利用DGGE-菌落原位杂交法分离土壤中精喹禾灵降解菌  

Isolation of Quizalofop-p-ethyl-degrading Bacteria from Soil by DGGE-Colony in situ Hybridization

作　　者：吕欣[1,2] 彭霞薇[1,2] 呼庆[2] 马安周[2] 江泽平[3] 魏远[3] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083 [2]中国科学院生态环境研究中心环境生物技术室,北京100085 [3]中国林业科学研究院林业研究所林木引种与植物地理研究室,北京100091

出　　处：《环境科学》2013年第1期263-270,共8页Environmental Science

基　　金：国家科技支撑计划项目(2012BAC09B03);国家自然科学基金项目(20707035)

摘　　要：从自然环境中分离到的可降解农药的土著微生物,因其对环境的友好性及原位修复的可行性,受到了高度关注.为从土壤中筛选精喹禾灵降解菌株,首先利用PCR-DGGE技术分析了除草剂精喹禾灵胁迫下土壤细菌群落结构及多样性的变化.结果表明,添加精喹禾灵后,土壤细菌群落结构发生了明显的改变.精喹禾灵使细菌多样性呈现出增加-减少-增加的变化趋势,其中第9 d变化最大,后期趋于稳定.根据DGGE图谱条带的测序结果推断,Pseudomonas、Massilia、Burkholderia等属中的细菌对精喹禾灵具有耐受性或降解潜力,这些微生物类群可作为减少农药残留的土著微生物资源进行分离筛选.根据条带的测序结果,合成了地高辛(Digoxigenin)标记的探针,并进行了菌落原位杂交,筛选到了3株具有降解潜力的菌株,其中L1可以利用精喹禾灵作为唯一碳源生长,经16S rRNA基因鉴定该菌株属于假单胞菌属(Pseudomonas sp.).利用高效液相色谱法测定了菌株L1在无机盐培养基中降解精喹禾灵的效果.结果表明,培养7 d后,精喹禾灵的含量减少了近50%,且随着精喹禾灵含量的降低,L1菌体数量增加,证实了菌株L1具有降解精喹禾灵的能力.这一结果为今后研究菌株L1降解精喹禾灵的机制、功能基因等奠定了基础.Naturally occurring bacteria isolates capable of metabolizing pesticides have received considerable attention because they offer the possibility of both environmentally friendly and in situ remediation.The effect of herbicide quizalofop-p-ethyl on bacterial community in soil was analyzed using the technique of PCR-DGGE for isolating strains biodegrading quizalofop-p-ethyl.Results indicated that the soil bacterial community structures significantly changed after adding quizalofop-p-ethyl.The bacterial diversity of soil showed an increasing-decreasing-increasing trend.The largest changes occurred in the 9th day and then became stabilized.According to the sequencing results of bands in DGGE profiles,it inferred that members of bacterial genera Pseudomonas,Massilia and Burkholderia had tolerance to quizalofop-p-ethyl,and the potential for degradation.These microbial groups could be used to isolate and screen as the indigenous microbial resources to reduce pesticide residues.Digoxigenin-labeled probes had been synthesized based on the sequencing results of bands in the DGGE profiles,and three bacterial strains capable of biodegrading quizalofop-p-ethyl were isolated from soil by colony in situ hybridization technique.The strain named L1 was able to utilize quizalofop-p-ethyl as the sole source of carbon.The strain was identified as Pseudomonas sp.,based on the phylogenetic analysis of 16S rRNA.The degrading ability of strain L1 in minimal medium with quizalofop-p-ethyl was investigated by HPLC.The quizalofop-p-ethyl content decreased by almost 50% after 7 days,and the biomass of strain L1 increased while the content of quizalofop-p-ethyl was decreased.This confirmed that the strain L1 had the capacity of degradation.This result provided a basis for future research on degradation mechanism and functional genes.

关 键 词：精喹禾灵 变性梯度凝胶电泳 菌落原位杂交 微生物降解 假单胞菌属 

分 类 号：X172[环境科学与工程—环境科学]