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Alternative TitleMolecular cloning and characterization of carp T-cell marker Genes
Thesis Advisor殷战 ; 郭琼林
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword转基因鲤鱼 抑制差减杂交 T细胞标志基因 斑马鱼 整体原位杂交 启动子
Abstract转“全鱼”生长激素(GH)基因能明显促进鲤鱼胸腺发育,有效延缓胸腺退化。应用抑制性差减杂交技术构建了4月龄F3代转全鱼生长激素(GH)基因鲤鱼 (Cyprinus carpio L.)胸腺发育差减cDNA文库。并随机挑取了200多个PCR检测呈阳性的克隆进行了斑点杂交,选取斑点杂交阳性的克隆进行测序,挑选6个斑点杂交阳性克隆进行了RT-PCR验证和7个斑点杂交阳性克隆进行了虚拟Northern杂交,这7个基因全部呈差异表达阳性。 通过RACE快速末端扩增得到了差异表达基因CD8β、CD4、TCRγ、CD3γ/δ 4个T细胞标志基因的cDNA全序列。RT-PCR检测这些已经得到全长cDNA的基因,研究它们在成年鲤鱼中的组织分布,在被检测的几种免疫相关的组织中这几个基因均检测到转录产物,其中胸腺、鳃、和小肠表达较强。 通过对斑马鱼早期胚胎发育阶段的观察,发现在注射了330pg的GH mRNA后, 斑马鱼胸腺发育标志基因Rag1的表达获得显著增强,表明和证实了GH 对胸腺早期发育的促进作用。 运用斑马鱼CD8α和CD8β 的反义RNA探针,对受精后第4天和第5天的斑马鱼幼苗进行整体原位杂交观察, 检测到其在肠泡和脊索部位的表达,且该部位的表达可为免疫抑制剂地塞米松所抑制,并为GH所增强,表明了CD8+细胞在早期斑马鱼发育阶段形成于肠道和脊索的特性。 构建了从500bp到11kb不同长度的斑马鱼CD8β5'基因组片段驱动EGFP表达的转基因报告斑马鱼,仅在脊索部位有明显的荧光表达。
Other AbstractSince the first transgenic fish was produced in 1985, various transgenic fish had been created to meet different needs for human beings. In 2003, Guo et al. demonstrated that “All-fish” growth hormone (GH)-transgene promotes thymus development and thymocyte proliferation, and retards thymus degeneration. In order to find the molecular mechanism beneath the effect of GH on thymus development, a subtractive cDNA library of thymus in F3 generation of 4-month transgenic carp was constructed by suppression subtractive hybridization (SSH) techniques. After pre-screening and dot blot analysis, seven cDNA clones were eventually identified as differential expressed gene fragments, which were further confirmed with RT-PCR or virtual northern hybridization. By using the technique of RACE, we have cloned complete cDNA sequences of differential expressed genes of CD8β、CD4、TCRγ、CD3γ/δ, which are all T cell marker genes. Tissue distribution of these genes in adult carp were analyzed by RT-PCR. Our results showed that they express predominantly in thymus, followed by intestine, gills. The effects of GH on the generation of lymphoid cells during zebrafish development was studied by analyzing the expression of recombination activating gene 1 (Rag1) using whole mount in situ hybridization(WISH). Our results have shown that the expression of Rag1 in thymus at 72hpf can be enhanced by the GH mRNA (330pg) injection compared with the one of the control group, which indicating the stimulation of GH administration on the thymus development. Detection with the CD8α and CD8β antisense RNA probes using WISH, the presence of CD8+ cells were found at intestine bulb and notochord in zebrafish at 4dpf and 5dpf stages. Furthermore, the corresponding signals could be largely suppressed by the treatment of 10μg/ml dexamethasone, an effective immune inhibitor, which suggesting the early CD8 positive cells developing in fish intestine and notochord area. Plasmids that EGFP was driven by zebrafish CD8β5' genomic DNA fragment region from 500bp to 11kb were constructed. The strong enhanced GFP fluorescence could be observed at notochord of the CD8β5':EGFP transgenic zebrafish under fluorescence microscope.
Document Type学位论文
Recommended Citation
GB/T 7714
孙晓凤. 鱼类T细胞标志基因的分子克隆及相关特性的研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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