新疆准东油田石油污染土壤萘双加氧酶基因的多态性
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摘要
为了分析新疆准东油田石油污染土壤中萘双加氧酶功能基因的多态性,采用无机盐培养基,以萘为唯一的碳源物质,对准东油田石油污染土壤中的萘降解菌进行液体富集培养,用高效液相色谱法(high performance liquid chromatography,简称HPLC)对降解产物进行检测,同时采用萘双加氧酶基因通用引物对萘双加氧酶基因片段进行扩增、测序及分析。高效液相色谱检测结果显示,萘被降解成小分子产物,证明液体培养基均富集培养获得了萘降解菌群。从准东油田共分离得到了49条萘双加氧酶基因片段,共编码35条氨基酸序列,GenBank登录号为KY304781～KY304829。获得的萘双加氧酶基因系统发育树结果显示,研究获得的萘双加氧酶基因分为4个类群。通过高效液相色谱分析及萘双加氧酶基因的分子检测,证明准东油田石油污染土壤中含有萘降解菌株,这些萘降解菌株是可以通过液体富集培养获得的,并且可以通过固体平板获得降解萘的单菌株。
引文
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[2]Ravelet C,Grosset C,Montuelle B,et al.Liquid chromatography study of pyrene degradation by two micromycetes in a freshwater sediment[J].Chemosphere,2001,44(7):1541-1546.
[3]罗洪君,王绪远,赵骞,等.石油污染土壤生物修复技术的研究进展[J].四川环境,2007,26(3):104-109.
[4]Liang Y T,Zhang X,Wang J,et al.Spatial variations of hydrocarbon contamination and soil properties in oil exploring fields across China[J].Journal of Hazardous Materials,2012,241(4):371-378.
[5]Yang Y Y,Wang J,Liao J Q,et al.Distribution of naphthalene dioxygenase genes in crude oil-contaminated soils[J].Microbial Ecology,2014,68(4):785-793.
[6]宋兴良,王江涛,张哲.多环芳烃蒽高效降解菌的筛选及其降解中间产物分析[J].海洋环境科学,2010,29(6):815-818.
[7]Gibson D T,Parales R E.Aromatic hydrocarbon dioxygenases in environmental biotechnology[J].Current Opinion in Biotechnology,2000,11(3):236-243.
[8]Abu-Omar M M,Loaiza A,Hontzeas N.Reaction mechanisms of mononuclear non-heme iron oxygenases[J].Chemical Reviews,2005,105(6):2227-2252.
[9]Visser S P,Kumar D.Iron-containing enzymes versatile catalysts of hydroxylation reactions in nature[M].London:Royal Society of Chemistry,2011:42-63.
[10]Iwai S,Johnson T A,Chai B L,et al.Comparison of the specificities and efficacies of primers for aromatic dioxygenase gene analysis of environmental samples[J].Applied and Environmental Microbiology,2011,77(11):3551-3557.
[11]王涛,蓝慧,田云,等.多环芳烃的微生物降解机制研究进展[J].化学与生物工程,2016,33(2):8-14.
[12]Hesham A,Wang Z Y,Zhang Y,et al.Isolationand identification of a yeast strain capable of degrading four and five ring aromatic hydrocarbons[J].Annals of Microbiology,2006,56(2):109-112.
[13]Qi J,Wang B,Li J,et al.Genetic determinants involved in the biodegradation of naphthalene and phenanthrene in Pseudomonas aeruginosa PAO1[J].Environmental Science and Pollution Research International,2015,22(9):6743-6755.
[14]Baldwin B R,Nakatsu C H,Nies L.Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR[J].Applied and Environmental Microbiology,2003,69(6):3350-3358.
[15]Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1870-1874.
[16]Yang Y Y,Wang J,Liao J Q,et al.Abundance and diversity of soil petroleum hydrocarbon-degrading microbial communities in oil exploring areas[J].Applied Microbiology and Biotechnology,2015,99(4):1935-1946.
[17]谭银萍,马媛,吕杰.石油污染对土壤细菌结构特征的影响[J].生物技术,2016,26(2):193-198.