本文選題：食烷菌　切入點：TRAP轉(zhuǎn)運　出處：《國家海洋局第三海洋研究所》2017年碩士論文　論文類型：學位論文

【摘要】：當前,海洋石油污染日益加重,海洋石油烴的降解機制研究受到人們的高度重視。本實驗室之前對典型的海洋烷烴降解菌Alcanivorax dieselolei B5烷烴的降解機制開展了深入研究,已獲得其烷烴外膜轉(zhuǎn)運系統(tǒng),經(jīng)實驗驗證為三個不同的OmpT蛋白,分別轉(zhuǎn)運不同鏈長的烷烴,但是其烷烴內(nèi)膜轉(zhuǎn)運系統(tǒng)的研究尚未開展。到目前為止,還未有關(guān)于烷烴轉(zhuǎn)運能力的內(nèi)膜轉(zhuǎn)運系統(tǒng)的報道。本文在A.dieselolei B5菌株中找到了一組TRAP內(nèi)膜轉(zhuǎn)運系統(tǒng),編號分別為B5T_03318(Dct P)、B5T_03317(Dct Q)及B5T_03316(Dct M)。將底物連接蛋白Dct P在大腸桿菌中大量表達以后,進行純化,先分析了其三維結(jié)構(gòu),發(fā)現(xiàn)其與已報道過的經(jīng)典的TRAP系統(tǒng)的底物連接蛋白有相似的結(jié)構(gòu),經(jīng)表面等離子共振(SPR)技術(shù)驗證了與C12與C16有非常好的親和性,初步斷定TRAP內(nèi)膜轉(zhuǎn)運系統(tǒng)在A.dieselolei B5菌株中參與了烷烴的內(nèi)膜轉(zhuǎn)運。本研究首次驗證了烷烴降解細菌的烷烴內(nèi)膜轉(zhuǎn)運系統(tǒng)。研究結(jié)果對于認識烷烴降解微生物的烷烴跨膜轉(zhuǎn)運機制具有十分重要的意義。本文對“大洋一號”第34航次在印度洋站點34I-SWIR-S025TVG13(49.54?E,37.88?S)表層沉積物富集和分離了一株可以降解石油烴的食烷菌屬中的新種MT13131進行新種鑒定。通過16SrDNA序列比較后發(fā)現(xiàn)相似性最高的菌株為Alcanivorax marinus R8-12T(KC415169),相似度為96.93%。該菌株用M2B平板培養(yǎng),為革蘭氏陰性菌,好氧,呈棒狀,長度大約為0.5~1.5μm,寬度在0.3~0.6μm,有周生菌毛。在M2B平板上,菌落形態(tài)微小,乳白色,半透明,表面光滑,邊緣規(guī)則,單菌落的直徑大約0.2mm。菌株可在10~42℃范圍內(nèi)生長,最適合其生長的溫度是28℃。其生長的pH范圍是3~10,最適生長pH范圍是6~8。其可生長的NaCl濃度范圍為1%~12%,最適生長的NaCl濃度為3%~4%。與模式種之間進行生理生化比較,發(fā)現(xiàn)其具備食烷菌屬的基本特征,同時也具有區(qū)別于其他食烷菌的特征,因此可判定該新菌為食烷菌屬的一個新種,命名為Alcanivorax movere,模式菌株為MT13131。菌株MT13131具有較寬的烷烴降解范圍,能在以C8~C32的偶數(shù)直鏈烷烴和支鏈烷烴姥鮫烷為唯一碳源的培養(yǎng)基上生長。由于其較寬的烷烴降解范圍,則其一定具有多套烷烴降解代謝網(wǎng)絡(luò),現(xiàn)已根據(jù)其基因組找出部分與烷烴降解相關(guān)的功能基因,功能基因的研究有待進一步進行。這些結(jié)果對于深入認識烷烴降解機制和更好的開發(fā)利用微生物的應(yīng)用潛力和資源價值都具有深遠的意義。
[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，

本文編號：1580921