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鳜和斑鳜金属硫蛋白-2基因的克隆及表达分析
Alternative TitleCloning and expression of metallothionein-2 in the mandarin fish (Siniperca chuatsi) and spotted mandarin fish (S. scherzeri)
高典
Subtype博士
2008-06-14
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 斑鳜 金属硫蛋白 基因克隆 荧光定量 免疫印迹 免疫组化
Abstract随着工农业的发展,重金属等污染物导致的水环境污染越来越严重,分子生物标记常常被用来指示和检测水体中重金属污染状况,金属硫蛋白(Metallothionein, MT)就是其中最重要的生物标记之一。鳜(Siniperca chuatsi Basilewsky, 1855)和斑鳜(S. scherzeri Steindachner, 1892) 是我国重要的名贵淡水养殖鱼类,对水质变化非常敏感,因此,本文以这两种鱼类为材料研究了鳜和斑鳜MT-2的基因结构,并检测了鳜组织中金属硫蛋白的表达情况,用以评价其在重金属污染检测中的应用。 通过RACE-PCR技术获得了鳜MT-2基因的cDNA全长,构建了系统发育进化树,PCR扩增了鳜和斑鳜MT-2的基因组序列,并采用TAIL-PCR的方法克隆了二者MT-2的启动子区域,对MT-2进行了基因组结构分析。 鳜MT-2 cDNA序列全长为388 bp。其中5′端、3′ 端非编码区分别为49bp和156bp,开放阅读框包含183 bp,编码60个氨基酸。获得的鳜MT基因组全长为865bp,包含三个外显子和两个内含子。扩增得到的启动子长1466bp,启动子区域含有典型的顺式作用元件-金属反应元件(Metal response element, MRE),并分为三簇;含有TATA盒和激活蛋白(Activator protein1, AP1),缺乏GC盒。 斑鳜MT-2基因组全长865bp,和鳜MT-2相比,只有外显子上少数几个碱基的差异,也包含有三个外显子和两个内含子。扩增得到的启动子长1167bp,包含的金属反应元件同样分成三簇,含有AP1和糖皮质激素应答元件(Glucocorticoid Response Element, GRE)等。 对鳜MT-2转录和表达产物的器官组织分布进行了详细的分析。real-time PCR的检测表明该基因受Cd2+诱导后,mRNA表达水平在鳃和脑组织中显著上升,在肌肉、肝脏和肾脏组织中表达水平变化不显著。构建了pET-32a(+)-MT-2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,对鳜的组织鳃、肝脏、心脏、肾脏、肌肉、头肾、脑、脾脏和肾脏等器官中的目的基因蛋白进行免疫印迹分析。Western blotting 结果显示,健康鳜组织中,鳃、肝脏、心脏、肾脏、肠道中MT-2表达量比较高,肌肉和脑组织中表达量比较低,头肾和脾脏中几乎检测不到MT-2的表达。对肝脏和肾脏组织进行免疫组织化学检测,在肝脏组织的染色中,MT-2分布在肝细胞的胞浆里;肾脏组织中,MT-2主要分布在肾脏近端小管和远端小管的上皮细胞胞浆内。
Other AbstractThe metallothionein-2 (MT-2) gene was cloned from the mandarin fish Siniperca chuatsi (Basilewsky, 1855) by using the method of rapid amplification of cDNA ends (RACE). The spliced cDNA sequence extended 388bp. The 5′ untranslated region (UTR) and 3′ UTR are 49 and 156 nt respectively. The polyadenylation signal (AATAAA) is found starting at 18 nt upstream from the polyA tail. The putative MT is predicted to be a peptide of 60 amino acids including highly conserved 20 cysteine residues. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothioneins. The amplified MT-2 genomic sequence is 865 bp in length, which consists of 3 exons and 2 introns. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The promoter region extended 1466 bp and contained 10 putative metal response elements (MREs) composing 3 clusters and TATA box, AP1, etc. The MT-2 genome from the spotted mandarin fish Siniperca scherzeri (Steindachner, 1892) was also amplified utilizing primers designed according to the mandarin fish MT-2 cDNA sequence. It extended 865bp consisting of 3 exons and 2 introns. The promoter region extended 1167bp including 6 MREs composing 3 clusters and GREs, AP1, etc. Real-time PCR analysis revealed that mandarin fish MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd2+-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine. It was weakly detected in the brain and muscle. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm of hepatocytes in the liver, and the cytoplasm of the proximal and distal tubular epithelium in the trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution.
Pages118
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12390
Collection学位论文
Recommended Citation
GB/T 7714
高典. 鳜和斑鳜金属硫蛋白-2基因的克隆及表达分析[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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