应用单细胞PCR方法研究胶州湾一株鳍藻的遗传特征

IOCAS-IR > 海洋环流与波动重点实验室

	应用单细胞PCR方法研究胶州湾一株鳍藻的遗传特征
	李萍
学位类型	博士
	2007-06-08
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	有害赤潮单细胞pcr 鳍藻 Lc-ms 亚历山大藻
摘要	近年来，分子生物学技术与方法被越来越多地应用于有害赤潮研究。其中，单细胞PCR方法是对难以室内培养的有害赤潮藻种进行遗传特征研究的一项重要技术。本实验尝试建立微藻的单细胞PCR方法，并将其应用于鳍藻研究。应用室内培养的亚历山大藻，研究了微藻的单细胞PCR方法，并对藻细胞的固定和保存方法进行了比较。结果表明，浮游植物研究中常用的甲醛固定方法只能用于短期保存样品（少于5天），而乙醇固定、鲁格氏液固定、或者在-20℃下冷冻保存的样品，在较长的时间后（60天）仍可以得到比较理想的PCR扩增结果。应用单细胞PCR方法，对青岛近海采集的鳍藻进行了研究，扩增并测定了包括核糖体大亚基（LSU）rDNA的5’端D1-D2区序列，以及5.8S rDNA和ITS区的部分序列信息。通过分析软件对所得到的鳍藻序列信息与基因库中已知的鳍藻序列信息进行了分析与对比。根据ITS和LSU序列信息构建的系统进化树都显示，本文采集的鳍藻藻种与国外报道的圆形鳍藻聚为一支，初步确定采集的鳍藻应为圆形鳍藻，对该藻种形态学特征的观察也支持这一结果。该藻种部分LSU rDNA序列与欧洲同种鳍藻相似度达到99%,与其它等鳍藻遗传距离在18%-20%之间。测定的ITS区序列与同种圆形鳍藻有62个碱基的差异，与LSU rDNA序列相比，ITS序列变异更大。应用LC-MS方法对该鳍藻的进行了DSP毒素分析，结果未检测到OA或DTX1毒素。这是我国首次应用单细胞PCR方法对鳍藻开展的研究工作，首次报道了我国鳍藻的核糖体部分序列信息。圆形鳍藻在青岛近海海域是初次报道，显示了单细胞PCR方法在鳍藻研究中的重要意义。
其他摘要	More and more molecular biological methods and techniques have been used in harmful-algal-bloom (HAB) studies on taxonomy and phylogenesis of harmful algae. The single cell PCR method, which has been developed recently, has many advantages in HAB studies, especially for those species which are difficult to culture in the laboratory. In this paper, the single cell PCR method was established in our lab and used to study the Dinophysis species collected from the sea area of Qingdao. Using the Alexandrium tamarense cultured in the laboratory, the single cell PCR method was developed and the sample preservation method was selected. The result indicated that formalin fixation (5%) method, which was often used in phytoplankton studies, was only suitable for the short-time preservation (<5days) of algae samples for single-cell PCR. The other methods, including ethanol fixation (95%), Lugol’s solution preservation, or storage under -20℃ could preserve the samples for a longer time (60 days in our experiment) for single cell PCR experiment. The method was used for the study of Dinophysis species collected from Qingdao. Partial sequence (D1-D2 region) of large subunit ribosomal RNA gene, and the internal transcribed spaces (ITS), were amplified and sequenced from the single Dinophysis cells. Phylogenetic studies were carried out using the sequences analyzed in this study and from the GeneBank. Based on the analytical results, the Dinophysis species from Qingdao was primarily identified as D. rotundata, which was later confirmed by morphological characteristics. Compared to the sequence information of the same species from other regions, the partial LSU sequence has 2 bp different, while the ITS sequence has 62 bp different. The LC-MS was used to analyze the dirrahetic shellfish poisoning toxins in the cells, but no OA or DTX1 was detected. This is the first study of Dinophysis using single-cell PCR method, and the ribosomal sequences of Dinophysis in China were first reported through this study. Meanwhile, the first report of D. rotundata in Qingdao indicates that the single cell PCR method is a promising method in HAB studies.
页数	71
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1067
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	李萍. 应用单细胞PCR方法研究胶州湾一株鳍藻的遗传特征[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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