3种鳢属鱼类ITS序列及系统进化分析

朱树人 孟庆磊 朱永安等

摘要[目的]阐明乌鳢、斑鳢、月鳢核基因序列（ITS）的遗传变异情况，并用ITS序列研究鳢属系统进化关系。[方法]利用PCR扩增3种鳢的ITS，经克隆、拼接得到ITS全长后进行分析。[结果]乌鳢、斑鳢和月鳢ITS全序列长度为902、 927、902或903 bp。3种鳢ITS的G+C含量较高，约占72%。3种鳢之间核苷酸差异明显大于种内，明显的差异性ITS片段可以鉴别3种鳢。采用NJ和ML方法建立的进化树都表明乌鳢和斑鳢的遗传距离最近，而乌鳢和月鳢遗传距离最远。[结论]该研究为鳢属的分类鉴定、系统进化关系以及种间杂交提供基础资料。

关键词乌鳢；斑鳢；月鳢；ITS序列；亲缘关系

中图分类号S961.6文献标识码A文章编号0517-6611（2015）31-047-03

Analysis on ITS Sequences and Phylogenetic Relationship of Three Kinds of Channa

ZHU Shuren1，2，3， MENG Qinglei1， ZHU Yongan1 et al

（1.Shandong Provincial Key Laboratory Freshwater Genetics and Breeding， Shandong Freshwater Fisheries Research Institute， Jinan，Shandong 250017； 2.Key Laboratory of Freshwater Aquatic Genetic Resources Gertificated by Ministry of Agriculture， Shanghai Ocean University， Shanghai 201306； 3.Department of Biology， East China Normal University， Shanghai 200241）

Abstract [Objective] The aim was to examine sequence variation in the internal transcribed spacer （ITS） isolates from Channa argus， C. maculata and C. asiatica， and to reconstruct their phylogenetic relationship using ITS sequences. [Method]The ITS sequences were amplified from each sample and the amplicons were cloned and spliced the full length of ITS sequences.[Result] The full length of ITS sequences of C. argus， C. maculata and C. asiatica were 902， 927， 902 or 903 bp. The content of G+C （about 72%） was higher than A+T. For three species of snakehead， interspecific differences were significantly greater than the intraspecific differences. The obvious difference of ITS fragments could identify three species of snakehead. Based on NJ and ML method， phylogenetic analysis showed that C. argus and C. maculata had the lowest genetic distance， while C. argus and C. asiatica had the highest genetic distance. [Conclusion]The results of this study could be useful for classification， phylogenetic relationship and interspecific hybrids of Channa.

Key words Channa argus； C. maculata ； C. asiatica； ITS sequences； Phylogenetic relationship

真核生物的核糖體都有3种rRNA，即18S rRNA、5.8S rRNA 和28S rRNA。这3种rRNA 基因都是以串联重复形式存在，编码这些rRNA的基因都按一定顺序排列在DNA上。ITS包括ITS1和ITS2，分别位于18S rRNA与5.8S rRNA，5.8S rRNA 和28S rRNA之间。由于ITS1和ITS2进化速度较快，两者广泛应用于鱼类物种的鉴定，特别用于分类较相似的鱼类之间[1-4]。可以根据ITS两边rRNA 具有很高的保守性，设计引物扩增ITS序列片段。

笔者对我国较为常见的乌鳢、斑鳢、月鳢种间的分子遗传距离和进化关系进行研究，旨在促进对其种内遗传多样性的认识，为分析鳢属种间杂交提供借鉴。

1材料与方法

1.1材料

3种鳢属鱼类样本于2013年5～10月份采集。乌鳢样本采集于山东微山湖，斑鳢和月鳢都采自广东。每种鳢采集5尾，样本均采自水上作业的渔民或附近的水产品市场，剪取少量胸鳍，保存于无水乙醇中，贴好标签放置于4 ℃冰箱备用。

1.2DNA提取

将胸鳍组织从乙醇中取出，充分漂洗晾干并剪碎，采用标准的酚-氯仿法提取基因组DNA。……