吉林克雷伯氏菌2N3降解氯嘧磺隆基因Kj-mhpC功能研究

Functional Analysis of Chlorimuron-ethyl Degrading Genes Kj-mhpC in Klebsiella jilinsis 2N3

采用λ-Red重组技术对氯嘧磺隆降解菌--吉林克雷伯氏菌2N3的2个水解酶mhp C基因Kj-mhp C-2096和Kj-mhp C-2289分别进行了敲除,获得了2个基因的突变体,并通过PCR对敲除突变体进行了验证。在含有5 mg/L氯嘧磺隆的液体基础培养基中,对突变体的生长速度以及对氯嘧磺隆的降解能力进行了检测。结果表明:10 h内野生型和突变体均生长至最高浓度,ΔKj-mhp C-2096生长速度最慢,最终细胞浓度最低,野生型生长速度最快。8 h内,ΔKj-mhp C-2096和ΔKj-mhp C-2289对氯嘧磺隆的降解率明显低于野生型,在8 h之后ΔKj-mhp C-2289对氯嘧磺隆的降解率逐渐与野生型相当,最终降解率>90%,而ΔKj-mhp C-2096在将氯嘧磺隆降解至约1 mg/L后,基本停止了降解。这表明mhp C的缺失对氯嘧磺隆的降解产生了一定的影响,导致菌株对氯嘧磺隆耐受能力降低,降解率与降解速度均降低,初步证明Kj-mhp C-2096参与了氯嘧磺隆的降解。

Two hydrolase mhpC genes Kj-mhpC-2096 and Kj-mhpC-2289 in chlorimuron-ethyl degrading bacteria Klebsiella jilinsis 2N3 were knocked out by X-Red recombination technique,mutants of the two genes were obtained,and the knockout mutants were verified by PCR.Growth rate of the mutants and degradation of chlorimuron-ethyl were detected in the liquid basal medium with 5 mg/L chlorimuron-ethyl.Strains WT and mutants ail grew to their highest concentrations at about 10 h.During the first 10 hours,Kj-mhpC-2096 had the slowest growth rate,the final cell concentration was the lowest,and the WT had the fastest growth rate.The degradation rate of△Kj-mhpC-2096 and△Kj-mhpC-2289 to chlorimuron-ethyl was significantly slower than that of the WT within 8 h.After 8 h,the degradation rate of△Kj-mhpC-2289 to chlorimuron-ethyl was equivalent to that of the WT,and the final degradation rate exceeded 90%,while almost no degradation of△Kj-mhpC-2096 was detected when the concentration of chlorimuron-ethyl dropped to 1 mg/L.These findings indicate that the deletion of Kj-mhpC weakens the degradation of chlorimuron-ethyl,which leads to the decrease of strain tolerance to chlorimuron-ethyl,and it is preliminarily proved that Kj-mhpC-2096 is involved in the degradation to chlorimuron-ethyl in Klebsiella jilinsis 2N3.