downstreamtransfer factor Gi were significant change occurs, in addition to these principal componentfactor, Sufu and GSK3beta feedback adjustment factor has changed dramatically; ClassicWnt signaling pathways in the significant changes of a total of eight factors, the major proteinWnt ligands and receptors frizzled, downstream transcription regulatory factors, fos, AP-1cyc D2and other seven kinds of signaling molecules expression capacity is significantlylower, negative feedback amount control factor GSK3B expression is significantly raised.5. After the geNorm and NormFinder program analysis, expression is relatively stable RPL40and GPx suitable for using in different growth period of pilose antler standardization analysisof target genes. RPL40as internal genes, the method of qPCR was used to detect mTORinhibitors, IHH and Wnt signaling pathways in significant changes in the signal molecules,results showed that the qPCR method are consistent with the transcriptome. Prove ouranalysis result basically reflects the pilose antler tissue within the gene expression of realdistribution in the signal path.Conclusion:1. Combined with the expression changes of signaling molecules bytranscriptome research technology and signal qPCR pathway in the expression of genes, canreflect the tissue pathway, to study specific signal transduction system contributes to theoverall system transfer pathway in tissues and its possible mechanism.2. Research shows that mTOR, IHH and Wnt signal transduction system playsan important role in the process of antler growth and development.