鱼类环境DNA研究中通用引物的筛选验证

为了筛选一个通用性和适用性良好的能够运用于环境DNA(eDNA)研究的鱼类引物,从相关文献中选取了鱼类线粒体基因组部分片段的5对引物,分别对线粒体D-loop区、16S rRNA基因、COI基因以及Cytb基因部分片段进行扩增。对千岛湖48种鱼类基因组DNA进行扩增后比较发现,引物16s和COI均可以取得良好的扩增效果,通用性优于其他几对引物。引物16s的扩增产物经凝胶电泳检测均出现明亮的目的条带,引物COI则有3种鱼的条带经凝胶电泳检测亮度较暗。利用上述引物对环境样品eDNA扩增时发现,只有16s和COI的引物具有良好的扩增效果,能够得到明显单一的亮带。对该两种引物的PCR产物克隆后测序比对发现,16s的PCR产物均为千岛湖常见鱼类物种的基因片段,COI的PCR产物则为细菌COI基因的部分片段。综上,我们认为引物16s在通用性和适用性上都更为适合作为鱼类群落结构eDNA研究的通用引物。

Universal primer screening and verification for fish environment DNA research

In order to screen the primer of well universality and applicability for eDNA research, five pairs of universal primer of fish mitochondrial genome fragment were chosen from the previous references to amplify the partial sequences of D-loop region, 16S rRNA gene, COI gene and Cytb gene.These primers were used to amplify genome of 48 species of fish in Qiandao Lake to test the universality, it was found that the 16s and COI were better than the other primers.Gel e-lectrophoresis showed that all 16s DNA bands of the 48 species of fish was light, but 3 COI gene bands of the fish species was dark.The eDNA from Qiandao Lake were amplified by these five pairs of primer, and the electrophoretogram showed that the PCR product of 16s DNA and COI gene presented a clear bright band.According to the cloned sequencing and BLAST results, the PCR product of 16s DNA was coincident to the fragments of fishes from Qiandao Lake, but the PCR product of COI gene was in accord with the COI gene of bacteria.In summary, the primer of 16S rRNA was the most suit-able universal primer to study on eDNA of fish community structure in the Qiandao Lake.