【作者】 王亚南；

【导师】 彭志英； 刘双江；

【作者基本信息】 华南理工大学 ， 食品科学， 2004， 博士

【摘要】 本文采用形态特征和生理生化特性为基础的传统分类学方法、结合以16S rDNA序列为基础的分子系统发育学分析的多相分类学技术，主要应用PCR扩增、16S rDNA限制性酶切(Amplified Ribosomal DNA Restriction Analysis，ARDRA)图谱分析、16S rDNA序列测定等分子生物学方法，研究和探讨了我国福建东山某近海养殖场底泥中各类群细菌数量和种群结构丰度。采用传统的平板菌落计数法(CFU)和最大可能数法(MPN)对底泥样品的各类微生物种群进行了分析和统计，并从中分离到不同形态的菌株106株。同时还探讨了应用ARDRA图谱聚类分析与细菌形态分类和生理生化分类相结合的方法，进行环境微生物生态研究的可行性。 从细菌形态类群、生理类群的研究，发现养殖场底泥中的细菌形态和生理表观特性具有一定的多样性；ARDRA图谱分析发现这些细菌存在基因细微结构类型的差别，ARDRA图谱聚类分析结果与生理生化和形态表观分类结果具有一定的一致性。 对分离菌株的进一步研究表明，养殖场底泥中87％的细菌种群为产芽孢细菌，占绝对优势；只有少量(13％)的细菌种群为非产芽孢的革兰氏阳性和阴性细菌。产芽孢细菌中主要是芽孢杆菌属(Bacillus)和盐芽孢杆菌属(Halobacillus)的细菌占优势，其种群在底泥中分布广泛，存在向底泥深处略有增多的现象；非芽孢细菌的种群有革兰氏阳性的微杆菌属、动性球菌属和其它的的革兰氏阳性非芽孢细菌，以及极少量的革兰氏阴性的细菌，并且这些非芽孢细菌是从底泥表层分离出来的。 分类及系统发育研究表明，有多株细菌的16S rDNA序列与GenBank中的已知序列相似性较低，可能是一些新属种的细菌。本论文对其中一株细菌2-2进行了详细的分类学研究，发现它与芽孢杆菌属的亲缘关系最近，其16S rDNA序列与B．pumilus的相似性为96％，生理生化和细胞壁成分分析都显示菌株2-2是芽孢杆菌属的一个新种，命名为Bacillus catenulatus。此外还发现这株细菌具有营养细胞的分化现象，通过细菌生长体细胞群落形态、个体细胞形态观察、16S rRNA基因序列的比对分析，证实了这一分化现象，并通过质粒提取、全细胞蛋白的SDS-PAGE分析等方法，拓宽了研究芽孢菌分化现象的思路。 本文还研究了养殖场底泥中细菌代谢各种生源要素的生理特征，分析了不同生理类群细菌的种群结构，以及它们在底泥中的分布情况。结果表明，养殖池底泥中，细菌代谢含碳有机化合物的能力比较强，且分解淀粉的细菌数量和种群均高更多还原

【Abstract】 Their taxonomy and systematic evolutionary are studied by their morphologic cheracteristics, physiological and biochemical properties, and 16S rDNA sequences. The molecular biological techniques, such as PCR, ARDRA pattern analysis and sequencing 16S rDNA, are used to study their polyphasic taxonomy. Applying analytical method of ARDRA pattern on environment microbical ecology is also discussed. Bacterial community structure and quantity of sediment from near-shore marine-cultural region located in DongShan, FuJian province of China are studied in the thesis. Based on the methods of CFU and MPN, quantity and community structure of bacteria in the samples of the sediment have been analyzed, from which 106 strains were isolated.The results indicate that there are morphologic, physiological and biochemical diversities among bacteria from the sediment. Diversity of 16S rDNA sequences of these strains is showed by ARDRA pattern analysis, these results are corresponded with that of morphologic cheracteristics, physiological and biochemical properties of the bacterial strains.The results show that 87% of isolates are the most aboundent populations and belong to endospore-forming bacteria, and the others are the lowest population and belong to non spore-forming bacteria. Bacillus, Halobacillus are the most aboundent populations among endospore-forming bacteria, and widely distribute in sediment. Microbium, Planococcus, a few gram-negative bacteria are the lowest populations, and mainly distributed on the top of sediment.Some strains might be novel genus or species, based on their 16S rDNA sequence. The bacterial strain 2-2 has been identified. The similarity of its 16S DNA sequence is lower than 96% to Bacillus pumilus, and its morphologic cheracteristics, physiological and biochemical properties, components of cell’s wall are also different from those of B. pumilus. These results strongly suggest that strain 2-2 is a novel species of the genus Bacillus and the name Bacillus catenulatus is proposed for this strain. The strain was found to produce the differential phenotypes of cells and this phenotypic difference is confirmed by growing-cells, colonies, 16S rDNA sequence contrasted. We try to widen the view to study the differential cell of Bacillus by extracting plasmid and analysised SDS-PAGE of cell’s protein.更多还原