海参核糖体基因间隔区2(ITS2)的克隆及序列分析

Clone and Sequence Analysis of the Internal Transcribed Spacers in Sea Cucumber Ribosomal(ITS2) DNA

为探讨刺参科海参和海参科海参的系统进化关系,本研究通过PCR技术获取19种刺参科和海参科海参的ITS2序列,从NCBI上获取瓜参(C. salma)的ITS2序列。结果表明ITS2序列具有长度多态性,从318 bp (绿刺参)到591 bp (白尼参属)。海参属的ITS2序列长度多态性高,ITS2的GC含量从56.7%(糙海参)到70.6%(瓜参)。海参ITS2序列保守性不高,仅有48个保守位点,其余均为变异位点。基于ITS2的系统进化树结果显示进化树主要分成两支,一支包括海参科的4个属:海参属、白尼参属、辐肛参属和格皮氏海参属。辐肛参属和格皮氏海参属为姐妹关系,二者聚在一起后与白尼参属聚为一支,随后再与海参属聚在一起。白尼参属和辐肛参属为单系,海参属为复系。另一支为C. salma和刺参科。梅花参属与刺参属聚为一支后,再与仿刺参属聚在一起,3个属都是单系。在20种海参中,S. naso与B. argus的遗传距离最大(6.415)。刺参属中,S. monotuberculatus和S. horrens遗传距离最近(0.012),海参属中,糙海参与H. fuscopunctata的遗传距离最大(3.24)。本研究为从分子水平上研究海参科和刺参科之间的系统进化关系奠定了基础。

In order to investigate the phylogenetic relationship between holothurians from family Holothuriidae and holothurians from family Stichopodidae, 19 ITS2 sequences of holothurians from family Holothuriidae and Stichopodidae were obtained with PCR technology. A ITS2 sequence of C. salma was obtained in NCBI. The result showed that ITS2 had length polymorphism, varied from 318 bp(Stichopus chloronotus) to 591 bp(Bohadschia).ITS2 sequence from Holothuria had high length polymorphism. The GC content of the ITS2 sequences was varied from 56.7%(Holothuria scabra) to 70.6%(C. salma). The conservation of ITS2 sequences from sea cucumber was not high, only 48 conservative loci were found and the others were variable site. The result of phylogenetic tree based on ITS2 showed that the phylogenetic tree was main divided into two clades. One clade included four genus of Holothuriidae: Holothuria, Bohadschia, Actinopyga and Pearsonothuria. Actinopyga and Pearsonothuria were sister relationship. Both of them were clustered together with Bohadschia, and then clustered with Holothuria.Bohadschia and Actinopyga were recovered as monophyletic, and Holothuria was paraphyletic. The other clade include Stichopodidae and C. salma. Thelenota and Stichopus were clustered together, and then clustered with Apostichopus. All of them were recovered as monophyletic. Among the 20 sea cucumbers, the genetic distance between S. naso and B. argus was highest(6.415). In Stichopus, the genetic distance between S. monotuberculatus and S. horrens was lowest(0.012). In Holothuria, the genetic distance between H. scabra and H. fuscopunctata was highest(3.24). This study would lay the foundation in molecular level for the research of phylogenetic relationships between family Holothuriidae and family Stichopodidae.