双盲口线虫ITS rDNA的PCR扩增及序列分析

PCR AMPLIFICATION AND SEQUENCE ANALYSIS OF ITS RDNA OF CONTRACAECUM OSCULATUM

以从波兰大西洋鳕体内采集的8条双盲口线虫样品为研究对象,利用保守引物通过PCR扩增其核糖体DNA内转录间隔区(internal transcribed spacers,ITS)ITS-1 rDNA、5.8S rDNA、ITS-2 rDNA后,将扩增片段纯化后克隆至pGEM-T Easy载体,经PCR及酶切鉴定后,对获得的阳性质粒DNA进行序列分析。结果显示,所获得的ITS rDNA及5.8S rDNA序列总长存在一定差异,ITS rDNA为885～927 bp,包含18S rDNA、28S rDNA部分序列及ITS-1 rDNA(441～449 bp)、5.8S rDNA(155 bp)和ITS-2rDNA(266 bp)全部序列。结果表明,双盲口线虫ITS序列种内相对保守(ITS-1 rDNA:0～2.2%;ITS-2 rDNA:0～3.4%),而种间差异较大(ITS-1 rDNA>20%;ITS-2 rDNA>35%),可作为种间遗传变异研究的标记。本研究结果为双盲口线虫的分子鉴定、种群遗传学以及双盲口线虫病的进一步研究提供了参考和依据。

The aim of this study was to amplify and analyze the sequences of internal transcribed spacers(ITS)of ribosomal DNA(rDNA)of Contracaecum osculatum isolated from Gadus morhua imported from Poland.The ITS rDNA sequences were amplified by PCR using primers NC5 and NC2.The amplicons were cloned into pGEM-T Easy vector and the inserts were sequenced.The results showed that the length of ITS rDNA in C.osculatum isolates varied(885~927 bp).Sequence analysis revealed that the ITS-1 rDNA,5.8S rDNA and ITS-2 rDNA of these samples were 441~449 bp,155bp and 266 bp in length,respectively.Furthermore,the intra-specific sequence variations within C.osculatum were 0~2.2%and 0~3.4%for ITS-1 rDNA and ITS-2 rDNA,while inter-species difference was obvious(ITS-1 rDNA>20%;ITS-2 rDNA>35%).The results indicated that the ITS rDNA sequences might be used as an useful genetic markers for molecular identification and population genetics studies of C.osculatum.