海洋丝状真菌石油高效降解菌株的筛选及其酯酶基因克隆

发布时间：2018-05-05 06:15

本文选题：海洋石油降解真菌 + 筛选和鉴定　；参考：《吉林大学》2015年硕士论文

【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。
[Abstract]:The ocean is an important resource on which human beings depend for survival. The ocean pollution is often caused by oil spill during the exploitation and transportation of offshore oil. According to preliminary statistics, there were about 3000 oil spills from ships in the coastal areas of China in the past 40 years, with an average pollution accident of 4-5 days. Among them, 95 oil spill accidents with a one-time leakage of more than 50 tons, an annual average of 2.5, the average oil spill per pollution accident 537 tons, the total amount of oil spill reached 38500 tons. Taking the oil spill accident in Penglai 19-3 oilfield as an example, a large number of petroleum pollutants entered the water body and sediment, resulting in a total of 870 square kilometers of sea water pollution around the Penglai 19-3 oilfield and its northwestern part. The oil content in seawater is 53 times higher than the background value. As a result of the oil spill accident, the species and diversity of plankton in the polluted sea area decreased, the larval larva of marine life and the larvae and larvae of fish eggs were damaged, and the content of petroleum hydrocarbons in benthos increased significantly. The marine habitat was damaged, and the 2014 China Marine Environment Bulletin showed that until 2014, the effects of oil spills were still present. Therefore, scientific and rapid control of offshore oil pollution has become the focus of domestic and foreign scholars. Bioremediation is an environmental protection, no re-pollution, safe and reliable marine oil pollution treatment measures, screening of high efficiency of marine oil pollution degradation strains is an important link. Because the marine water resources belong to high salt environment, the marine petroleum degrading bacteria must have the characteristics of salt resistance, easy floating and combining with oil, and the ability to use oil as carbon source and to make use of it to survive. It has been proved that filamentous fungi may possess the above three properties, so screening filamentous fungi in the ocean is the premise of oil spill treatment. In this study, four oil-degrading filamentous fungi were isolated from petroleum polluted marine waters, and one of them, which grew rapidly, was used for gene cloning and prokaryotic expression of esterase, and a recombinant esterase with high activity was obtained. The main results are as follows: 1. Isolation of marine fungi from oil contaminated area of Bohai Bay. Using petroleum as the sole carbon source, four strains of fungi were screened. The four fungi were identified by biological observation and sequencing. They were Eurotium rubrum SH58, Fusarium culmorum S21, Colletotrichum gloeosporioides SH4 and Rhizomucor pusillus CC2. The growth rate of rubrum SH58 is the fastest and the biological characteristics are stable. Therefore, the strain was selected to further study. 2. On the basis of studying the biological characteristics of Eurotium rubrum SH58, the esterase of the strain was studied. Using m RNA of E.rubrum SH58 as material, a esterase gene Erlipase was synthesized by reverse transcription PCR, and its prokaryotic expression and purification were carried out. It was proved that the prokaryotic expression product of the gene had high esterase activity through the determination of esterase degradation activity. These results suggest that E. rubrum SH58 has the characteristics of petroleum degradation, which is related to the expression of its Erlipase gene. The study not only provides biomaterials for the treatment of marine oil pollution, but also lays a foundation for environmental pollution treatment and genetic improvement of related microorganisms.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172;X55

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