几种赤潮微藻的形态和系统进化分析及荧光原位杂交检测

IOCAS-IR > 海洋环流与波动重点实验室

	几种赤潮微藻的形态和系统进化分析及荧光原位杂交检测
其他题名	Morphological and phylogenetic analysis and detection by fluorescentin situ hybridization of several red tide causative microalgae
	陈国福
学位类型	博士
	2007-06-13
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	赤潮形态系统进化 Lsu Its 荧光原位杂交鉴定检测
摘要	赤潮也称红潮，通常是指由于一些海洋浮游生物在水体中过度繁殖或聚集而使海水变色的现象。赤潮特别是有害赤潮造成了严重的生态环境问题，给水产养殖业和滨海旅游业造成了巨大损失，并可直接危害人类健康。研究赤潮，进而预防和控制赤潮，首先要对引发赤潮的生物种类进行准确鉴定并对自然水域的赤潮生物进行监控，并建立赤潮藻的快速鉴定与检测方法。本文分别对几株赤潮微藻进行了形态和系统进化分析，并探讨了荧光原位杂交在赤潮检测中的应用。分别对5株分离自中国沿海不同水域的中肋骨条藻[Skeletonema costatum (Greville) Cleve]类似种 (SK-BH、SK-FQ、 SK-HH、SK-DH和SK-XM) 进行光镜和扫描电镜观察，并PCR扩增了转录内间隔区 (含5.8S rDNA)(ITS) 和核糖体大亚基 (D1-D2)区 (LSU)，获得的序列与其它已报道的骨条藻的同源序列进行了进化分析，以探讨５株骨条藻与已报道的骨条藻之间的进化关系。5株骨条藻在形态上各不相同，其中，只有1株 (SK-XM)被鉴定为中肋骨条藻，而其余4株皆与已报道的骨条藻的形态学特征不符。ITS树和LSU树具有不同的拓扑结构，并表明5株骨条藻至少分属3个不同的种。遗传距离分析提示了在地理距离上靠近的种，在进化上也可能靠近。此外，还可以观察到这5株藻之间的细微的形态学“进化”关系。所有结果表明了中国沿海骨条藻属种的多样性。对1株分离自赤潮水域的裸甲藻 (Gymnodinium)类似种进行了形态学分析，并探讨了该藻与裸甲藻、凯伦藻(Karena)、旋沟藻(Gyrodinium)、下沟藻(Karlodinium)和共生甲藻(Symbiodinium)的进化关系。光镜观察表明该藻具有裸甲藻的一些典型的形态学特征，而我们没能获得细胞形态保存完好的电镜样品；进化分析初步鉴定该藻为一种共生甲藻。获得了赤潮异湾藻[Heterosigma akashiwo (Hada) Hada]的LSU和ITS序列，设计了以胞质rRNA和胞核rDNA为靶序列的特异性探针，建立了赤潮异湾藻的全细胞和细胞核荧光原位杂交技术，对探针的特异性进行了验证，并考察了杂交信号和检测率在整个细胞周期的变化情况。探针能分别使整个细胞和细胞核呈现明亮的绿色荧光。探针是特异性的，不与其它受试藻进行交叉反应。杂交信号在整个细胞周期内变化不明显，且检测率为70%–80%。整个检测过程不到1 ｈ，能实现赤潮异湾藻的快速、准确、特异和半定量检测。获得了海洋原甲藻(Prorocentrum micans Ehrenberg)的LSU和ITS序列，设计了以胞质rRNA为靶序列的特异性探针，建立了海洋原甲藻的全细胞荧光原位杂交技术，并对探针的特异性进行了验证。探针能使整个细胞呈现强烈的绿色荧光。探针不与其它受试藻种进行交叉反应，表明是特异性的。
其他摘要	So-called red tide is an ecologic phenomenon that seawater turns red, brown, or yellow, when oceanic plankton overly propagate or congregate. Red tide, especially harmful algae blooms have neagatively impacted the aquaculture, fisheries and tourism industries as well as the environment and public health. To understand red tide and further control and prevent the occurring of HABs, it is foremost and essential to ascertain the causative species, while the efficient detection methods should be developed to accelerate the identification and quantification aiming at monitoring the composition of organisms in the natural water. In this study, we performed morphological and phylogenetic analysis of several red tide caustative microalgae, and explore the application of fluorescent in situ hybridization (FISH) in the detection of causative species. Five Skeletonema costatum (Greville) Cleve-like diatoms isolated from different areas along the coast of the China Sea (SK-BH, SK-FQ, SK-HH, SK-DH and SK-XM) were studied with light microscopy (LM) and scanning electron microscopy (SEM), and internal transcribed spacers (containing 5.8S rDNA) (ITS) and partial large subunit ribosomal DNA (D1-D2) (LSU) sequences were obtained by PCR amplification, and included in a phylogenetic analysis with published sequences of other Skeletonema spp., aiming at exploring the relationships between our isolates and previously described Skeletonema species. The five isolates differed in morphology, only one (SK-XM) being identified as Skeletonema costatum sensu stricto. The other four isolates did not correspond morphologically to any described Skeletonema species. The LSU and ITS trees provided different topologies and phylogenetic inferences suggested that the five isolates belonged to at least three different species. Sequence distance analysis indicated that genetic similarity between the isolates paralleled their geographical location. Additionally, subtle morphological revolution could be detected among the five species. Together, all of results above proved the diversities of genus Skeletonema along the China Sea coast. Morphological investigation was perfomed on a Gymnodinium-like species isolated from a sample collected from where red tide occurred, and the relationships between this species and Gymnodinium, Gyrodinium, Karena, Karlodinium and Symbiodinium were also explored. This species was characterized with typical features similar to Gymnodinium in LM, while we failed to obtain well-preserved cells for SEM with integrative morphology. Phylogenetic analysis suggested this Gymnodinium-like species is a Symbiodinium species. We obtained LSU and ITS sequences from Heterosigma akashiwo (Hada) Hada, designed species specific probes against cytoplasmic rRNA and nuclear rDNA, established whole cell and nuclear FISH protocols for the detection of H. akashiwo, tested specificity of the probes and examined the change in the hybridization signal and detection efficiency in the whole cell cycle. The probes were specific against H. akashiwo, showing no cross-reactivity with four other test microalgae. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA targeted probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo. Similarily, we obtained LSU and ITS sequences from Prorocentrium micans Ehrenberg, designed rRNA targeted probe, established whole cell hybridization detection protocol, and test the specificy of the probe. P. micans cells labled with the probe displayed intensive fluorescence. The designed probe did not react with other test microalgae, proving its specifiy against with P. micans.
页数	150
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/817
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	陈国福. 几种赤潮微藻的形态和系统进化分析及荧光原位杂交检测[D]. 海洋研究所. 中国科学院海洋研究所,2007.

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