三株赤潮硅藻5.8S rDNA及转录间隔区(ITS)的克隆及序列分析

The cloning and sequence analysis of the 5.8S rDNA and its region from three strains of marine diatoms

对引发赤潮的3株硅藻———1株尖刺拟菱形藻(Pseudo-nitzshia pungens)和2株中肋骨条藻(Skeletonema costatum)的5.8S rDNA和ITS(internal transcribed spacers)序列进行了PCR扩增、克隆和序列测定,并分析了硅藻门10株赤潮藻(7株从GenBank获得)的系统进化关系.研究结果表明,尖刺拟菱形藻的ITS和5.8S rDNA的长度为693bp,SK-1(分离自东海赤潮暴发区)测序得到715bp,除ITS和5.8SrDNA外,还包含部分18S rDNA和28S rDNA;SK-2(分离自青岛养殖场)的ITS和5.8S rDNA的长度为331bp,尖刺拟菱形藻与从GenBank中获得的2株尖刺拟菱形藻相似程度最高,为100%,与该属的多列拟菱形藻相似程度稍低,为82.9%.SK-2的ITS序列与SK-1的相似程度很低,只有51%,但与拟中肋骨条藻的ITS序列相似程度高,为95.5%.SK-1的ITS序列与拟中肋骨条藻的相似程度也低,为56.7%.系统进化树反映的结果与相似性反映的结果一致.研究的该株尖刺拟菱形藻从根据ITS序列研究的结果与形态鉴定的结果看是一致的;SK-2可能属于拟中肋骨条藻;SK-1比较特殊,有待于用其他的方法进一步研究确定其分类地位.

Pseudo-nitzschia pungens and Skeletonema costatum are two strains of diatoms which cause harmful algal bloom in China, SK-1 and SK-2 are two strains of diatoms, assumably species of S. costatum by morphological characteristics. Three strains, SK-1 which was isolated from the East China Sea and SK- 2 which was isolated from the Aquatic Farm of Qingdao and one strain of Pseudo-nitzschia pungens which was from the East China Sea, were studied based on rDNA data. The sequences of 5.8S rDNA and ITS regions of three strains of diatoms were amplified, cloned and sequenced. Meanwhile, seven additional ITS sequences and 5.8S rDNA were included in the data analysis. The results were as follows： （1） The ITS sequence and 5.8S rDNA of P. pungens were 693 bp while the sequence of SK-1 was 715 bp, which contained complete ITS and 5.8S rDNA , partial 18S rDNA and 28S rDNA; The ITS and 5. 8S rDNA sequences of SK-2 were 331bp; （2） the sequence of P. pungens had very high level of similarity with that of two strains of P. pungens retrieved from GenBank, and the similarity was 100% ; while it showed 82. 9% of identity compared with P. multiseries which also belonged to the genus of Pseudo-nitzschia ; the ITS sequence similarity of SK-2 and SK-1 was very low, showing 51.5M of identity, but it had very high level of similarity with that of Skeletonema pseudocostatum （95. 5 %） ; the ITS sequence of SK-1 had lower similarity with S. pseudocostaturn; （3） results of evolutionary tree coincided with similarity analysis of all three strains; it was suggested that recognition of P. pungens by morphological characteristics was supported with rDNA and ITS regions; （4） the findings demonstrated that classifications of SK-2 and SK-1 based on morphological criteria maybe were incongruous. SK-2 should be belonged to S. pseudocostaturn and more data are needed to classify which groups SK-1 is belonged to.