Functional Analysis Of Chlorimuron-ethyl Degradation Gene Kj-gst

Chlorimuron-ethyl is a sulfonylurea herbicide.Due to its high efficiency and economic characteristics,it has been widely used to control broad-leaved weeds in the world in recent years.However,chlorimuron-ethyl has the characteristic of long residual effect period due to the structural characteristics of the chlorimuronethyl and the high chlorimuron-ethyl residue in soil has strong toxic effect on a plurality of crops after planting,so the degradation of the chlorimuron-ethyl under the natural condition is particularly important.At present,three degradation methods of chlorimuron-ethyl have been reported,including hydrolysis,photolysis and microbial degradation.Among them,microbial degradation is considered to be the most likely degradation mode to solve the residual problem of chlorimuronethyl.Klebsiella jilinsis 2N3 is a gram-negative bacterium that can degrade chlorimuron-ethyl with high efficiency.the degradation rate of chlorimuron-ethyl can reach 92.5% by K.jilinsis2N3 under the appropriate conditions.In order to in-depth explore the main mechanism of chlorimuron-ethyl degradation by K.jilinsis 2N3 2N3,we have conducted whole genome sequencing of K.jilinsis 2N3 and transcriptome sequencing with and without the difference of chlorimuron-ethyl addition.After analyzing the transcriptome data,the chlorimuron-ethyl degradation gene Kj-gst was finally obtained through screening and experimental verification in this study.Gene knockout and catch-up experiments showed that K.jilinsis 2N3 2N3 without the gene Kj-gst had a significantly decreased ability to degrade chlorimuron-ethyl.On the basis of gene knockout,we prokaryotically expressed the gene Kj-gst and obtained the protein Kj-GST.The degradation experiments showed that the protein Kj-GST had a significant degradation effect on chlorimuron-ethyl.Under the condition of 16℃,p H=7,the initial concentration of 50 mg /L was degraded by 42.91%.LC-MS analysis of the degradation product of chlorimuron-ethyl by protein revealed that two degradation products of chlorimuron-ethyl were identified,namely,osulfonic acid benzoate imine and 2-amino-4-chloro-6-methoxypyrimidine.In order to further explore the main mechanism of degradation of chlorimuron-ethyl by protein Kj-GST,we conducted a protein point mutation experiment and successfully confirmed that the GSH-H site was the main functional region of degradation of chlorimuron-ethyl by protein Kj-GST.In addition,we also explored the function of ABC transporter in the degradation of chlorimuronethyl.This part of the experiment laid a foundation for completely revealing the process from absorption of chlorimuron-ethyl into bacteria to transport of products out of bacteria after hydrolysis.