应用PCR-DGGE技术研究锰污染农田土壤微生物群落遗传多样性
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摘要
重金属在环境中的排放、迁移、富集能造成极大的危害。研究重金属污染土壤中微生物的群落结构组成和变化能揭示生物对重金属污染环境的适应进化策略,为保护环境提供有用的信息。
聚合酶链式反应-变性梯度凝胶电泳技术(简称PCR-DGGE技术)是分子生物学方法的一种,用于检测DNA突变。它的分辨精度高,最高可以检测到一个核苷酸水平的差异。将PCR-DGGE技术应用于微生物研究,能较好地克服传统微生物培养方法的局限性,能获得更多微生物群落结构和遗传多样性的信息,结合克隆和序列分析,在环境诊断领域将有着广阔的应用前景。
本文简介了重金属污染的危害和土壤微生物的国内外研究成果,综述了研究土壤微生物群落组成的分子生物学方法最新进展,指出了研究土壤微生物群落组成的意义,总结了目前研究中存在的问题,提出了以后进一步研究的方向。
采集了湖南省湘潭市锰矿尾渣坝附近受不同程度锰污染的7个水稻田土壤样品,总锰浓度范围为114~4611 mg/kg,有效态锰浓度为61~1258 mg/kg。有效态锰浓度与总锰浓度随着距离污染源的远近有着一致的变化趋势。对土壤样品做理化分析,土壤pH值与锰浓度有正相关性。测得各样点有机碳、总氮、阳离子交换量,都不同程度的受到锰胁迫的影响。实验揭示锰在矿山周边农田土壤中的化学形态分布规律。同时根据培养细菌计数,锰污染对土壤微生物数量有明显的抑制。
提取土壤细菌总DNA,用通用引物对其中的16S rDNA进行扩增,然后进行变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis),根据电泳图谱分析长期受锰污染的水稻田土壤微生物群落结构和遗传多样性变化,存在于污染最严重样点中的细菌种群对锰有较高的耐受性,不同锰污染的样点群落结构发生变化并且有菌种的消亡和新菌种的产生,锰污染土壤微生物的组成产生了明显的影响。根据UPGMA结果显示,随着离锰矿尾渣坝距离的增加,锰污染的变化,土壤微生物群落结构有一个逐渐演替的过程。不同程度的锰污染不仅影响了组成微生物群落的细菌种群数目,还使微生物群落结构以及优势种群发生了改变。不同种类的微生物不仅是对锰的敏感性不同,对锰污染程度的敏感性也不同。锰污染直接和间接地改变了水稻田土壤微生物群落结构和遗传多样性。
Heavy metals in environment lead to great harm to organism and human being through discharging, transferring and gathering. Research on the effect of long-term exposure to heavy metals on structure and diversity of microbial community in soils could reveal the tolerance and adaptation of microbes and supply useful information for environment monitoring.
Denaturing gradient gel electrophoresis (DGGE) is one kind of molecular biology methods for detecting mutation of DNA. Since traditional methods of cultivating require the use of media or substrates on which only 1.0-10% of soil microrganicsms can grow, applications of DGGE technique has been applied to soil ecosystem in recent years and has enabled the researchers to study microbial diversity at the molecular level.
In this paper, the research findings and up-to-date research progress in here and abroad were introduced. It pointed out the importance of the microbial community structure and diversity in soil, and summarized the problems in the recent research. Furthermore, the developing trends of the technology were discussed in the end.
Seven soil samples were collected from seven different sites near an abandoned manganese ore mine in Xiangtan. The contents of total manganese were 114~4611 mg/kg and the contents of extractable manganese were 61~1258 mg/kg. Manganese influenced the pH, organic C, total N and cation exchange capacity (CEC). The speciation of manganese in soil at different sites was studied. Bacteria were found being influenced negatively by manganese pollution.
The profiles of amplified 16S rDNA sequences obtained from community DNA by denaturing gradient gel electrophoresis (DGGE) reflected the altered community structure and diversity along the manganese gradient as expressed in terms of the position and number of bands. Based on the profiles of DGGE, five bacterial populations were found in all of the soil-sampling sites. That observation may indicate that these five species are manganese resistant. Population and structure varied in bacterial richness among the low contamination sites, but with no sharp difference. The observed changes in the different soil microbial populations probably result from the combined direct and indirect effects of manganese contamination
聚合酶链式反应-变性梯度凝胶电泳技术(简称PCR-DGGE技术)是分子生物学方法的一种,用于检测DNA突变。它的分辨精度高,最高可以检测到一个核苷酸水平的差异。将PCR-DGGE技术应用于微生物研究,能较好地克服传统微生物培养方法的局限性,能获得更多微生物群落结构和遗传多样性的信息,结合克隆和序列分析,在环境诊断领域将有着广阔的应用前景。
本文简介了重金属污染的危害和土壤微生物的国内外研究成果,综述了研究土壤微生物群落组成的分子生物学方法最新进展,指出了研究土壤微生物群落组成的意义,总结了目前研究中存在的问题,提出了以后进一步研究的方向。
采集了湖南省湘潭市锰矿尾渣坝附近受不同程度锰污染的7个水稻田土壤样品,总锰浓度范围为114~4611 mg/kg,有效态锰浓度为61~1258 mg/kg。有效态锰浓度与总锰浓度随着距离污染源的远近有着一致的变化趋势。对土壤样品做理化分析,土壤pH值与锰浓度有正相关性。测得各样点有机碳、总氮、阳离子交换量,都不同程度的受到锰胁迫的影响。实验揭示锰在矿山周边农田土壤中的化学形态分布规律。同时根据培养细菌计数,锰污染对土壤微生物数量有明显的抑制。
提取土壤细菌总DNA,用通用引物对其中的16S rDNA进行扩增,然后进行变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis),根据电泳图谱分析长期受锰污染的水稻田土壤微生物群落结构和遗传多样性变化,存在于污染最严重样点中的细菌种群对锰有较高的耐受性,不同锰污染的样点群落结构发生变化并且有菌种的消亡和新菌种的产生,锰污染土壤微生物的组成产生了明显的影响。根据UPGMA结果显示,随着离锰矿尾渣坝距离的增加,锰污染的变化,土壤微生物群落结构有一个逐渐演替的过程。不同程度的锰污染不仅影响了组成微生物群落的细菌种群数目,还使微生物群落结构以及优势种群发生了改变。不同种类的微生物不仅是对锰的敏感性不同,对锰污染程度的敏感性也不同。锰污染直接和间接地改变了水稻田土壤微生物群落结构和遗传多样性。
Heavy metals in environment lead to great harm to organism and human being through discharging, transferring and gathering. Research on the effect of long-term exposure to heavy metals on structure and diversity of microbial community in soils could reveal the tolerance and adaptation of microbes and supply useful information for environment monitoring.
Denaturing gradient gel electrophoresis (DGGE) is one kind of molecular biology methods for detecting mutation of DNA. Since traditional methods of cultivating require the use of media or substrates on which only 1.0-10% of soil microrganicsms can grow, applications of DGGE technique has been applied to soil ecosystem in recent years and has enabled the researchers to study microbial diversity at the molecular level.
In this paper, the research findings and up-to-date research progress in here and abroad were introduced. It pointed out the importance of the microbial community structure and diversity in soil, and summarized the problems in the recent research. Furthermore, the developing trends of the technology were discussed in the end.
Seven soil samples were collected from seven different sites near an abandoned manganese ore mine in Xiangtan. The contents of total manganese were 114~4611 mg/kg and the contents of extractable manganese were 61~1258 mg/kg. Manganese influenced the pH, organic C, total N and cation exchange capacity (CEC). The speciation of manganese in soil at different sites was studied. Bacteria were found being influenced negatively by manganese pollution.
The profiles of amplified 16S rDNA sequences obtained from community DNA by denaturing gradient gel electrophoresis (DGGE) reflected the altered community structure and diversity along the manganese gradient as expressed in terms of the position and number of bands. Based on the profiles of DGGE, five bacterial populations were found in all of the soil-sampling sites. That observation may indicate that these five species are manganese resistant. Population and structure varied in bacterial richness among the low contamination sites, but with no sharp difference. The observed changes in the different soil microbial populations probably result from the combined direct and indirect effects of manganese contamination
引文
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