【作者】 顾朝辉；

【导师】 曾甫清；

【作者基本信息】 华中科技大学 ， 泌尿外科， 2010， 博士

【摘要】 第一部分：生物信息数据挖掘新基因预测平台的建立摘要目的：依据最新生物信息学数据库和数据挖掘技术,充分挖掘现有生物数据库内蕴含信息,建立基于生物信息和数据挖掘技术的新基因克隆和功能预测平台。方法：首先,通过检索词从dbEST数据库检索,下载研究相关目标序列,将所选序列进行Blast分析,比对出相应的Unigene同源簇,下载上述同源簇或通过数字差异显示工具NCBI_DDD检索组织特异性Unigene同源簇；其次,通过Biolign软件进行EST clusters的拼接、延伸和组装,应用Blast检索程序进行EST clusters拼接的contigs序列基因组相似性分析,校正contigs组装产生的误差；第三,应用Genscan程序分析基因组同源序列的编码区(CDS)；第四,DNAssist预测上述CDS序列的开放阅读框(open reading frame, ORF);最后,Premier 5.0设计针对每个ORF的对应引物。结果：通过数字差异显示工具分析RIKEN full-length enriched mouse cDNA library和Epididymis cDNA library间的表达序列标签的差异序列,得到一个在RIKEN full-length enriched mouse cDNA library高丰度表达的同源簇Mm.5168,下载其对应的同源簇为UGID:258077;进行后续EST组装、延伸和contigs序列校对,同时预测基因组同源性编码区和开放阅读框,并设计相应的引物。结论：生物信息数据挖掘新基因克隆和功能预测平台能有效的发现组织特异性Unigene同源簇,为未知新基因的克隆和功能预测提供了很好的方向。第二部分：小鼠睾丸特异表达基因TSEG-3的克隆、组织定位及表达谱分析目的：应用生物信息数据挖掘新基因克隆和功能预测平台,预测可能参与睾丸精子发生和男性不育相关睾丸基因,并通过实验验证预测的准确性。方法：首先,利用数字差异显示工具Digital Differential Display检索与EST片段BY706707.1的同源簇,按前文提出的生物信息数据挖掘新基因克隆和功能预测流程,预测出一个新的编码区序列；其次,通过RT-PCR验证,证实其确实存在于成年小鼠睾丸；第三,通过T／A克隆,进行测序分析其与设计序列的一致性和同源性,验证了新基因预测平台的可靠程度；通过原位杂交分析分析TSEG.3表达的细胞类型,同时以用Northern blotting分析转录本大小和RT-PCR分析TSEG-3多器官和不同发育阶段表达谱序列。结果：成功从小鼠睾丸组织中扩增出TSEG-3,测序结果与预测结果一致,将基因序列提交Genbank数据库,获得Genbank登录号：EU477370(GI：186892498),命名为睾丸特异表达基因3(TSEG-3)。原位杂交结果显示TSEG-3表达于精原细胞、少量精母细胞和精子细胞等,Northern Blotting印迹证实转录长度为1023bp左右,小鼠TSEG-3多组织表达谱分析显示TSEG-3特异地表达于睾丸组织,在出生后2月龄时达到表达峰值。结论：TSEG-3是睾丸特异性表达基因,具有发育阶段特异性特征,可能参与小鼠生精过程,深入研究TSEG-3的功能有助于阐明精子发生机制和男性不育机制。第三部分：小鼠睾丸特异表达基因TSEG-3生物信息学分析及TSEG-3多克隆抗体制备与鉴定目的：应用生物信息数据挖掘工具,进行TSEG-3蛋白生物信息学分析,为进一步研究TSEG-3蛋白的功能提供必要方向。方法：应用序列信息挖掘软件,分析TSEG-3蛋白质物理特性、疏水区域和亲水区、跨膜区、蛋白质二级结构、蛋白质三级结构、拓朴树分析、特异性磷酸化位点、抗原位点及肽段、亚细胞定位及TSEG-3蛋白功能注释；设计TSEG-3抗原多肽并合成多肽,制备抗TSEG-3蛋白血清,Western Blotting鉴定TSEG-3蛋白分子量大小。结果：通过序列分析显示TSEG-3位于第17号染色体17A3.3区,有六个外显子组成。TSEG-3属于DFU634家族基因,在TSEG-3的5’侧翼区域有六个启动子序列；miRNA检索发现16个可能相关miRNA;并在TSEG.3第277AA.285AA预测出一个非稳定区段(disordered regions)序列为：QSLHEALFG；TSEG-3属于非跨膜蛋白,47.8%可能胞质的：21.7%定位于细胞核；理论等电点为7.15.吸光系数为23680；具有10个丝氨酸(Serine)位点,3个酪氨酸(Tyrosine)位点,在第211到221间存在一个PKC位点,第28氨基酸残基处为信号肽区,第65个氨基酸为一个精氨酸／赖氨酸前肽切割位点(Arg(R)／Lys(K))等；TSEG-3蛋白预测理论分子量为38,52976KD和Western Blotting检测结果一致；功能预测提示TSEG-3可能参与生殖细胞的分化和凋亡。结论：生物信息数据挖掘工具为新基因TSEG-3功能研究提供了丰富的研究内容,极大减少了TSEG-3功能研究的盲目性,加快了基因功能鉴定的速度。第四部分：过表达TSEG-3基因对小鼠精母细胞系GC-2 spd增殖、凋亡的影响目的：探讨TSEG-3过表达对小鼠精母细胞系GC-2 spd(ts)的增殖和凋亡的影响。方法：通过构建pEGFP-TSEG-3载体和体外培养小鼠精母细胞系GC-2 spd(ts),观察融合蛋白EGFP-TSEG-3的亚细胞定位；采用MTT法检测TSEG-3过表达对GC-2spd(ts)细胞增殖的影响；应用流式细胞仪分析TSEG-3过表达对GC-2 spd(ts)细胞周期的作用；AO/EB双重染色法、Hoechst 33258/PI双染、Annexin V/PI双染法和JC-1染色分析TSEG-3过表达对GC-2 spd(ts)细胞凋亡率的影响；Real-Time RT-PCR分析TSEG-3过表达对凋亡相关蛋白的影响。结果：融合蛋白EGFP-TSEG-3定位于细胞核,MTT法结果提示TSEG-3过表达抑制GC-2 spd(ts)细胞增殖；TSEG-3过表达诱导GC-2 spd(ts)细胞出现G1和G2／M期捕获。TSEG-3过表达诱导GC-2 spd(ts)细胞凋亡,Real-Time RT-PCR显示TSEG-3过表达诱导Fas上调,同时Bcl-2/Bax比率下调,说明Fas-Fas通路和Bcl-2/Bax均参与TSEG-3过表达诱导GC-2 spd(ts)细胞凋亡。结论：TSEG-3过表达抑制GC-2 spd(ts)细胞增殖,诱导GC-2 spd(ts)细胞凋亡,Fas/Fas通路和Bcl-2/Bax均参与TSEG-3过表达诱导GC-2 spd(ts)细胞凋亡途径。第五部分：TSEG-3过表达诱导小鼠睾丸精原细胞凋亡及其睾丸疾病模型表达谱分析目的：整体水平探讨TSEG-3过表达对小鼠睾丸精原细胞的影响,并分析其在睾丸疾病模型表达谱。方法：制备in vivo-jetPEITM-pEGFP-TSEG-3聚合物：构建TSEG-3过表达模型,HE染色和Tunel分析TSEG-3过表达对小鼠生殖细胞的影响；同时构建手术隐睾模型,分析TSEG-3转录水平与细胞凋亡率的关系,分析温度对TSEG-3转录水平的影响；复制17β雌二醇诱导隐睾模型,检测17β雌二醇诱导隐睾模型中TSEG-3转录水平,明确17β雌二醇对TSEG-3转录的影响。结果：荧光显微镜观察提示In Vivo JetPEITM是一种高效、可靠安全的DNA转染试剂,能有效的携带外源DNA入生殖细胞。TSEG-3过表达使睾丸组织生精小管官腔内细胞密度减少,生精小管官腔变薄,大量精原细胞及精母细胞缺失,未发现成熟精子。Tunel结果提示TSEG-3过表达可能诱导生精细胞的凋亡。手术隐睾模型结果显示：TSEG-3的转录激活与手术组小鼠睾丸内生精细胞凋亡率增加呈正相关关系,进一步提示TSEG-3可能参与睾丸组织生精过程中细胞凋亡过程。17β-雌二醇诱导隐睾模型原位杂交结果提示17β-雌二醇可能参与抑制不同阶段生殖细胞中TSEG-3的转录。结论：TSEG-3过表达诱导小鼠睾丸生殖细胞凋亡,温度可能诱导TSEG-3的转录,17β-雌二醇可能参与抑制不同阶段生殖细胞中TSEG-3的转录。更多还原

【Abstract】 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.更多还原