培养与非培养方法对青藏高原土壤细菌多样性的研究
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摘要
本文采用传统的细菌分离培养方法和DGGE非培养方法对青藏铁路沿线23个不同地区土壤的细菌群落多样性及群落动态变化进行了研究。
利用多种不同类型的培养基(R2A、嗜盐、嗜碱、无氮阿须贝等),通过传统的细菌分离培养方法从23种不同的土壤中分离筛选可培养细菌2249株,根据菌落菌体形态及地域特征,选择了1043株菌进行了16S rRNA基因序列的扩增并序列测序,通过与GenBank已知序列进行比对分析。结果表明,1043株细菌分属于83个类群,其中,假单孢菌属、芽孢杆菌属、节杆菌属和链霉菌属为最常见类群。本文章根据采样地点土壤质地、植被利用方式及气候特点的不同,对各个地区的细菌数量、主要细菌类群及多样性进行了较为系统的分析。
利用细菌通用引物,对从青藏高原23个地区土壤样品中提取的总DNA进行16S rRNA基因的聚合酶链式反应(PCR)扩增,采用变性梯度凝胶电泳(DGGE)技术对PCR产物进行分析,以揭示青藏高原土壤中细菌群落结构的多样性。DGGE图谱中的主要条带的序列分析结果显示,青藏高原23个土壤样品中细菌群落大多数为非培养细菌(Uncultured bacterium)。DGGE图谱分析结果显示,青藏高原土壤细菌群落多样性受土壤类型,植被特点及海拔高度等因素的影响。
纯培养方法从青藏高原铁路周边23种不同地区土壤中得到的细菌群落与DGGE检测到的群落多样性相比,发现利用非培养检测到的群落较培养方法得到的明显丰富。纯培养得到常见菌群为假单孢菌属、芽孢杆菌属、节杆菌属和链霉菌属。而对DGGE图谱中的主要条带序列分析结果显示,非培养细菌(Uncultured bacterium),根瘤菌(Sinorhizobium),不动杆菌属(Acinetobacter),黄单胞菌属(Xanthomonas)为优势菌属。可见非培养方法与培养方法结果差别较大。
可培养的细菌中,编号为R43,R101,R720三株菌其16S rRNA基因序列在NCBI Blast中比对发现与Sphingomonas属的Sphingobium vermicomposti同源性最高为97.4—97.7% ,且分离生境差异较大。以Sphingobium vermicomposti的模式菌ACCC05496=DSM21299~T为参比菌株,采用多相分类的方法,对其形态学特征、生理生化特征、API鉴定系统、Biolog、脂肪酸成分组成分析(MIDI)、G+Cmol%测定及16S rRNA基因序列的系统发育分析等进行分析。实验结果初步证实,R43,R101,R720三株菌代表Sphingobium属中的新种。
In this paper, bacterial diversity of soil was explored with culture-dependent and culture-independent methods. Two ways were mainly used with the traditional methods of bacterial isolation and DGGE test to research the bacterial diversity and community dynamics around the railway on the Qinghai-Tibet Plateau of 23 different soil samples.
2249 strains were isolated through the traditional methods with different media(R2A, halophilic, alkaliphilic, non-nitrogen etc.) from 23 different types of soil, and amplified 16S rRNA gene sequences of 1043 strains according to the Morphological of colony and geographical characteristics. Drew the phylogeny tree with the known of sequences alignment in the Genbank and the result showed that 1043 strains belonged to 83 groups. Among them, Pseudomonas, Bacillus, Arthrobacter and Streptomyces were the most common groups; This article is based on the the different of textures, land using patterns and climate characteristics of soil sampling, had a more systematic study on the number of bacteria, the major bacterial populations and the diversity of the different area.
Soil samples were collected from 23 soil samples from the Qinghai-Tibet Plateau. Total DNA was extracted from those samples with the aid of bacterial universal primer and amplified by means of PCR and its product analyzed with the DGGE technique to explore diversity of bacterial communities. Seqence analysis of main bands in the DGGE patterns indicated unculture bacteria was the most frequently encountered general between 23 soil samples. Soil microbial diversity in Qinghai-Tibet Plateau was influenced by soil type, altitude and vegetation characteristics and other factors.
The community isolated and cultured from Qinghai-Tibet Plateau railway region with 23 different soil samples compared with that obtained by DGGE atlas and the result found that the culture-independent approach detected a richer bacterial communities than that by culture-dependent method. and Uncultured bacterium was the most frequently encountered general between 23 soil samples. Pseudomonas, Bacillus, Arthrobacter, Streptomyces were the most frequently encountered generas in the culture-dependent collection. However, 16S rRNA sequence analysis of main bands in the DGGE patterns of the same sample indicated that Uncultured bacterium, Sinorhizobium, Acinetobacter, Xanthomonas were the dominant generas of bacterial communities. The results obtained by culture-dependent and culture-independent approaches were quiet distinct from each other.
The strains of R43, R101, R720 from all isolated strains of Qinghai-Tibet Plateau soil samples, maybe the new species because the similarity were 97.4—97.7% in the NCBI Balst and belonged to Sphingomona vermicomposti and the habitats of the three strains were different with each other. Tested with the closest type strain ACCC05496=DSM21299~T by the test of polyphase sort method: morphology, physiology and biochemistry characteristics, API identify system, Biolog, analysing of fatty acid composes (MIDI), GCmol% and phylogenetic degeneration analysing of 16S rRNA gene sequence. The result showed that, R43, R101, R720 were the new species of the Sphingobium.
利用多种不同类型的培养基(R2A、嗜盐、嗜碱、无氮阿须贝等),通过传统的细菌分离培养方法从23种不同的土壤中分离筛选可培养细菌2249株,根据菌落菌体形态及地域特征,选择了1043株菌进行了16S rRNA基因序列的扩增并序列测序,通过与GenBank已知序列进行比对分析。结果表明,1043株细菌分属于83个类群,其中,假单孢菌属、芽孢杆菌属、节杆菌属和链霉菌属为最常见类群。本文章根据采样地点土壤质地、植被利用方式及气候特点的不同,对各个地区的细菌数量、主要细菌类群及多样性进行了较为系统的分析。
利用细菌通用引物,对从青藏高原23个地区土壤样品中提取的总DNA进行16S rRNA基因的聚合酶链式反应(PCR)扩增,采用变性梯度凝胶电泳(DGGE)技术对PCR产物进行分析,以揭示青藏高原土壤中细菌群落结构的多样性。DGGE图谱中的主要条带的序列分析结果显示,青藏高原23个土壤样品中细菌群落大多数为非培养细菌(Uncultured bacterium)。DGGE图谱分析结果显示,青藏高原土壤细菌群落多样性受土壤类型,植被特点及海拔高度等因素的影响。
纯培养方法从青藏高原铁路周边23种不同地区土壤中得到的细菌群落与DGGE检测到的群落多样性相比,发现利用非培养检测到的群落较培养方法得到的明显丰富。纯培养得到常见菌群为假单孢菌属、芽孢杆菌属、节杆菌属和链霉菌属。而对DGGE图谱中的主要条带序列分析结果显示,非培养细菌(Uncultured bacterium),根瘤菌(Sinorhizobium),不动杆菌属(Acinetobacter),黄单胞菌属(Xanthomonas)为优势菌属。可见非培养方法与培养方法结果差别较大。
可培养的细菌中,编号为R43,R101,R720三株菌其16S rRNA基因序列在NCBI Blast中比对发现与Sphingomonas属的Sphingobium vermicomposti同源性最高为97.4—97.7% ,且分离生境差异较大。以Sphingobium vermicomposti的模式菌ACCC05496=DSM21299~T为参比菌株,采用多相分类的方法,对其形态学特征、生理生化特征、API鉴定系统、Biolog、脂肪酸成分组成分析(MIDI)、G+Cmol%测定及16S rRNA基因序列的系统发育分析等进行分析。实验结果初步证实,R43,R101,R720三株菌代表Sphingobium属中的新种。
In this paper, bacterial diversity of soil was explored with culture-dependent and culture-independent methods. Two ways were mainly used with the traditional methods of bacterial isolation and DGGE test to research the bacterial diversity and community dynamics around the railway on the Qinghai-Tibet Plateau of 23 different soil samples.
2249 strains were isolated through the traditional methods with different media(R2A, halophilic, alkaliphilic, non-nitrogen etc.) from 23 different types of soil, and amplified 16S rRNA gene sequences of 1043 strains according to the Morphological of colony and geographical characteristics. Drew the phylogeny tree with the known of sequences alignment in the Genbank and the result showed that 1043 strains belonged to 83 groups. Among them, Pseudomonas, Bacillus, Arthrobacter and Streptomyces were the most common groups; This article is based on the the different of textures, land using patterns and climate characteristics of soil sampling, had a more systematic study on the number of bacteria, the major bacterial populations and the diversity of the different area.
Soil samples were collected from 23 soil samples from the Qinghai-Tibet Plateau. Total DNA was extracted from those samples with the aid of bacterial universal primer and amplified by means of PCR and its product analyzed with the DGGE technique to explore diversity of bacterial communities. Seqence analysis of main bands in the DGGE patterns indicated unculture bacteria was the most frequently encountered general between 23 soil samples. Soil microbial diversity in Qinghai-Tibet Plateau was influenced by soil type, altitude and vegetation characteristics and other factors.
The community isolated and cultured from Qinghai-Tibet Plateau railway region with 23 different soil samples compared with that obtained by DGGE atlas and the result found that the culture-independent approach detected a richer bacterial communities than that by culture-dependent method. and Uncultured bacterium was the most frequently encountered general between 23 soil samples. Pseudomonas, Bacillus, Arthrobacter, Streptomyces were the most frequently encountered generas in the culture-dependent collection. However, 16S rRNA sequence analysis of main bands in the DGGE patterns of the same sample indicated that Uncultured bacterium, Sinorhizobium, Acinetobacter, Xanthomonas were the dominant generas of bacterial communities. The results obtained by culture-dependent and culture-independent approaches were quiet distinct from each other.
The strains of R43, R101, R720 from all isolated strains of Qinghai-Tibet Plateau soil samples, maybe the new species because the similarity were 97.4—97.7% in the NCBI Balst and belonged to Sphingomona vermicomposti and the habitats of the three strains were different with each other. Tested with the closest type strain ACCC05496=DSM21299~T by the test of polyphase sort method: morphology, physiology and biochemistry characteristics, API identify system, Biolog, analysing of fatty acid composes (MIDI), GCmol% and phylogenetic degeneration analysing of 16S rRNA gene sequence. The result showed that, R43, R101, R720 were the new species of the Sphingobium.
引文
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