付泉洁 范超 谢国驷 叶仕根 史成银

摘要 [目的]对严重危害斑石鲷鱼苗的卵鞭虫病病原——渤海分离株（Bohai-1407 isolate）进行分子生物学鉴定和系统发育分析。[方法]设计4对特异性引物，应用PCR方法克隆并测定渤海分离株的核糖体DNA（rDNA）序列；应用Blast比對分析渤海分离株rDNA的结构；依据rDNA序列，分别构建10种胚沟科鞭毛虫和19株眼点淀粉卵涡鞭虫分离株/克隆的系统发育树，分析渤海分离株的系统分类地位。[结果]扩增出了渤海分离株的4个DNA片段，拼接出长度为6 530 bp的rDNA操纵子序列；该操纵子由74 bp的部分外转录间隔区（ETS）、1 813 bp的小亚基（SSU）、352 bp的内转录间隔区1（ITS1）、159 bp的5.8S、698 bp的内转录间隔区2（ITS2）和3 388 bp的大亚基（LSU）串联而成，3′端还有46 bp的部分非转录间隔区（NTS）；渤海分离株与眼点淀粉卵涡鞭虫宁德株（Ningde1412）的rDNA序列相似性高达99.5%；依据SSU序列建立了包含10种胚沟科鞭毛虫的系统发育树，渤海分离株与世界各地分离到的眼点淀粉卵涡鞭虫聚类在一起，将其鉴定为眼点淀粉卵涡鞭虫；依据ITS1和ITS2序列建立了包含19株眼点淀粉卵涡鞭虫的2个系统发育树，渤海分离株与从美国弗罗里达盐水池塘中分离到的墨西哥湾分离株FL_21（DQ490260.1）总是聚类在一起。[结论]斑石鲷卵鞭虫病的病原——渤海分离株可以鉴定为眼点淀粉卵涡鞭虫，该分离株与墨西哥湾分离株FL_21的亲缘关系最近。

关键词 斑石鲷；眼点淀粉卵涡鞭虫；核糖体DNA；分子鉴定；系统发育分析

中图分类号 S941.5 文献标识码 A 文章编号 0517-6611（2017）13-0135-06

Molecular Identification and Phylogenetic Analysis of a Pathogenic Dinoflagellate Isolated from Spotted Knifejaw （Oplegnathus puncatus）

FU Quan-jie1， 2， FAN Chao2， 3， XIE Guo-si2， SHI Cheng-yin2* et al

（1. College of Fisheries and Life Science， Dalian Ocean University， Dalian， Liaoning 116023；2. Key Laboratory of Maricultural Organism Disease Control， Ministry of Agriculture， Yellow Sea Fisheries Research Institute， Chinese Academy of Fishery Sciences， Qingdao， Shandong 266071；3. College of Fisheries and Life Science， Shanghai Ocean University， Shanghai 201306）

Abstract [Objective] A pathogenic dinoflagellate （the Bohai-1407 isolate） was isolated from diseased Oplegnathus puncatus. To perform molecular identification and phylogenetic analysis of the Bohai-1407 isolate. [Method] Four pair of specific PCR primers were used to amplify the ribosomal DNA （rDNA） fragments of the Bohai-1407 isolate. The rDNA fragments were cloned and sequenced. The sequences of cloned rDNA fragments were assembled and then analyzed by Blast. The systematic taxonomy of the Bohai-1407 isolate was analyzed by constructed phylogenetic trees based on the rDNA sequences of 10 dinoflagellates in family Blastodiniidae and the rDNA sequences of 19 isolates/clones in species Amyloodinium ocellatum. [Result] Four ribosomal DNA （rDNA） fragments of the Bohai-1407 isolate were amplified. Sequence analysis showed that the sequenced rDNA operon were 6 530 bp in length. The rDNA operon was composed of 74 bp of partial external transcribed spacer （ETS）， 1 813 bp of 18S （also called small subunit， SSU）， 352 bp of internal transcribed spacers 1 （ITS1）， 159 bp of 5.8S， 698 bp of internal transcribed spacers 2 （ITS2） and 3388 bp of 23S （also called large subunit， LSU）， followed by a 46 bp of partial non-transcribed spacer （NTS） in the 3 end. The sequence identity between the Baohai-1407 isolate and the Ningde1412 strain of A. ocellatum was as high as 99.5%. Based on the SSU sequence of 10 dinoflagellates in family Blastodiniidae， a phylogenetic tree was constructed and the Bohai-1407 isolate clustered with isolates of A. ocellatum. Two phylogenetic trees based on the ITS1 and ITS2 sequences of 19 isolates/clones in species A. ocellatum were also constructed. The Bohai-1407 isolate always clustered with the Gulf of Mexico isolate FL_21 （DQ490260.1）. [Conclusion] The pathogenic dinoflagellate （the Bohai-1407 isolate） isolated from diseased O. puncatus was A. ocellatum according to the molecular identification results of rDNA. The Bohai-1407 isolate was most closely related to the Gulf of Mexico isolate FL_21 of A. ocellatum.

Key words Oplegnathus puncatus；Amyloodinium ocellatum；rDNA；Molecular identification；Phylogenetic analysis

斑石鲷（Oplegnathus puncatus）是一种具有较高经济价值的海水鱼类，其抗逆性强，生长迅速，营养价值高，具有良好的发展前景。……