沙福芽孢杆菌多铜氧化酶CotA蛋白锰氧化活性研究

doi:10.7668/hbnxb.20195936

摘要/Abstract

摘要：

沙福芽孢杆菌ST7菌株具有较强的锰氧化活性,其中多铜氧化酶(MCO)蛋白家族基因BSL056-RS03010 (cotA)在锰胁迫下的表达量显著上调,可能参与Mn(Ⅱ)氧化,使菌株具有较强的锰耐受能力。为了验证沙福芽孢杆菌多铜氧化酶cotA基因的锰氧化能力,克隆cotA基因并构建表达载体,通过转化大肠杆菌进行异源表达,研究cotA 基因过表达对大肠杆菌锰氧化活性及锰去除率的影响,从而验证cotA基因功能,为构建高效锰氧化菌株治理锰污染提供技术手段。利用PCR、RT-qPCR和SDS-PAGE等方法克隆沙福芽孢杆菌ST7 cotA基因,连接到pET28a(+)表达载体,转化大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,并测定cotA 基因转化菌株的生长曲线、锰耐受浓度、锰氧化活性和锰去除率。基因克隆和序列分析显示,沙福芽孢杆菌ST7 cotA基因编码区为1 533 bp,编码510 aa,分子质量约为58.8 ku。重组质粒pET28a-cotA在大肠杆菌BL21(DE3)细胞中表达量显著上调且不抑制菌体生长,在0,250,1 000 mg/L锰胁迫条件下,cotA基因高效表达并呈现时间与浓度依赖性。重组菌株的锰氧化活性较对照组提高0.9倍,锰耐受浓度提高1.0倍。沙福芽孢杆菌CotA蛋白具有锰氧化活性,cotA基因的过表达提高大肠杆菌细胞对Mn(Ⅱ)的氧化活性、耐受能力和去除效率,对构建高效cotA基因的锰氧化菌株治理锰污染具有重要意义。

关键词: 沙福芽孢杆菌, cotA基因, 基因克隆, 锰氧化, 锰污染修复

Abstract:

The Bacillus safensis ST7 strain has strong manganese oxidation activity,in which the expression of the multicopper oxidase protein family gene BSL056-RS03010(cotA)is significantly upregulated under manganese stress,which may be involved in Mn(Ⅱ)oxidation and make the strain have strong manganese tolerance.In order to verify the manganese oxidation ability of the cotA in B.safensis,the cotA gene was cloned and an expression vector was constructed,and then transformed to Escherichia coli for heterologous expression to study the effect of cotA gene overexpression on the manganese oxidation activity and removal rate of E.coli,so as to validate the function of the cotA gene,and to provide a technological means for constructing highly efficient manganese oxidizing strains to treat manganese pollution.Using PCR,RT-qPCR and SDS-PAGE,the cotA gene of B.safensis ST7 was cloned,ligated into the pET28a(+)expression vector,and efficiently expressed in E.coli BL21(DE3)induced by isopropyl thiogalactoside.The growth curve,manganese tolerance concentration,manganese oxidation activity and manganese removal rate of the cotA gene transformed strain were determined.The cloning and sequence analysis showed that the coding region of the cotA gene of B.safensis ST7 was 1 533 bp,encoding 510 aa with a molecular mass of about 58.8 ku.The expression of the recombinant plasmid pET28a-cotA was significantly up-regulated in E.coli BL21(DE3)cells without inhibition of the growth of the bacterium.The cotA gene was highly efficiently expressed under 0,250,1 000 mg/L manganese stress with time and concentration dependence.The manganese oxidizing activity of the recombinant strain was 0.9-fold higher than that of the control,the manganese tolerance concentration was 1.0-fold higher.The CotA protein of B.safensis has manganese oxidizing activity,and overexpression of the cotA gene can improve the oxidizing activity,tolerance and removal efficiency of Mn(Ⅱ)in E.coli cells,which is of great significance for the construction of manganese oxidizing strains with high efficient cotA gene for manganese pollution management.

Key words: Bacillus safensis, cotA gene, Gene cloning, Manganese oxidizing, Manganese pollution remediation

中图分类号:

S939.97

苏成丽, 牛熙, 黄世会, 李升, 冉雪琴, 王嘉福. 沙福芽孢杆菌多铜氧化酶CotA蛋白锰氧化活性研究[J]. 华北农学报, 2026, 41(1): 230-238. doi: 10.7668/hbnxb.20195936.

SU Chengli, NIU Xi, HUANG Shihui, LI Sheng, RAN Xueqin, WANG Jiafu. Study on the Manganese Oxidation Activity of CotA Protein of Multicopper Oxidase from Bacillus safensis[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 230-238. doi: 10.7668/hbnxb.20195936.

http://www.hbnxb.net/CN/Y2026/V41/I1/230

图/表 9

表1 引物信息

Tab.1 Primer information

引物名称 Primer name	引物序列(5'— 3') Primer sequence (5'— 3')	产物长度/bp Product size
cotA-F	CGC GGATCCATGAACCTAGAAAAATTTGTTG	1 551
cotA-R	CCC AAGCTTTTACTGAATGATATCCATAGGC
cotA-qF	TCACACGTCCGCTAAAAGGG	134
cotA-qR	GGCGGCCGTATTTATCCTGT

图1 cotA基因扩增与生物信息学分析 A.扩增DNA片段;B.DNA与氨基酸序列;C.CotA结构域。

Fig.1 Amplification and bioinformatics analysis of the cotA gene A.Amplified DNA fragment;B.DNA and amino acid sequence;C.CotA structural domain.

图2 重组质粒pET28a-cotA鉴定 A.菌落PCR验证重组质粒;B.质粒PCR验证重组质粒;C.酶切验证重组质粒。M1—M3.DL2000 DNA Marker;M4.1 kb DNA Ladder;1—8.cotA基因片段;9.表达载体空质粒;10.重组质粒pET28a-cotA双酶切。

Fig.2 Identification of recombinant plasmid pET28a-cotA A.Colony PCR validation of recombinant plasmid;B.Plasmid PCR validation of recombinant plasmid;C.Enzymatic validation of recombinant plasmid.M1—M3.DL2000 DNA Marker;M4.1 kb DNA Ladder;1—8.cotA gene fragment;9.Expression vector empty plasmid;10.Recombinant plasmid pET28a-cotA double enzyme digestion.

图3 IPTG诱导不同时间相对表达量与表达倍数比 A.诱导不同时间表达量;B.诱导不同时间表达倍数比。不同小写字母表示0.05水平差异显著。图5,6同。

Fig.3 Relative expression levels and expression-fold ratios at different times of IPTG induction A.Induction of expression at different times;B.Expression multiplicity ratio by induction of different time. Different lowercase letters indicate significant difference at 0.05 level.The same as Fig.5,6.

图4 SDS-PAGE检测CotA蛋白表达 M.蛋白Marker;1—2.大肠杆菌BL21/pET28a-cotA诱导上清、诱导沉淀;3—4.大肠杆菌BL21/pET28a-cotA未诱导上清、未诱导沉淀;5—6.大肠杆菌BL21-pET28a未诱导全菌、诱导全菌;7—9.大肠杆菌BL21(DE3)未诱导上清、诱导上清、诱导沉淀。

Fig.4 Detection of CotA protein expression by SDS-PAGE M.Protein Marker;1—2.E.coli BL21/pET28a-cotA induced supernatant and precipitation;3—4.E.coli BL21/pET28a-cotA uninduced supernatant and precipitate;5—6.E.coli BL21-pET28a uninduced holobacteria,induced holobacteria;7—9.E.coli BL21(DE3)uninduced supernatant,induced supernatant and precipitate.

图5 不同浓度锰胁迫下cotA基因表达量

Fig.5 Expression of cotA gene under different concentration manganese stresses

图6 锰氧化活性检测 A.液体锰氧化;B.大肠杆菌BL21(DE3)与大肠杆菌BL21/pET28a-cotA锰氧化活性比较;C、D.固体锰氧化。

Fig.6 Manganese oxidizing activity test A.Liquid manganese oxidation;B.Comparison of manganese oxidizing activity between E.coli BL21(DE3)and E.coli BL21/pET28a-cotA;C,D.Solid manganese oxidation.

图7 不同菌株金属锰耐受浓度检测

Fig.7 Manganese metal tolerance concentration test of different strains

图8 菌株对Mn²⁺的去除率随培养时间的变化 A.培养基中;B.水溶液中;C.锰污染土壤中。

Fig.8 Changes in the removal rate of Mn²⁺ by bacterial strains over cultivation time A.In medium;B.In aqueous solution;C. In manganese contaminated soil.

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