rpoB、gyrA、cheA基因在芽孢杆菌鉴定上的应用

Application of rpoB,gyrA and cheA genes in identifying Bacillus genus

为确定rpoB、gyrA和cheA基因在芽孢杆菌近缘种鉴定上的有效性,通过Blast-N算法,比对菌肥常用芽孢杆菌模式菌16S rRNA、rpoB、gyrA、cheA基因的序列差异,确定其应用效力。据此,提出一种根据菌株原始信息选择对应的基因引物扩增其序列,通过序列相似度和系统发育树快速、准确鉴定芽孢杆菌菌种的方法,并以19株可用于微生物肥料生产的芽孢杆菌分离株为材料,验证该方法的可行性。结果显示:地衣芽孢杆菌(Bacillus licheniformis)与枯草芽孢杆菌亚种(Bacillus subtilis subsp.subtilis、Bacillus subtilis subsp.inaquosorum、Bacillus subtilis subsp.spizizenii)、解淀粉芽孢杆菌亚种(Bacillus amyloliquefaciens、Bacillus velezensis)之间rpoB、gyrA、cheA基因序列的一致性分别为84.93%~86.57%、低于78.21%和78.25%~78.58%,枯草芽孢杆菌和解淀粉芽孢杆菌亚种之间gyrA基因序列的一致性为81.01%~95.82%,cheA基因序列的一致性为94.50%~95.83%,蜡样芽孢杆菌亚种(Bacillus cereus、Bacillus thuringiensis)之间gyrA基因序列的一致性为94.54%,巨大芽孢杆菌(Bacillus megaterium)和阿氏芽孢杆菌(Bacillus aryabhattai)cheA基因序列的一致性为94.55%。序列差异表明,rpoB、gyrA和cheA基因可以区分地衣芽孢杆菌、枯草芽孢杆菌组和解淀粉芽孢杆菌组,gyrA和cheA基因可以鉴定出枯草芽孢杆菌组、解淀粉芽孢杆菌组的亚种,gyrA基因能够鉴定蜡样芽孢杆菌组的亚种,cheA基因能区分巨大芽孢杆菌和阿氏芽孢杆菌。应用所提方法,将19株芽孢杆菌分离株鉴定为茹氏短芽孢杆菌(Brevibacillus reuszeri)、Bacillus mesonae、五大连池芽孢杆菌(Bacillus wudalianchiensis)、大猩猩芽孢杆菌(Bacillus massiliogorillae)、枯草芽孢杆菌枯草亚种(Bacillus subtilis subsp.subtilis)、蜡样芽孢杆菌、阿氏芽孢杆菌和贝莱斯芽孢杆菌(Bacillus velezensis)。

To determine the discriminatory power of rpoB,gyrA and cheA genes in Bacillus identification,comparative sequence analysis of rpoB,gyrA and cheA genes were conducted through Blast-N algorithm.Based on this,a rapid and accurate identification method for Bacillus genus was proposed.The genomic DNA of a purified strain was extracted and amplified by using specific primers which were selected by the original taxonomy stated on the packaging.Then,the final bacterial taxonomy was determined by sequence identities and phylogenetic analysis.A total of 19 Bacillus isolates were adopted in the present study to verify the feasibility of this proposed method.It was shown that sequence identities of rpoB,gyrA and cheA genes within B.licheniformis and B.subtilis group(including Bacillus subtilis subsp.subtilis,Bacillus subtilis subsp.inaquosorum,Bacillus subtilis subsp.spizizenii),and operational group B.amyloliquefaciens(including Bacillus amyloliquefaciens,Bacillus velezensis)were 84.93%-86.57%,less than 78.21%,and 78.25%-78.58%,respectively.Sequence identities of gyrA and cheA genes within B.subtilis group and operational group B.amyloliquefaciens were 81.01%-95.82%and 94.50%-95.83%,respectively.B.cereus was 94.54%identical to B.thuringiensis by gyrA gene,while B.megaterium was 94.55%identical to B.aryabhattai by cheA gene.It was summarized that B.licheniformis,B.subtilis group and operational group B.amyloliquefaciens could be distinguished by rpoB,gyrA and cheA gene.The subspecies within B.subtilis group and operational group B.amyloliquefaciens could be identified by gyrA and cheA genes.Moreover,gyrA gene was sufficient to distinguish B.cereus from B.thuringiensis,and cheA gene could differentiate B.megaterium from B.aryabhattai.By using the proposed method,19 Bacillus isolates were identified as Brevibacillus reuszeri,Bacillus mesonae,Bacillus wudalianchiensis,Bacillus massiliogorillae,Bacillus subtilis subsp.subtilis,Bacillus cereus,Bacillus aryabhattai and Bacillus velezensis.