五种/株原甲藻核糖体小亚基(18S rRNA)基因克隆及序列分析

CLONING AND SEQUENCES ANALYSIS OF 18S rRNA GENE OF FIVE PROROCENTRUM SPECIES/STRAINS

采用分子克隆及序列比对的方法,对五种/株赤潮原甲藻18SrRNA基因全长序列进行扩增、克隆和序列测定,并从GenBank上下载13个原甲藻18SrRNA基因接近全长的序列,用NJ法和ME法构建了原甲藻属的系统树。结果表明,五种/株原甲藻18SrRNA基因扩增序列长度为1782—1783bp,其中来自南海(中国海域)和来自美国海域的两株微小原甲藻(Prorocentrumminimum)的序列完全一致;东海的赤潮原甲藻(Prorocentrumsp·)与具齿原甲藻(P·dentatum)的序列也完全一致,与微小原甲藻只有5个碱基的差异;而海洋原甲藻(P·micans)与微小原甲藻和具齿原甲藻的序列差异较大,分别为27个和28个碱基。通过NJ法和ME法构建的系统树基本一致。由系统树可以看到:原甲藻属大致分为两支,本实验的微藻全部分布在同一支上。18SrRNA基因序列还将有助于有害赤潮藻快速鉴定的特异性分子探针的研制。

In recent years, red tide or HAB broke out frequently in maritime China. Most HABs were caused by genus Prorocentrum. We carried out this molecular study aiming at revealing the phylogenesis of Prorocentrum, and also developing a molecular detection method to determine the HAB causer. Using gene cloning, sequencing and muhiple alignment, we cloned and sequenced 18S rRNA genes in five species/ strains of Prorocentrum. Thirteen of them can be retrieved in GenBank. After that, the phylogenetic trees of Prorocentrum were constructed in styles of NJ （neighbor-joining） and ME （ minimum evolution）. The results demonstrate that the 18S r[INA genes of five strains are 1782-1783bp in length. The sequences of P. minimum from the South China Sea and American coast were identical, and Prorocentrum sp. separated from the East China Sea had the same 18S rRNA sequences as that of the P. dentatum, which is different from P. minimum in 5 nucleotide sites. P. micans had 27 different nucleotide sites from that of P. minimum and 28 from that of P. dentatum. The phylogenetic trees in NJ and ME manners are almost identical. The 18S rRNA sequences of these Prorocentrum clustered in two clades. One includes P. lima, P. arenarium and others;and they are benthic type. Another clade contains P. minimum, P. panamense and others and they are planktonic or bentho-planktonic type. All those we secluenced were within a clade. Therefore, we believed that the benthic Prorocentrum species should be singled out from the genus Prorocentrum. Through the analysis on five strains of P. minimum and three strains of P. micans, we found that the 18S rRNA gene sequences among the same species are highly conservative. Prorocentrum sp., which caused large HAB in the East China Sea these years, has the same 18S rRNA gene sequence to that of P. dentatum（ CCMP 1517）and they should belong to the same species. In sequence alignment, species-specific sequences can be found according which molecular probing technique could be developed, for rapid detection of the HAB species.