定量PCR方法与流式成像技术在亚历山大藻藻华研究中的应用

	定量PCR方法与流式成像技术在亚历山大藻藻华研究中的应用
	高岩1,2,3
学位类型	博士
导师	于仁成 ; 周名江 ; Donald Anderson ; Michael Brosnahan
	2015-05-21
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生态学
关键词	有害藻华塔玛亚历山大藻实时荧光定量pcr（qpcr）流式细胞成像技术（imaging Flowcytobot）
摘要	有害藻华（harmful algal bloom, HAB）是近海常见的一类海洋生态灾害现象。部分有害藻华原因种能够产生藻毒素，其中，麻痹性贝类毒素（paralytic shellfish toxins, PST）在全球分布最广、危害也最为严重。甲藻中的有毒亚历山大藻是海洋中麻痹性贝类毒素的重要来源，在亚历山大藻研究中，如何针对有毒藻种开展特异性检测，获取长期、连续的观测资料，对于剖析亚历山大藻藻华成因、防控藻华危害效应至关重要。塔玛亚历山大藻复合种（Alexandrium tamarense species complex）是一类重要的有害藻华原因种，在我国近海多次形成藻华。本研究针对我国近海的有毒塔玛亚历山大藻复合种，首次将针对特定产毒藻种和麻痹性贝类毒素产毒基因 qPCR 检测方法应用于近海亚历山大藻藻华研究；并在留学基金委支持下，在美国开展了应用 Imaging FlowCytobot（IFCB）进行亚历山大藻藻华原位观测的研究，取得了以下认识：（1）针对中国近海塔玛亚历山大藻复合种的两种有毒藻种 -- A. fundyense （I 核糖体型）和 A. pacificum（IV 核糖体型），分别建立了基于 TaqMan 探针的 qPCR 检测方法，并对该方法进行了特异性、灵敏度和准确性验证。将 qPCR 方法与传统的显微镜检测方法及麻痹性贝类毒素高效液相色谱分析技术相结合，在亚历山大藻藻华多发的长江口邻近海域及黄渤海海域开展了调查和研究。根据 2011 年春季和 2012 年春季在长江口邻近海域及黄渤海的调查结果，发现 A. pacificum 主要分布在长江口邻近海域，是该海域亚历山大藻中的优势藻种和麻痹性贝类毒素的主要来源，另外在渤海秦皇岛附近海域也有少量该藻种存在；而 A. fundyense 是黄海海域亚历山大藻的优势种，主要分布在 34^oN 以北海域。该研究首次阐明了两种有毒亚历山大藻（A. fundyense 和 A. pacificum）在我国近海不同的分布特征，也显示了qPCR 检测方法良好的特异性、灵敏度及其在亚历山大藻生态学研究中的应用潜力。（2）将一种针对产毒基因sxtA4的qPCR检测方法应用于黄海和长江口邻近海域的麻痹性贝类毒素产毒藻检测，并与该海域有毒藻种检测结果和麻痹性贝类毒素分析结果进行了对比。发现基于sxtA产毒基因的qPCR方法检测结果与有毒塔玛亚历山大藻复合种（A. fundyense和A. pacificum）的丰度显著相关（r 值为0.857，p < 0.05），表明有毒塔玛亚历山大藻复合种是调查期间该海域主要的产毒藻种。调查海域麻痹性贝类毒素的分布状况与两种qPCR方法得到的有毒藻分布状况基本一致，但藻毒素水平与有毒藻丰度之间缺乏良好的相关性。该研究首次将基于产毒基因的检测方法应用于我国近海麻痹性贝类毒素有毒藻种检测，结果表明该方法适合于我国近海麻痹性贝类毒素产毒藻的监测，具有良好的应用前景，该方法与毒素化学分析手段相结合，将更有利于对有毒藻种的早期监测和预警。（3）应用IFCB，开展了美国Cape Cod地区Salt Pond中有毒亚历山大藻A. fundyense藻华动态的研究，并对比分析了64子集和80子集分类系统对藻华种类的识别情况。结果发现，64子集分类系统对A. fundyense的自动检测结果好于80子集分类系统，与手动校正结果比较，R²值可达0.958。根据IFCB检测结果，获得了A. fundyense藻华全程观测数据，揭示了藻华末期A. fundyense的有性生殖过程及寄生甲藻对藻华消亡的影响。此外，还发现了由异帽藻（Heterocapsa triquetra）形成的藻华，最高丰度可达10⁷cells L^-1。上述结果表明，IFCB是一种具有良好应用潜力的藻华原位观测技术，其应用有助于认识藻华动态过程、探索藻华生消机制。总之，本研究将针对特定有毒藻种和产毒基因的qPCR方法应用于渤、黄、东海的有毒塔玛亚历山大藻研究，首次阐明了两种麻痹性贝类毒素产毒藻在该海域的分布格局，揭示了该海域麻痹性贝类毒素的主要来源，也证明了分子生物学方法在有害藻华生态学研究中的意义和价值。此外，应用全自动原位观测设备IFCB开展了亚历山大藻藻华动态的探索性研究，揭示了A. fundyense 有性生殖过程及寄生甲藻对藻华消亡的影响。研究结果深化了对我国近海有毒亚历山大藻藻华的认识，也为开展亚历山大藻藻华的监测和研究提供了重要的方法学依据。
其他摘要	Harmful algal bloom (HAB) is a common ecological disaster. Some HAB species are toxigenic and paralytic shellfish toxins (PST) are the most widespread phycotoxin with severe impacts. The most important PST source in seawater is toxic species in Alexandrium genus (dinoflagellate). Therefore, the species-specific detection for toxic Alexandrium species and in-situ observation of Alexandrium blooms are significant issues in studies of harmfu algal blooms. Alexandrium tamarense species complex is a group of important HAB causative species in China. In this study, species-specific and PST-related gene detecting assays were applied into toxic A. tamarense species complex detection along Chinese coastal waters for the first time. In addition, the in-situ study of Alexandrium bloom dynamics monitoring using Imaging FlowCytobot (IFCB) was conducted in US under the support of Chinese Scholarship Council. The summary is as follows: (1) Two TaqMan-based qPCR assays targeted at large subunit ribosomal DNA gene (LSU rDNA) of A. fundyense and A. pacificum belonging to A. tamarense species complex were established and tested for their specificity, sensitivity and accuracy. Those methods were applied to the detection of A. fundyense and A. pacificum in parallel with microscopy counting for Alexandrium population quantitation and high performance liquid chromatography (HPLC) for PST level measurements in Changjiang River estuary, Yellow Sea and the Bohai Sea. It is indicated that A. pacificum mainly presented in the sea area adjacent to the Changjiang River estuary in Spring 2011. Additionally, a small amount of A. pacificum was also found in the coastal waster of Qinhuangdao in the BS. A. fundyense mainly distributed northern to the sea area of 34^oN in the YS. This is the first report of the distribution pattern of A. fundyense and A. pacificum in the coastal waters of China. Meanwhile, the significant specificity and sensitivity of qPCR-based assays were exhibited which will be great potential in Alexandrium ecological study. (2) A SYBR Green qPCR assay targeted at the unique gene sxtA4 in saxitoxin biosynthesis was established and applied to monitor PST-producing algae in the YS in Spring 2012 and in sea area adjacent to the Changjiang River estuary in Spring 2013. In addition, TaqMan-based qPCR assays were employed to analyze toxic A. tamarense species complex and HPLC was used to analyze PST in parallel. It was found that the distribution pattern of sxtA gene coincided with those of A. fundyense/A. pacificum and PST, and sxtA-based qPCR results correlated well with abundances of toxic A. tamarense species complex derived from TaqMan-based qPCR assays (r=0.857 in YS and 0.720 in Changjiang River estuary respectively). These suggest that toxic A. tamarense species complex were the major PST producers during the sampling season, and sxtA-based qPCR is a promising method to monitor PST-producing algae along Chinese coastal waters. The correlation between PST levels and sxtA-based qPCR results was less significant (r=0.552 in YS), implying that sxtA-based qPCR is not enough to reflect the toxicity and potential impacts of PST-producing blooms. Hence, the combination of sxtA-based qPCR assay with chemical means could offer an even stronger approach in early-warning of toxic algal blooms. IFCB was applied into the study of toxic A. fundyense bloom dynamics in Salt Pond of Cape Cod, US. Two versions of classifier were built and compared for the automated quantitation of A. fundyense. The results showed that the 64 sub-classes version was superior to the 80 sub-classes version. There was significant relationship between automated classification results of 64 sub-classes classifier and corrected results (R² =0.958). During the deployment, IFCB recorded the entire A. fundyense bloom process. In addition, it was the first time that Heterocapsa triquetra also bloomed during A. fundyense bloom and the cell abundance could reach 10⁷cells L^-1. It showed that IFCB is an important harmful algal bloom detecting technology with great potential to improve the understanding of bloom mechanisms. In general, this study employed species-specific and sxtA-based qPCR assays into the detection of toxic Alexandrium in the BS, YS and East China Sea. It clarified the distribution pattern of two toxic Alexandrium species and indicated the source of PST in the investigated sea areas. It also verified the great value of molecular biological technology in HAB studies. In addition, the in-situ study of Alexandrium bloom using IFCB supported by China Scholarship Council indicated the sexual recombination phase and the role of the parasite Amoebophrya in the termination of the bloom. Those results improved the understanding of toxic Alexandrium bloom along Chinese coastal waters and provide important methodological basis for Alexandrium study.
学科领域	海洋生物学 ; 海洋生态与环境科学 ; 海洋生物技术
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/23277
专题	海洋生态与环境科学重点实验室
作者单位	1.中国科学院海洋研究所 2.中国科学院大学 3.美国伍兹霍尔海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	高岩. 定量PCR方法与流式成像技术在亚历山大藻藻华研究中的应用[D]. 北京. 中国科学院大学,2015.

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