石油烃厌氧降解基因masD和bamA实时荧光定量PCR方法的建立及应用
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摘要
masD和bamA基因分别是烷烃和芳烃厌氧降解的关键基因。本研究建立了这两种基因的SYBR Green Ⅰ实时荧光定量PCR检测方法。通过参考相关石油烃厌氧降解菌株的Gen Bank序列,利用Primer express软件设计烷烃和芳烃厌氧降解基因的扩增引物masD-f、masD-r和bamA-f、bamA-r。经过常规PCR扩增分别得到片段大小为389和354 bp扩增产物,经测序并在NCBI数据库查询,确定为masD和bamA片段。通过实时荧光定量PCR构建测定这两种基因的标准曲线。优化后的扩增体系(25μL)为:12.5μL 2×Trans Start Top Green qPCR Super Mix,引物浓度为0. 2μmol/L,masD和bamA基因最适退火温度分别为52℃和56℃。建立的两种基因的实时荧光定量PCR检测方法具有非常好的重复性,其灵敏度比传统PCR技术高100倍。对于氧化石墨烯促进石油烃的厌氧降解体系中厌氧基因的定量检测显示,添加不同浓度的石墨烯均促进了bamA拷贝数的增加,但对masD的拷贝数无显著影响。
The masD and bamA are key genes for anaerobic degradation of alkanes and aromatic hydrocarbons,respectively. In this study,the SYBR Green I real-time quantitative polymerase chain reaction(Real Time-qPCR) method was established with the two genes as targets. With reference to the GenBank sequence of related degraded oil strains,the anaerobic degradation gene amplification primers masD-f,masD-r and bamA-f,bamA-r for alkanes and aromatic hydrocarbons were designed and synthesized. Two pairs of primers were amplified by conventional PCR to obtain 389 bp and 354 bp fragments,respectively. The amplified products were confirmed as fragments of masD and bamA by being sequenced and queried in the NCBI database. The positive clone plasmid was extracted and serially diluted to construct a real-time qPCR standard curve. The best reaction conditions for the 25-μL amplification system were as follows: 0.2 μmol/L pre-primer and post-primer,12. 5 μL of 2 × Trans Start Top Green q PCR Super Mix,52℃ of annealing temperatures for masD and 56℃ for bamA genes. Real Time-qPCR technology showed high sensitivity and repeatability,which was 100 times higher than traditional PCR technology. The quantitative detection of anaerobic genes in the anaerobic degradation system of petroleum hydrocarbons promoted by graphene oxide showed that the addition of different concentrations of graphene oxide increased the copy numbers of bamA gene,but had no significant effect on the copy numbers of masD gene.
The masD and bamA are key genes for anaerobic degradation of alkanes and aromatic hydrocarbons,respectively. In this study,the SYBR Green I real-time quantitative polymerase chain reaction(Real Time-qPCR) method was established with the two genes as targets. With reference to the GenBank sequence of related degraded oil strains,the anaerobic degradation gene amplification primers masD-f,masD-r and bamA-f,bamA-r for alkanes and aromatic hydrocarbons were designed and synthesized. Two pairs of primers were amplified by conventional PCR to obtain 389 bp and 354 bp fragments,respectively. The amplified products were confirmed as fragments of masD and bamA by being sequenced and queried in the NCBI database. The positive clone plasmid was extracted and serially diluted to construct a real-time qPCR standard curve. The best reaction conditions for the 25-μL amplification system were as follows: 0.2 μmol/L pre-primer and post-primer,12. 5 μL of 2 × Trans Start Top Green q PCR Super Mix,52℃ of annealing temperatures for masD and 56℃ for bamA genes. Real Time-qPCR technology showed high sensitivity and repeatability,which was 100 times higher than traditional PCR technology. The quantitative detection of anaerobic genes in the anaerobic degradation system of petroleum hydrocarbons promoted by graphene oxide showed that the addition of different concentrations of graphene oxide increased the copy numbers of bamA gene,but had no significant effect on the copy numbers of masD gene.
引文
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2 Burgin A J,Hamilton S K.Front.Ecol.Environ.,2007,5(2):89-96
3 Chakraborty R,Coates J D.Appl.Microbiol.Biot.,2004,64(4):437-446
4 WANG Jian,ZHANG Xu,LI Guang-He.Environmental Science,2012,33(4):1352-1360王坚,张旭,李广贺.环境科学,2012,33(4):1352-1360
5 Callaghan A V,Davidova I A,Savage-Ashlock K,Parisi V A,Gieg L M,Suflita J M,Kukor J J,Wawrik B.Environ.Sci.Technol.,2010,44(19):7287-7294
6 Aitken C M,Jones D M,Maguire M J,Gray N D,Sherry A,Bowler B F J,Ditchfield A K,Larter S R,Head I M.Geochim.Cosmochim.Acta,2013,109(109):162-174
7 Peters F,Shinoda Y,Mcinerney M J,Boll M.J.Bacteriol.,2007,189(3):1055-1060
8 Boll M.J.Mol.Microb.Biotech.,2005,10(2-4):132-142
9 Gennaro P D,Moreno B,Annoni E,Garcia-Rodriguez S,Bestetti G,Benitez E.J.Hazard.Mater.,2009,172(2-3):1464-1469
10 HU Dan-Dan,GU Jin-Gang,JIANG Rui-Bo,DONG Jin-Gao.Journal of Plant Nutrition and Fertilizer,2007,13(3):520-525胡丹丹,顾金刚,姜瑞波,董金皋.植物营养与肥料学报,2007,13(3):520-525
11 N7lvak H,Truu M,Truu J.Sci.Total Environ.,2012,426(2):351-358
12 Liu Z Y,Wang F,Yuan L J,Zhang X X,Ying Q K,Yu L,Zhang L,Cheng L F,Zhang F L,Lu J G,Wu X A.Int.J.Mol.Med.,2016,38(3):951-960
13 Toth C R A,Gieg L M.Front.Microbiol.,2018,8:2610
14 Yoshida N,Miyata Y,Mugita A,Iida K.Materials,2016,9(9):742
15 Li L,Liu Q,Wang Y X,Zhao H Q,He C S,Yang H Y,Gong L,Mu Y,Yu H Q.Sci.Rep.,2016,6:30082
16 Wang J,Wang D,Liu G F,Jin R F,Lu H.J.Chem.Technol.Biot.,2014,89(5):750-755
17 Toral-Sanchez E,Valdes J A A,Aguilar C N,Cervantes F J,Rangel-Mendez J R.Carbon,2016,99:456-465
18 Hu X G,Ouyang S H,Mu L,An J,Zhou Q.Environ.Sci.Technol.,2015,49(18):10825-10833
19 Kimes N E,Callaghan A V,Aktas D F,Smith W L,Sunner J,Golding B T,Drozdowska M,Hazen T C,Suflita J M,Morris P J.Front.Microbiol.,2013,4:50
20 Staats M,Braster M,Roling W F.Environ.Microbiol.,2011,13(5):1216-1227
21 LIU Qing-Long,TANG Jing-Chun,WAN Xiao-Tong.Chinese J.Anal.Chem.,2014,42(9):1348-1353刘庆龙,唐景春,万晓彤.分析化学,2014,42(9):1348-1353
22 Powell S M,Ferguson S H,Bowman J P,Snape I.Microb.Ecol.,2006,52(3):523-532
23 Wang L Y,Gao C X,Mbadinga S M,Zhou L,Liu J F,Gu J D,Mu B Z.Int.Biodeter.Biodegr.,2011,65(3):444-450
24 ZOU Wen,YANG Xu,YANG Li-Ping,LAI Li-Ying,LEI Jian-Hua,LUO Hong-Yu,ZHANG Yong-Hong.Chinese Journal of Hepatology,2004,12(7):444邹文,杨旭,杨立平,赖力英,雷建华,罗红雨,张永红.中华肝脏病杂志,2004,12(7):444
25 ZHAO Li,CUI Bao-An,CHEN Hong-Ying,WEI Zhan-Yong,ZHENG Lan-Lan,LV Xiao-Li,JIA Yan-Yan,ZHAO XuYong.Chinese Journal of Biotechnology,2008,24(7):1149-1154赵丽,崔保安,陈红英,魏战勇,郑兰兰,吕晓丽,贾艳艳,赵绪永.生物工程学报,2008,24(7):1149-1154