污染土壤中主要石油降解基因AlkB和Nah定量检测方法的建立和应用  

Establishment of Analysis Method for Detection of Petroleum Degrading Genes AlkB and Nah in Contaminated Soil and Its Application

作　　者：刘庆龙[1] 唐景春[1] 万晓彤 

机构地区：[1]南开大学环境科学与工程学院环境污染过程与基准教育部重点实验室天津市城市生态环境修复与污染防治重点实验室,天津300071

出　　处：《分析化学》2014年第9期1348-1353,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.31270544);博士点基金项目(博导类)(No.20120031110015);863重大项目(No.2013AA06A205);天津市自然科学基金项目(No.10JCYBJC05600)资助~~

摘　　要：建立了SYBR Green I实时荧光定量聚合酶链式反应(Real Time-qPCR)检测油田污染土壤中烷烃降解基因AlkB和萘降解基因Nah的方法。比对相关降解石油菌株的GenBank序列,设计合成针对烷烃和萘降解基因扩增引物AlkBf/AlkBr和Nahf/Nahr。将纯化的常规PCR胶回收产物与pEASY-T1载体连接,转化到感受态细胞培养。提取并梯度稀释阳性克隆质粒,构建Real Time-qPCR标准测定曲线。25μL扩增体系最佳反应条件:前后引物终浓度为0.2μmol/L,12.5μL 2×TransStart Top Green qPCR SuperMix,AlkB和Nah基因最适退火温度分别为50℃和57℃。Real Time-qPCR技术显示出很高的灵敏性和重复性,比传统PCR技术灵敏度高100倍。对采集于某油田3个功能区的14土壤样品中AlkB定量检测显示,石油污染严重的采油区含有最高的AlkB拷贝数,污染较轻的生活区AlkB拷贝数最少;Nah基因分布均匀。SYBR Green I Real Time-qPCR method （alkanes degradation gene） and Nah （naphthalene was developed to quantify dioxygenase degradation ge the numbers of eopyies ne） functional degradation of AlkB on gene corresponding to alkanes and aromatic hydrocarbons degradation. Two pairs of primers AlkBf/AlkBr and Nahf/ Nahr were designed for AlkB and Nah amplification respectively, according to the nucleotide sequences of related degradation microorganisms published in GenBank. The purified recovery products of traditional PCR were combined with pEASY-T1 vectors and transformed in competent cells to amplify. The recombinant plasmids were extracted and used as positive templates to create standard curve through gradient dilution. The conditions for the real time PCR were as the follows： the final concentration of forward and reverse primers were 0.2μmol/L, 2×TransStart Top Green qPCR SuperMix, and the annealing temperatures of AlkB and Nah PCR were 50 ℃ and 57 ℃, respectively. The method showed a sensitivity of 100 times higher than that of the traditional PCR method and good repeatability. The numbers of copies of AlkB in three functional regions of an oilfield indicated that oil producing zone with serious oil pollution had the highest AlkB copy numbers, and residential zone with lighter oil pollution had the lowest AlkB copy numbers. Nah degradation gene distribution was more uniform.

关 键 词：石油烃降解基因 荧光 聚合酶链式反应 SYBR Green I 烷烃降解基因 萘降解基因 

分 类 号：X835[环境科学与工程—环境工程] X172