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题名: 鲫鱼低氧反应基因的筛选及血红素加氧酶-1 基因的特征分析
作者: 王丹
答辩日期: 2008-05-28
导师: 桂建芳
专业: 遗传学
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 鲫鱼囊胚细胞 ; 差减cDNA文库 ; 低氧反应基因 ; 血红素加氧酶-1 ; 诱导表达 ; 过表达 ; 亚细胞定位
其他题名: Screening of hypoxia-induced genes and characterization of heme oxygenase-1 in Goldfish (Carassius auratus L.)
摘要: 随着经济和人口的快速增长,生活污水和工业废水大量排入水体,高密度养殖等现状导致养殖水体富营养化日益严重,由此引起的低氧问题已成为制约水产养殖可持续发展的主要因素之一。 本研究选用鲫鱼囊胚细胞(goldfish (Carassius auratus L.) blastulae embryonic cells, CAB)进行诱导,运用抑制性差减杂交技术(suppression subtractive hybridization, SSH)成功构建了低氧诱导的鲫鱼囊胚细胞差异表达基因的差减cDNA文库。用RT-PCR的方法从差减cDNA文库中随机挑选了1300个克隆进行序列分析,得到1117个有效ESTs。将所得的ESTs片段在GenBank中进行同源搜索,发现其代表260个基因,有39个为未知基因,221个为已知或已命名基因。在已知或已命名的基因中,有38个基因与哺乳动物低氧诱导基因有同源性,其中13个在鱼类中已被鉴定,25个在鱼类中为首次鉴定。另外183个基因在鱼类低氧的适应性反应中也未见报道。 在对其中13个基因进行RT-PCR差异表达检测时发现血红素加氧酶-1(HO-1)具有非常明显的上调表达作用,所以我们对其进行了克隆鉴定、诱导表达和初步功能分析。根据文库中筛选的EST 序列,设计特异引物获得了全长cDNA 1247bp,编码272aa 的CaHO-1 基因。CaHO-1 具有同源HO-1 蛋白的基本结构,其中包括:N 端含有血红素加氧酶结构域,血红素结合标签,C端另有一个预测的跨膜区。系统进化分析显示, CaHO-1 与斑马鱼的HO-1同源性最高。 RT-PCR和实时定量PCR分析结果表明CaHO-1 基因广泛表达于健康鲫鱼的后肾、头肾、鳃和肠中,且在后肾组织中有明显的低氧诱导表达增强。为了进一步验证低氧诱导上调表达作用,我们同样对CAB细胞及鲫鱼幼苗进行不同氧气浓度的低氧处理,结果再一次证明低氧对CaHO-1基因的诱导作用。GFP融合蛋白亚细胞定位分析表明该基因的蛋白不但定位在细胞质中而且与结构预测相吻合,同时定位在细胞膜上。与此同时,我们构建了稳定表达HO-1的细胞系CAB/pcDNA3.1-HO-1 和空白对照细胞系CAB/pcDNA3.1,经4天低氧处理(1% O2)后发现CAB/pcDNA3.1-HO-1细胞系较对照细胞系CAB/pcDNA3.1 可以很好的抑制低氧处理所引起的大量细胞脱落死亡。另外,利用CCK-8试剂盒检测两种细胞系在低氧处理和常氧恢复实验中细胞生存力状况时也发现了两种细胞耐受低氧方面的明显不同。因此,我们推测CaHO-1基因可能对CAB细胞抵抗低氧压力方面起到重要的保护重要。
英文摘要: With the rapid growth of human population and economy, the problem of eutrophication has become worse in the recent years. Hypoxia caused by eutrophication has been one of the main factors that restrict the continuing development of aquiculture. In this study, a subtractive cDNA library was successfully constructed with total RNA made from goldfish blastulae embryonic (CAB) cells treated with 1% O2 for 24 h and control cells using suppression subtractive hybridization (SSH). From this subtractive cDNA library, 1117 ESTs were identified through randomly screening 1300 colonies by PCR. Sequencing analysis revealed 260 genes in which 38 genes had been found to be hypoxia-induced genes in mammals and 208 genes were firstly reported to be hypoxia-responsive genes in fish. We noted that heme oxyenase-1 was induced greatly by hypoxia when selected 13 genes of all for hypoxia-induced analysis using semi-quantitive RT-PCR. So, we aimed to study this gene under hypoxic stress. In this report, a cDNA encoding Carassius auratus HO-1 (CaHO-1) was isolated from an SSH cell line, C. auratus blastulae embryonic (CAB) cells, exposed to 1% O2 for 24h. The CaHO-1 cDNA is 1247bp in length that encodes 272 amino acid residues. CaHO-1 is homologous to mammalian HO-1 in the primary structure, characterized by heme oxygenase domain, a heme oxygenase signature motif and a putative trans-membrane domain. Phylogenetic analysis shows that CaHO-1 had the highest homology with zebrafish HO-1 in amino acid level. RT-PCR and real-time PCR analysis indicated that CaHO-1 was predominantly transcribed in posterior kidney, head kidney, gill and intestine, and significantly predominant induction expression was observed in posterior kidney under hypoxic stress. Moreover, the significantly hypoxia-induced expression was confirmed in goldfish larvae and in the in vitro cultured CAB cells. Green fluorescence of HO-1-GFP fusion protein revealed its cytoplasm and plasma membrane localization, which was consistent with the putative trans-membrane structure. Subsequently, we established stably transfected CAB/pcDNA3.1-HO-1 cell line and control CAB/pcDNA3.1 cell line, and found that the death cells were obviously reduced in the pcDNA3.1-HO-1 transfected cells under 4 days of hypoxia (1% O2) treatments in comparison with numerous detached death cells in the control pcDNA3.1 transfected cells. Furthermore, a significant cell viability difference between two kinds of transfected cells during hypoxia treatment and reoxygenation was revealed by using Cell Counting Kit-8 for cell viability assay. Therefore, the data suggested that fish HO-1 might play a significant protection role for the cells in response to hypoxic stress.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12346
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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鲫鱼低氧反应基因的筛选及血红素加氧酶-1 基因的特征分析.王丹[d].中国科学院水生生物研究所,2008.20-25
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