【作者】 石伟；

【导师】 柳参奎；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2011， 博士

【摘要】 松嫩平原是世界三大苏打盐碱土集中分布区之一,主要盐成分以Na2CO3和NaHCO3为主。在松嫩平原重度盐碱地区域内分布着大量无植物覆盖的裸地,常称为“碱斑”,碱斑上除有时零星生长耐盐碱的碱蓬、碱蒿、碱地肤等外,大多是寸草不生的光碱斑。碱斑是普通生物不能生存的极端盐碱土壤环境,微生物是该环境中主要的生物成分。在极端盐碱土壤中微生物由于受到长期自然选择的压力,形成了稳定的生理生态适应机制,是宝贵的抗盐碱基因资源。对极端盐碱土壤微生物的分离鉴定和抗性特性的研究有利于获得优良的抗盐碱基因资源,从而为分子选育抗盐碱植物新品种提供丰富的耐盐碱基因。另外,目前在土壤生态学领域内,为了改善和利用极端盐碱土壤,开展了许多盐生植物生理生态的科学研究,而对极端盐碱土壤细菌的系统研究较少,尤其是针对松嫩平原这一生态环境中细菌资的源系统研究更是罕见报道。松嫩平原主要分布着是碳酸盐水解而形成的盐碱地,与氯化物相比,具有高pH值的Na2CO3和NaHCO3对微生物的影响更加复杂。本文以极端盐碱土壤(Extreme soda saline-alkali soil)为研究材料,以非盐碱普通土壤(non-saline soil)为对照,在培养基中分别使用了不同浓度的pH、Na2CO3、NaHCO3作为胁迫因素,比较分析了盐碱土壤与非盐碱土壤细菌的抗盐碱性水平差异,揭示了极端盐碱土壤环境微生物资源的整体特征,在此基础上,使用NaCl、pH、Na2CO3作为筛选压,对土壤中的高抗盐碱的细菌进行筛选。利用实体显微镜、扫描电子显微镜对细菌的菌落和细胞形态特征进行观察,从菌落和菌体形态特征两方面揭示了极端盐碱土壤细菌种类的多样性。利用细菌的16S rDNA序列分析技术,初步对分离获得的细菌进行了鉴定,并对潜在的新物种进行了多相分类学的研究,分析了极端盐碱土壤中高抗盐碱细菌的多样性。同时研究了该环境中的细菌分别在不同浓度NaC1、pH、Na2CO3胁迫下的生长特性,根据抗性特征对此环境细菌进行分类,其结果揭示了极端盐碱土壤中细菌群落组成特征。本研究首次针对松嫩平原内无植物覆盖的极端盐碱土壤环境特点、及其中生活的细菌种类、细菌抗性等方面特征进行了较系统的研究,其成果有利于抗性基因资源的获取,对极端盐碱土壤的恢复治理具有很实际的指导意义。上述研究主要成果如下：1.总体上讲,极端盐碱土壤(soda saline-alkali soil)生存着大量的高耐盐碱的细菌,而普通非盐碱土壤(non-saline soil)中耐盐碱细菌相对较少,两种类型的土壤存在一定的差异。在培养基中Na2CO3含量超过50mM或pH高于10.0时,从非盐碱土壤多次培养都分离不到细菌,而对于极端盐碱土壤,即便培养基含有高达200mM的Na2CO3或pH值为10.0时仍能从中分离到大量的细菌；同样利用含有较高浓度NaHCO3的培养基从极端盐碱土壤中分离获得的细菌数量高于从非盐碱土壤中获得的细菌数量。相反,在含有低pH或低Na2CO3含量或低NaHCO3培养条件下,从非盐碱土壤中分离到的细菌数量高于极端盐碱土壤,可见,在极端盐碱土壤中分布着大量的喜盐碱细菌,非耐盐碱细菌不占有优势。2.本研究分离筛选获得28株具有代表性的细菌,通过对其观察结果显示,这些菌株的菌落大小、颜色和边缘都有一定的差异,其个体形态以杆状的细菌为主,仅有一株为球状。可见,菌落和菌体形态的差异特征反应了28个菌株蕴含了不同的种类,暗示着极端盐碱土壤细菌具有丰富的多样性。3.对具有代表性的28个菌株进行基于16S rDNA序列的系统发育分析,结果共检测到了21个种,分布于细菌域的4个大的系统发育类群(Bacteroidetes, Firmicutes, Actinobacteria, Gamma-Proteobacteria)的5个科(Micrococcaceae, Bacillaceae, Cytophagaceae, Halomonadaceae, Xanthomonadaceae)及Gamma-Proteobacteria门中一个未定的科(Alkalimonas属)、7个属(Bacillus, Nesterenkonia, Zhihengliuella, Halomonas, Stenotrophomonas, Alkalimonas, Litoribacter)。多数菌株属于Firmicutes和Gamma-Proteobacteria门,其次是Bacteroidetes门和Actinobacteria门。4.对分离到的潜在新种H1进行了多项分类学鉴定,结果显示,菌株H1是革兰氏阴性、严格好氧、不运动的可形成桔红色菌落的细菌。该菌株生长温度范围是10～30℃、pH范围7.5～9.2、NaCl范围是0.5～4%。16S rDNA序列系统发育分析结果显示,菌株H1与Litoribacter ruber YIM CH208T同源性最近,其相似性为93.2%。该菌株DNAG+C含量为39.1mo1%。主要的脂肪酸是C15：0iso(27.50%)、C17:0iso3OH(13.95), summed feature9(C17:1iso w9c or C16:010-methyl10.63%)和C17:1iso w9c10.63%。基于表型,化学分类和系统发育的数据的基础上,菌株H1可归为Bacteroidetes门中一个新属中的新菌种,命名为Andtalea andensis,模式菌株为ANESC-ST(原名称为H1)(=CICC10485T=NCCB100412T)。5.以细菌对碳酸钠和氯化钠的抗性为依据,对碱斑环境中的细菌提出以下分类系统,把松嫩平原碱斑中的细菌分为强耐碳酸钠嗜盐菌、中度耐碳酸钠嗜盐菌、轻度耐碳酸钠嗜盐菌,强耐碳酸钠耐盐菌、中度耐碳酸钠耐盐菌、轻度耐碳酸钠耐盐菌六类。更多还原

【Abstract】 Songnen Plain is one of the world’s three concentrated regions of saline-alkali soil, and its main salt compositions are sodium carbonate and sodium bicarbonate. In the heavily saline-alkali region of Songnen Plain, all are the contiguous saline-alkali spots most of which are bare spots except for sporadic growth of some seepweed, Artemisia, Kochia scoparius and so on. The environment which common creature cannot survive in is termed extreme environment, and saline-alkali spot soil environment is extremely alkaline. Extreme environment is often the main habitat of extremophiles. Due to the long-term pressure of natural selection, microorganisms in extreme saline-alkali soil environments enjoy the special microbial species and their associated functions which just lack of in the general environmental, and its saline-alkali tolerant characteristics suggest a wealth of saline-alkali tolerant genes. These resources will be useful for selective breeding saline-alkali resistance new varieties of plants. Therefore, the related research of soil microorganisms and their resistance characteristics in this extreme saline-alkali environment is a prerequisite to obtain good gene resistant to saline-alkali.Compared with NaCl stress, the impact of Na2CO3and NaHCO3stress on microorganism is more complex. In this paper, extreme saline soil and non-saline soil were choosed as experimental material and control respectively, and we compared and analyzed the different tolerance level of bacteria between these two soils. Based on three types of selection pressure(high salt and low pH, high salt and high pH, high pH), we screened a variety of bacteria with high saline-alkali tolerance from extreme saline soil. And then, through microscopes and scanning electron microscopy, the colony morphology and individual morphology of bacteria obtained were analyzed, and by bacterial16S rDNA sequence analysis, the molecular identification of the bacteria isolated was completed. Morver, we studied the resistance characteristics of these strains on NaCl, pH and Na2CO3in details. This study reveals the extreme saline soil microbial resources in the overall characteristics of bacterial community composition and resistance characteristics. The result has practically instructional significance on understanding of physiological and ecological mechanisms of salt-tolerance of bacteria. More importantly, it is the first time to complete a systematic study in the resistance characteristics of bacteria which survived in extreme saline soil without plant cover of Songnen Plain soda saline-alkali soil on saline-alkali stress, and this study laid a theoretical foundation for microbial resistance resource development of Songnen plain.Main results of the above research are as follows:1. In conclusion, there were more high salinity bacteria in extreme soda saline-alkali soil than in non-saline soil. There were significant differences between these two soil types, when take Na2CO3and pH as stress factors. The bacteria were disappeared in repeated cultivated non-saline soil, but many bacterial colonies were survived in extreme soda saline-alkali soil when the concentration of Na2CO3was higher than30mmol/L and pH value was higher than10.0. When take NaC1as a stress factor, most of bacteria was moderately salt-tolerant bacteria in extreme soda saline-alkali soil. Compared with bacteria in extreme soda saline-alkali soil, the higher concentrate of NaHCO3has inhibited bacteria in non-saline soil, but the inhibitory potency was lighter than the other inhibiting factors.2. Twenty-eight of bacteria which had high saline-alkali resistance were selected through various filter selection pressure. Physical microscope image showed that there were some certain differences in these bacteria’s size, color and edges. Scanning electron microscope showed that the individual morphological characteristics of the bacteria from alkali spot soil were mainly rod-shaped, only one spherical bacterium was separated.3. Twenty-eight of the isolated strains were analyzed by the method which based on phylogenetic development of16S rRNA gene sequence. The results showed that the isolates are members of twenty one species of five families(Micrococcaceae, Cytophagaceae, Halomonadaceae, Xanthomonadaceae) and an unclassified family in the Gamma-Proteobacteria group of seven genera(Bacillus, Nesterenkonia, Zhihengliuella, Halomonas, Stenotrophomonas, Alkalimonas, Litoribacter) in four major phylogenetic groups(Bacteroidetes, Firmicutes, Actinobacteria, Gamma-Proteobacteria). Most of the strains belonged to Firmicutes and Gamma-Proteobacteria category, followed by Bacteroidetes and Actinobacteria category.4.Strains H1was characterized using a polyphasic approach. Strain H1was Gram- negative, strictly aerobic and non-motile and formed orange red colonies. Range growth conditions were10～37℃, pH7.5-9.2and0.5～4.0%NaCl. Phylogenetic analysis based on16S rRNA gene sequence comparisons showed that the isolate formed a distinct line within a clade containing the genus Litoribacter in the phylum Bacteroidetes and was related to the species Litoribacter ruber YIM CH208T(sequence similarity93.2%). The DNA G+C content was39.1mol%. The predominant cellular fatty acids were C15:0iso(27.50%)C17:0iso3OH(13.95), summed feature9(C17:1iso w9c or C16:010-methyl10.63%) and C17:1iso w9c10.63%. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain H1represents a novel species of a new genus in the Bacteroidetes.5.Based on the resistance to sodium carbonate and sodium chloride, the bacteria under alkaline conditions of Songnen Plain could be divided into six types:halophilic bacteria of strong resistance to sodium carbonate, halophilic bacteria of moderate resistance to sodium carbonate, halophilic bacteria of slight resistance to sodium carbonate, salt-tolerant bacteria of strong resistance to sodium carbonate, salt-tolerant bacteria moderate resistance to sodium carbonate, salt-tolerant bacteria of slight resistance to sodium carbonate.更多还原