典型养殖水体中细菌群落结构的DGGE分析及其与环境因子相关性研究
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摘要
对虾养殖是我国水产养殖的支柱产业之一,随着水产养殖结构的调整及可持续性发展道路的要求,走生态养殖道路迫在眉睫。水体生态环境优劣是水产养殖成败的关键,水体理化因子与微生物,以及水生生物之间的关系,对形成良性循环的健康生态养殖体系有至关重要的作用。因此,研究养殖水体中各因素之间的变化规律,对水产业的发展有着重大的意义。
本文针对珠三角地区典型对虾养殖水体建立了PCR-DGGE技术实验平台,并将优化后的方法应用于研究养殖水的时空变化取样,最终通过对DGGE指纹图谱的分析结合统计学分析方法,对菌群结构变化规律及其与对应理化指标的关系进行了初步探讨。为今后对养殖水体的细化研究奠定了方法基础,提供了参考数据。
主要结果如下:
a.优化了针对养殖水样的全套PCR-DGGE系统方法,有效地提高了电泳图谱的效果:采用CTAB法提取总DNA,确定了降落式PCR(td-PCR)的较优条件,DGGE变性剂梯度范围为40%～55%,在电压75V温度57℃下电泳18小时左右,并采用银染法染色及克隆测序。
b.应用Quantity One软件对DGGE图谱进行排除背景干扰,识别泳道条带,全自动相对定量和相似性分析,平行样品证明了DGGE实验能够区分养殖水样中细微差别的样品,并且有良好的复现性。
c.利用优化后的PCR-DGGE系统方法对不同时空的养殖水样本进行了检测,DGGE指纹图谱分析显示,对虾养殖水体中微生物结构复杂,不同时空的样品在菌群结构和含量上都有较大变化。
d. UPGMA聚类分析和多维尺度分析(MDS)的结果显示,在一定程度上时间变化比空间差异对菌落结构影响更大。此外,不同时间样品按顺序的类聚,反映出同一养殖场的细菌群落组成随时间推移很可能产生了连贯性的变化,而微生物制剂可能对菌落结构有较大影响。
e.对DGGE图谱中条带的回收测序显示,β-变形菌纲、α-变形菌纲、芽孢杆菌纲和梭菌纲很可能是样品养殖水中的优势菌种。硝化螺旋菌、双歧杆菌、硝化杆菌、脱硫弧菌、蛭弧菌可能是养殖水中的常见菌种。检测到的细菌大部分为不可培养菌种,而它们之间的亲缘关系都比较远。
f.应用主成分分析(PCA)和典型相应分析(CCA)结果显示,本研究所选取的理化指标基本涵盖了与细菌种群相关性较高的所有环境因素的信息(94%和76%),这些理化指标相关性较高,可以归纳为三、四个主成分因素。而温度、pH值、氨氮、总磷、叶绿素a、溶氧、亚硝酸盐和透明度在本研究中对养殖水细菌群落结构起着非常重要的作用。
With the restructuring of aquaculture and sustainable development, the requirement of ecological farming is around the corner. Shrimp aquaculture is one of the mainstay industries in China. As we know the key to the success of aquaculture is water environment. Physicochemical factors, microorganisms, and the relationship between aquatic organisms play a crucial role in the health of the ecological farming system.
In this paper, the typical shrimp water in the Canton River Delta was studied by PCR-DGGE technology. Experiment platform has been established and optimized. The DGGE fingerprint analysis combined with statistical analysis methods were applied to study the temporal and spatial changes of bacteria group structure in aquaculture water. And its relationship with the corresponding physical and chemical indicators was discussed. This study lays the foundation of the methods and provides reference data for the future study on aquaculture water.
The main results are as follows:
a. Optimiziton of the PCR-DGGE system for aquaculture water was proved effectively improved the result of DGGE: CTAB method used to extract total DNA, the conditions of Touchdown PCR (td-PCR)was optimized, DGGE was defined to be 40%-55%, 75V, 57℃, 18 hours, and silver staining, clonning and sequencing were used.
b. Quantity One 4.6.2 was applied with the DGGE profiles to move background noise, identificate lanes and bands, automatic analyze the relative quantity and similarity. Parallel samples proved DGGE can distinguish subtle differences in samples and show good reproducibility.
c. The aquaculture water samples of different space and different date were detected by the optimized PCR-DGGE system. DGGE fingerprint analysis revealed that there was a complex microbial species in shrimp pools. The bacterial structure and quantity showed great change in different samples.
d. UPGMA cluster analysis and multidimensional scaling (MDS) results indicated that time change showed a greater impact on the bacterial structure than the spatial change. In addition, microbial preparation may have an impact on the microbiologic population.
e. Sequencing results showed that beta Proteobacteria, alpha Proteobacteria, Bacillus and Clostridia is likely to be the dominant species in aquaculture water. Nitrospirae, Bifidobacterium, Nitrobacter, Desulfovibrio, Bdellovibrio may be common species in aquaculture water. Most detected bacteria were uncultured strains, and their genetic relationships were far from each other.
f. Results of Principal Component Analysis (PCA) and Canonical Correspondence Analysis (CCA) showed that enviromental indicators selected in this study covering most environmental factors associated with bacterial structure (94% and 76%). The relevance of these indicators can be summarized in three or four principal component factors. And the temperature, pH, ammonia nitrogen, total phosphorus, chlorophyll a, dissolved oxygen, nitrite and transparency played a very important role in the changes of bacterial community structure.
本文针对珠三角地区典型对虾养殖水体建立了PCR-DGGE技术实验平台,并将优化后的方法应用于研究养殖水的时空变化取样,最终通过对DGGE指纹图谱的分析结合统计学分析方法,对菌群结构变化规律及其与对应理化指标的关系进行了初步探讨。为今后对养殖水体的细化研究奠定了方法基础,提供了参考数据。
主要结果如下:
a.优化了针对养殖水样的全套PCR-DGGE系统方法,有效地提高了电泳图谱的效果:采用CTAB法提取总DNA,确定了降落式PCR(td-PCR)的较优条件,DGGE变性剂梯度范围为40%～55%,在电压75V温度57℃下电泳18小时左右,并采用银染法染色及克隆测序。
b.应用Quantity One软件对DGGE图谱进行排除背景干扰,识别泳道条带,全自动相对定量和相似性分析,平行样品证明了DGGE实验能够区分养殖水样中细微差别的样品,并且有良好的复现性。
c.利用优化后的PCR-DGGE系统方法对不同时空的养殖水样本进行了检测,DGGE指纹图谱分析显示,对虾养殖水体中微生物结构复杂,不同时空的样品在菌群结构和含量上都有较大变化。
d. UPGMA聚类分析和多维尺度分析(MDS)的结果显示,在一定程度上时间变化比空间差异对菌落结构影响更大。此外,不同时间样品按顺序的类聚,反映出同一养殖场的细菌群落组成随时间推移很可能产生了连贯性的变化,而微生物制剂可能对菌落结构有较大影响。
e.对DGGE图谱中条带的回收测序显示,β-变形菌纲、α-变形菌纲、芽孢杆菌纲和梭菌纲很可能是样品养殖水中的优势菌种。硝化螺旋菌、双歧杆菌、硝化杆菌、脱硫弧菌、蛭弧菌可能是养殖水中的常见菌种。检测到的细菌大部分为不可培养菌种,而它们之间的亲缘关系都比较远。
f.应用主成分分析(PCA)和典型相应分析(CCA)结果显示,本研究所选取的理化指标基本涵盖了与细菌种群相关性较高的所有环境因素的信息(94%和76%),这些理化指标相关性较高,可以归纳为三、四个主成分因素。而温度、pH值、氨氮、总磷、叶绿素a、溶氧、亚硝酸盐和透明度在本研究中对养殖水细菌群落结构起着非常重要的作用。
With the restructuring of aquaculture and sustainable development, the requirement of ecological farming is around the corner. Shrimp aquaculture is one of the mainstay industries in China. As we know the key to the success of aquaculture is water environment. Physicochemical factors, microorganisms, and the relationship between aquatic organisms play a crucial role in the health of the ecological farming system.
In this paper, the typical shrimp water in the Canton River Delta was studied by PCR-DGGE technology. Experiment platform has been established and optimized. The DGGE fingerprint analysis combined with statistical analysis methods were applied to study the temporal and spatial changes of bacteria group structure in aquaculture water. And its relationship with the corresponding physical and chemical indicators was discussed. This study lays the foundation of the methods and provides reference data for the future study on aquaculture water.
The main results are as follows:
a. Optimiziton of the PCR-DGGE system for aquaculture water was proved effectively improved the result of DGGE: CTAB method used to extract total DNA, the conditions of Touchdown PCR (td-PCR)was optimized, DGGE was defined to be 40%-55%, 75V, 57℃, 18 hours, and silver staining, clonning and sequencing were used.
b. Quantity One 4.6.2 was applied with the DGGE profiles to move background noise, identificate lanes and bands, automatic analyze the relative quantity and similarity. Parallel samples proved DGGE can distinguish subtle differences in samples and show good reproducibility.
c. The aquaculture water samples of different space and different date were detected by the optimized PCR-DGGE system. DGGE fingerprint analysis revealed that there was a complex microbial species in shrimp pools. The bacterial structure and quantity showed great change in different samples.
d. UPGMA cluster analysis and multidimensional scaling (MDS) results indicated that time change showed a greater impact on the bacterial structure than the spatial change. In addition, microbial preparation may have an impact on the microbiologic population.
e. Sequencing results showed that beta Proteobacteria, alpha Proteobacteria, Bacillus and Clostridia is likely to be the dominant species in aquaculture water. Nitrospirae, Bifidobacterium, Nitrobacter, Desulfovibrio, Bdellovibrio may be common species in aquaculture water. Most detected bacteria were uncultured strains, and their genetic relationships were far from each other.
f. Results of Principal Component Analysis (PCA) and Canonical Correspondence Analysis (CCA) showed that enviromental indicators selected in this study covering most environmental factors associated with bacterial structure (94% and 76%). The relevance of these indicators can be summarized in three or four principal component factors. And the temperature, pH, ammonia nitrogen, total phosphorus, chlorophyll a, dissolved oxygen, nitrite and transparency played a very important role in the changes of bacterial community structure.
引文
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