建立用于环境及土壤微生物群落分析的基因芯片技术
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摘要
微生物的生态多样性在各种生态系统的功能和持续都起着综合的和独特的作用，如它对持续农业系统的作用为许多近期研究所证实。微生物在全球物质循环中起作重要作用，因而关系到地球在物质平衡、大气构成、地质形成等方面的变化，如微生物所进行的反硝化作用使土壤、海洋的氮预算失去平衡，所形成的中间产物NO和N：O的积累则是全球气候变暖和臭氧层的破坏的主要原因之一。另外，微生物在环境的生物治理中有作极为重要的作用。所以弄清楚微生物群落的组成、结构和功能；它们对毒素污染、气候变化、农业和工业活动等环境变化的反应和适应，对于持续和恢复理想的尘态功能是非常必要的。然而，仅有小于1％的微生物能人为培养，微生物群落的定性和对其在自然环境中的种群的检测是长期困扰着微生物学家和生态学家的一大难题。
传统的微生物群落分析建立在细菌的培养上，这已经很不适应现在的研究要求。近10年发展的分子生物学方法使微生物群落研究有了显著的进展，如16S rRNA基因分析方法。但是这些方法仍有其局限，不能满足对微生物种群进行全面了解和监测的高信息输出、简单、快速、及时和经济等方面的要求。
基因芯片是最近发展起来的基因组研究技术。现已表明它是在基因组规模上研究基因表达和调控的一种强有力的研究工具，同时它也是研究原核和真核生物基因多念性的极好方法。与传统的以膜为基础的杂交方法比较，以载玻片为支撑物的基因芯片技术具有高密度、快速检测、基于多荧光素标记的平行检测等多种优势，并还有相对低成本、自动化、低背景噪音等特点。可以预计，基因芯片将是研究微生物群落的一种潜在有力的方法，但是其关节技术和应用都没有得到认真研究，尤其没有就环境样品的复杂性对基因芯片技术进行过研究。
近来发现在研究特定功能种群时功能基因可作为标记基因而代替16S rRNA基因，如研究氮循环细菌可采用硝化和反硝化作用有关的基因。另外，在膜上的染色体DNA杂交被证明具有高的分辨率。
本研究中，我们在分离新的反硝化菌株、克隆亚硝酸还原酶基因的基础上构建和测试了功能基因芯片(FGA)和群落染色体芯片(CGA)，建立了用这两种芯片进行的杂交检测的技术方法，并探索了它们在自然细菌群落的分析上的应用。其主要研究结果如下：1、反硝化细菌分离、亚硝酸还原酶基因克隆及太平洋北部海洋沉积物中的反硝化细菌的多样性分析
通过在厌氧和反硝化条件下进行富集培养和随后的平板分离，结合rep—PCR指纹分析筛选和硝酸还原活性测定，从华盛顿大陆架6个样品站(两个地点)及Puget Sound海洋沉积物中共获得反硝化分离株30个。从其中12个分离株中分离和测序了含有铁作为辅基的nirs基因和从5个分离株中分离测序了nirk基因。经16S心rRNA基因测序和与基因数据库比较，确定30个分离株分别属于7个不同的细菌届或9个不同的细菌种。目前基因数据库中从纯培养获得的nirS和nirK基因主要是与Pseudomonax相联系的，我们从另外多个属获得了这些基因，从而丰富了这些可作为分子标记的基因的序列信息。
用PCR放大的方法从puget Sound和华盛顿大陆架两个地点的海洋沉积物样品中克隆了nirS和nirK基因。nirS基因可从两个样品点所获的样品中放大，而nirK基因却仅从华盛顿大陆架样品中检测到。为研究nir基因在这些样品中的基本结构，两种基因的克隆被进行限制性内切片段长度多态性分析。稀有度分析揭示，在Puget Sound样品中nirS基因克隆具有特别高的多样性，而华盛顿大陆架样品中nirS和nirK基因克隆
的多样性稍低。在＂irK克隆中发现有一个优势群，在两处生境的沉积物样品中均没有
发现nfrt的优势群，只发现少数克隆的丰度较高。经杂交实验证实除一个克隆外所有
假定的228个njtti克隆确为njrt基因，其核苦酸同一性低至45．3％。系统发育分析将
mIS克隆归群到m6基因分类树中的3个明显不同的亚族并与他们来源的两个生境相
对应。这些序列与己知的灯 基因序列或从华盛顿大陆架样品中所获分离株（大多与
Pseudomonas stutzeri有关）的mIS基因序列的关系甚小。nfth克隆彼此之间比较类
似，其核音酸同一性达78．6％或更高；仅有微弱杂交信号的克隆与己知的nfln基因序列
无关。所有nfrk克隆也被归群到一个明显不同的亚族，它不能和任何己知 ＂irk序列的
菌株放到一起。这些发现表明即便在一个微小的样品中nj。序列的多样性也非常高，同
时这些新的其中有些与己知序列很不同的灯 基因族是可培养的反硝化细菌中所没有
的。
2、功能基因芯片（Functional gene array，FGA）
我们用来自于纯培养和海洋沉积物克隆的亚硝酸还原酶基因（m6和 njrk）、馁单
加氧酶基因（amoa）及甲烷单加氧酶基因（moa）这些涉及到氮循环过程的基因构建了一种
功能基因DNA芯片。在高紧迫度（65℃）下获得了该芯片对不同基因的特异杂交。在纯培
吩 养染色体DNA中对灯 基因的检测灵敏度大约为lug，在土壤群落DNA中对这一基因的
检测灵敏?
Microbial diversity plays an integral and unique role in variety of ecosystem, for example, this has been proven in agricultural ecosystem by many recent research. Microorganisms play a key role in global cycle of carbon, nitrogen, sulfur, and heavy metal and so on, thus it affects the material balance, the composition of the atmosphere and geochemistry procedure in the global scale. Understanding the structure and composition of microbial communities and their responses and adaptations to environmental perturbations such as toxic contaminants, climate change, and agricultural and industrial practices is critical in maintaining or restoring desirable ecosystem functions. However, because less than 1 % of microorganisms can be cultured, characterization and detection of microbial populations in natural environments present a great challenge to microbial ecologists. Current methods for analyzing microbial communities, especially their key functions, are too cumbersome. Rapid, simple, reliable, quantitative, and cost-effective tools that can be operated in real-time and in heterogeneous field-scale environments are needed.
Microarrays (or microchips) are a recently developed genomic technology that has been shown to be a powerful tool for studying gene expression and regulation on a genomic scale and detecting genetic polymorphisms in both eukaryotes and prokaryotes. Compared to conventional membrane-based hybridization, glass slide-based microarrays offer the additional advantages of high sensitivity, rapid detection, lower cost, automation, and low background levels. Although microchip-based genomic technology is potentially an extremely powerful tool for characterizing microbial communities and their biological functions, the concept and performance of microarray hybridization have not been rigorously tested and validated with complex environmental samples.
A recent discovery is that some functional genes, such as nitrite reductase gene can be used as marker genes to analyze special functional group of microorganisms in replacing 16S rRNA gene. In addition, quantitative Reverse Sample Genome Probing (RSGP), which permits the identification of bacteria based on genomic DNA hybridization, has been employed in many environments to monitor bacterial population in response to environmental change.
In this study, new denitrifiers were isolated and new nir genes were cloned from marine sediments samples to enrich the sequence information of the functional gene in denitrifying bacteria. Based on this, two kinds of microarraies: functional gene microarray (FGA) and community genomic microarray (CGA), were constructed and
tested. And the application of these microarraies were explored using both surface soil and marine sediment samples. The key results obtained from this study are as bellow.
1. Isolation of denitrifying isolates, cloning of nir genes and the diversity investigation of denitrifying bacteria in Pacific Northwest Marine Sediment communities
30 denitrifiers were isolated from the marine sediment sample from Puget Sound and two sites of Washington margin by enriching in nutrient broth and plating on agar anaerobically under denitrifying conditions, screening using rep-PCR finger print analysis and nitrate depletion testing. 12 nirS gene and 5 nirK genes were amplified by PCR and sequenced from these isolates, some of which were new for the genebank. The phylogenetic analysis indicated that these isolates are very diverse that they are distributed to 7 genera or 9 species.
nirK and nirS genes were cloned from the marine sediment samples from Puget Sound and two sites on the Washington continental margin by PCR approaches. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially
传统的微生物群落分析建立在细菌的培养上，这已经很不适应现在的研究要求。近10年发展的分子生物学方法使微生物群落研究有了显著的进展，如16S rRNA基因分析方法。但是这些方法仍有其局限，不能满足对微生物种群进行全面了解和监测的高信息输出、简单、快速、及时和经济等方面的要求。
基因芯片是最近发展起来的基因组研究技术。现已表明它是在基因组规模上研究基因表达和调控的一种强有力的研究工具，同时它也是研究原核和真核生物基因多念性的极好方法。与传统的以膜为基础的杂交方法比较，以载玻片为支撑物的基因芯片技术具有高密度、快速检测、基于多荧光素标记的平行检测等多种优势，并还有相对低成本、自动化、低背景噪音等特点。可以预计，基因芯片将是研究微生物群落的一种潜在有力的方法，但是其关节技术和应用都没有得到认真研究，尤其没有就环境样品的复杂性对基因芯片技术进行过研究。
近来发现在研究特定功能种群时功能基因可作为标记基因而代替16S rRNA基因，如研究氮循环细菌可采用硝化和反硝化作用有关的基因。另外，在膜上的染色体DNA杂交被证明具有高的分辨率。
本研究中，我们在分离新的反硝化菌株、克隆亚硝酸还原酶基因的基础上构建和测试了功能基因芯片(FGA)和群落染色体芯片(CGA)，建立了用这两种芯片进行的杂交检测的技术方法，并探索了它们在自然细菌群落的分析上的应用。其主要研究结果如下：1、反硝化细菌分离、亚硝酸还原酶基因克隆及太平洋北部海洋沉积物中的反硝化细菌的多样性分析
通过在厌氧和反硝化条件下进行富集培养和随后的平板分离，结合rep—PCR指纹分析筛选和硝酸还原活性测定，从华盛顿大陆架6个样品站(两个地点)及Puget Sound海洋沉积物中共获得反硝化分离株30个。从其中12个分离株中分离和测序了含有铁作为辅基的nirs基因和从5个分离株中分离测序了nirk基因。经16S心rRNA基因测序和与基因数据库比较，确定30个分离株分别属于7个不同的细菌届或9个不同的细菌种。目前基因数据库中从纯培养获得的nirS和nirK基因主要是与Pseudomonax相联系的，我们从另外多个属获得了这些基因，从而丰富了这些可作为分子标记的基因的序列信息。
用PCR放大的方法从puget Sound和华盛顿大陆架两个地点的海洋沉积物样品中克隆了nirS和nirK基因。nirS基因可从两个样品点所获的样品中放大，而nirK基因却仅从华盛顿大陆架样品中检测到。为研究nir基因在这些样品中的基本结构，两种基因的克隆被进行限制性内切片段长度多态性分析。稀有度分析揭示，在Puget Sound样品中nirS基因克隆具有特别高的多样性，而华盛顿大陆架样品中nirS和nirK基因克隆
的多样性稍低。在＂irK克隆中发现有一个优势群，在两处生境的沉积物样品中均没有
发现nfrt的优势群，只发现少数克隆的丰度较高。经杂交实验证实除一个克隆外所有
假定的228个njtti克隆确为njrt基因，其核苦酸同一性低至45．3％。系统发育分析将
mIS克隆归群到m6基因分类树中的3个明显不同的亚族并与他们来源的两个生境相
对应。这些序列与己知的灯 基因序列或从华盛顿大陆架样品中所获分离株（大多与
Pseudomonas stutzeri有关）的mIS基因序列的关系甚小。nfth克隆彼此之间比较类
似，其核音酸同一性达78．6％或更高；仅有微弱杂交信号的克隆与己知的nfln基因序列
无关。所有nfrk克隆也被归群到一个明显不同的亚族，它不能和任何己知 ＂irk序列的
菌株放到一起。这些发现表明即便在一个微小的样品中nj。序列的多样性也非常高，同
时这些新的其中有些与己知序列很不同的灯 基因族是可培养的反硝化细菌中所没有
的。
2、功能基因芯片（Functional gene array，FGA）
我们用来自于纯培养和海洋沉积物克隆的亚硝酸还原酶基因（m6和 njrk）、馁单
加氧酶基因（amoa）及甲烷单加氧酶基因（moa）这些涉及到氮循环过程的基因构建了一种
功能基因DNA芯片。在高紧迫度（65℃）下获得了该芯片对不同基因的特异杂交。在纯培
吩 养染色体DNA中对灯 基因的检测灵敏度大约为lug，在土壤群落DNA中对这一基因的
检测灵敏?
Microbial diversity plays an integral and unique role in variety of ecosystem, for example, this has been proven in agricultural ecosystem by many recent research. Microorganisms play a key role in global cycle of carbon, nitrogen, sulfur, and heavy metal and so on, thus it affects the material balance, the composition of the atmosphere and geochemistry procedure in the global scale. Understanding the structure and composition of microbial communities and their responses and adaptations to environmental perturbations such as toxic contaminants, climate change, and agricultural and industrial practices is critical in maintaining or restoring desirable ecosystem functions. However, because less than 1 % of microorganisms can be cultured, characterization and detection of microbial populations in natural environments present a great challenge to microbial ecologists. Current methods for analyzing microbial communities, especially their key functions, are too cumbersome. Rapid, simple, reliable, quantitative, and cost-effective tools that can be operated in real-time and in heterogeneous field-scale environments are needed.
Microarrays (or microchips) are a recently developed genomic technology that has been shown to be a powerful tool for studying gene expression and regulation on a genomic scale and detecting genetic polymorphisms in both eukaryotes and prokaryotes. Compared to conventional membrane-based hybridization, glass slide-based microarrays offer the additional advantages of high sensitivity, rapid detection, lower cost, automation, and low background levels. Although microchip-based genomic technology is potentially an extremely powerful tool for characterizing microbial communities and their biological functions, the concept and performance of microarray hybridization have not been rigorously tested and validated with complex environmental samples.
A recent discovery is that some functional genes, such as nitrite reductase gene can be used as marker genes to analyze special functional group of microorganisms in replacing 16S rRNA gene. In addition, quantitative Reverse Sample Genome Probing (RSGP), which permits the identification of bacteria based on genomic DNA hybridization, has been employed in many environments to monitor bacterial population in response to environmental change.
In this study, new denitrifiers were isolated and new nir genes were cloned from marine sediments samples to enrich the sequence information of the functional gene in denitrifying bacteria. Based on this, two kinds of microarraies: functional gene microarray (FGA) and community genomic microarray (CGA), were constructed and
tested. And the application of these microarraies were explored using both surface soil and marine sediment samples. The key results obtained from this study are as bellow.
1. Isolation of denitrifying isolates, cloning of nir genes and the diversity investigation of denitrifying bacteria in Pacific Northwest Marine Sediment communities
30 denitrifiers were isolated from the marine sediment sample from Puget Sound and two sites of Washington margin by enriching in nutrient broth and plating on agar anaerobically under denitrifying conditions, screening using rep-PCR finger print analysis and nitrate depletion testing. 12 nirS gene and 5 nirK genes were amplified by PCR and sequenced from these isolates, some of which were new for the genebank. The phylogenetic analysis indicated that these isolates are very diverse that they are distributed to 7 genera or 9 species.
nirK and nirS genes were cloned from the marine sediment samples from Puget Sound and two sites on the Washington continental margin by PCR approaches. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially
引文
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