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
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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。
This study aims to construct polycyclic aromatic hydrocarbons engineering bacteria with geneticstability 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 therecombinant vector pUTmini-Tn5-C23 O was obtained. Secondly, the C230 gene of pUTmini-Tn5-C23 O wasintegrated into the chromosomal DNA of Pseudomonas sp. wp4 with the triparental conjugation effect of miniTn5 transposition, and finally the gene engineering bacteria wp4-C23 O was obtained. The result of contrastexperiment to degrade pyrene solution(50 mg?L~(-1)) for 7 days indicated that the optimum environment conditionsfor strain wp4 and strain wp4-C23 O were 37 ℃ and pH 7.5. Under these conditions, the engineering strain wp4-C23 O 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 removePAHs from petroleum-contaminated soil.
This study aims to construct polycyclic aromatic hydrocarbons engineering bacteria with geneticstability 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 therecombinant vector pUTmini-Tn5-C23 O was obtained. Secondly, the C230 gene of pUTmini-Tn5-C23 O wasintegrated into the chromosomal DNA of Pseudomonas sp. wp4 with the triparental conjugation effect of miniTn5 transposition, and finally the gene engineering bacteria wp4-C23 O was obtained. The result of contrastexperiment to degrade pyrene solution(50 mg?L~(-1)) for 7 days indicated that the optimum environment conditionsfor strain wp4 and strain wp4-C23 O were 37 ℃ and pH 7.5. Under these conditions, the engineering strain wp4-C23 O 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 removePAHs from petroleum-contaminated soil.
引文
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[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.
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[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.
[16]胡凤钗,苏振成,孙健,等.高效芘降解菌N12的分离鉴定与降解特性[J].应用生态学报,2011,22(6):1566-1572.
[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.
[19]JIN J,YAO J,ZHANG Q,et al.Biodegradation of pyrene by pseudomonas sp.JPN2 and its initial degrading mechanism study by combining the catabolic nahAc gene and structure-based analyses[J].Chemosphere,2016,164:379-386.
[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.
[16]胡凤钗,苏振成,孙健,等.高效芘降解菌N12的分离鉴定与降解特性[J].应用生态学报,2011,22(6):1566-1572.
[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.
[19]JIN J,YAO J,ZHANG Q,et al.Biodegradation of pyrene by pseudomonas sp.JPN2 and its initial degrading mechanism study by combining the catabolic nahAc gene and structure-based analyses[J].Chemosphere,2016,164:379-386.