Establishing Of Novel Gene Prediction Platform Based On Bioinformatics And Data Mining And The Identification Of TSEG-3

PART I Establishing of novel gene prediction platform based on bioinformatics data miningObjective:According to the latest development of bioinformatics databases and data mining techniques, to fully exploit the valuable information of the existing biological database and build the new technology platforms of cloning and functional prediction based on bioinformatics and data mining.Method:Search dbEST database and download target sequence involved in the research. The analysis of the selected sequence will be alignmented using the program of Blast in mouse genomic. Download the clusters of Unigene and the splicing, extension and assembly of EST clusters were carried out using Biolign software. The assembled contigs of EST clusters are analyzed by Blast_N in mouse genomics. Coding region sequences (CDS) of the homologous sequences of the assembled contigs in Genome were predicted via Genscan program. ORF sequences of the CDS were predicted by DNAssist. Each primers for ORF were designed by Primer Premier 5.0.Results:The cDNA library of Mm.5168 was searched by the digital differential display between RIKEN full-length enriched mouse cDNA library and Epididymis cDNA library. The sequences of Unigene UGID:258077 for Mm.5168 was download. Some new CDS was predicted by Genescan and the open reading frame for each CDS was analyzed by the DNAssist.Conclusion:The new gene prediction platform based on bioinformatics and data mining is a effective tool in searching the novel unknown genes and predicting the function of novel gene and provides a good direction for study of the novel gene. PARTⅡThe cloning, cellular location of mouse testis-specific gene 3 and the analysis of expression profiles for TSEG-3Objective:To predict the novel testis genes involved in spermatogenesis and male infertility using the novel gene prediction platform based on bioinformatics data mining and accuracy of prediction will be verified by RT-PCR.Method:the homology EST clusters of BY706707.1 was found by using Digital Differential Display (DDD). The CDS for a novel gene was predicted by the novel gene prediction platform based on bioinformatics data mining. We detected the expression of the novel gene by RT-PCR. The cDNA of the novel gene was cloned into pGM-T and The cDNA of the novel gene was sequenced. The cellular location of the novel gene was analyzed by in situ hybridization. The length of transcript for the novel gene TSEG-3 was verified by nothern blotting. The expression profiles of the novel gene was analyzed by RT-PCR.Results:A novel gene was amplified from adult mouse testis and named TSEG-3. No sequences were consistent with the sequence of TSEG-3 in Genebank database. The sequence of the novel gene TSEG-3 was consistent with the sequence of prediction. Brown Sediment for Hybridization signal of TSEG-3 mRNA was found in spermatogonia, spermatocytes, and a small amount of sperm cells, etc. The length of transcript of TSEG-3 is 1023bp by northern blotting. The transcript of TSEG-3 was detected only in testis tissue and up to peak at postnatal 2 months.Conclusion:TSEG-3 is a testis specifically expressed gene and specific devolopment stages gene. TSEG-3 may participate in spermategensis and the study of TSEG-3 maybe help to eluciate the molecular mechanism of male infertility. PART III Bioinformatics analysis of mouse testis-specific gene 3, the production of TSEG-3 polyclonal antibody and identification of TSEG-3 protein.Objective:To provide the necessary research directions for TSEG-3 and to further study the function of TSEG-3 protein, the bioinformatics analysis of the mouse testis-specific gene 3 was executed via the tool based on bioinformatics & data mining tools.Method:Physical property, hydrophobic regions, hydrophilic regions, transmembrane region, protein secondary structure, protein structure, phylogenetic tree, specific phosphorylation sites, antigenic sites and the peptides, subcellular localization and TSEG-3 protein functional annotation was predicted using the sequences'data mining software. Results:The theoretical molecular weight of TSEG-3 protein was 38529.76 Dalton, and its theoretical isoelectric point was 7.15. A putative conserved domain DUF634 was detected between 110 and 310 residues of TSEG-3 by National Biological Information Center (NCBI) blast_p program. The phylogenetic tree and homology tree indicated that TSEG-3 had a closest genetic relationship with rat LOC294301, and close genetic relationships with human C6orf81, Canis LOC474886, Bos taurus LOC540812, and Equus caballus LOC100053594. The nucleic acid and amino acid sequences of TSEG-3 were 99% homologous to those of 4930511I11Rik. NetPhos 1.0 Server showed a protein kinase C (PKC) phosphorylation site at the 224th residue of TSEG-3 protein. A propeptide cleavage site was found at the 56th residue of TSEG-3 protein by ProPv.l.Ob program. A signal peptide site was noted at the 28th residue of TSEG-3 protein by SignalP 3.0 Server. The PSORTⅡprogram showed that TSEG-3 protein localized in cytoplasm (47.8% possibility) or nuclei (21.7% possibility). The prediction of the PFP Server indicated that TSEG-3 might participate in cell differentiation and induction of apoptosis.Conclusion:The function study of TSEG-3 protein was provided in different directions using bioinformatics & data mining tools, greatly reducing the blindness of the TSEG-3 function study, accelerated the pace of the identification of the novel gene function. PART IV Overexpression of TSEG-3 inhibited mouse spermatogonial cell lines GC-2 spd cell proliferation and induce apoptosisObjective:To analyze the regulation of proliferation and apoptosis for mouse spermatogonial cell lines GC-2 spd(ts) cell post-overexpression of TSEG-3 in vitro.Method:Firstly, to watch the subcellular of TSEG-3 protein, the plasmid pEGFP-TSEG-3 was constructed. The proliferation of GC-2 spd(ts) cell was evaluated by MTT post-overexpression of TSEG-3. The cell cycle of GC-2 spd(ts) cell was detected by FCM after overexpression of TSEG-3. Apoptosis ratio of GC-2 spd(ts) cell was analyzed by FCM via AO/EB double staining, Hoechst 33258/PI double staining, Annexin V/PI double staining and the change of Mitochondrial membrane potential was evaluated by FCM via JC-1 staining. The transcript of apoptosis pathway related genes was detected by real-time quantitative RT-PCR.Results:EGFP-TSEG-3 fusion protein is located in the nuclear of cos-7,239 cell and GC-2 spd(ts) cell. The proliferation of GC-2 spd(ts) cell was inhibited post overexpression of TSEG-3 via MTT. G2 and G2/M of GC-2 spd(ts) cell was arrested by FCM via PI staining after overexpression of TSEG-3. Overexpression of TSEG-3 induced the apoptosis of GC-2 spd(ts) cell. The transcript of Fas was up-regulated and the ratio of Bcl-2/Bax was down-regulted post overexpression of TSEG-3.Conclusion:Overexpression of TSEG-3 inhibits the proliferation of GC-2 spd (ts) cell, and induces the apoptosis of GC-2 spd (ts) cells. Fas/Fas pathway and Bcl-2/Bax were involved in the process that overexpression of TSEG-3 induced the apoptosis of GC-2 spd (ts). PART V TSEG-3 overexpression induced mouse testis germ cell apoptosis in vivo and expression pattern analysis of TSEG-3 in different cryptorchidism modelsObjective:To explore the changes of mouse testis germ cell via the overexpression of TSEG-3, analyze the relationship between TSEG-3 and apoptosis of mouse testis germ cell in vivo and detected the expression profiles in different cryptorchidism models.Method:Firstly, the polymer of in vivo-jetPEI TM-pEGFP-TSEG-3 was prepared according to standard protocol. The histological changes and apoptosis cells was evaluated by HE staining and Tunel after 72 hours post TSEG-3 overexpression in mouse testis. Secondly, Surgical cryptorchidism model was constructed according to the previous procedure. The TSEG-3 transcription level in testis of surgical cryptorchidism model was detected by real-time RT-PCR. The relationship between germ cell apoptosis and testis of surgical cryptorchidism was analyzed via Tunel staining. Thirdly, the 17β-estradiol induced cryptorchidism model was build using previous method. The transcript of TSEG-3 in testis tissues was analyzed by in situ hybridzation and real-time RT-PCR.Results:In Vivo JetPEITM is an efficient, reliable and safe transfection reagent for plasmid DNA via fluorescence microscope and can effectively carry exogenous DNA into the germ cells. The number and cell density of seminiferous tubules reduced in testis of TSEG-3 overexpression model. Seminiferous tubule in testis of TSEG-3 overexpression model is thinner than control group. There were the loss of spermatogonia and spermatocyte, and not any mature sperm. Tunel results suggest TSEG-3 overexpression may induce germ cell apoptosis. In surgical cryptorchidism model, the transcript of TSEG-3 was up-regulated and positive correlation with testicular germ cell apoptosis. These resluts showed TSEG-3 may be involved in the process of testicular germ cell apoptosis. But, results of in situ hybridization and real-time RT-PCR suggest 17β-estradiol inhibited the TSEG-3 transcription in different stages of germ cells.Conclusion:TSEG-3 overexpression induced germ cells apoptosis in vivo. Temperature may induce TSEG-3 transcription and 17β-estradiol maybe inhibit the TSEG-3 transcription in different stages of germ cells.