不同引物扩增对东海陆架沉积物真核微型生物多样性的影响

摘要

采用三对通用引物(ITS1/ITS4,EF3/EF4,NS1/NS4)检测其对东海陆架DH-13站点表层沉积物真核微型生物的扩增特异性和多样性差异.对323个有效克隆子进行OTU分型和多样性分析,结果表明,NS1/NS4引物扩增所获得的文库多样性最高,EF3/EF4其次,ITS 1/ITS4最后.通过NCBI比对和分类学研究,发现优势种群主要为原生生物界,包括丝足虫门(Cercozoa,占所有OTU的36.4％)、横裂甲藻纲(Dinophyceae,占14.2％)、不等鞭毛门(Stramenopiles,占7.4％)和纤毛虫门(Ciliophora,占6.3％);另含部分真菌界如子囊菌门(Ascomycota,占7.4％)和壶菌门(Chytridiomycota,占6.3％).原生生物方面,NS1/NS4引物表现的多样性最高,适合于扩增丝足虫门、横裂甲藻纲和不等鞭毛门,并扩增到独有的眼虫门(Euglenozoa)、隐藻门(Cryptophyta)和顶复门(Apicomplexa);ITS1/ITS4适合于扩增甲藻和纤毛虫门;EF3/EF4则适合于丝足虫门.真菌方面,ITS1/ITS4适合于扩增子囊菌门;EF3/EF4适合于子囊菌门和壶菌门,而NS1/NS4适合于壶菌门.系统发育分析表明,三对引物的原生生物系统树中的相似序列多来自海洋环境样品,真菌序列则分别来自陆地土壤真菌和海洋真菌,说明三对引物更适合于海洋沉积环境微型原生生物的分子生态分析.%Three pairs of universal primer (ITS1/ITS4,EF3/EF4 and NS1/NS4) were applied to determine the amplification specificity and diversity difference in microeukaryotes isolated from surface sediment at station DH-13,on the East China Sea shelf.After the OTU classification and diversity analysis on 323 valid clones,we demonstrate that the library generated by the amplification of primer NS1/NS4 obtains the highest diversity,followed by the EF3/EF4,and then the ITS 1/ITS4.Through comparison with NCBI and taxonomical study,we found that the dominant species mainly belong to the Protista,which includes Cercozoa (accounting for 36.4％ of the total OTU),Dinophyceae (14.2％),Stramenopiles (7.4％) and Ciliophora (6.3％); meanwhile,a part of the species belongs to Fungi,such as Ascomycota (7.4％) and Chytridiomycota (6.3％).When it comes to the Protista,primer NS 1/NS4 demonstrates the highest diversity,and it fits for the amplification about species of Cercozoa,Dinophyceae and Stramenopiles.Moreover,exclusive species was also amplified,such as Euglenozoa,Cryptophyta and Apicomplexa.In the course of amplification ITS 1/ITS4 fits for the species of Dinophyceae and Ciliophora,while EF3/EF4 is suitable for Cercozoa.In terms of fungus,ITS1/ITS4 fits for the species ofAscomycota; EF3/EF4 fits for the species of Ascomycota and Chytridiomycota,while NS1/NS4 merely fits for the genus Chytridiomycota.The result of phylogenetic analysis reveals that most of the similar sequences in the phylogenetic tree of Protista amplified by the three pairs of primer were derived from marine environmental samples,whereas the fungal sequences were derived from both of the soil and ocean,suggesting that three pairs of the primer are fit for the molecular ecological analysis of Protista in marine sedimentary environment.