斑石鲷卵鞭虫病病原的分子鉴定与系统发育分析

摘要

[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 of5.8S,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 ofA.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 ofA.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 ofA.ocellatum.%[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比对分析渤海分离株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74bp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(TTS1)、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46bp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.