沈抚灌区广宿主石油烃代谢质粒的分离及鉴定  

Isolation and characterization of petroleum catabolic broad-host-range plasmids from Shen-Fu wastewater irrigation zone

作　　者：王亚菲[1,2] 李慧[1] 李小彬[1,2] 

机构地区：[1]中国科学院沈阳应用生态研究所森林与土壤生态国家重点实验室,沈阳110164 [2]中国科学院大学,北京100049

出　　处：《应用生态学报》2013年第11期3289-3299,共11页Chinese Journal of Applied Ecology

基　　金：国家自然科学基金项目(31070102)资助

摘　　要：本研究采用"三亲配对外源分离法",从沈抚灌区土壤、底泥和水样中共计分离得到8个广宿主(BHR)石油烃代谢质粒,并通过对其进行抗生素抗性检测和抗性遗传标记,将其转移至大肠杆菌(Escherichia coli)EC100宿主中进行操作.不相容性群分析结果表明:pS3-2C、pS4-6G为Inc P质粒;pS3-2G、pW22-3G、pA15-7G为Inc N质粒;pS7-2G为Inc W质粒;pA23-1G和pA10-1C为Inc Q质粒.采用PCR扩增已报道的石油烃污染物降解基因的方法初步分析其石油烃代谢功能,质粒pS3-2G、pS7-2G、pA23-1G、pW22-3G和pA10-1C上含有编码芳香环羟化双加氧酶基因(phdA)和甲苯单加氧酶基因(touA)的片段;pA15-7G含有编码甲苯双加氧酶和甲苯单加氧酶基因片段;pS3-2C含有编码芳香环羟化双加氧酶、苯双加氧酶和甲苯双加氧酶基因片段;pS4-6G仅含有编码芳香环羟化双加氧酶基因片段.通过宿主范围检测,除质粒pS3-2C外,其余7个质粒均可在变形菌纲(Proteobacteria)α-、β-、γ-亚纲的代表性菌株根瘤农杆菌(Agrobacterium tumefaciens)C58、钩虫贪铜菌(Cupriavidus necator)JMP228、大肠杆菌EC100间进行转移并稳定传代.Based on triparental mating, we isolated a total of eight broad host range （BHR） petro- leum hydrocarbon catabolic plasmids from the soils, sediments, and wastewater samples in the Shen-Fu irrigation zone. The antibiotic resistance of the plasmids was tested, and then, the plas- raids were transferred to Escherichia coli EC100. The plasmids carrying no antibiotic resistance were tagged by miniTn5 transposon consisting of antibiotic resistant genes. The PCR-based incompatibili- ty test revealed that the pS3-2C and pS4-6G belonged to Inc P group, the pS3-2G, pW22-3G, and pA15-7G belonged to Inc N group, the pST-2G was identified as Inc W plasmid, and the pA23-1G and pA10-1C were placed into Inc Q group. By adopting the reported PCR amplification methods of petroleum hydrocarbon-degrading catabolic genes, the petroleum-degrading capability of these BHR plasmids were preliminarily analyzed. The plasmids pS3-2G, pS7-2G, pA23-1G, pW22-3G, and pA10-1C carried aromatic ring- hydroxylating dioxygenase gene phdA and toluene monooxygenase gene touA; the plasmid pAIS-7G carried touA and toluene dioxygenase gene rod; the plasmid pS3- 2C carried ben, phdA, and tod; whereas the pS4-6G only carried ben. The host range test showed that all the isolated plasmids except pS3-2C could be transferred and maintained stably in the repre- sentative strains Agrobacteriura tumefaciens C58, Cupriavidus necator JMP228, and E. coli EC100 of the α-, β-, and γ-Proteobacteria. respectively.

关 键 词：水平基因转移 三亲配对外源质粒分离 广宿主质粒 石油烃降解 生物修复 

分 类 号：X171.5[环境科学与工程—环境科学]