矿物风化细菌的筛选及谷氨酰胺抑制菌株S71风化黑云母机制研究

发布时间：2018-02-17 00:05

本文关键词： 谷氨酰胺乙酸合成基因矿物风化黑云母抑制作用　出处：《南京农业大学》2015年博士论文　论文类型：学位论文

【摘要】：矿物风化是地球表面广泛发生的地质现象,对地表矿质元素循环和环境等产生重要的影响。目前对于细菌风化矿物的研究主要集中在细菌资源发掘,矿物风化细菌的生态多样性与分布,石质文物保护以及使用矿物风化细菌进行土壤营养改良,细菌风化矿物机制等,但是有关细菌风化矿物的分子机制有待深入研究。本研究以黑云母为供试矿物,从酸模根际土壤、根与茎样品中分离筛选黑云母风化细菌,对细菌风化黑云母的效应、矿物风化细菌多样性进行了研究;对高效矿物风化细菌S71风化黑云母的影响因素、矿物风化生理机制等进行了分析;同时对谷氨酰胺影响菌株S71风化黑云母的分子机制进行研究。从酸模根际土壤、根与茎样品中分离纯化得到121株细菌,其中96.7%的细菌具有风化黑云母的能力;扩增核糖体DNA限制性分析(Amplified ribosomal DNArestriction analysis,ARDRA)表明,分离自酸模根际土壤、根与茎样品中的菌株分别有16、11、6个ARDRA类型。系统发育分析表明,酸模根际土壤、根和茎细菌分属于9,7,和4个菌属,其中菌株S71被鉴定为Enterobacter cloocae。菌株S71在风化黑云母过程中主要产生乙酸(乙酸浓度达1.0g/L)。另外,矿物风化细菌Myroides sp.TH-19的生理生化特征与遗传学分析表明,菌株TH-19与同属的Myroidesodoratimimus JCM 7460T,Myroides profundi D25T和Myroides marinus JS-08T的16SrDNA的同源性为98.7%-97.5%。菌株TH-19主要呼吸醌为MK-6,主要脂肪酸为C15:0iso,C17:0iso 3-OH与C13:0iso,DNA G+C含量为37.2 mol%,与3株模式菌Myroides odoratimimus JCM 7460T,Myroides profundi D25T和Myroides marinus JS-08T的DNA-DNA杂交同源性均小于70%。因此,菌株TH-19被鉴定为Myroides属一个新种,命名为Myroide xuanwuensis sp.nov.。本文主要研究了谷氨酰胺对菌株S71风化黑云母的影响及其分子机制。结果表明,谷氨酰胺能显著提高菌株S71发酵液pH与氨氮含量,抑制其产乙酸及对黑云母的风化作用。另外,谷氨酰胺同时也能抑制芽孢杆菌属、节杆菌属、伯克霍尔德氏菌属、克雷伯氏菌属等矿物风化细菌对黑云母的风化作用。通过氯甲酸乙酯衍生化方法,使用GC-MS对菌株S71胞内代谢物进行测定与分析,结果表明,添加谷氨酰胺处理菌株S71细胞内甘氨酸、丙氨酸、缬氨酸、苹果酸与琥拍酸等代谢产物含量显著高于未添加谷氨酰胺处理。添加谷氨酰胺后,细胞中乙酸合成受到抑制,3-磷酸甘油酸和丙酮酸转化为丙氨酸、缬氨酸和甘氨酸等氨基酸量增加;未添加谷氨酰胺时,细胞中的3-磷酸甘油酸和丙酮酸主要合成乙酸。分别构建了菌株S71的ackA、pta与poxB等乙酸合成基因突变株,并比较了突变株与其野生型菌株风化黑云母的能力、乙酸产量。结果表明,与野生型菌株S71相比,poxB和pta基因突变株风化黑云母能力无显著变化,而ackA基因突变株产乙酸以及风化黑云母能力均显著下降。
[Abstract]:Mineral weathering is a widely occurring geological phenomenon on the surface of the earth, which has an important effect on the circulation of mineral elements and the environment on the surface. At present, the research on bacterial weathering minerals is mainly focused on the exploration of bacterial resources. Ecological diversity and distribution of mineral weathering bacteria, preservation of stone cultural relics, soil nutrition improvement by mineral weathering bacteria, mechanism of bacterial weathering minerals, etc. However, the molecular mechanism of bacterial weathering minerals needs further study. Biotite was used as the tested mineral in this study. Biotite weathering bacteria were isolated and screened from rhizosphere soil, root and stem samples, and their effects on bacterial weathering biotite were studied. The diversity of weathering bacteria in minerals was studied, and the factors affecting the weathering biotite of highly efficient weathering bacteria S71 and the physiological mechanism of weathering of minerals were analyzed. At the same time, the molecular mechanism of weathering biotite of strain S71 was studied. 121 strains of bacteria were isolated and purified from rhizosphere soil, root and stem samples. 96.7% of them had the ability of weathering biotite. Amplified ribosomal DNArestriction analysis (ARDRAA) showed that the strains isolated from rhizosphere soil were 161.11 and 6 ARDRA types, respectively. Phylogenetic analysis showed that rhizosphere soil, root and stem bacteria belong to 9, 7 and 4 genera, respectively. Among them, strain S71 was identified as Enterobacter cloocae. Strain S71 mainly produced acetic acid (acetic acid concentration up to 1.0 g / L) during weathering biotite. In addition, the physiological and biochemical characteristics and genetic analysis of Myroides sp.TH-19, a mineral weathering bacterium, showed that, The homology of 16s rDNA between strain TH-19 and Myroidesodoratimimus JCM 7460Troides profundi D25T and Myroides marinus JS-08T is 98.7- 97.5.The main respiratory quinone of strain TH-19 is MK-6, the main fatty acid is C15: 0isoquinone, and the contents of C17: 0iso3-OH and C13: 0isoDNAG C are 37.2 mol mol / mol, respectively, with the DNA-DNA hybrids of Myroidesodoratimimus JCM 7460T Myroides profundi D25T and Myroides marinus JS-08T. The homology is less than 70%. The strain TH-19 was identified as a new species of Myroides, named Myroide xuanwuensis sp.nov.In this paper, the effects of glutamine on the weathering biotite of strain S71 and its molecular mechanism were studied. The results showed that glutamine could significantly increase pH and ammonia nitrogen content in fermentation broth of strain S71. Inhibition of acetic acid production and weathering of biotite. Glutamine also inhibits Bacillus, Arthrobacter, Birkholderia, The weathering of biotite by mineral weathering bacteria of Klebsiella. The intracellular metabolites of strain S71 were determined and analyzed by GC-MS by the method of ethyl chloroformate derivation. The contents of glycine, alanine, valine, malic acid and succinic acid in S71 cells treated with glutamine were significantly higher than those without glutamine treatment. The synthesis of acetic acid was inhibited by the conversion of glyceric acid and pyruvate to alanine, and the amount of amino acids such as valine and glycine increased. Acetic acid was mainly synthesized by glyceric acid 3-phosphate and pyruvate. The acetic acid synthesis gene mutants of strain S71 were constructed, and the ability of weathering biotite and the yield of acetic acid were compared between the mutant and its wild-type strain. Compared with wild type strain S71, there was no significant change in the ability of weathering biotite, but the ability of ackA mutant to produce acetic acid and weathered biotite decreased significantly.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：P512.1;Q93

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