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题名: 烟草尼古丁降解细菌多样性与假单胞杆菌尼古丁代谢相关基因的研究
作者: 魏海雷
学位类别: 博士
答辩日期: 2009-05-21
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 刘培贵
关键词: 烟草 ; 尼古丁 ; 生物降解 ; 多样性 ; 节杆菌属 ; 假单胞杆菌属 ; 剑菌属 ; Tn5转座
学位专业: 植物学
中文摘要: 尼古丁,俗称烟碱,是烟草生物碱的主要成分,也是烟草生产废弃物中的主要有害物质,对人体呼吸系统和交感神经具有潜在的危害。利用微生物降低烟叶烟碱含量和消除烟碱污染是一项具有广阔研究和开发前景的课题。然而,尼古丁降解微生物资源的匮乏和尼古丁代谢机制研究的滞后,严重制约了尼古丁微生物降解的开发和应用。本研究在系统调查和比较烟草根围、烟草内生尼古丁降解细菌多样性的基础上,对其中重要的假单胞杆菌尼古丁代谢相关基因进行了克隆和功能分析。本论文所获得的主要结果如下: 1) 以尼古丁为唯一碳源,分别从烟草根围和烟草叶片内分离到56和40株尼古丁降解细菌。内生降碱细菌中,19株(47.5%)细菌降解效率超过90%。根围降碱细菌中,39株(69.6%)细菌降解效率超过90%。内生降碱细菌中,40个菌株分布在10个属内,其中Pseudomonas spp.是优势种群,占62.5%。根围降碱细菌中,56个菌株分布在6个属内,其中Arthrobacter spp.是优势种群,占60.7%。烟草内生降碱细菌比根围降碱细菌的丰富度高,同时多样性系数和优势度也高。而根围降碱细菌具有较高的均匀度。本研究首次从Ensifer、Sinorhizobium、Sphingomonas、Massilia、Erwinia、Brevundimonas、Paenibacillus、Cellulosimicrobium属中分离到具有降解尼古丁功能的细菌。 2) 从筛选菌株中选取了降解能力较强的J5和N7菌株对其分类地位和降解能力进行了深入研究。通过形态观察、生理生化特征分析以及16S rDNA系统发育分析,将菌株J5和N7分别鉴定为Pseudomonas putida和Ensifer sp.。J5菌株在24 h能够完全降解3.0 g/L的尼古丁,最适生长尼古丁浓度为2.0 g/L;N7菌株在24 h能够完全降解2.0 g/L的尼古丁,最适生长尼古丁浓度为2.0 g/L。J5、N7菌株的生长和降解均呈线性关系。分别用108cfu/ml的J5和N7发酵液处理烟草叶片,尼古丁含量降低了11.7%和16.0%,由此说明利用J5和N7可以降低烟草中的尼古丁含量,具有应用和开发潜力。 3) 利用Tn5转座技术,构建了J5菌株的随机突变体库,并从16324个转化子中筛选到28个完全丧失尼古丁降解能力的突变体。这28个突变体均为单拷贝插入,利用鸟枪法克隆了所有突变体的突变位点基因。对28个突变体的克隆序列进行分析,可以将其分为6类:Ⅰ为氧化还原酶类;Ⅱ为蛋白或金属离子转运系统;Ⅲ为蛋白酶或肽酶类;Ⅳ为转录或翻译调控因子;Ⅴ为未知功能蛋白;Ⅵ类转座子插入在了两个基因的间隔区。其中M728、M430和M9502均突变在钼离子转运系统(ModABC)组成基因上。而节杆菌代谢尼古丁的第一个催化酶就具有严格的钼离子依赖特性。M2022突变位点与节杆菌的6-羟基-L-尼古丁氧化酶(6HLNO)同源性为33%。6HLNO是节杆菌第二个尼古丁催化酶,负责6-羟基-L-尼古丁的氧化。M10突变位点与大肠杆菌的酮泛酸羟甲基转移酶(PanB)同源。对这些基因的进一步研究有助于了解尼古丁在假单杆菌中的转化和代谢。 4) 根据M10的插入位点序列,克隆了突变位点panB基因全长。该基因全长798 bp,编码265个氨基酸的肽链,与E. coli 酮泛酸羟甲基转移酶(PanB)同源性达54%。利用同源重组对J5菌株的panB基因进行了定位敲除,突变体J5ΔB丧失了对尼古丁、丙酮酸和酮异戊酸的利用能力,而经过基因互补后能够恢复到野生水平。PanB是泛酸合成途径的关键酶,负责从酮异戊酸合成酮泛解酸,而丙酮酸是泛酸合成的直接前体。假单胞杆菌可以将尼古丁代谢为2,5-二羟基吡啶,而2,5-二羟基吡啶可以进一步转化为丁烯二酸和丙酮酸。由此推测,在J5菌株中尼古丁经过一系列酶促降解后参与了泛酸和CoA的合成,为生理代谢提供基质。 论文对尼古丁微生物降解资源的调查研究不但具有重要的科学意义,还将为开发新的降解菌株,获得自主知识产权奠定基础。对假单胞杆菌尼古丁代谢相关基因的鉴定和分析是对尼古丁代谢途径、降解关键酶及分子生物学研究的有益补充,为更加充分和合理的利用微生物资源提供理论支持。
英文摘要: Nicotine [1-methyl-2-(3-pyridyl-pyrrolidine), C10H14N2] is the main alkaloid component of cigarettes and the main non-recyclable powdery waste in tobacco manufacturing process. Biological treatments with micro-organisms have potential for manipulation of nicotine content in cigarettes production and detoxification of tobacco wastes containing high concentrations of nicotine. In particular, the bacterial community residing in the tobacco rhizosphere has presumably adapted to use nicotine as a growth substrate and has developed biochemical strategies to decompose this organic heterocyclic compound. But the exploitation and application of nicotine degradation with micro-organisms are restricted by the nicotine-degrading microbial resources and their metabolic mechanisms. In this thesis, we fix our attention on two primary contents, diversity of tobacco-associated nicotine-degrading bacteria and identification of genes involved in nicotine degradation in Pseudomonads. The main results of this study are as follows: A total of 96 nicotine-degrading bacterial strains were isolated with nicotine as the sole carbon source, in which 56 strains from tobacco rhizosphere and 40 from tobacco leaves. The degrading efficiency of 19 endophytic strains (47.5%) and 39 strains (69.6%) from rhizosphere exceeded 90%. The endophytic nicotine-degrading bacterial strains were distributed into 10 genera, in which 62.5% were Pseudomonas spp. and 12.5% were Arthrobacter spp. The nicotine-degrading bacterial strains in tobacco rhizosphere were distributed into 6 genera, in which 60.7% were Arthrobacter spp. and 25.0% were Pseudomonas spp. The species richness, diversity and dominance index of endophytic nicotine-degrading bacterial community were higher than that of rhizosphere. But the evenness index of nicotine-degrading bacteria in tobacco rhizosphere was higher compared with endophytic nicotine-degrading bacteria. In this thesis, we firstly reported nicotine-degrading Ensifer, Sinorhizobium, Sphingomonas, Massilia, Erwinia, Brevundimonas, Paenibacillus, and Cellulosimicrobium. Strain J5 and N7 were the most effectual nicotine-degrading bacteria isolated from tobacco rhizosphere. The optimum nicotine concentration for the growth of strain J5 and N7 were 2.0 g/l. 3 g of nicotine/l could be fully decomposed being treated with strain J5 for 24 h. And there was no more nicotine detected in the medium containing 2.0 g nicotine/l after N7 growth for 24 h. There were statistically significant linear relationships between nicotine degradation and biomass of strain J5 and N7. Based on morphology, flagella dyeing, physiological characteristics, and 16S rDNA sequence analysis, strain J5 and N7 were identified as Pseudomonas putida and Ensifer sp., respectively. When strain J5 and N7 cell suspensions (108 CFU/ml) were applied to treat tobacco leaves, the nicotine concentration was decreased by 11.7% and 16.0%, respectively. These results suggest that the novel strain J5 and N7 may be useful for nicotine biodegradation. A mini-Tn5 mutagenesis library of P. putida J5 was constructed, and 28 mutants that failed to grow in the M9 medium with 1.0 g nicotine/l as the sole carbon source were screened from 16324 transformants. The flanking sequences of Tn5 transposon were cloned with shotgun method from the mutant genome. The mutant sites could be divided into six groups as follows: oxidoreductases, proteins and metals transport systems, proteases and peptidases, transcriptional and translational regulators, unknown protein, and interspaces between two genes. The Tn-5 inserted genes of M728, M430 and M9502 were identical to molybdenum transport system (modABC), which regulated the expression of nicotine dehydrogenase in A. nicotinovorans. A homolog of 6-hydroxy-L-nicotine oxidase, the second enzyme of nicotine metabolism in A. nicotinovorans, was identified from the mutant M2022. Furthermore, a homolog of ketopantoate hydroxymethyltransferase, the first enzyme for biosynthesis of vitamin pantothenate, was identified from the mutant M10. The further research of these mutants and the Tn5-inserted genes will help to find out the mechanism of transformation and metabolism of nicotine in P. putida J5. Sequence analysis revealed that the Tn5 cassette of mutant M10 was inserted into a putative open reading frame of about 801 bp and encoding a polypeptide of 266 amino acids with a molecular mass of 27.8 kDa. The deduced amino acid sequence showed 54% identity to ketopantoate hydroxymethyltransferase (PanB) of E. coli K-12, in which PanB initiates the first reaction of pantothenate bosynthesis. The insertion occurred at the site of 185 amino acids of PanB. In-frame deletion of the panB gene abolished the ability to utilize nicotine, pyruvic acid and ketoisovalerate as the sole carbon resources, respectively, while complementation with panB from P. putida J5 and E. coli K-12 restored the mutant to the wild-type level. These results suggest that ketopantoate hydroxymethyltransferase is a crucial enzyme in nicotine metabolism in P. putida J5. In E. coli, Ketopantoate hydroxymethyltransferase is a key enzyme for synthesis of the vitamin pantothenate, a vital and central metabolic compound in all organisms. PanB initiates the first reaction by using ketoisovalerate to generate ketopantoate, which is reduced to D-pantoic acid. In the synthesis of pantothenate, pyruvic acid is the direct precursor of ketoisovalerate in many bacteria such as Escherichia, Salmonella, and Pseudomonas. Alternatively, pyruvic acid could be the end product of nicotine catabolism in Pseudomonas via 2,5-dihydroxypyridine and maleic acid. Given the known reaction mechanism and general chemical considerations, we propose that nicotine, when used as the carbon source by P. putida J5, is catabolized to pyruvic acid via pyrrolidine pathway and then participates in the synthesis of vitamin pantothenate and CoA, which supply the energy for the normal functioning. Investigation of nicotine-degrading bacterial resources not only has basilic scientific significance, but also lays an experimental foundation for exploitation of new nicotine-degrading bacterial agents and owning the independent intellectual property rights. Identification of nicotine metabolic genes of Pseudomonas will reinforce the research on molecular pathway of nicotine metabolism, and also provide theoretical support for fully and reasonably utilizing the microbial resources.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/420
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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Recommended Citation:
烟草尼古丁降解细菌多样性与假单胞杆菌尼古丁代谢相关基因的研究.魏海雷[d].中国科学院昆明植物研究所,2009.20-25
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