荧光假单胞CLW17菌株对草甘膦的降解及其机制初探

doi:10.11924/j.issn.1000-6850.casb2022-1170

中国农学通报 ›› 2022, Vol. 38 ›› Issue (30): 108-117.doi: 10.11924/j.issn.1000-6850.casb2022-1170

所属专题：生物技术

荧光假单胞CLW17菌株对草甘膦的降解及其机制初探

曹永清¹^,²(), 刘艳¹^,², 张丽慧¹^,², 晋婷婷², 任嘉红²()

¹山西师范大学生命科学学院,太原 030000
²长治学院生命科学系,山西长治 046011

收稿日期:2021-12-06 修回日期:2022-03-15 出版日期:2022-10-25 发布日期:2022-10-27
通讯作者: 任嘉红
作者简介:曹永清,女,1996年出生,山西大同人,硕士研究生,研究方向：资源微生物。通信地址：046011 山西省长治市潞州区太行东街街道长治学院北校区长治学院,Tel：13994361141,E-mail： 1051946513@qq.com。
基金资助:
国家自然科学基金“AM真菌和溶磷细菌互作对南方红豆杉生长及紫杉醇合成的效应研究”(31100471);山西省“1331工程”资助项目“太行山药用植物生物肥料工程研究中心”(1331KSC)

Pseudomonas fluorescens Strain CLW17: Degradation of Glyphosate and Its Mechanism

CAO Yongqing¹^,²(), LIU Yan¹^,², ZHANG Lihui¹^,², JIN Tingting², REN Jiahong²()

¹College of Biological Sciences, Shanxi Normal University, Taiyuan 030000
²Department of Biological Sciences, Changzhi College, Changzhi, Shanxi 046011

Received:2021-12-06 Revised:2022-03-15 Online:2022-10-25 Published:2022-10-27
Contact: REN Jiahong

摘要/Abstract

摘要：

探讨根际促生长细菌荧光假单胞菌(Pseudomonas fluorescens)CLW17对草甘膦的降解作用及其分子机制。利用平板培养法和钼锑抗比色法对CLW17菌株降解草甘膦能力进行研究。采用Plackett-Burman(PB)与Central Composite Design(CCD)试验联用确定CLW17降解草甘膦的最佳条件。对降解草甘膦关键基因thiO进行生物信息学分析,并构建敲除株(CLW17ΔthiO)及回补株(ΔthiO/thiO),研究其对草甘膦降解的影响。荧光假单胞菌CLW17菌株耐受草甘膦的最大浓度为0.7%。在降解草甘膦最优条件下,野生株CLW17、敲除株CLW17ΔthiO和回补株ΔthiO/thiO对草甘膦降解率分别为72.81%、32.78%和54.33%。生物信息学分析显示thiO基因编码366个氨基酸,拥有信号肽和跨膜结构域。CLW17具有较强的降解草甘膦能力,且thiO基因与降解草甘膦能力密切相关。研究结果可为草甘膦污染的治理及土壤修复提供良好的菌种资源。

关键词: 荧光假单胞菌, 草甘膦, 生物降解, 优化, thiO基因

Abstract:

The subject is to study the degradation of glyphosate by the rhizobacteria-promoting strain Pseudomonas fluorescens CLW17 and the molecular mechanism of the strain. The glyphosate degradation ability of strain CLW17 was evaluated by plate culture method and molybdenum antimony resistance colorimetric method. An optimized condition of degrading glyphosate by strain CLW17 was achieved by employing Plackett-Burman (PB) design, and central composite design (CCD). Bioinformatics analysis was carried out for understanding the key gene thiO in glyphosate degradation. Deletion mutant strain (CLW17ΔthiO) and complement strain (ΔthiO/thiO) were constructed, and their effects on glyphosate degradation were studied. The maximum concentration of glyphosate tolerance of strain CLW17 was 0.7%. The degradation rate of CLW17, CLW17ΔthiO and ΔthiO/thiO was 72.81%, 32.78% and 54.33%, respectively, under the optimal condition for glyphosate degradation. Bioinformatics analysis revealed that thiO gene encoded 366 amino acids and included signal peptide and transmembrane domain. CLW17 was capable of degrading glyphosate effectively. Moreover, thiO gene was closely related to glyphosate degradation ability. These findings can provide valuable bacterial resources for glyphosate pollution control and soil remediation.

Key words: Pseudomonas fluorescens, glyphosate, biodegradation, optimization, thiO gene

中图分类号:

曹永清, 刘艳, 张丽慧, 晋婷婷, 任嘉红. 荧光假单胞CLW17菌株对草甘膦的降解及其机制初探[J]. 中国农学通报, 2022, 38(30): 108-117.

CAO Yongqing, LIU Yan, ZHANG Lihui, JIN Tingting, REN Jiahong. Pseudomonas fluorescens Strain CLW17: Degradation of Glyphosate and Its Mechanism[J]. Chinese Agricultural Science Bulletin, 2022, 38(30): 108-117.

图/表 13

参考文献 28

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Primers	Sequences (5’→3’)
CLW17_04770FW (Hind Ⅲ)	CCCAAGCTTCTGAGTCCAGTGGCAACATCAC
CLW17_Del04770FW	AGGAAGAAAAACTGACTAGATGGTCATGTTTGATCCAACCGT
CLW17_Del04770RV	ACGGTTGGATCAAACATGACCATCTAGTCAGTTTTTCTTCCT
CLW17_04770RV (EcoR I)	CCGGAATTCACGTGATCGAACTGCGTGTG
CLW17_04770SQFW	ACTACGCGGTGCTCGACACTAACGCTGAT
CLW17_04770SQRV	CCAACAGGAAACCGTGGTCGATGTG
CLW17_Comp04770FW -EcoR Ⅰ	CCGGAATTCACGGTTGGATCAAACATG
CLW17_Comp04770RV -Spe I	CGGACTAGTCTAGATCCGTCCGGCCGGTG
pBAD-Forward	ATGCCATAGCATTTTTATCC
T7	TAATACGACTCACTATAGGG

Primers	Sequences (5’→3’)
CLW17_04770FW (Hind Ⅲ)	CCCAAGCTTCTGAGTCCAGTGGCAACATCAC
CLW17_Del04770FW	AGGAAGAAAAACTGACTAGATGGTCATGTTTGATCCAACCGT
CLW17_Del04770RV	ACGGTTGGATCAAACATGACCATCTAGTCAGTTTTTCTTCCT
CLW17_04770RV (EcoR I)	CCGGAATTCACGTGATCGAACTGCGTGTG
CLW17_04770SQFW	ACTACGCGGTGCTCGACACTAACGCTGAT
CLW17_04770SQRV	CCAACAGGAAACCGTGGTCGATGTG
CLW17_Comp04770FW -EcoR Ⅰ	CCGGAATTCACGGTTGGATCAAACATG
CLW17_Comp04770RV -Spe I	CGGACTAGTCTAGATCCGTCCGGCCGGTG
pBAD-Forward	ATGCCATAGCATTTTTATCC
T7	TAATACGACTCACTATAGGG

因素	回归系数	效果	平方和	贡献值
intercept	34.04
X₁	9.25	18.49	1025.78	23.09
X₂	-2.29	-4.58	62.92	1.42
X₃	6.19	12.38	459.52	10.34
X₄	0.74	1.48	6.54	0.15
X₅	-11.01	-22.01	1453.54	32.72
X₆	-8.69	-17.38	906.4	20.40
X₇	2.32	4.65	64.75	1.46
X₈	4.37	8.74	229.32	5.16
X₉	-1.28	-2.57	19.76	0.44
X₁₀	2.87	5.75	99.15	2.23
X₁₁	3.10	6.19	115.05	2.59

因素	回归系数	效果	平方和	贡献值
intercept	34.04
X₁	9.25	18.49	1025.78	23.09
X₂	-2.29	-4.58	62.92	1.42
X₃	6.19	12.38	459.52	10.34
X₄	0.74	1.48	6.54	0.15
X₅	-11.01	-22.01	1453.54	32.72
X₆	-8.69	-17.38	906.4	20.40
X₇	2.32	4.65	64.75	1.46
X₈	4.37	8.74	229.32	5.16
X₉	-1.28	-2.57	19.76	0.44
X₁₀	2.87	5.75	99.15	2.23
X₁₁	3.10	6.19	115.05	2.59

实验组数	编码/预测值			实际值	预测值
实验组数	X₁/℃	X₅/(g/L)	X₆/(g/L)	实际值	预测值
1	-1/26	-1/8	-1/0.3	61.2962	59.81
2	1/30	-1/8	-1/0.3	64.2350	64.62
3	-1/26	1/12	-1/0.3	64.0738	64.43
4	1/30	1/12	-1/0.3	67.1785	65.49
5	-1/26	-1/8	1/0.7	67.0071	67.93
6	1/30	-1/8	1/0.7	66.0919	64.97
7	-1/26	1/12	1/0.7	68.8709	67.73
8	1/30	1/12	1/0.7	60.3094	61.03
9	-2/24.64	0/10	0/0.5	66.3589	66.80
10	2/31.36	0/10	0/0.5	64.5662	65.21
11	0/28	-2/6.64	0/0.5	63.2265	63.63
12	0/28	2/13.36	0/0.5	63.5127	64.18
13	0/28	0/10	-2/0.16	62.4065	63.49
14	0/28	0/10	2/0.84	66.5652	66.56
15	0/28	0/10	0/0.5	73.4263	72.78
16	0/28	0/10	0/0.5	74.3185	72.78
17	0/28	0/10	0/0.5	70.0747	72.78
18	0/28	0/10	0/0.5	73.5353	72.78
19	0/28	0/10	0/0.5	72.5685	72.78
20	0/28	0/10	0/0.5	72.9370	72.78

荧光假单胞CLW17菌株对草甘膦的降解及其机制初探

Pseudomonas fluorescens Strain CLW17: Degradation of Glyphosate and Its Mechanism

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实验组	X₁	X₂	X₃	X₄	X₅	X₆	X₇	X₈	X₉	X₁₀	X₁₁	降解率/%
1	1	1	-1	1	1	1	-1	-1	-1	1	-1	10.2175
2	-1	1	1	-1	1	1	1	-1	-1	-1	1	7.7161
3	1	-1	1	1	-1	1	1	1	-1	-1	-1	56.8252
4	-1	1	-1	1	1	-1	1	1	1	-1	-1	14.1815
5	-1	-1	1	-1	1	1	-1	1	1	1	-1	13.3826
6	-1	-1	-1	1	-1	1	1	-1	1	1	1	26.5896
7	1	-1	-1	-1	1	-1	1	1	-1	1	1	50.2848
8	1	1	-1	-1	-1	1	-1	1	1	-1	1	37.3735
9	1	1	1	-1	-1	-1	1	-1	1	1	-1	62.5904
10	-1	1	1	1	-1	-1	-1	1	-1	1	1	58.4321
11	1	-1	1	1	1	-1	-1	-1	1	-1	1	42.4330
12	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	-1	28.4748

差异来源	平方和	df	自由度	F值	P值
Model	342.46	9	38.05	17.01	<0.0001
X₁	3.04	1	3.04	1.36	0.2705
X₅	0.39	1	0.39	0.18	0.6842
X₆	11.42	1	11.42	5.11	0.0474
X₁X₅	6.99	1	6.99	3.13	0.1075
X₁X₆	30.11	1	30.11	13.46	0.0043
X₅X₆	11.62	1	11.62	5.19	0.0459
X₁²	82.75	1	82.75	36.99	0.0001
X₅²	141.80	1	141.80	63.38	<0.0001
X₆²	108.32	1	108.32	48.42	<0.0001
Residual	22.37	10	2.24
Lack of fit	11.64	5	2.33	1.08	0.4660
Pure error	10.74	5	2.15
Cor total	364.84	19

基因编号	蛋白	KO号	功能
GE04770	甘氨酸氧化酶thiO	K03153	甘氨酸氧化酶活性
GE04748	phnA	K06193	磷酸酯水解酶活性
GE01859	phnB	K04750	乙醛酶/博莱霉素抗性蛋白/双加氧酶
GE01316	phnC	K02041	ABC转运系统,核苷酸结合蛋白
GE01318	phnE	K02042	ABC转运系统跨膜蛋白
GE05347	phnN	K05774	核糖1,5-二磷酸磷酸激酶
GE02201	phnR	–	参与2-氨基乙基膦酸酯降解操纵子的转录调控
GE00269	phnX	K05306	磷酸酯水解酶活性
GE01317	htxC	K02042	磷酸酯ABC转运蛋白渗透酶
GE04402	肌氨酸氧化酶SoxD	K00304	肌氨酸氧化酶活性
GE03301	草甘膦乙酰转移酶gat	K03823	N-乙酰基转移酶活性

菌株	降解率/%
CLW17	72.81±0.88
CLW17ΔthiO	32.78±1.87
ΔthiO/thiO	54.33±3.14

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