ARDRA分型测定刺参养殖环境中蛭弧菌多样性

ARDRA analysis of diversity of Bdellovibrio-and-like organisms in mariculture environment of Apostichopus japonicus

摘要:

【目的】研究刺参养殖环境中蛭弧菌类微生物的多样性及其组成特征。【方法】对刺参养殖用水进行过滤，获得微生物并提取其总DNA，分别采用两对特异性引物Per和Bac扩增蛭弧菌类微生物相应的16S rRNA基因目的片段。通过核糖体DNA扩增片段限制性内切酶分析(ARDRA)方法，使用Hae Ⅲ和Msp Ⅰ，双酶切各60个目的基因的单克隆，选不同条带克隆进行测序并对测序结果的多重序列分析比对。【结果】确定两对引物分别存在8和9种碱基差异序列，均属于噬菌弧菌属，分属于3个类群中。类群Ⅰ、Ⅱ为优势类群，且均为已知类群，类群Ⅲ为潜在的新类群；Per1 (KP214541)和Bac44 (KP214551)代表菌株分别为优势种。【结论】刺参养殖环境中蛭弧菌具有较高的多样性，包括已知类型和未知类型的蛭弧菌；可进一步进行蛭弧菌的分离、培养，用于刺参养殖中病害防治研究。

Abstract:

[Objective] To study the diversity and composition of Bdellovibrio-and-like organisms (BALOs) in mariculture environment of Apostichopus japonicus. [Methods] The aquaculture water of A. japonicus was filtered to obtain the microorganisms. Then the total DNA of microorganisms was extracted. The purpose fragments of 16S rRNA gene of BALOs were amplified using two pairs of primers Per and Bac by PCR, respectively. Different patterns were determined with restriction enzymes Hae Ⅲ and Msp I by amplified ribosomal DNA restriction analysis (ARDRA). Positive clones were verified from 60 monoclonal strains. [Results] Eight and nine differences base sequences were obtained from two pairs of primers, respectively, and which were all belong to Bacteriovorax sp. and affiliated with three different clusters. Clusters Ⅰ and Ⅱ are dominant type which are known clusters; Cluster Ⅲ is a new one; The representative strains of Per1 (KP214541) and Bac44 (KP214551) are the dominant species. [Conclusion] The BALOs presented high diversity in mariculture environment of A. japonicus, including known and unknown, and the BALOs should be further isolated and cultured for the study of disease control in A. japonicus culture.