段魏魏1,
徐苗1,
吴盼云1,
肖璐梅1,
马腾飞1,
晁群芳1,
1.新疆大学生命科学与技术学院,乌鲁木齐 830046
基金项目: 国家自然科学基金资助项目(31460027)
Integration and expression of catechol-2,3-dioxygenase gene in pyrene degrading bacteria genome
ZHAO Yaguang1,,DUAN Weiwei1,
XU Miao1,
WU Panyun1,
XIAO Lumei1,
MA Tengfei1,
CHAO Qunfang1,
1.College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
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摘要:为了构建能够稳定遗传且高效降解多环芳烃的工程菌,利用PCR技术对Pseudomonas songnenensis wp3-1的邻苯二酚-2, 3-双加氧酶(C23O)基因进行克隆,并将其与自杀性载体pUTmini-Tn5连接,得到重组载体pUTmini-Tn5-C23O。在三亲接合作用下,经mini-Tn5转座子将重组载体pUTmini-Tn5-C23O中的C23O基因整合到菌株Pseudomonas sp. wp4的染色体DNA中,最终得到基因工程菌wp4-C23O。在不同pH、温度下,菌株wp4和工程菌wp4-C23O对浓度为50 mg?L-1的芘进行降解7 d。2株菌降解最适温度为37 ℃、最适pH为7.5。在此条件下,工程菌wp4-C23O对芘降解率显著高于wp4菌株(P<0.05),降解率提高11.45%。以PAHs降解优势菌株为受体构建工程菌可以去除石油污染土壤中的PAHs。
关键词: 多环芳烃(PAHs)/
邻苯二酚-2,3-双加氧酶(C23O)基因/
三亲接合转化/
基因工程菌
Abstract:This study aims to construct polycyclic aromatic hydrocarbons engineering bacteria with genetic stability and highly efficient degradation. Firstly, the catechol-2,3-dioxygenase (C23O) gene of Pseudomonas songnenensis wp3-1 was cloned by PCR technique, and was linked to suicide vector pUTmini-Tn5, then the recombinant vector pUTmini-Tn5-C23O was obtained. Secondly, the C230 gene of pUTmini-Tn5-C23O was integrated into the chromosomal DNA of Pseudomonas sp. wp4 with the triparental conjugation effect of mini-Tn5 transposition, and finally the gene engineering bacteria wp4-C23O was obtained. The result of contrast experiment to degrade pyrene solution (50 mg?L-1) for 7 days indicated that the optimum environment conditions for strain wp4 and strain wp4-C23O were 37 ℃ and pH 7.5. Under these conditions, the engineering strain wp4-C23O showed significantly higher degradation rate of pyrene than wp4 strain (P<0.05), and it increased by 11.45%. The engineering bacteria taking PAHs degrading dominant strain as acceptor can effectively remove PAHs from petroleum-contaminated soil.
Key words:polycyclic aromatic hydrocarbons(PAHs)/
catechol-2,3-dioxygenase (C23O) gene/
triparetal conjugation/
genetically engineering bacteria.
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[1] | LEE J, MIN K R, KIM Y. Cloning and overexpression of methylcatechol-2, 3-dioxygenase gene from toluene-degrading Pseudomonas putida, mt-2 (pWWO)[J]. Archives of Pharmacal Research, 1992, 15(4): 360-364. |
[2] | 张杰, 刘永生, 冯家勋, 等. 邻苯二酚-2,3-双加氧酶基因克隆、定位和高效表达[J]. 应用与环境生物学报, 2003, 9(5):542-545. |
[3] | 胡日查, 孙立波. 低温-1,2,4-TCB降解菌的选育、降解特性及邻苯二酚-1,2-双加氧酶基因表达水平[J]. 环境工程学报, 2013, 7 (2): 777-782. |
[4] | BALDERAS-HERNANDEZ V E, TREVINO-QUINTANILLA L G, HERNANDEZ-CHAVEZ G, et al. Catechol biosynthesis from glucose in Escherichia coli anthranilate-overproducer strains by heterologous expression of anthranilate-1,2-dioxygenase from Pseudomonas aeruginosa PAO1[J]. Microbial Cell Factories, 2014, 13(1): 1-11. |
[5] | ARAUJO S S, NEVES C M, GUIMARAES S L, et al. Structural and kinetic characterization of recombinant 2-hydroxymuconate semialdehyde dehydrogenase from Pseudomonas putida G7[J]. Archives of Biochemistry & Biophysics, 2015, 579(1):8-17. |
[6] | LIN J, MILASE R N. Purification and characterization of catechol-1,2-dioxygenase from Acinetobacter sp. Y64 strain and Escherichia coli transformants[J]. Protein Journal, 2015, 34(6): 1-13. |
[7] | LEE N, KWON D Y.Characteristics of a recombinant-2,3-dihydroxybiphenyl-1,2-dioxygenase from Comamonas sp. expressed in Escherichia coli[J]. Indian Journal of Microbiology, 2016, 56(4): 1-9. |
[8] | 杜宏伟, 武俊, 肖琳, 等. 聚磷激酶基因在假单胞菌中的整合和表达[J]. 环境科学, 2009, 30(10): 3011-3015. |
[9] | 蔡丰聚, 徐英黔. 基因工程菌稳定性的若干影响因素[J]. 生命的化学, 2006, 26(5): 451-453. |
[10] | NAGAYAMA H, SUGAWARA T, ENDO R, et al. Isolation of oxygenase genes for indigo-forming activity from an artificially polluted soil metagenome by functional screening using Pseudomonas putida strains as hosts[J]. Applied Microbiology and Biotechnology, 2015, 99(10): 4453-4470. |
[11] | 陈志丹, 晁群芳, 杨滨银, 等. 一株芘降解菌B2的降解条件优化及降解基因[J]. 环境工程学报, 2012, 6(10): 3795-3800. |
[12] | 谢云. 高效石油烷烃降解菌及原油降解基因工程菌构建研究[D]. 西安: 西北大学, 2014. |
[13] | 聂麦茜, 张志杰, 雷萍. 优势短杆菌对多环芳烃的降解性能[J]. 环境科学, 2001, 22(6): 83-85. |
[14] | 杨轩, 张威, 李师翁, 等. 多环芳烃降解菌的分离鉴定及其生理特性研究[J]. 环境科学学报, 2012, 32(5): 1033-1040. |
[15] | 卫昆, 陈烁娜, 尹华, 等. 蜡状芽胞杆菌对芘的降解特性及降解酶研究[J]. 环境科学学报, 2016, 36(2): 506-512. |
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[17] | HU F C, LI X Y, SU Z C, et al. Identification and degradation capability of three pyrene-degrading Gordonia sp.strains [J]. Chinese Journal of Applied Ecology, 2011, 22(7): 1857-1862. |
[18] | AL-THUKAIR A A, MALIK K. Pyrene metabolism by the novel bacterial strains Burkholderia fungorum (T3A13001) and Caulobacter sp (T2A12002) isolated from an oil-polluted site in the Arabian Gulf[J]. International Biodeterioration & Biodegradation, 2016, 110: 32-37. |
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邻苯二酚-2,3-双加氧酶基因在芘降解菌基因组的整合与表达
赵亚光1,,段魏魏1,
徐苗1,
吴盼云1,
肖璐梅1,
马腾飞1,
晁群芳1,
1.新疆大学生命科学与技术学院,乌鲁木齐 830046
基金项目: 国家自然科学基金资助项目(31460027)
关键词: 多环芳烃(PAHs)/
邻苯二酚-2,3-双加氧酶(C23O)基因/
三亲接合转化/
基因工程菌
摘要:为了构建能够稳定遗传且高效降解多环芳烃的工程菌,利用PCR技术对Pseudomonas songnenensis wp3-1的邻苯二酚-2, 3-双加氧酶(C23O)基因进行克隆,并将其与自杀性载体pUTmini-Tn5连接,得到重组载体pUTmini-Tn5-C23O。在三亲接合作用下,经mini-Tn5转座子将重组载体pUTmini-Tn5-C23O中的C23O基因整合到菌株Pseudomonas sp. wp4的染色体DNA中,最终得到基因工程菌wp4-C23O。在不同pH、温度下,菌株wp4和工程菌wp4-C23O对浓度为50 mg?L-1的芘进行降解7 d。2株菌降解最适温度为37 ℃、最适pH为7.5。在此条件下,工程菌wp4-C23O对芘降解率显著高于wp4菌株(P<0.05),降解率提高11.45%。以PAHs降解优势菌株为受体构建工程菌可以去除石油污染土壤中的PAHs。
English Abstract
Integration and expression of catechol-2,3-dioxygenase gene in pyrene degrading bacteria genome
ZHAO Yaguang1,,DUAN Weiwei1,
XU Miao1,
WU Panyun1,
XIAO Lumei1,
MA Tengfei1,
CHAO Qunfang1,
1.College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
Keywords: polycyclic aromatic hydrocarbons(PAHs)/
catechol-2,3-dioxygenase (C23O) gene/
triparetal conjugation/
genetically engineering bacteria
Abstract:This study aims to construct polycyclic aromatic hydrocarbons engineering bacteria with genetic stability and highly efficient degradation. Firstly, the catechol-2,3-dioxygenase (C23O) gene of Pseudomonas songnenensis wp3-1 was cloned by PCR technique, and was linked to suicide vector pUTmini-Tn5, then the recombinant vector pUTmini-Tn5-C23O was obtained. Secondly, the C230 gene of pUTmini-Tn5-C23O was integrated into the chromosomal DNA of Pseudomonas sp. wp4 with the triparental conjugation effect of mini-Tn5 transposition, and finally the gene engineering bacteria wp4-C23O was obtained. The result of contrast experiment to degrade pyrene solution (50 mg?L-1) for 7 days indicated that the optimum environment conditions for strain wp4 and strain wp4-C23O were 37 ℃ and pH 7.5. Under these conditions, the engineering strain wp4-C23O showed significantly higher degradation rate of pyrene than wp4 strain (P<0.05), and it increased by 11.45%. The engineering bacteria taking PAHs degrading dominant strain as acceptor can effectively remove PAHs from petroleum-contaminated soil.