变性梯度凝胶电泳技术在微藻鉴定及浮游植物群落结构研究中的应用
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摘要
变性梯度凝胶电泳技术(denaturing gradient gel electrophoresis, DGGE)是一种重要的分子多态性技术,在物种鉴定及浮游生物群落多样性研究方面有着广泛的应用。本研究将DGGE技术运用到微藻的鉴定中,并运用该技术对东海、黄海海域及长江干流的浮游植物多样性进行了研究,探讨了多样性组成与环境因子的关系,得出以下结果:
(1)运用DGGE技术,分别针对核糖体序列的18S rDNA、28S rDNA及ITS区序列设计引物,优化了PCR条件,并根据扩增效果选择18S rDNA和28S rDNA的片段作为特征区域,通过垂直电泳和时间进程实验进一步优化了DGGE电泳条件,从而获得了我国沿海43种常见微藻的特征性条带,建立了特征性图谱,为我国沿海常见微藻的鉴定提供了一种新的手段。
(2)运用DGGE技术对东海和黄海海域2007年秋季航次的24个站位表层样品的浮游植物群落多样性进行了研究。各站位聚类分析的结果与其所属海域的环境特征较为一致;运用典范对应分析(Canonical Correspondence Analysis, CCA)对群落多样性与理化因子的关系进行了探讨,结果表明浮游植物的种类组成主要受温度、硅酸盐和溶解无机氮的影响。
(3)2006年4月(平水期)及9月(丰水期)对长江干流进行调查,对表层样品的浮游植物的群落多样性进行了DGGE分析,探讨了浮游植物多样性与环境因子的关系。分析结果表明,各站位均呈现较高的浮游植物多样性;基于各站位的物种组成聚类的结果很好地反映了它们所属站位的空间分布格局;4月份浮游植物的组成与硅酸盐和悬浮颗粒物相关性较大,而9月份则主要受硅酸盐和铵氮的影响较大。
Denaturing gradient gel electrophoresis (DGGE) is an important molecular polymorphism technique. It has been widely used in the study of species identification and plankton community composition. In this study, DGGE was applied to the identification of microalgae. It was also applied to the analysis of phytoplankton community diversity in the East China Sea, Yellow Sea and Yangtze River. Additionally, the linkage of environmental factors and phytoplankton community was analyzed by the ordination technique of canonical correspondence analysis (CCA). The results are summarized as below:
(1) Specific primers for amplification of the 18S rDNA, 28S rDNA and rDNA ITS gene were developed for the analysis of microalgae by DGGE. By optimization of PCR reaction conditions, partial fragments of the 18S rDNA and 28S rDNA sequences were selected as characteristic gene domain. Denaturing gradient and electrophoresis time were optimized by perpendicular electrophoresis and time travel experiment. Fortunately, unique bands were obtained for most microalgae studied here. A database was constructed for microalgae of China coast, providing a new method for microalgae identification.
(2) Phytoplankton samples were collected from 24 stations of the East China Sea and Yellow Sea in November 2007. Genetic diversity of phytoplankton communities were investigated with DGGE fingerprinting. It was suggested that each group resulted from UPGMA clustering analysis of DGGE profiles showed good consistency with the eco-environmental characteristics of the sea area they belonged to. The Relationship of phytoplankton diversity to environmental factors was statistically analyzed with CCA. Temperature, DIN and silicate-Si were found to be significantly related to the phytoplankton community composition.
(3) Surveys were conducted during April (dry season) and September (rainy season) 2006 along 25 stations of the Yangtze River. Phytoplankton community structure was analyzed by DGGE of 18S rRNA genes. It was shown that application of DGGE could reflect regional phytoplankton community distribution in the Yangtze River. CCA was further applied to reveal the relationships between phytoplankton diversity and environmental factors. Results showed that the phytoplankton community was primarily correlated with silicate-Si and SPM in April. In September, silicate-Si and Ammonium-N were found to be significantly related to the phytoplankton community composition.
(1)运用DGGE技术,分别针对核糖体序列的18S rDNA、28S rDNA及ITS区序列设计引物,优化了PCR条件,并根据扩增效果选择18S rDNA和28S rDNA的片段作为特征区域,通过垂直电泳和时间进程实验进一步优化了DGGE电泳条件,从而获得了我国沿海43种常见微藻的特征性条带,建立了特征性图谱,为我国沿海常见微藻的鉴定提供了一种新的手段。
(2)运用DGGE技术对东海和黄海海域2007年秋季航次的24个站位表层样品的浮游植物群落多样性进行了研究。各站位聚类分析的结果与其所属海域的环境特征较为一致;运用典范对应分析(Canonical Correspondence Analysis, CCA)对群落多样性与理化因子的关系进行了探讨,结果表明浮游植物的种类组成主要受温度、硅酸盐和溶解无机氮的影响。
(3)2006年4月(平水期)及9月(丰水期)对长江干流进行调查,对表层样品的浮游植物的群落多样性进行了DGGE分析,探讨了浮游植物多样性与环境因子的关系。分析结果表明,各站位均呈现较高的浮游植物多样性;基于各站位的物种组成聚类的结果很好地反映了它们所属站位的空间分布格局;4月份浮游植物的组成与硅酸盐和悬浮颗粒物相关性较大,而9月份则主要受硅酸盐和铵氮的影响较大。
Denaturing gradient gel electrophoresis (DGGE) is an important molecular polymorphism technique. It has been widely used in the study of species identification and plankton community composition. In this study, DGGE was applied to the identification of microalgae. It was also applied to the analysis of phytoplankton community diversity in the East China Sea, Yellow Sea and Yangtze River. Additionally, the linkage of environmental factors and phytoplankton community was analyzed by the ordination technique of canonical correspondence analysis (CCA). The results are summarized as below:
(1) Specific primers for amplification of the 18S rDNA, 28S rDNA and rDNA ITS gene were developed for the analysis of microalgae by DGGE. By optimization of PCR reaction conditions, partial fragments of the 18S rDNA and 28S rDNA sequences were selected as characteristic gene domain. Denaturing gradient and electrophoresis time were optimized by perpendicular electrophoresis and time travel experiment. Fortunately, unique bands were obtained for most microalgae studied here. A database was constructed for microalgae of China coast, providing a new method for microalgae identification.
(2) Phytoplankton samples were collected from 24 stations of the East China Sea and Yellow Sea in November 2007. Genetic diversity of phytoplankton communities were investigated with DGGE fingerprinting. It was suggested that each group resulted from UPGMA clustering analysis of DGGE profiles showed good consistency with the eco-environmental characteristics of the sea area they belonged to. The Relationship of phytoplankton diversity to environmental factors was statistically analyzed with CCA. Temperature, DIN and silicate-Si were found to be significantly related to the phytoplankton community composition.
(3) Surveys were conducted during April (dry season) and September (rainy season) 2006 along 25 stations of the Yangtze River. Phytoplankton community structure was analyzed by DGGE of 18S rRNA genes. It was shown that application of DGGE could reflect regional phytoplankton community distribution in the Yangtze River. CCA was further applied to reveal the relationships between phytoplankton diversity and environmental factors. Results showed that the phytoplankton community was primarily correlated with silicate-Si and SPM in April. In September, silicate-Si and Ammonium-N were found to be significantly related to the phytoplankton community composition.
引文
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