海洋科学  2020, Vol. 44 Issue (4): 90-95   PDF    
http://dx.doi.org/10.11759/hykx20191018002

文章信息

傅奇, 林俊杰, 甘美裕, 卢源钦, 肖玉娟. 2020.
FU Qi, LIN Jun-jie, GAN Mei-yu, LU Yuan-qin, XIAO Yu-juan. 2020.
16S与gyrB基因联合建树快速鉴定海水中的一株地衣芽胞杆菌
Identification of a Bacillus licheniformis strain from seawater using a phylogenetic tree based on 16S and gyrB gene sequences
海洋科学, 44(4): 90-95
Marine Sciences, 44(4): 90-95.
http://dx.doi.org/10.11759/hykx20191018002

文章历史

收稿日期:2019-10-18
修回日期:2019-11-08
16S与gyrB基因联合建树快速鉴定海水中的一株地衣芽胞杆菌
傅奇, 林俊杰, 甘美裕, 卢源钦, 肖玉娟     
厦门华厦学院环境与公共健康学院, 福建 厦门361024
摘要:在保守序列高度相似的细菌鉴定中,单独使用16S rDNA/RNA序列进行比对和构建进化树通常无法准确鉴定到种,需要增加测序基因数并对多基因进行分析。为实现快速鉴定,课题组对16S与gyrB基因联合建树的方法进行了研究,将海洋来源的一株杆菌,分别用通用引物扩增16S和gyrB基因并测序,在GeneBank进行序列比对后,选择各菌种保藏中心16S和gyrB基因均相似的菌株,取16S和gyrB基因序列,采用Paup*4.0构建进化树。使用16S与gyrB拼接序列构建的进化树中属于同一种的菌株均很好的聚合在一枝,种间分枝自展值均高于98,分类结构准确,筛选得到的杆菌与地衣芽胞杆菌(Bacillus licheniformis)聚合在一枝,自展值为100,鉴定为地衣芽胞杆菌。经生理生化试验验证,该菌株与地衣芽胞杆菌特征完全一致,使用16S和gyrB基因联合建树得到的鉴定结果准确且快速简便。
关键词16S    gyrB    进化树    地衣芽胞杆菌(Bacillus licheniformis)    
Identification of a Bacillus licheniformis strain from seawater using a phylogenetic tree based on 16S and gyrB gene sequences
FU Qi, LIN Jun-jie, GAN Mei-yu, LU Yuan-qin, XIAO Yu-juan     
College of Environment and Public Health, Xiamen Huaxia University, Xiamen 361024, China
Abstract: For identifying bacteria with highly similar conserved sequences, 16S rDNA does not provide adequate resolution for comparative sequence analysis; therefore, more DNA sequences and analysis models are required for classification. To develop a rapid identification method, phylogenetic trees based on 16S and gyrB gene sequences were investigated. A strain of bacillus was isolated from seawater, and the 16S and gyrB genes of the strain were amplified with universal primers and sequenced. Using BLAST in GenBank, other strains with similar 16S and gyrB genes were selected. Phylogenetic trees based on 16S and gyrB genes were constructed using PAUP* 4.0. In the tree constructed using sequences of both 16S and gyrB genes, strains belonging to the same species were clustered together, the self-expanding values of the branches were higher than 98, and the classification structure was accurate. The isolated strain polymerized with Bacillus licheniformis in the phylogenetic tree, and the self-expanding value was 100. Physiological and biochemical tests verified that the strain belongs to the species B. licheniformis. Thus, the identification of this strain of B. licheniformis obtained by construction of a phylogenetic tree based on the combination of 16S and gyrB genes was accurate, rapid, and simple.
Key words: 16S    gyrB    phylogenetic tree    Bacillus licheniformis    

芽胞杆菌(Bacillus)是一个重要的微生物类群, 包括枯草芽胞杆菌(Bacillus subtilis)、地衣芽胞杆菌(Bacillus licheniformis)、凝结芽胞杆菌(Bacillus coagulans)、炭疽芽胞杆菌(Bacillus anthracis)、蜡样芽胞杆菌(Bacillus cereus)等与发酵工业和生活密切相关的多个菌种[1]。其中地衣芽胞杆菌由于生物安全性好、产生多种功能性代谢产物, 在药品[2]、功能食品[3]、饲料[4]和发酵工业[5-7]得到广泛应用, 其中海洋来源的地衣芽胞杆菌在海水污染处理、新型生物活性物质开发方面具有重要潜力[8, 9]。但在地衣芽胞杆菌的筛选过程中, 由于芽胞杆菌类群保守序列的高度相似性, 无法单独依靠常用的16S rDNA/RNA测序来准确定位到种, 通常需要结合生理生化鉴定[10, 11]或者其他序列, 如5'端16S-23S ITS核苷酸序列[12]gyrB基因[13]gyrA[14]甚至全基因序列[15, 16]进行分析。gyrB基因编码DNA促旋酶的B亚单位, 在细菌中广泛存在, 序列相对保守, 但进化速度快于16S, 目前在细菌鉴定中的使用逐渐增加[17], 积累了较为丰富的比对数据库, 但单独使用时部分芽胞杆菌也难以准确定位, 而16S与gyrB序列分别比对建树的方法容易出现进化树结构冲突, 干扰定位的情况。利用16S与gyrB基因联合建树, 以较少的序列数和进化树数实现对筛选菌株快速鉴定的方法, 可提高数据的利用率, 减少数据冲突, 为芽胞杆菌种群的分子生物学鉴定方法开发提供参考。

1 材料与方法 1.1 样本

分离菌株样本为海泥, 采集于厦门市集美区红树林区。

1.2 材料与试剂

2216E(液体)、2216E琼脂、营养琼脂、Ⅴ-P试剂盒、卵黄琼脂培养基、色氨酸肉汤、克氏柠檬酸盐培养基、苯丙氨酸脱羧酶培养基、明胶生化管、硝酸盐还原试剂盒、Kovacs氏靛基质试剂盒等培养基与鉴定试剂均由青岛海博生物科技有限公司生产; 细菌基因组DNA提取试剂盒、胶回收试剂盒、PCR试剂由生工生物工程(上海)股份有限公司生产; 16S通用引物27F/1492R、gyrB扩增引物UP-1/UP-2r、gyrB测序引物UP-1S/UP-2Sr[18]由生工生物工程(上海)股份有限公司合成。

1.3 仪器与设备

PCR仪:新加坡ESCO公司; 核酸电泳仪:美国GE公司; 摇床:上海苏坤公司; 生化培养箱:上海一恒公司。

1.4 方法 1.4.1 样品处理方法

取海泥25 g加入225 mL 2216E液体培养基均质后稀释涂布2216E琼脂平板, 30℃培养48 h, 取单菌落进行纯化。

1.4.2 16S与gyrB序列提取

用细菌基因组DNA抽提试剂盒, 提取菌株的总DNA; 分别用16S与gyrB扩增引物扩增, 所得PCR产物电泳纯化后, 送由生工生物工程(上海)股份有限公司测序。

16S扩增条件: 94℃预变性4 min; 94℃变性30 s, 65℃退火40 s, 72℃延伸90 s, 共35个循环; 72℃延伸10 min。

gyrB扩增条件[18]: 94℃预变性4 min; 94℃变性1 min, 60℃退火1 min, 72℃延伸2 min, 共35个循环; 72℃延伸10 min。

1.4.3 16S与gyrB序列比对与进化分析

将测序结果在Genbank进行比对。为保障数据的准确性, 取gyrB相似度较高的1 000个序列中的各菌种保藏中心菌株进行比对, 包括ATCC、NCTC、BCRC、DSM 4个菌种保藏中心菌株。下载相似菌株的16S与gyrB序列, 分别用BioEdit[19]和Clustal X[20]进行比对和编辑, 取有效区间, 分别使用16S、gyrB和16S与gyrB线形拼接序列建树[21], 采用最大简约算法, 用Paup* 4.0构建进化树[22, 23], 选择大肠杆菌ATCC 25922[24]作为外群。

1.4.4 生理生化验证

参考《伯杰细菌鉴定手册》[1]和行业标准《饲料微生物添加剂地衣芽孢杆菌》[25]进行验证。

2 结果与分析 2.1 菌株分离纯化

从海泥中筛选得到一株革兰氏阳性杆菌, 产芽胞, 菌落湿润, 呈白色, 编号为HX2019004。

2.2 16S与gyrB基因测序

测序所得16S与gyrB序列拼接后有效区域分别为1 430 bp和1 115 bp。

2.3 序列比对与进化树构建

16S序列比对结果中, 高相似度菌株过多, 且多数菌株无法获得更多的遗传标记序列; 而gyrB序列比对结果中, 多数菌株同时可获取16S序列, 因此, 以gyrB相似菌株作为建树群, 下载16S与gyrB基因序列(表 1), 比较分别使用16S、gyrB和16S与gyrB线形拼接序列建树结果。

表 1 GenBank中拥有相似16S与gyrB序列的菌株 Tab. 1 Bacterial strains in GenBank with similar 16S and gyrB sequences
序号 菌种 菌株编号 序列号
16S gyrB
1 Bacillus licheniformis ATCC 14580 CP034569.1 CP034569.1
2 NCTC 10341 LR134392.1 LR134392.1
3 ATCC 9789 CP023729.1 CP023729.1
4 BCRC 14353[13] AY167869.1 DQ309324.1
5 BCRC 11702[13] NR116023.1 DQ309295.1
6 BCRC 15413[13] DQ993676.1 DQ309325.1
7 NCTC 8721 LR134165.1 LR134165.1
8 BCRC 12826[13] EF423608.1 DQ309323.1
9 Bacillus pumilus ATCC 27142[26] AY876287.1 AY167870.1
10 NCTC 10337 LT906438.1 LT906438.1
11 ATCC 7061[27] AY876289.1 KF194263.1
12 Bacillus sonorensis BCRC 17532[13] DQ993679.1 DQ309304.1
13 BCRC 17416[13] EF433411.1 DQ309300.1
14 Bacillus paralicheniformis ATCC 9945a[28] CP005965.1 CP005965.1
15 Bacillus jeotgali DSM 18226 CP025025.1 CP025025.1

16S比对后去除头尾不整齐片段, 保留中心区域1 440 bp(含gap); gyrB比对后去除头尾不整齐片段, 保留中心区域1 056 bp(含gap)。构建进化树如图 1所示, A树基于16S序列构建, B树基于gyrB序列构建, C树基于16S与gyrB拼接序列构建, 计算次数为1 000, 参数如表 2所示。

图 1 基于16S与gyrB序列的进化树(A基于16S序列, B基于gyrB序列, C基于16S与gyrB拼接序列) Fig. 1 Phylogenetic trees based on sequences of 16S and gyrB (A for 16S, B for gyrB, C for merged sequences of 16S and gyrB)

表 2 基于16S与gyrB序列的进化树参数表 Tab. 2 Parameters of phylogenetic trees based on the combined sequences of 16S and gyrB
进化树 树长 一致性指数 保留指数 同型指数
A 755 0.964 0.863 0.036
B 981 0.843 0.855 0.157
C 1741 0.893 0.852 0.107

图 1中可以看出, 仅使用16S序列构建的进化树A分枝自展值低, 无法准确定位HX2019004菌株与Bacillus licheniformisBacillus sonorensisBacillus paralicheniformis的进化关系; 仅使用gyrB序列构建的进化树B整体分枝自展值较高, 但一致性指数(CI)较低, 且HX2019004菌株与Bacillus paralicheniformis的分类关系存在疑义, 与A树存在较大的结构冲突; 使用16S与gyrB拼接序列构建的进化树C中, 同一类菌种均很好的聚合在一枝, 种间分枝自展值均高于98, 表明分类结构准确, HX2019004菌株与地衣芽胞杆菌聚合在一枝, 自展值为100, 表明该菌株为地衣芽胞杆菌可能性高。

2.4 生理生化验证

菌株HX2019004可液化明胶、水解淀粉、还原硝酸盐, 可利用柠檬酸盐做为碳源, 可利用葡萄糖、阿拉伯糖、木糖、甘露醇产酸, 可分解酪素, 可使牛奶胨化, 可在pH 5.5~8.7范围内、0.001%溶菌酶、7%NaCl、厌氧琼脂中生长, Ⅴ-P反应、接触酶反应阳性; 不能使苯丙氨酸脱氨, 不能使牛奶凝固, 不能分解酪氨酸, 卵黄反应和吲哚反应呈阴性。试验结果与地衣芽胞杆菌生理生化特征吻合[25], 与进化分析结果一致, 命名为Bacillus licheniformis HX2019004。

3 结论

利用16S与gyrB基因联合建树的方法, 课题组快速将一株从海水中筛选得到的芽胞杆菌HX2019004准确鉴定为地衣芽胞杆菌, 表明该方法在保守序列高度相似菌种间的分类鉴定中具有深入研究的价值。与常用的增加测序基因数的方法相比, 该方法测序量小, 时间短, 数据利用率高; 与多基因分别建树的方法相比, 可避免进化树结构之间的冲突和数据紊乱, 提高了进化树准确性。课题组下一步将继续研究该方法在其他菌群鉴别中的有效性。

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