【作者】 陈绍兴；

【导师】 谢志雄；

【作者基本信息】 武汉大学 ， 微生物学， 2005， 硕士

【摘要】 本文采用CAS检测方法检测Pseudomonas sp.SPF-1菌株产铁载体的基本特征,并且通过测定铁载体圈或者荧光圈直径与菌落直径的比值法、紫外-可见光连续波长扫描法、△OD₆₃₀、OD₆₀₀、OD₄₀₀光谱吸收等方法对Pseudomonas sp.SPF-1菌株产铁载体条件、铁离子对其产铁载体能力的影响进行了研究,并且分离纯化了Pseudomonas sp.SPF-1菌株铁载体并对其性质进行了初步分析。结果显示: Pseudomonas sp.SPF-1菌株具有在通用CAS平板较快、较强的产圈特性;产铁载体的最适的温度为28-30℃,最适的pH值为7.5-8.0,并且通气量越充分,铁载体分泌量越大;通过与其它7株假单胞菌产铁载体比较,发现不同株假单胞菌存在较大的差异,Pseudomonas sp.SPF-1菌株在产铁载体特别在产荧光铁载体能力方面较强;通过在培养基配方的比较研究,发现蔗糖-天冬氨酸（MSA）培养基较通用CAS培养基更加灵敏,特别适于荧光铁载体的检测。 超过1μM的铁离子可以促进Pseudomonas sp.SPF-1菌株的生长但是严重抑制其铁载体的分泌,其中在液体培养基内效果比固体培养基内更加明显;同时2-4μM的铁离子螯合剂可以促进铁载体的分泌,而二价阳离子螯合剂没有效果;并且发现荧光铁载体与非荧光铁对铁离子的应答存在差异,且荧光铁载体在铁离子应答方面较非荧光铁载体灵敏; 用Sephadex G-25对Pseudomonas sp.SPF-1铁载体进行分离纯化,初步的性质研究显示:其分泌的铁载体是水溶性,具有很高热稳定性;在400nm处有特异的吸收峰,pH值影响其最大的吸收波长;紫外线照射下发荧光,与铁离子结合荧光萃灭,而400nm处的吸收峰仍存在;分离纯化的铁载体与Pseudomonas sp.SPF-1一样能在通用CAS检测平板上形成铁载体晕圈;通过琼脂糖电泳与薄层色谱发现Pseudomonas sp.SPF-1分泌3种荧光铁载体,且极性差异较大;波长扫描发现其与荧光素在光谱吸收（490nm）、运输铁离子方面存在本质区别。 研究结果说明我们建立的MSA-CAS检测方法是一种高灵敏的、特别适于荧光铁载体检测的方法;发现荧光与非荧光铁载体在铁离子应答差异,且前者较后者灵敏的现象,这对更深入地探讨假单胞菌铁载体合成与调控机制提供依据。更多还原

【Abstract】 In this study, universal CAS assay was used to screen basic characteristic of Pseudomonas sp. SPF-1 which was isolated from the East-lake of Wuhan. The siderophroe of Pseudomonas sp. SPF-1 was analysed by mensurating the ratio of the diameter of the siderophore and the colony, running wave scanning under ultraviolation to visible light and determining the optical density at 600 nm, 400 nm and the changing of the optical density at 630 nm after the siderophore combined with Fe3+. The siderophore was purified by Sephadex G-25, and the characteristic of which was determined.The biosynthesis condition of siderophore from Pseudomonas sp. SPF-1 was compared with other 7 species of Pseudomonas. And the results showed as the followings.The optimal conditions of producing siderophores were the temperature at 28-30 °C, the pH value at 7.5-8.0 andunder better condition of aeration. Found the difference of siderophore yield between these 8 strains of Pseudomonas, and secreted two kinds of siderophroes which were fluorescent and non-fluorescent. Compared thedifference responsion to iron between these two kinds of siderophroes, the result showed that the fluorescent siderophore was more sensitive than non-fluorescent siderophore.When the concentration of iron was exceeded to 1μM on which the growth was promoted and the yield of siderophroe of Pseudomonas sp. SPF-1 was inhibited seriously, which was more obviously on agar plate than in liquid medium. However 2-4 uM of iron chelators could accelerate the secretion of siderophores, whereas the chelators of calcium and magnesium did not. And we also found that the MSA medium was moreyared and suitable to detect fluorescent siderophores from Pseudomonas.The fluorescent siderophore was purified by using the resin of Sephdex-G25 and the basic chartersitics of fluorescent siderophore was determined. The results showed that the fluorescent siderophore a water soluble organism with a high temperature stability and proper absorbance at 400 nm. The maximal absorbance changed went with pH value. The fluorescence was disappeared but the optimal optical density at 400 nm was also existed when fluorescent siderophore combined with iron. The purified siderophore could producd halo on universal CAS agar which was the same to Pseudomonas sp. SPF-1. Three groups of fluorescent siderophores were detected by electrophoresis and thin-layer chromatogram. There were much moredifference of these three groups of these three groups of fluorescent on polarity. We had compared with fluorescent siderophoreand flurescein, and the results showed that there were difference betweenthem on the aspects of spectrum absorbance and transportation of iron(III).The results of this study showed that the MSA-CAS detection agar which we constructed in this study, was a high-sensitive detection method on siderophres, especially for fluorescent siderophore. And we found the different response to iron(III) between fluorescent and non-fluorescent siderophores. Which would proceed the research of siderophores from Pseudomonas sp. SPF-1.更多还原