Sequencing And De Novo Analysis Of The Chinese Sika Deer Antler Transcriptome And Screening Differentially Expressed Genes During The Rapid Growth Process

Purpose Antler tips tissue between the first branch anther (10days) and rapid growthperiod (60days) were using for sequencing and analysising depth transcriptome datasbaesd on high-throughput sequencing technology. We screened significantdifference expression of related genes during the antler rapid growth process andexplored the mechanism of growth identified foundation for further studying theantler rapid growth and discovering functional genes.Methods10days and60days antler tips of total RNA was isolated using TRIzolreagent and RNA quality was tested using agarose gel electrophoresis, BioSpec nanouv vis spectrophotometer and Agilent Technologies2100Bioanalyzer analyzer.Weget unigene sequence datas based on sequencing original data (clear reads) after theassembly splicing used Trinity software,using Illumina/Solexa high-throughputsequencing technology for antler transcriptome depth sequencing. Those Unigeneswere used for blast search against an protein database nr, Swiss Prot, KEGG and COGfor sequence alignment, and GO functional annotation and KEGG metabolismpathway analysis. According to genes about the different growth period had adifferentially expressed analysis and screened fast growth related differentiallyexpressed genes. Verify the accuracy of transcriptome sequencing using qPCR.Results1. Total RNA extracted from the wild peach period (LA) and fast growth period (LC)of antler tips tissue were used for the non degeneration agarose electrophoresis and28s and18s band is clear, BioSpec-nano trace uv-vis spectrophotometer testing andsample density were2560.85ng/μ l and1986.23ng/μ l, Agilent Technologies2100Bioanalyzer detection and RIN value were7.4and8.5. Comply with the requirementof building database.2. Using Illumina/Solexa high-throughput sequencing technologies to removecarrier sequence, low quality sequence, we obtained40,721,676clean reads from LAperiod, the total base number was3,664,950,840nt,97.25%of the sequence sequencing quality value was in Q20above, the GC content was53.07%.40,721,676clean reads of LA period were assembled into112,343contigs using soapdenovosoftware and the contigs were assembled into67,580unigenes, average length is709nt, N50is1292nt. Through the protein database nr blast,33,451unigenes (49.5%)of LA were used for blast search against an nr database known protein.The samples ofLA had a GO function annotation, COG function classification and KEGGmetabolism path analysis.10,667unigenes is classified into50GO functionalcategory,10,370unigenes were categorized into25a COG function classification,21,767unigenes were categorized into223KEGG metabolic pathways.3. We obtain39,787,196clean reads from LC period, the total base number was3,580,847,640nt,97.05%of the sequence sequencing quality value was in Q20above,the GC content was51.45%.39,787,196clean reads of LC period were assembledinto107,848contigs and the contigs were assembled into63,253unigenes, averagelength is639nt, N50is1072nt.32,247unigenes (50.98%) of LC were used for blastsearch against an nr database known protein. The samples of LC had a GO functionannotation, COG function classification and KEGG metabolism path analysis.11,936unigenes is classified into52GO functional category,9,119unigenes werecategorized into25COG function classification,22,561unigenes were categorizedinto223KEGG metabolic pathways.4. The sample of LA and LC had a GO function enrichment analysis and KEGGpathway expressed differentially enrichment analysis, a total of15,539genesexpressed differentially were categorized into GO functional category,5,244genesexpressed differentially were categorized into235KEGG metabolism pathway.5. The samples of LA and LC had a transcriptome differentially expressed genesanalysis and we screened significant10，027difference expression genes. Combinedwith the GO function enrichment analysis and KEGG metabolic pathway enrichmentanalysis, we obtained significant differences expressed genes including18kinds oftranscription factor,17kinds of growth factors,16kinds of extracellular matrixgenes and27significant differences expressed genes which were related tochondrocyte proliferation, differentiation and hypertrophy in the process of antler rapid growth.6. we use qPCR to detect eight kinds of differentially genes expressed whichfound in our data random.qPCR results was consistent with transcription group data.Conclusion1.Through the analysis of high throughput sequencing and data information, weestablished a comprehensive antler transcriptome database during the rapid growthperiod and expanded antler data information in gene level and protein level.2.Through the antler transcription groups differentially expressed gene analysis duringthe different growth period, we screened out a lot of antler regulation genes relatedrapid growth process and lay the foundation for researching antler growth mechanismand excavating functional genes.