【作者】 蔡文婷；

【导师】 汝少国；

【作者基本信息】 中国海洋大学 ， 生态学， 2008， 硕士

【摘要】 卵黄原蛋白(Vitellogenin,VTG)是检测水环境雌激素的重要生物标志物。雄性鱼类VTG mRNA是一种检测环境雌激素的新兴生物标志物。我国水环境生物监测主要采用传统的群落学方法和生物化学检测,应用分子生物学方法检测水环境雌激素的报道较少。久效磷(Monocrotophos, MCP)是一种常用的有机磷农药,已经在我国造成了严重的农药残留污染。本文选用在我国广泛分布的鱼类——金鱼作为模式生物,研究久效磷对雄性金鱼VTG mRNA表达的影响。实验室邴欣等已成功分离纯化17β-雌二醇(17β-estradiol,E2)暴露的雄性金鱼血清VTG;用非变性聚丙烯酰胺电泳(PAGE)测得VTG的分子量约为440kDa;用Western-blot技术证明在0.01 mg·L-1、0.10 mg·L-1、1.00mg·L-1三个久效磷浓度暴露组都能够诱导雄性金鱼分泌VTG。基于实验室已经完成的工作,本文采用RT-PCR检测金鱼VTG mRNA的表达,克隆编码金鱼VTG蛋白的部分cDNA片段,并以此为模板制备地高辛标记的RNA探针,用这种分子生物学技术检测环境雌激素对金鱼的雌激素效应,建立了新的筛选环境雌激素的分子生物学方法。实验所用金鱼购自青岛南山花鸟市场,驯养两周后开始实验。取雌鱼肝脏提取总RNA,根据已知氨基酸序列的鱼种设计兼并引物,RT-PCR扩增金鱼VTG基因片段,得到一条1135bp的特异性条带。将所得DNA进行测序,其序列用NCBI的Blast进行比对。结果显示,扩增所得片段与鲤鱼(Cyprinus carpio)的同源性为94%,与金鱼(Carassius auratus)同源性为93%,与鲦鱼(Pimephales promelas)同源性为90%,并且在系统发生树中与金鱼最为接近,由此可以初步认为这段序列是编码金鱼VTG的基因序列。为了进一步验证这段序列就是编码金鱼VTG的基因序列,同时建立检测环境雌激素的新方法,将所得片段转入大肠杆菌进行增殖,提取质粒,内切酶SphⅠ将质粒线性化,纯化得到高纯的线性DNA。选取Sp6 RNA聚合酶,以DNA为模板合成DIG标记的cRNA探针。用所得探针(615bp)进行Northern杂交实验,结果显示,0.01 mg L-1、0.10 mg L-1和1.00 mg L-1三个久效磷浓度组表达了与每条鱼0.2μg的17-β雌二醇暴露的金鱼相同大小的VTG mRNA。证明鱼类在久效磷诱导下能够产生与内源雌激素刺激下相同的VTG基因,进一步验证了久效磷是一种环境雌激素。本实验建立了RT-PCR与Northern杂交相结合的以VTG mRNA为生物标志物的筛选环境雌激素的分子生物学方法。更多还原

【Abstract】 Vitellogenin (VTG) is an important biomarker for monitoring environmental estrogens (EEs) in aquatic environment. VTG mRNA in male fish is a new kind of biomarker. In our country, we often choose community and chemistry methods to detect EEs and reports of molecular biology methods in detecting EEs are seldom. Monocrotophos (MCP), a kind of organophosphorus pesticide in common use in the past, has made serious pollution of pesticide residues. We use goldfish treated by MCP as a model animal to study the influence to the expression of VTG mRNA.In our laboratory, Bing Xin has isolated and purified vitellogenin in serum of 17β-estradiol treated goldfish and measured molecular weight of vitellogenin of 440kDa by native-PAGE. In addition, MCP, with concentration of 0.01 mg L-1, 0.01 mg·L-1、0.10 mg·L-1、1.00mg·L-1 can induce the production of vitellogenin in male goldfish serum. Based on the production, we use RT-PCR to detect expression of goldfish VTG mRNA and use the segment of partial cDNA as the template to synthesize DIG labeled RNA probe.Goldfish is purchased from a local ornamental fish supplier. All fish were maintained indoor in dechlorinated tap water for two weeks. Total liver RNA was extracted from the liver by Trizol kit. According to the conserved domains of amino acids, we design degenerate primers for RT-PCR and get a segment of 1135bp. Special bands was purification and cloned in EcoliⅠ. Clone was sequenced and compared with other species in Blast on NCBI net. As a result, this sequence has homology of 94 percent with common carp (Cyprinus carpio), 93% percent with goldfish (Carassius auratus) and 90% percent with fathead minnows (Pimephales promelas). In the present study, part of goldfish vitellogenin genes have been isolated and used as a biomarker to evaluate the influence of MCP.The induction of vitellogenesis is a complex process requiring coordinated control and expression of many hepatic gene products, such as vitellogenin (VTG). The authors have isolated a partial cDNA encoding goldfish VTG by RT-PCR. This cDNA sequence showed high homology with the VTG of other fishes. DIG-labled RNA probe was synthesized and Northern blot analysis yielded a VTG transcript from MCP treated goldfish hepatic tissue. We develop a new model of molecular biology to screen environmental estrogens.更多还原