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目的探讨口虾蛄肠道细菌种群的多样性。方法通过不依赖于分离培养的分子生物学分析方法,以直接提取虾蛄肠道细菌的总DNA为模板经过PCR扩增16S DNA,然后经与T载体连接建立质粒文库。用限制性内切酶(BsuRⅠ和Hin6Ⅰ)对阳性克隆的PCR扩增产物进行限制性酶切片段长度多态性(RFLP)分析,选取有代表性的克隆进行测序。结果16S DNA序列通过CLUSTALX进行多序列比对及NCBI数据库中的BLAST分析后发现口虾蛄肠道细菌主要分成4类:未培养细菌,未培养支原体科细菌,未培养δ变形菌和马特斯支原体。结论口虾蛄肠道细菌种类较为简单,且多为未培养的细菌。
Objective To explore the diversity of intestinal flora in the mouth shrimp. Methods The 16S DNA was amplified by direct DNA extraction from the gut bacteria in shrimp culture using a molecular biological method independent of isolation and culture. The plasmid was ligated with T vector to construct a plasmid library. Restriction endonuclease fragment length polymorphism (RFLP) analysis of the PCR products of the positive clones was carried out by restriction endonucleases (BsuRⅠand Hin6Ⅰ), and representative clones were selected for sequencing. Results Multiple sequence alignment of 16S DNA sequence by CLUSTALX and BLAST analysis in NCBI database revealed that intestinal bacteria were mainly divided into four groups: uncultured bacteria, uncultured Mycoplasma bacteria, unformed Delta bacteria and Mattes Mycoplasma. Conclusion The species of intestinal bacteria in the mouth and shrimp is relatively simple, and most of them are uncultured bacteria.