本文關(guān)鍵詞： 谷氨酰胺 乙酸合成基因 礦物風(fēng)化 黑云母 抑制作用　出處：《南京農(nóng)業(yè)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：礦物風(fēng)化是地球表面廣泛發(fā)生的地質(zhì)現(xiàn)象,對地表礦質(zhì)元素循環(huán)和環(huán)境等產(chǎn)生重要的影響。目前對于細菌風(fēng)化礦物的研究主要集中在細菌資源發(fā)掘,礦物風(fēng)化細菌的生態(tài)多樣性與分布,石質(zhì)文物保護以及使用礦物風(fēng)化細菌進行土壤營養(yǎng)改良,細菌風(fēng)化礦物機制等,但是有關(guān)細菌風(fēng)化礦物的分子機制有待深入研究。本研究以黑云母為供試礦物,從酸模根際土壤、根與莖樣品中分離篩選黑云母風(fēng)化細菌,對細菌風(fēng)化黑云母的效應(yīng)、礦物風(fēng)化細菌多樣性進行了研究;對高效礦物風(fēng)化細菌S71風(fēng)化黑云母的影響因素、礦物風(fēng)化生理機制等進行了分析;同時對谷氨酰胺影響菌株S71風(fēng)化黑云母的分子機制進行研究。從酸模根際土壤、根與莖樣品中分離純化得到121株細菌,其中96.7%的細菌具有風(fēng)化黑云母的能力;擴增核糖體DNA限制性分析(Amplified ribosomal DNArestriction analysis,ARDRA)表明,分離自酸模根際土壤、根與莖樣品中的菌株分別有16、11、6個ARDRA類型。系統(tǒng)發(fā)育分析表明,酸模根際土壤、根和莖細菌分屬于9,7,和4個菌屬,其中菌株S71被鑒定為Enterobacter cloocae。菌株S71在風(fēng)化黑云母過程中主要產(chǎn)生乙酸(乙酸濃度達1.0g/L)。另外,礦物風(fēng)化細菌Myroides sp.TH-19的生理生化特征與遺傳學(xué)分析表明,菌株TH-19與同屬的Myroidesodoratimimus JCM 7460T,Myroides profundi D25T和Myroides marinus JS-08T的16SrDNA的同源性為98.7%-97.5%。菌株TH-19主要呼吸醌為MK-6,主要脂肪酸為C15:0iso,C17:0iso 3-OH與C13:0iso,DNA G+C含量為37.2 mol%,與3株模式菌Myroides odoratimimus JCM 7460T,Myroides profundi D25T和Myroides marinus JS-08T的DNA-DNA雜交同源性均小于70%。因此,菌株TH-19被鑒定為Myroides屬一個新種,命名為Myroide xuanwuensis sp.nov.。本文主要研究了谷氨酰胺對菌株S71風(fēng)化黑云母的影響及其分子機制。結(jié)果表明,谷氨酰胺能顯著提高菌株S71發(fā)酵液pH與氨氮含量,抑制其產(chǎn)乙酸及對黑云母的風(fēng)化作用。另外,谷氨酰胺同時也能抑制芽孢桿菌屬、節(jié)桿菌屬、伯克霍爾德氏菌屬、克雷伯氏菌屬等礦物風(fēng)化細菌對黑云母的風(fēng)化作用。通過氯甲酸乙酯衍生化方法,使用GC-MS對菌株S71胞內(nèi)代謝物進行測定與分析,結(jié)果表明,添加谷氨酰胺處理菌株S71細胞內(nèi)甘氨酸、丙氨酸、纈氨酸、蘋果酸與琥拍酸等代謝產(chǎn)物含量顯著高于未添加谷氨酰胺處理。添加谷氨酰胺后,細胞中乙酸合成受到抑制,3-磷酸甘油酸和丙酮酸轉(zhuǎn)化為丙氨酸、纈氨酸和甘氨酸等氨基酸量增加;未添加谷氨酰胺時,細胞中的3-磷酸甘油酸和丙酮酸主要合成乙酸。分別構(gòu)建了菌株S71的ackA、pta與poxB等乙酸合成基因突變株,并比較了突變株與其野生型菌株風(fēng)化黑云母的能力、乙酸產(chǎn)量。結(jié)果表明,與野生型菌株S71相比,poxB和pta基因突變株風(fēng)化黑云母能力無顯著變化,而ackA基因突變株產(chǎn)乙酸以及風(fēng)化黑云母能力均顯著下降。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：P512.1;Q93

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本文編號：1516750