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淡水甲藻的形态鉴定和分子鉴定方法探索
Alternative TitleMORPHOLOGICAL IDENTIFICATION OF FRESHWATER DINOFLAGELLATES AND EXPLORATION ON THEIR MOLECULAR IDENTIFICATION
胡圣
Subtype硕士
Thesis Advisor胡征宇
2008-06-17
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword波兰多甲藻 毒素 水华 单细胞pcr 多重置换扩增 全基因组扩增
Abstract甲藻门藻类是一类重要的浮游植物,广泛分布在淡水和海水中。它们也是形成水华的主要优势种类之一,其中不少种类还能产生毒素,海洋藻毒素种类中的73%-75%由甲藻类产生;淡水产毒甲藻种类稀少,目前确切记载的只有波兰多甲藻(Peridinium polonicum)一种。通过光学显微镜和电子扫描显微镜观察,我们确认在湖北香溪河高阳镇河段发现的一种甲藻为波兰多甲藻,中国新记录,这种甲藻可以在水库或池塘形成水华,造成鱼类死亡,因此其对环境和生态的影响值得关注。 一直以来,基于形态学的甲藻的类鉴定都非常困难,对甲藻进行分子系统学研究,一般需要大量的甲藻纯培养物,由于淡水甲藻实验室培养非常困难,我们尝试用单细胞PCR技术扩增甲藻的某些分子标记,实验结果表明,冻融法是最适合单细胞PCR的细胞裂解方法,在单细胞PCR扩增酶系统的选择中,TakaRa Ex Taq及其配套系统具有明显的优势。从材料的保存方法来看,80%乙醇是单细胞PCR最好的材料保存方法。细胞数量并不是影响单细胞PCR结果的主导因素。 单细胞直接PCR技术虽然有许多优越性,但是其成功率较低,每次挑选的细胞只能用于一次分析,难以分析多个位点,为此我们使用多重置换扩增法从甲藻单细胞中扩增出了甲藻全基因组DNA,产物片段大,产量和纯度高,足以满足绝大多数基因组分析需求。另外,我们还对比了单细胞直接扩增和先进行多重置换扩增然后进行二次PCR扩增结果的差异,试验表明,在某些容易扩增的区域,两种方法可以达到相当的效果,但是对某些较难扩增的区域,则只有先通过全基因组扩增后才能获得理想的结果。
Other AbstractThe dinoflagellates are an important group of phytoplankton in marine and fresh water, they are also a group of the dominant bloom species, and some of them even can produce toxins. Though approximately 73% -75% marine algal toxins are produced by dinoflagellates, it is seldom to find toxin production dinoflagellates in freshwater while Peridinium polonicum was the only recorded toxin production species. By observation of optical microscope and scanning electron microscopy, we firstly identified the species found in Xiangxi River, Hubei Province as Perdinium polonicum, a new record in China. Considering Perdinium polonicum’s toxin producing and water bloom forming characteristics which often causes mass mortality of fish, its impaction on environment and ecology need to be further concerned. For a long time, morphology-based identification of dinoflagellates has always been a difficult work, and it always demands a large number of pure cultures of algae to carry out the molecular systematics research though most freshwater dinoflagellates can't be cultured in laboratory. For all of these inconveniences, we attempt to amplify some molecular markers with single-cell PCR. Our experiments show that freezing-thawing is the most suitable cell lysis method for single-cell PCR, and TakaRa Ex Taq with its supporting system has obvious advantages over others when make the decision on which amplification system to choose. As for the cell preservation, 80% ethanol is obviously the best choice in single-cell PCR. We also attempt to investigate the relations between cell amount and the result of single-cell PCR, which proved that cell amount just has slightly influence on the result of PCR in such a small cell amount. Though single-cell PCR has many advantages, it also has very low success rate , at the same time, sorted cells can only be used once which causes the attempt to analyze more sites become impossible.So we amplified the whole genome DNA from single dinoflagellate by multiple displacement amplification and obtained large fragment, high purity and high yield products which were sufficient to meet the demands of most majority of genome analysis. In addition, we also compared the result obtained from directly single-cell PCR and reamplification of genome DNA from multiple displacement amplification. These experiments shows that both methods can achieve identical result in some chromosomal region while in others it was only firstly amplified by multiple displacement amplification cell that can get the ideal result.
Pages74
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12328
Collection学位论文
Recommended Citation
GB/T 7714
胡圣. 淡水甲藻的形态鉴定和分子鉴定方法探索[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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