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)

Integration and expression of catechol-2,3-dioxygenase gene in pyrene degrading bacteria genome

ZHAO Yaguang^1,,
DUAN Weiwei¹,
XU Miao¹,
WU Panyun¹,
XIAO Lumei¹,
MA Tengfei¹,
CHAO Qunfang^1,
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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刊出日期:2019-01-08

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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。

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