斑石鯛卵鞭蟲(chóng)病病原的分子鑒定與系統(tǒng)發(fā)育分析

付泉潔 范超 謝國(guó)駟 葉仕根 史成銀

摘要 [目的]對(duì)嚴(yán)重危害斑石鯛魚(yú)苗的卵鞭蟲(chóng)病病原——渤海分離株（Bohai-1407 isolate）進(jìn)行分子生物學(xué)鑒定和系統(tǒng)發(fā)育分析。[方法]設(shè)計(jì)4對(duì)特異性引物，應(yīng)用PCR方法克隆并測(cè)定渤海分離株的核糖體DNA（rDNA）序列；應(yīng)用Blast比對(duì)分析渤海分離株rDNA的結(jié)構(gòu)；依據(jù)rDNA序列，分別構(gòu)建10種胚溝科鞭毛蟲(chóng)和19株眼點(diǎn)淀粉卵渦鞭蟲(chóng)分離株/克隆的系統(tǒng)發(fā)育樹(shù)，分析渤海分離株的系統(tǒng)分類(lèi)地位。[結(jié)果]擴(kuò)增出了渤海分離株的4個(gè)DNA片段，拼接出長(zhǎng)度為6 530 bp的rDNA操縱子序列；該操縱子由74 bp的部分外轉(zhuǎn)錄間隔區(qū)（ETS）、1 813 bp的小亞基（SSU）、352 bp的內(nèi)轉(zhuǎn)錄間隔區(qū)1（ITS1）、159 bp的5.8S、698 bp的內(nèi)轉(zhuǎn)錄間隔區(qū)2（ITS2）和3 388 bp的大亞基（LSU）串聯(lián)而成，3′端還有46 bp的部分非轉(zhuǎn)錄間隔區(qū)（NTS）；渤海分離株與眼點(diǎn)淀粉卵渦鞭蟲(chóng)寧德株（Ningde1412）的rDNA序列相似性高達(dá)99.5%；依據(jù)SSU序列建立了包含10種胚溝科鞭毛蟲(chóng)的系統(tǒng)發(fā)育樹(shù)，渤海分離株與世界各地分離到的眼點(diǎn)淀粉卵渦鞭蟲(chóng)聚類(lèi)在一起，將其鑒定為眼點(diǎn)淀粉卵渦鞭蟲(chóng)；依據(jù)ITS1和ITS2序列建立了包含19株眼點(diǎn)淀粉卵渦鞭蟲(chóng)的2個(gè)系統(tǒng)發(fā)育樹(shù)，渤海分離株與從美國(guó)弗羅里達(dá)鹽水池塘中分離到的墨西哥灣分離株FL_21（DQ490260.1）總是聚類(lèi)在一起。[結(jié)論]斑石鯛卵鞭蟲(chóng)病的病原——渤海分離株可以鑒定為眼點(diǎn)淀粉卵渦鞭蟲(chóng)，該分離株與墨西哥灣分離株FL_21的親緣關(guān)系最近。

關(guān)鍵詞 斑石鯛；眼點(diǎn)淀粉卵渦鞭蟲(chóng)；核糖體DNA；分子鑒定；系統(tǒng)發(fā)育分析

中圖分類(lèi)號(hào) S941.5 文獻(xiàn)標(biāo)識(shí)碼 A 文章編號(hào) 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

斑石鯛（Oplegnathus puncatus）是一種具有較高經(jīng)濟(jì)價(jià)值的海水魚(yú)類(lèi)，其抗逆性強(qiáng)，生長(zhǎng)迅速，營(yíng)養(yǎng)價(jià)值高，具有良好的發(fā)展前景。……