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鳜凋亡相关基因的克隆与功能研究
高原
Subtype博士
Thesis Advisor聂品
2006-07-05
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细胞凋亡 鳜肿瘤坏死因子相关的凋亡诱导配体 鳜肿瘤坏死因子相关的凋亡诱导配体的诱骗受体 鳜mst3和sok1相关激酶 基因超表达
Abstract鳜(Siniperca chuatsi)是我国一种重要的淡水养殖鱼类,但是各种疾病的发生严重影响着其产量,免疫防治是控制疾病的重要而有效的途径。细胞凋亡是鱼类非特异性免疫调节的一个重要方面,本文对鳜凋亡相关基因进行了克隆鉴定及功能研究。 通过构建SMART cDNA文库并结合RACE技术首次在鳜中克隆到三个凋亡相关基因,并采用PCR和Genome Walking的方法克隆这三个基因及其启动子区域。鳜肿瘤坏死因子相关的凋亡诱导配体(ScTRAIL)基因cDNA全长1359 bp,5′和3′非翻译区分别为112 bp和395 bp,开放阅读框包含852 bp,编码283 aa,基因全长9021 bp,含有6个外显子和5个内含子。启动子区域732 bp,缺乏典型的TATA或CAAT结构,但存在Oct-1、Sp-1、NF-1、RAP-1、C/EBPalp、NF-kappaB和AP-1等潜在的转录因子结合位点。 鳜肿瘤坏死因子相关的凋亡诱导配体的诱骗受体(ScDcTRAILR)基因,cDNA全长2494 bp,5′和3′非编码区分别为106 bp和1326 bp,开放阅读框包含1062 bp,编码353 aa,基因全长10437 bp,含有10个外显子和9个内含子。启动子区域804 bp,缺乏典型的TATA框或CAAT盒,但有一些特异的转录因子结合位点,如C/EBPalp、Sp-1、Oct-1和GATA-1等。 鳜Mst3和SOK1相关激酶(ScMASK)基因,cDNA全长1743 bp,5′和3′非编码区分别为161 bp和364 bp,开放阅读框包含1218 bp,编码405 aa,基因全长8561 bp,包含有9个外显子和8个内含子。启动子区域1526 bp,缺乏典型的TATA框或CAAT盒,存在Oct-1、Sp-1、NF-1、RAP-1、C/EBPalp、NF-kappaB和AP-1等潜在的转录因子结合位点。Southern blotting分析表明在鳜基因组中,ScTRAIL基因为单拷贝基因,在此基础上采用荧光定量PCR方法揭示ScDcTRAILR和ScMASK基因也是单拷贝基因。 对三个基因转录和表达产物的器官组织分布进行了详细的分析。RT-PCR表明这三个基因在健康鳜组织中呈组成型表达,且三者具有不同的组织分布模式。分别构建ScTRAIL、ScDcTRAILR和ScMASK基因的原核表达质粒,通过原核表达、表达蛋白纯化、并制备多克隆抗体,对头肾、肾脏、肝脏、脾脏、肠、皮肤、心脏、脑、鳃和心脏等器官中的目的基因蛋白进行的免疫印迹,发现ScTRAIL蛋白分布于各个组织中,在头肾、性腺中显示较强的带,在脑中的带较弱。ScDcTRAILR蛋白在脾脏、脑、心脏和鳃中有明显的分布,在其它组织中带很弱或没有。ScMASK蛋白在鳃、肝脏、脑中有明显的带被标记。 模拟病原感染的情况,采用荧光定量PCR中的相对定量方法分析这三个基因在LPS或PolyI:C诱导后变化表达,发现ScTRAIL的表达在诱导前后变化不明显。ScDcTRAILR在LPS诱导组的头肾中呈上调表达,在肝脏、脾脏、肾脏、皮肤和心脏中下调;在PolyI:C诱导组,该基因表达量在头肾、肾脏中有显著的上调,在肝脏、心脏中有较大的下调。ScMASK基因的表达在两个诱导组中的头肾和心脏中有明显的上调,在Poly I:C刺激下,肝脏和脾脏中也检测到该基因的上调表达,在其它组织中则有下调趋势。 利用荧光显微镜观察、核酸电泳以及流式细胞术等方法研究了ScTRAIL、ScDcTRAILR和ScMASK在Hela细胞中超表达对细胞的影响。ScTRAIL和ScMASK基因具有促进肿瘤细胞凋亡的作用。ScDcTRAILR在Hela细胞中不能引起细胞发生凋亡现象。
Other AbstractThe mandarin fish Siniperca chuatsi is an important species in China’s aquaculture. The culture of the mandarin fish has been threatened seriously by many kinds of diseases. It is thus important and necessary to develop some effective methods for the control of diseases. The understanding of immune system in this fish will provide a base for such development. The present study aimed to clone some apoptosis-related genes for the mandarin fish, with their apoptotic function analyzed in cell lines. Three apoptosis related genes were cloned for the first time in the mandarin fish, i.e. TNF-related apoptosis-inducing ligand (TRAIL), a TRAIL decoy receptor (DcScTRAILR) and Mst3 and SOK1-related kinase (MASK) which are named as ScTRAIL, and ScDcTRAIL, and ScMASK. ScRAIL gene with the full-length cDNA of 1359 bp contains an open reading frame coding for a 283 aa protein; it’s full length genome has 9021 bp which contains six exons and five introns. The promoter region of ScTRAIL is 732 bp, which contains some putative transcription factor binding sites such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPalp, NF-kappaB and AP-1; however, it lacks typical TATA or CAAT box. The ScDcTRAILR gene with the full-length cDNA of 2494 bp contains an open reading frame coding for a 353 aa protein; its full-length genome sequence is 10437 bp which contains ten exons and nine introns. The promoter region of ScDcTRAILR is 804 bp, which contains some putative transcription factor binding sites such as C/EBPalp、Sp-1、Oct-1 and GATA-1, but it lacks typical TATA or CAAT box. The ScMASK gene with the full-length cDNA of 1743 bp contains an open reading frame coding for a 405 aa protein; its full-length genome sequence is 8561 bp, which contains nine exons and eight introns. The promoter region of ScMASK is 1526 bp, and lacks typical TATA or CAAT box, but it contains some putative transcription factor binding sites such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPalp, NF-kappaB and AP-1. Southern blotting analyses revealed that in mandarin fish genome, the ScTRAIL gene is present as a single copy; based on this result, the ScDcTRAILR and ScMASK genes were also found as single copy genes, as analyzed by real-time PCR. The organ distribution of the three genes was analyzed both in the transcription and protein levels. By RT-PCR analysis, the transcription products of the cloned genes were investigated in different tissues of healthy mandarin fish, and the three genes had different patterns of tissue distribution. Western blotting analyses revealed that ScTRAIL protein existed broadly in examined tissues with the highest expression level in head kidney and gonad while lowest in brain. ScDcTRAILR protein had a relatively higher expression level in spleen, brain, heart and gill, while ScMASK protein was just detected in gill, liver and brain. Furthermore, expression of the three genes was analyzed to mimic pathogen attacking by real-time quantitative PCR. Obvious changes in expression level of ScTRAIL were not observed when fish were stimulated with PolyI:C or LPS. ScDcTRAILR gene expression in LPS induced group was up-regulated in head kidney, and in PolyI:C stimulated group its expression level increased in head kidney and kidney, while decreased in liver and heart. The expression level of ScMASK in LPS stimulated group was up-regulated in head kidney and heart, and its expression level increased in liver and spleen when stimulated with PolyI:C. Apoptosis effect of the three genes was investigated by over-expression of three genes in Hela cell line, respectively. Apoptosis were detected by several methods including fluorescence microscopy, agarose gel electrophoresis and flow cytometry. The existence of apoptotic bodies, DNA ladder and apoptosis peaks in the ScTRAIL and ScMASK transfected Hela cells indicated that these two genes induced apoptosis. However in the ScDcTRAILR gene transfected Hela cells, no apoptotic characters were observed, suggesting that this gene may not induce cell apoptosis and may be probably a decoy death receptor.
Pages120
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/11968
Collection学位论文
Recommended Citation
GB/T 7714
高原. 鳜凋亡相关基因的克隆与功能研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2006.
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