美洲大蠊肠道无色杆菌的分离鉴定及其抑菌活性的初步研究

Isolation and identification of intestinal endogenic Achromobacter from Periplaneta americana and preliminary study on its antibacterial activity

目的从美洲大蠊肠道中分离、鉴定4株内生无色杆菌,并初步测定其抑菌活性和鉴定其关键次级代谢生物合成基因。方法采用平板划线法、16S rDNA PCR扩增、Blast比对和最大似然值法构建系统发育树,对4株美洲大蠊肠道内生无色杆菌进行分离、鉴定。采用牛津杯法初步测定4株美洲大蠊肠道内生无色杆菌次级代谢产物的抗菌活性。采用PCR扩增、琼脂糖凝胶电泳法鉴定4株美洲大蠊肠道内生无色杆菌的关键次级代谢生物合成基因PKS I、NRPS和卤化酶基因FADH2。结果从美洲大蠊肠道中分离得到了4株内生无色杆菌,且4株美洲大蠊肠道内生无色杆菌与已知无色杆菌Achromobacter xylosoxidans、Achromobacter aegrifaciens和Achromobacter marplatensis的相似度均在99.3%以上。抑菌实验结果表明,4株美洲大蠊肠道内生无色杆菌对枯草芽孢杆菌、金黄色葡萄球菌、黑曲霉菌等病原菌均具有一定的选择抑制作用。琼脂糖凝胶电泳结果显示,均能从4株美洲大蠊肠道内生无色杆菌的关键次级代谢生物合成基因PKS I、NRPS,且在菌株WA5-1-54的检测到卤化酶基因FADH2。结论美洲大蠊肠道内生无色杆菌可能具有产抑菌活性物质的能力。

In order to isolate and identify four Achromobacter from the Periplaneta american gut,and detect the antimicrobial activity and the key secondary metabolite biosynthesis gene of the Periplaneta americana intestinal endogenic Achromobacter.Four Periplaneta Americana intestinal endogenic Achromobacter were isolated and identified by means of streak plate,16S rDNA PCR,Blast comparison and construction of phylogenetic tree by maximum-likelihood method.The antimicrobial activity of 4 strains were tested preliminary by means of Oxford cup method.The key secondary metabolite biosynthesis gene PKS I and NRPS and the halogenase gene FADH2 were detected by means of PCR and agarose gel electrophoresis method.The results showed that the four strains of the Achromobacter from the Periplaneta american gut were similar to the known Achromobacter and the similarities were both more than 99.3%.And the results of antimicrobial activity assay showed that both the Periplaneta americana intestinal endogenic Achromobacter had selective inhibition ability to the Bacillus subtilis,Staphylococcus aureus,Aspergillus niger and other pathogenic bacteria.The results of agarose gel electrophoresis showed that the key secondary metabolite biosynthesis gene PKS I and NRPS were both detected in the four strains Periplaneta americana intestinal endogenic Achromobacter,the halogenase gene FADH2 was only detected in the strain WA5-1-54.This study revealed that the Periplaneta Americana intestinal endogenic Achromobacter might have the ability to produce the anti-bacterial and anti-fungi substances.