Alcanivorax Dieselolei B5烷烃内膜转运机制的研究及一株食烷菌的分类鉴定

发布时间：2018-03-07 20:56

  本文选题：食烷菌　切入点：TRAP转运　出处：《国家海洋局第三海洋研究所》2017年硕士论文　论文类型：学位论文

【摘要】：当前,海洋石油污染日益加重,海洋石油烃的降解机制研究受到人们的高度重视。本实验室之前对典型的海洋烷烃降解菌Alcanivorax dieselolei B5烷烃的降解机制开展了深入研究,已获得其烷烃外膜转运系统,经实验验证为三个不同的OmpT蛋白,分别转运不同链长的烷烃,但是其烷烃内膜转运系统的研究尚未开展。到目前为止,还未有关于烷烃转运能力的内膜转运系统的报道。本文在A.dieselolei B5菌株中找到了一组TRAP内膜转运系统,编号分别为B5T_03318(Dct P)、B5T_03317(Dct Q)及B5T_03316(Dct M)。将底物连接蛋白Dct P在大肠杆菌中大量表达以后,进行纯化,先分析了其三维结构,发现其与已报道过的经典的TRAP系统的底物连接蛋白有相似的结构,经表面等离子共振(SPR)技术验证了与C12与C16有非常好的亲和性,初步断定TRAP内膜转运系统在A.dieselolei B5菌株中参与了烷烃的内膜转运。本研究首次验证了烷烃降解细菌的烷烃内膜转运系统。研究结果对于认识烷烃降解微生物的烷烃跨膜转运机制具有十分重要的意义。本文对“大洋一号”第34航次在印度洋站点34I-SWIR-S025TVG13(49.54?E,37.88?S)表层沉积物富集和分离了一株可以降解石油烃的食烷菌属中的新种MT13131进行新种鉴定。通过16SrDNA序列比较后发现相似性最高的菌株为Alcanivorax marinus R8-12T(KC415169),相似度为96.93%。该菌株用M2B平板培养,为革兰氏阴性菌,好氧,呈棒状,长度大约为0.5~1.5μm,宽度在0.3~0.6μm,有周生菌毛。在M2B平板上,菌落形态微小,乳白色,半透明,表面光滑,边缘规则,单菌落的直径大约0.2mm。菌株可在10~42℃范围内生长,最适合其生长的温度是28℃。其生长的pH范围是3~10,最适生长pH范围是6~8。其可生长的NaCl浓度范围为1%~12%,最适生长的NaCl浓度为3%~4%。与模式种之间进行生理生化比较,发现其具备食烷菌属的基本特征,同时也具有区别于其他食烷菌的特征,因此可判定该新菌为食烷菌属的一个新种,命名为Alcanivorax movere,模式菌株为MT13131。菌株MT13131具有较宽的烷烃降解范围,能在以C8~C32的偶数直链烷烃和支链烷烃姥鲛烷为唯一碳源的培养基上生长。由于其较宽的烷烃降解范围,则其一定具有多套烷烃降解代谢网络,现已根据其基因组找出部分与烷烃降解相关的功能基因,功能基因的研究有待进一步进行。这些结果对于深入认识烷烃降解机制和更好的开发利用微生物的应用潜力和资源价值都具有深远的意义。
[Abstract]:At present, the pollution of marine oil is becoming more and more serious, and the degradation mechanism of marine petroleum hydrocarbons has been paid more attention to. The degradation mechanism of typical marine alkane degrading bacteria Alcanivorax dieselolei B5 has been studied deeply in our laboratory. The outer membrane transport system of alkanes has been obtained. It has been proved by experiments that three different OmpT proteins transport alkanes with different chain lengths respectively. However, the study on the inner membrane transport system of alkanes has not been carried out. In this paper, we found a group of TRAP endomembrane transport systems in A. dieselolei B5 strain, numbered B5T03318 Dct (B5T03317Dct Q) and B5TSn03316Dct MtQ. after expressing the substrate conjugate protein Dct P in Escherichia coli, After purification, the three-dimensional structure was analyzed, and it was found that it had similar structure with the reported substrates of the TRAP system. The surface plasmon resonance (SPR) technique proved that it had very good affinity with C12 and C16. It was preliminarily concluded that the TRAP intimal transport system was involved in the internal transport of alkanes in A. dieselolei B5 strain. This study first verified the inner transport system of alkane degradation bacteria. The transmembrane transport mechanism is of great significance. This paper deals with the 34th voyage of Oceanic 1 at the Indian Ocean station 34I-SWIR-S025TVG1349.54? E,37.88? S) the surface sediment enriched and isolated a new species of MT13131 from the genus Dioscorea, which can degrade petroleum hydrocarbon. By comparing 16s rDNA sequence, it was found that the most similar strain was Alcanivorax marinus R8-12TKC415169, and the similarity was 96.93%. The strain was cultured on M2B plate. Gram-negative bacteria, aerobic, rod-shaped, about 0.5 渭 m in length, 0.30.60 渭 m in width, with peripheral fimbriae. On the M2B plate, the colony is small, milky white, translucent, smooth surface, regular edge. The diameter of the single colony is about 0.2 mm. the strain can grow in the range of 10 ~ 42 鈩，

本文编号：1580921