用荧光原位杂交（FISH）技术鉴定赤潮甲藻的研究

Identification of two species of dinoflagellate using fluorescence in situ hybridization

探索了用荧光原位杂交(Fluorescence In Situ Hybridization,FISH)技术检测赤潮藻的可行性.用FISH技术鉴定了2株已知的赤潮甲藻--东海原甲藻(Prorocentrum donghaiense)和塔马亚历山大藻(Alexandrium tamarense).荧光标记的DNA探针是东海原甲藻的核糖体转录单元内间隔区(ITS)序列.研究结果如下:(1)在荧光显微镜下观察与荧光探针杂交后的东海原甲藻细胞,在细胞顶端可见绿色的荧光,而作为对照的塔马亚历山大藻在与标记的探针杂交后没有产生绿色荧光,说明探针只与目标株东海原甲藻反应,不与对照组反应,表明根据ITS序列设计的探针是高度特异的,(2)材料固定后,经蓝光激发,均未产生细胞内源的叶绿素和甲藻素等色素荧光干扰杂交信号的现象,说明固定材料的方法正确.以上的结果表明,FISH技术在鉴定赤潮微藻方面具有其它技术无法比拟的优越性:准确、快速、简单,将其应用到现场定性、定量检测赤潮微藻方面将具有很大的潜力.

To explore the feasibility of applying fluorescence in situ hybridization （FISH） to detect HAB species, two species of dinoflagellate were detected by FISH technique in this study. Fluorescent DNA probes were designed according to the ITS sequence of Prorocentrum donghaiense. The control was one strain of Alexandrium tamarense. The results were as follow： （1） Green fluorescence from the tip of cells of P. donghaiense was observed under epifluorescent microscopy, while there was no green fluorescence in cells of A. tamarense observed under epifluorescent microscopy. That is to say, probes react with target strain of P. donghaiense, and do not react with A. tamarense, showing that the probes are species-specific, and ITS sequence is a good region for species-specific probes design. （2） After being fixed and excited by blue light, there was no autofluorescence from algal cell chlorophyll and phycopyrrin observed under epifluorescent microscopy, which show that the fixation is valid. Above results show that the technique of FISH is superior to other techniques in sensitivity, simplicity and quickness, and could be applied to detecting HAB species in future.