【作者】 王军；

【导师】 张世宏；

【作者基本信息】 吉林大学 ， 植物保护（专业学位）， 2015， 硕士

【摘要】 海洋是人类赖以生存的重要资源,海洋石油开采和运输过程常因石油泄漏造成海洋严重污染。据初步统计,我国沿海近四十年来(1973-2011年)发生船舶溢油事故约3000起,平均4-5天发生一起污染事故。其中,一次性泄漏50吨以上的溢油事故95起,年均2.5起,平均每起污染事故溢油量537吨,溢油总量达38500吨。以蓬莱19-3油田溢油事故为例,大量石油类污染物进入水体和沉积物,造成蓬莱19-3油田周边及其西北部总计870平方公里海域海水受到严重污染,其海水中石油类含量超过背景值53倍之多。该次溢油事故导致污染海域的浮游生物种类和多样性降低,海洋生物幼虫幼体及鱼卵仔稚鱼受到损害,底栖生物体内石油烃含量明显升高,海洋生物栖息环境遭到破坏,《2014年中国海洋环境状况公报》显示,直到2014年,溢油造成的影响仍然存在。因此,开展海洋石油污染科学快速治理研究成为国内外学者关注的焦点。生物修复是环保、无再次污染、安全可靠的海洋石油污染处理措施,筛选获得高效的海洋石油污染降解菌株是重要环节。由于海洋水资源属于高盐环境,海洋石油降解菌必须具备抗盐特性、易于漂浮水面并与石油结合特性,以及以石油为碳源并利用其生存的能力。实践证明,丝状真菌可能具有上述三个特性,因此筛选海洋中的丝状真菌是泄漏石油处理的前提。本研究从石油污染海洋水域中筛选获得4株石油降解丝状真菌,以其中一株生长速度快的真菌为材料,进行了酯酶的基因克隆和原核表达,获得了高活性的重组酯酶。主要研究结果如下:1、对渤海湾石油污染区的海洋真菌进行了分离。利用石油作为唯一碳源的培养基,筛选了4株可利用石油生存的真菌。通过生物学观察、IST序列扩增和测序鉴定,确定了该4种真菌为赤散囊菌(Eurotium rubrum SH58)、大刀镰刀菌(Fusarium culmorum S21)、胶孢炭疽菌(Colletotrichum gloeosporioides SH4)和微小根毛霉(Rhizomucor pusillus CC2)。其中,Eurotium rubrum SH58的生长速度最快,生物学特性稳定。因此,选择该菌株进行了进一步深入研究。2、在对Eurotium rubrum SH58生物学特性研究基础上,本研究对其酯酶进行了研究。以E.rubrum SH58的m RNA为材料,通过反转录PCR合成c DNA,我们克隆了一个酯酶基因Erlipase;并对其进行了原核表达和融合蛋白的纯化。通过对酯类降解的活性测定,证明该基因的原核表达产物具有较高的酯酶活性。以上结果表明,E.rubrum SH58具有石油降解特性,并且这种特性这与其Erlipase基因的表达相关。研究不仅提供了可用于海洋石油污染治理的生物材料,同时也为环境污染处理和相关微生物的遗传改良奠定了基础。更多还原

【Abstract】 The oceans are important to the survival of humans resource, Marine oil exploitation and transportation is often Marine pollution caused by oil spill.According to preliminary statistics, nearly four decades along the coast of China(1973-2011), some 3000 of ship oil spill accident, the average 4-5 days a pollution accident occurs.Among them, the disposable leak 95, 50 tons of oil spill by an average of 2.5, the average pollution accident-- 537 tons, total of 38500 tons of oil spill.In penglai 19-3 oil spill accident, for example, a large number of petroleum pollutants into the water and sediment, caused the penglai 19-3 oil field and its surrounding a total of 870 square kilometers northwest waters severely contaminated seawater, the water content of full 53 times more than the background value.The oil spill accident results in the decrease of the phytoplankton species and diversity of Marine pollution, Marine biological larvae damage of larvae and eggs larvae, benthic organisms petroleum hydrocarbon content increased significantly, Marine biological habitat destruction, the 2014 China ocean environment bulletin shows that it was not until 2014 that the impact of oil spill is still there.Therefore, research on Marine oil pollution science quickly become the focus of scholars both at home and abroad.Bioremediation is environmental protection, no pollution, safe and reliable again of Marine oil pollution treatment measures, to obtain the efficient Marine oil pollution degradation strains is an important link.Due to sea water belongs to the high salt environment, offshore oil degradation bacteria must have resistance to salt, easy to float the water and oil combined with characteristics, and oil as carbon source and use its ability to survive.Practice has proved that may have the above three characteristics of filamentous fungi, therefore screening in the sea is the precondition of leaking oil processing of filamentous fungi.This study We got 4 filamentous fungi degradating petroleum from ocean waters the oil pollution polluted by petroleum in the ocean waters to get 4 of oil degradation filamentous fungi, with one of the fast growth of fungus strains as the material, the esterase gene cloning and prokaryotic expression, won the highly active reorganization of esterase.The main results were as follows:1.the bohai bay oil pollution in Marine fungi were separated.To use oil as sole carbon source of culture medium, 4 strains screened in the available oil fungus of survival.By observation of biology, IST sequence amplification and sequencing identification, identify the 4 kinds of fungi to red powder capsule(Eurotium rubrum SH58), broadsword sickle bacteria(Fusarium culmorum S21), glue spore anthrax(Colletotrichum gloeosporioides SH4) and tiny hair mildew(Rhizomucor pusillus CC2).Among them, the Eurotium rubrum SH58 fastest growth, stable biological characteristics.Therefore, choose the strain for the further study.2.the Eurotium rubrum SH58 the basis of studying the biological characteristics, this research studied the esterase.With e. rubrum SH58 m RNA for materials, synthesis c DNA by reverse transcription PCR, we cloned a Erlipase esterase gene;And the prokaryotic expression and purification of fusion protein.Through to the esters degradation activity determination, proved that the prokaryotic expression of gene product has the high activity of esterase.The above results show that e. rubrum SH58 oil degradation characteristics, and the characteristic that is related to its Erlipase gene expression.Research not only provides the Marine oil pollution control biological materials are available, and also for the microbial genetic improvement of environmental pollution treatment and relevant laid a foundation.更多还原