16S与gyrB基因联合建树快速鉴定一株蜡样芽孢杆菌

Rapid Identification of a Bacillus cereus Strain with Phylogenetic Tree Based on Combination of 16S and gyrB Gene

摘要:

开发一种可用于保守序列相似度较高的菌群快速鉴定的方法，并用其鉴定一株从土壤采集的芽孢杆菌。从厦门市同安国家农业科技园区施用动物肥的种植区采集土壤，稀释涂布平板，分离纯化菌株后，扩增16S与gyrB基因序列并测序，在Genbank选择序列相似的ATCC菌株进行比对，将16S序列与gyrB序列线性拼接后，利用Paup* 4.0构建进化树，通过生理生化实验对鉴定结果进行验证。在16S序列与gyrB基因序列单独建树均无法鉴定到种的情况下，通过16S与gyrB碱基线性拼接序列联合建树，将土壤中分离得到的芽孢杆菌HX2016002鉴定为蜡样芽孢杆菌，该方法所构建的进化树自展值高，鉴定结果与生理生化实验一致。对Genbank中已知的蜡样芽孢杆菌序列建树分析表明，该方法在蜡样芽孢杆菌中具有普适性。利用16S与gyrB基因拼接序列联合建树，在保守序列相似度高的属内菌种鉴定中具有进一步研究的价值。

Abstract:

This work was aimed to develop a method for rapid identification of bacteria with high similarity of conserved sequences in DNA, and to identify a Bacillus strain collected from soil. The prokaryote strain was isolated and purified from the soil after diluting and coating the plate, which collected from the planting area with animal fertilizer in Tong'an National Agricultural Science and Technology Park, Xiamen. The 16S and gyrB gene of the strain were amplified by PCR and sequenced. The ATCC strains with similar sequences selected from Genbank were compared. After the 16S sequence and the gyrB sequence were linearly spliced, the phylogenetic tree was constructed with Paup*4.0, and the identification results were verified by physiological and biochemical methods. The 16S sequence and gyrB gene sequence were constructed separately, however, the strain was failed to be identified to specie level. After the joint construction, the Bacillus HX2016002 isolated from soil was identified as Bacillus cereus. This method obtained a high self-expanding phylogenetic tree, and the identification results were consistent with the physiological and biochemical experiments. The sequence analysis with the known Bacillus cereus in Genbank showed that the method was generally applicable in Bacillus cereus identification. The phylogenetic tree based on the combination of 16S and gyrB gene has the value of further research in the identification of genus strains with high conserved sequence similarity.