rpoB、gyrA、cheA基因在芽孢杆菌鉴定上的应用

doi:10.3969/j.issn.1004-1524.2022.01.16

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (1): 128-140.DOI: 10.3969/j.issn.1004-1524.2022.01.16

rpoB、gyrA、cheA基因在芽孢杆菌鉴定上的应用

颜静婷(), 乔凯, 蔡燕飞^*()

华南农业大学资源环境学院,广东广州 510642

收稿日期:2021-01-10 出版日期:2022-01-25 发布日期:2022-02-05
作者简介:* 蔡燕飞,E-mail: yanfeicai@scau.edu.cn
颜静婷(1996—),女,江西萍乡人,硕士研究生,主要从事芽孢杆菌微生物肥研究。E-mail: 939015374@qq.com
通讯作者: 蔡燕飞
基金资助:
国家自然科学基金(41977035);广东省重点领域研发计划(2019B020218009)

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

YAN Jingting(), QIAO Kai, CAI Yanfei^*()

College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, China

Received:2021-01-10 Online:2022-01-25 Published:2022-02-05
Contact: CAI Yanfei

摘要/Abstract

摘要：

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)。

关键词: 芽孢杆菌, 鉴定, rpoB基因, gyrA基因, cheA基因

Abstract:

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.

Key words: Bacillus, identification, rpoB, gyrA, cheA

中图分类号:

S182

颜静婷, 乔凯, 蔡燕飞. rpoB、gyrA、cheA基因在芽孢杆菌鉴定上的应用[J]. 浙江农业学报, 2022, 34(1): 128-140.

YAN Jingting, QIAO Kai, CAI Yanfei. Application of rpoB, gyrA and cheA genes in identifying Bacillus genus[J]. Acta Agriculturae Zhejiangensis, 2022, 34(1): 128-140.

图/表 14

参考文献 46

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菌种 Species	模式菌 Reference strain	GenBank序列号 GenBank accession No.	序列长度 Sequence length/bp				菌株来源 Strain source
菌种 Species	模式菌 Reference strain	GenBank序列号 GenBank accession No.	16S rRNA	rpoB	gyrA	cheA	菌株来源 Strain source
巨大芽孢杆菌Bacillus megaterium	ATCC 14581^T	CP035094.1	1 552	3 564	2 511	1 980	未知Unknown
阿氏芽孢杆菌Bacillus aryabhattai	B8W22^T	FMZY01000001.1	1 533	—	—	1 980	未知Unknown
地衣芽孢杆菌Bacillus licheniformis	ATCC 14580^T	NC_006322.1	1 549	3 582	2 469	2 022	未知Unknown
胶质芽孢杆菌Paenibacillus mucilaginosus	3016^T	NC_016935.1	1 545	3 555	2 508	—	根际土Rhizosphere soil
多黏类芽孢杆菌Paenibacillus polymyxa	ATCC 842^T	NZ_GL905390.1	1 564	3 546	2 463	2 097	未知Unknown
解淀粉芽孢杆菌Bacillus amyloliquefaciens	DSM7^T	FN597644.1	1538	3 582	2 460	2 013	发酵厂Fermentation plant
贝莱斯芽孢杆菌Bacillus velezensis	FZB42^T	CP000560.2	1 553	3 582	2 460	2 019	发病甜菜根际土
							Rhizosphere soil of diseased beet
枯草芽孢杆菌枯草亚种	168^T	NC_000964.3	1 554	3 582	2 466	2 019	诱变土壤Mutagenic soil
Bacillus subtilis subsp. subtilis
Bacillus subtilis subsp. inaquosorum	KCTC 13429^T	NZ_CP029465.1	1 554	3 582	2 466	2 010	未知Unknown
枯草芽孢杆菌斯氏亚种	TU-B-10^T	CP002905.1	1 538	3 582	2 466	2 016	未知Unknown
Bacillus subtilis subsp. spizizenii
苏云金芽孢杆菌Bacillus thuringiensis	ATCC 10792^T	CP020754.1	1 556	3 612	2 472	2 019	动物组织Animal tissue
蜡样芽孢杆菌Bacillus cereus	ATCC 14579^T	CP034551.1	1 555	3 534	2 472	2 019	未知Unknown

菌种 Species	模式菌 Reference strain	GenBank序列号 GenBank accession No.	序列长度 Sequence length/bp				菌株来源 Strain source
菌种 Species	模式菌 Reference strain	GenBank序列号 GenBank accession No.	16S rRNA	rpoB	gyrA	cheA	菌株来源 Strain source
巨大芽孢杆菌Bacillus megaterium	ATCC 14581^T	CP035094.1	1 552	3 564	2 511	1 980	未知Unknown
阿氏芽孢杆菌Bacillus aryabhattai	B8W22^T	FMZY01000001.1	1 533	—	—	1 980	未知Unknown
地衣芽孢杆菌Bacillus licheniformis	ATCC 14580^T	NC_006322.1	1 549	3 582	2 469	2 022	未知Unknown
胶质芽孢杆菌Paenibacillus mucilaginosus	3016^T	NC_016935.1	1 545	3 555	2 508	—	根际土Rhizosphere soil
多黏类芽孢杆菌Paenibacillus polymyxa	ATCC 842^T	NZ_GL905390.1	1 564	3 546	2 463	2 097	未知Unknown
解淀粉芽孢杆菌Bacillus amyloliquefaciens	DSM7^T	FN597644.1	1538	3 582	2 460	2 013	发酵厂Fermentation plant
贝莱斯芽孢杆菌Bacillus velezensis	FZB42^T	CP000560.2	1 553	3 582	2 460	2 019	发病甜菜根际土
							Rhizosphere soil of diseased beet
枯草芽孢杆菌枯草亚种	168^T	NC_000964.3	1 554	3 582	2 466	2 019	诱变土壤Mutagenic soil
Bacillus subtilis subsp. subtilis
Bacillus subtilis subsp. inaquosorum	KCTC 13429^T	NZ_CP029465.1	1 554	3 582	2 466	2 010	未知Unknown
枯草芽孢杆菌斯氏亚种	TU-B-10^T	CP002905.1	1 538	3 582	2 466	2 016	未知Unknown
Bacillus subtilis subsp. spizizenii
苏云金芽孢杆菌Bacillus thuringiensis	ATCC 10792^T	CP020754.1	1 556	3 612	2 472	2 019	动物组织Animal tissue
蜡样芽孢杆菌Bacillus cereus	ATCC 14579^T	CP034551.1	1 555	3 534	2 472	2 019	未知Unknown

菌株 Strain	菌种 Species	分离年份与地点 Identification year and source	促生特性 Growth-promoting properties
YC001	枯草芽孢杆菌枯草亚种 Bacillus subtilis subsp. subtilis	2017,根际土,华南地区 2017,rhizosphere soil,south China	溶磷 Phosphorus-dissolving
YC002	枯草芽孢杆菌枯草亚种 Bacillus subtilis subsp. subtilis	2017,根际土,华南地区 2017,rhizosphere soil,south China	溶磷 Phosphorus-dissolving
YC003	甲基营养型芽孢杆菌 Bacillus methylotrophicus	2016,根际土,华南地区 2016,rhizosphere soil,south China	生物防治土传病害 Biological control of soil-borne diseases
YC004	甲基营养型芽孢杆菌 Bacillus methylotrophicus	2018,蔬菜土,华南地区 2018,vegetable soil,south China	生物防治土传病害 Biological control of soil-borne diseases
YC005	解淀粉芽孢杆菌 Bacillus amyloliquefaciens	2018,蔬菜土,华南地区 2018,vegetable soil,south China	生物防治土传病害 Biological control of soil-borne diseases
YC006	苏云金芽孢杆菌 Bacillus thuringiensis	2015,蔬菜土,华南地区 2015,vegetable soil,south China	溶磷 Phosphorus-dissolving
YC007	壁芽孢杆菌 Bacillus muralis	2015,火炉山,华南地区 2015,Huolu Mountain,south China	溶磷 Phosphorus-dissolving
YC008	德林芽孢杆菌 Bacillus drentensis	2015,火炉山,华南地区 2015,Huolu Mountain,south China	溶磷 Phosphorus-dissolving
YC009	巨大芽孢杆菌 Bacillus megaterium	2016,农场土,华南地区 2016, farmland soil,south China	溶磷 Phosphorus-dissolving
YC010	阿氏芽孢杆菌 Bacillus aryabhattai	2016,农场土,华南地区 2016, farmland soil,south China	溶磷 Phosphorus-dissolving
YC011	阿氏芽孢杆菌 Bacillus aryabhattai	2019,根际土,华南地区 2019,rhizosphere soil,south China	溶磷 Phosphorus-dissolving
YC012	阿氏芽孢杆菌 Bacillus aryabhattai	2015,根际土,华南地区 2015,rhizosphere soil,south China	产生长素 Auxin-producing
YC013	阿氏芽孢杆菌 Bacillus aryabhattai	2019,甘蔗土,华南地区 2019,sugarcane soil,south China	产生长素 Auxin-producing
YC014	巨大芽孢杆菌 Bacillus megaterium	2019,根际土,华南地区 2019,rhizosphere soil,south China	产生长素 Auxin-producing
YC015	巨大芽孢杆菌 Bacillus megaterium	2015,根际土,华南地区 2015,rhizosphere soil,south China	产生长素 Auxin-producing
YC016	巨大芽孢杆菌 Bacillus megaterium	2015,根际土,华南地区 2015,rhizosphere soil,south China	产生长素 Auxin-producing
YC017	巨大芽孢杆菌 Bacillus megaterium	2017,根际土,华南地区 2017,rhizosphere soil,south China	产生长素 Auxin-producing
YC018	五大连池芽孢杆菌 Bacillus wudalianchiensis	2019,生菜土,华南地区 2019,lettuce soil,south China	产生长素 Auxin-producing
YC019	根内芽孢杆菌 Bacillus endoradicis	2019,农场土,华南地区 2019,farmland soil,south China	产生长素 Auxin-producing

菌株 Strain	菌种 Species	分离年份与地点 Identification year and source	促生特性 Growth-promoting properties
YC001	枯草芽孢杆菌枯草亚种 Bacillus subtilis subsp. subtilis	2017,根际土,华南地区 2017,rhizosphere soil,south China	溶磷 Phosphorus-dissolving
YC002	枯草芽孢杆菌枯草亚种 Bacillus subtilis subsp. subtilis	2017,根际土,华南地区 2017,rhizosphere soil,south China	溶磷 Phosphorus-dissolving
YC003	甲基营养型芽孢杆菌 Bacillus methylotrophicus	2016,根际土,华南地区 2016,rhizosphere soil,south China	生物防治土传病害 Biological control of soil-borne diseases
YC004	甲基营养型芽孢杆菌 Bacillus methylotrophicus	2018,蔬菜土,华南地区 2018,vegetable soil,south China	生物防治土传病害 Biological control of soil-borne diseases
YC005	解淀粉芽孢杆菌 Bacillus amyloliquefaciens	2018,蔬菜土,华南地区 2018,vegetable soil,south China	生物防治土传病害 Biological control of soil-borne diseases
YC006	苏云金芽孢杆菌 Bacillus thuringiensis	2015,蔬菜土,华南地区 2015,vegetable soil,south China	溶磷 Phosphorus-dissolving
YC007	壁芽孢杆菌 Bacillus muralis	2015,火炉山,华南地区 2015,Huolu Mountain,south China	溶磷 Phosphorus-dissolving
YC008	德林芽孢杆菌 Bacillus drentensis	2015,火炉山,华南地区 2015,Huolu Mountain,south China	溶磷 Phosphorus-dissolving
YC009	巨大芽孢杆菌 Bacillus megaterium	2016,农场土,华南地区 2016, farmland soil,south China	溶磷 Phosphorus-dissolving
YC010	阿氏芽孢杆菌 Bacillus aryabhattai	2016,农场土,华南地区 2016, farmland soil,south China	溶磷 Phosphorus-dissolving
YC011	阿氏芽孢杆菌 Bacillus aryabhattai	2019,根际土,华南地区 2019,rhizosphere soil,south China	溶磷 Phosphorus-dissolving
YC012	阿氏芽孢杆菌 Bacillus aryabhattai	2015,根际土,华南地区 2015,rhizosphere soil,south China	产生长素 Auxin-producing
YC013	阿氏芽孢杆菌 Bacillus aryabhattai	2019,甘蔗土,华南地区 2019,sugarcane soil,south China	产生长素 Auxin-producing
YC014	巨大芽孢杆菌 Bacillus megaterium	2019,根际土,华南地区 2019,rhizosphere soil,south China	产生长素 Auxin-producing
YC015	巨大芽孢杆菌 Bacillus megaterium	2015,根际土,华南地区 2015,rhizosphere soil,south China	产生长素 Auxin-producing
YC016	巨大芽孢杆菌 Bacillus megaterium	2015,根际土,华南地区 2015,rhizosphere soil,south China	产生长素 Auxin-producing
YC017	巨大芽孢杆菌 Bacillus megaterium	2017,根际土,华南地区 2017,rhizosphere soil,south China	产生长素 Auxin-producing
YC018	五大连池芽孢杆菌 Bacillus wudalianchiensis	2019,生菜土,华南地区 2019,lettuce soil,south China	产生长素 Auxin-producing
YC019	根内芽孢杆菌 Bacillus endoradicis	2019,农场土,华南地区 2019,farmland soil,south China	产生长素 Auxin-producing

引物名称 Primers name	上游引物序列 Forward primer sequence (5'→3')	下游引物序列 Reverse primer sequence (5'→3')	PCR产物长度 PCR product length/bp
16S rRNA	AGAGTTTGATCMTGGCTCAG	GGTTACCTTGTTACGACTT	1 465
rpoB-bs	GGAAACCGCCGTTTACGTTC	CCATGAGGCACACGAAGAGA	1 433
gyrA-bs	GCGATCCTTGACATGAGGCT	AGACGCACACCTTGAGTGAC	1 106
gyrA-ba	TTGCCAGAACGGGTTTAATCG	CTTCGGTTTCTTCCGGCTCT	1 239
gyrA-bc	GCGTCTGCAACGTTTAACTGG	TGTCGCTACCTCTTGCTCATC	1 084
cheA-bm	ACACCCGGCAGATAATGACC	CGTTAATGACCAGCTAATGCGT	1 842

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

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菌株Strain	B. am	B. v	B. sub	B. ina	B. spi	B. lich	B. t	B. c	B. m	B. ar
B. v	99.61
B. sub	99.48	99.68
B. ina	99.42	99.49	99.74
B. spi	99.48	99.54	99.87	99.87
B. lich	98.18	98.06	98.26	98.14	98.38
B. t	94.30	94.16	94.22	94.29	94.29	94.08
B. c	94.01	94.09	94.16	94.29	94.29	94.02	99.74
B. m	93.47	93.77	93.84	93.78	93.73	93.71	94.62	94.55
B. ar	93.36	93.55	93.62	93.62	93.55	93.49	94.52	94.48	99.80
P. muci	88.03	88.04	81.12	87.95	88.00	88.15	88.13	88.00	88.57	88.15
P. poly	88.02	87.99	88.11	88.07	88.03	88.08	88.87	88.79	89.17	88.92

菌株Strain	B. am	B. v	B. sub	B. ina	B. spi	B. lich	B. t	B. c
B. v	98.44
B. sub	90.23	90.31
B. ina	90.29	90.26	97.88
B. spi	90.09	90.17	97.52	97.88
B. lich	86.34	86.57	84.93	85.19	85.38
B. t	78.48	78.63	78.92	79.23	78.94	76.91
B. c	78.28	78.69	78.97	79.31	78.96	76.97	97.37
B. m	79.47	79.63	80.49	80.41	80.56	78.18	81.68	80.70

菌株Strain	B. am	B. v	B. sub	B. ina	B. spi	B. lich	B. t	B. c	B. m
B. v	94.65
B. sub	NF	NF
B. ina	NF	NF	94.50
B. spi	NF	NF	95.00	95.83
B. lich	78.21	NF	NF	NF	NF
B. t	NF	NF	NF	NF	NF	NF
B. c	NF	NF	NF	NF	NF	NF	99.70
B. m	NF	NF	NF	NF	NF	NF	NF	NF
B. ar	NF	NF	NF	NF	NF	NF	NF	NF	94.55

菌株Strain	B. am	B. v	B. sub	B. ina	B. spi	B. lich	B. t	B. c
B. v	95.08
B. sub	81.16	NF
B. ina	81.16	81.01	94.00
B. spi	81.58	81.26	94.12	95.82
B. lich	78.27	78.39	NF	78.58	78.25
B. t	NF	NF	NF	NF	NF	NF
B. c	72.17	NF	NF	NF	NF	NF	94.54
B. m	NF	NF	NF	NF	NF	NF	NF	75.57

分离株 Bacillus isolates	枯草芽孢杆菌枯草亚种 Bacillus subtilis subsp. subtilis	Bacillus subtilis subsp.inaquosorum	枯草芽孢杆菌斯氏亚种 Bacillus subtilis subsp. spizizenii	解淀粉芽孢杆菌 Bacillus amyloliquefaciens	贝莱斯芽孢杆菌 Bacillus velezensis	地衣芽孢杆菌 Bacillus licheniformis
YC001	99.56	97.27	97.19	87.89	88.40	83.80
YC002	88.44	88.31	88.44	97.68	98.58	84.94
YC003	88.86	88.88	89.01	97.97	98.95	85.36
YC004	88.77	88.71	89.00	97.64	98.52	85.32
YC005	88.82	88.84	89.97	98.22	99.41	85.77

分离株 Bacillus isolates	巨大芽孢杆菌 Bacillus megaterium	阿氏芽孢杆菌 Bacillus aryabhattai
YC009	94.06	98.42
YC010	94.75	95.77
YC011	96.90	94.64
YC012	94.99	96.11
YC013	94.86	95.94
YC014	94.31	97.97
YC015	96.42	94.61
YC016	94.29	98.76
YC017	94.92	95.99

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