Development of genetic diagnosis method of toxic microalgae

近年来，由形态特征非常相似的有毒甲藻引起的麻痹性贝类中毒，给水产养殖场和公众健康带来了严重的问题。本研究旨在建立有毒甲藻亚历山大藻的分子鉴定方法。(1)基于18S rDNA序列和28S rDNA中的D_1/D_2区的系统发育分析，对有毒甲藻、卡他沙门氏菌、塔米亚瓦氏A、奥氏甲藻和无毒甲藻、兄弟甲藻、中甲藻、假甲藻进行了系统发育分析。(2)建立了一种快速、准确鉴定有毒甲藻的方法。利用荧光原位杂交(FISH)技术，设计、评价和建立了快速检测和定量检测塔玛和卡氏甲藻营养细胞和包囊(休眠合子)的实时荧光定量分析方法。(4)利用差异显示和差减杂交技术，建立了塔玛和卡特氏菌在不同阶段表达的差异表达基因的筛选方法。结果检测到一些有性融合和萌发阶段特异的基因。

Recent paralytic shellfish poisoning caused by toxic dinoflagellates which have very similar morphological features are serious problems in aquatic farms and public health. The purpose of this study are the establishment of molecular identification of toxic dinoflagellates Alexandrium spp. and development of genetic diagnosis method of toxic dinoflagellates(1) Phylogenetic analysis based on 18S rDNA sequences and the D1/D2 region in 28S rDNA were examined among the many isolates of toxic A.tamarense, A.catenella, A.tamiyavanichii, A.ostenferdii and nontoxic A.affine, A.fraterculus, A.insuetum, A.psedogonyaulax.(2) A rapid and precise identification method was established using a fluorescent, rRNA-targeted, oligonucleotide probes for toxic A.tamiyavanichii which are recently observed in Harima-Nada and the nontoxic A.affine and A.frateruculus. Each probe was species specific when applied using fluorescence in situ hybridization (FISH).(3) Real-time PCR assay was designed, evaluated and established for rapid detection and quantification of the vegetative cells and cysts (hypnozygotes) of toxic dinoflagellates A.tamarense and A.catenella.(4) The screening methods of the specific genes expressed in differential stages, such as cyst formation and germination of A.tamarense and A.catenella, were developed by use of differential display and subtractive hybridization methods. As a result, some genes specific for the sexual fusion and germination stages were detected.