百菌清降解菌的分离鉴定及功能基因分析

Isolation,identification and functional gene analysis of chlorothalonil degrading bacteria

百菌清是一种广谱非内源性农药,在土壤中难以降解,已经成为农业环境污染的主要污染源之一。因此,其对环境的污染和被污染土壤的修复技术越来越受到关注。土壤环境中原位降解细菌的多样性对于评价环境毒理学、生物降解性、自净化能力和污染物的修复潜力具有重要价值。该研究从长期被百菌清污染的土壤中收集大量样本,分离到了14种能够明显降解百菌清的细菌。根据菌株形态和rDNA同源性分析,将它们分为假单胞菌属(Pseudomonas sp.)、无色杆菌属(Achromobacter sp.)、苍白杆菌属(Ochrobactrum sp.)、青枯菌属(Ralstonia sp.)和溶杆菌属(Lysobacter sp.)。其中溶杆菌属是该研究中新发现的具有百菌清降解能力的功能菌属,该发现扩大了已知的百菌清降解菌的菌属范围。通过进一步鉴定及生理生化分析,确定了该降解菌的分类地位及理化特征。此外,该研究通过基因文库法克隆到了发挥关键降解作用的水解酶基因,并发现该基因与转座子原件IS-Olup相连,二者组成代谢转座子,具有水平转移的分子基础。通过揭示降解基因在细菌间的漂移机制,初步明确了降解菌的功能基因及其分布规律。

Chlorothalonil(2,4,5,6-tetrachloroisophthalonitrile,TPN)was used as a broad-spectrum and non-systemic fungicide in China.However,this pesticide has been classified as a“probable human carcinogen”by the U.S.Environment Protection Agency(US EPA),due to its highly toxic to birds,fish,and aquatic invertebrates.Alternatively,bioremediation can be expected to degrade,even remove organic pollutants,with the promising application prospects.The diversity of in situ degrading bacteria in a polluted environment is critical to evaluate environmental toxicology,biodegradability,self-purification ability,and remediation potential of pollutants.In this study,an attempt was made to apply the biodegradation for the control of pollution.Firstly,the soil samples were collected from the long-term chlorothalonil-contaminated field.Fourteen chlorothalonil-degrading bacteria producing transparent halos were isolated using the plate culture and chlorothalonil-selective medium.Using the morphology and 16S rDNA homology,the bacteria were then classified to genus Pseudomonas sp.,Achromobacter sp.,Ochrobactrum sp.,Ralstonia sp.and Lysobacter sp.Lysobacter sp.The RB-31 and RB-38 were newly discovered strains with chlorothalonil degradation ability.Two strains were determined into species level as Lysobacter daejeonensis.And their specific physiological properties were studied.Secondly,the genomic library of strain daejeonensis RB-38 was successfully constructed in the pUC19 vector using E.coil DH10B as the host strain,where about 10000 clones were obtained from selective culture.A 3494 bp of desired fragment was isolated from the library using the functional ability to degrade chlorothalonil.In the desired fragment,three open reading frames(ORFs)were tentatively identified by ORF findings and BLAST alignment on NCBI.Specifically,ORF3 encoded a hydrolytic dehalogenase chd.Through subcloning of this reading frame,it was proved that the degradation function of chlorothalonil was catalyzed by the enzyme encoded in this region chd,and no other regulation regions were required for its expression.Two ORFs upstream of chd gene showed that ORF1 encoded a transposase,whereas,ORF2 encoded on IstB-like ATP-binding protein.Two ORFs were flanked by 20 bp terminal inverted repeat sequences(IR).The complete sequence presented a perfect structural similarity to IS21 transposon family members that all contain transposase coding region,ATP-binding protein coding region,and flanked by inverted repeat sequences.A new member of this family was discovered and designated as IS-Olup.The chd gene was closely associated with the insertion sequence,to construct a catabolic transposon.Finally,the chd gene and the upstream IS-Olup fragment were cloned and identified from several genomic DNA of chlorothalonil-degrading bacteria using a PCR strategy.It infers that the sequence element of IS-Olup was the molecular basis for the horizontal transfer in the chd genes,leading that the gene exchange can occur among these degrading species.This study can enrich the chlorothalonil-degrading bacterial library,and to clone hydrolytic enzyme genes that played a key role in degrading from the genetic level.A dispersing mechanism of degrading gene was also proposed among different genus bacteria.This preliminarily clarified the functional gene and its distribution in degrading bacteria.