【作者】 刚锰；

【导师】 熊德琪；

【作者基本信息】 大连海事大学 ， 环境科学， 2013， 博士

【摘要】 随着各国对石油需求量的不断增加,促使海上石油开采业和运输业蓬勃发展,导致海上溢油事件频繁发生,石油污染已严重威胁到海洋生态系统和海洋生物。本文选取了经典的模式生物—马粪海胆为研究对象,应用环境化学、分子生物学等技术检测石油烃污染对马粪海胆的毒理效应,探讨污染物对海洋生物的损伤机制,为海上石油烃污染风险评估提供依据。研究结论如下：(1)海胆胚胎暴露在不同浓度的石油烃中的表现各不相同,在低浓度的石油烃影响下,表现出诱导效应,但是暴露浓度越大,抑制效果越显著；不同暴露浓度对海胆胚胎的4种酶均有不同程度的影响,但是对于CAT和SOD的影响要明显小于对GPx和GST的影响；石油烃对CAT、SOD、GPx及GST这4种酶活性的影响都很明显,而囊胚期最为敏感。(2)利用单细胞凝胶电泳技术(SCGE)研究石油烃对马粪海胆管足DNA的损伤情况,研究结果表明：在油浓度为5、20、30、40mg·L-1的影响下,都没有发现DNA损伤,但是在油浓度为50mg·L-1时,发现了明显的DNA损伤情况。该暴露浓度下,随着暴露时间的增加,马粪海胆管足细胞DNA损伤百分比、彗尾DNA百分含量和DNA平均迁移长度都在增加。(3)利用单链构象多态性分析技术(SNPS)研究石油烃对马粪海胆体内SPLOX.WNT8和HNF—6这三种基因的损伤情况,结果表明：SPLOX基因在编码区第41位上发生碱基A—G的变化,而对应的氨基酸则由赖氨酸变成精氨酸；WNT8基因在编码区706位上T—C,对应的氨基酸由苯丙氨酸变为亮氨酸；HNF—6基因在编码区1119位上G—C,对应的氨基酸由丝氨酸变为苏氨酸。利用高效液相色谱研究马粪海胆在石油烃的影响下,体内DNA甲基化的发生情况,结果发现,暴露时间和甲基化程度二者呈正比关系。(4)通过经典的随机扩增多态性DNA技术(RAPD)研究了石油烃对马粪海胆DNA的损伤情况,优化了RAPD反应的最佳条件并且探讨了马粪海胆的多态性,筛选最佳引物研究损伤情况。具体结果为：Taq酶、引物、dNTP浓度分别为2U、100μmol·L-1、2500μmol·L-1, DNA按照80倍稀释,最后的25μL体系为：Taq酶、10×Buffer、dNTP、DNA、引物和ddH2O分别为0.2μL、2.5μL、2μL、1μL、1μL和18.3μL。45个个体之间的最大、最小和平均遗传距离分别为0.5731、0.2569和0.3254。讨论石油烃对海胆管足DNA损伤情况,发现引物S65、Sll、S49均出现了目标带的缺失或者明暗度的变化。更多还原

【Abstract】 Nowadays, in many countries, the petroleum industry is the main economic source, and the technology of exploitation transport production the technology are more and more mature. With the increasing demand of petroleum and petrole um industry development, oil spill events occurr frequently all over the world. It has a serious impact on the marine and estuarine environment. In the paper, He micentrotus pulcherrimus was selected as the test object. Molecular biology techn ology was used to find out the toxicological effects of diesel dispersed liquid on Hemicentrotus pulcherrimus. The results are as follows:(1) Sea urchin embryos was exposed in different concentrations of diesel dispersed liquid. In the low concentration, the induced effect occurred. But the higher the oil concentration was, the earlier the induced and deducted time occurred. The effects of the four enzymes on the embryos were very different, and the effects on CAT and SOD were significant than the effects on GPx and GST. In the cell growth periods that we researched, the blastocyst stage was the most sensitive.(2) In order to find out the damage on DNA of Hemicentrotus pulcherrimus tube, single cell gel electrophoresis (SCGE) was used. The results show that:in the concentrations of5,20,30and40mg-L-1, the damage on DNA wre not found, but in the concentration of50mg·L-1, the damage on DNA was significant. The longer the exposure time was, the more serious the damage was.(3) From the molecular level, SPLOX gene mutation was detected through single-strand conformation polymor-phism (SNP) technique and sequence determination. The results suggest that single base mutation has taken place in coding region of the41, with the conversion of A-G, where amino acid changed from lysine into arginine. WNT8gene mutation detection results show that single base mutation has taken place in coding region of the706, with the conversion of T-C, where amino acid changed from phenylalanine into leucine. The effects on HNF-6gene was that:single base mutation has taken place in coding region of the1119, with the conversion of G-C, where amino acid changed from serine into threonine. The globle genomic DNA was detected by high performance liquid chromatography and the longer the exposure time was, the more serious the methylation was.(4) This paper analyses the Hemicentrotus pulcherrimus’genetic diversity using the technology of RAPD. First, optimize the conditions of the test by taking some single factor designs to the4factors-Taq enzyme, primer, dNTP, DNA which will affect the result of the test to find the optimal reacting concentration. Through optimization, the writer found the optimal reacting concentration of the4factors:2U of Taq enzyme,100μmol·L-1of primer,2500μmol·L-1of dNTP, diluted80-fold of DNA. Next, the writer analyses the Hemicentrotus pulcherrimus’ genetic diversity in the sea area of Dalian in the optimal reacting conditions. Fragment PCR amplification to the DNA of45Hemicentrotus pulcherrimuses using15double primers. The maximum, minimum and average genetic distance between45individuals were0.5731,0.2569and0.3254. So, there are some diversities in the Hemicentrotus pulcherrimuses. In the concentration of30mg·L-1, the primer65, the DNA was damaged, in the concentration of40mg·L-1, the primer11, the DNA was damaged and in the concentration of50mg·L-1, the primer49and the primer67, the DNA was also damaged.更多还原