Laccase-mediated Degradation Of Atrazine And Isoproturon Residues In Rice

Laccase which has specific redox sites is a kind of copper polyphenol oxidase involved in plant growth,development and catalytic exogenous substances such as biotic and abiotic stresses.It is thought of playing an importent role in detoxification of xenobiotics toxic compounds such as herbicides.Atrazine belongs to the triazine herbicide family of selective uptake herbicide before seedlings or postemergence.The mechanism of atrazine is interfering photosynthesis,destorying of chlorophyll synthesis,reducing carbohydrate synthesis and deoxidation of CO2 resulting in weeds death.Isoproturon is a kind of internal conduction type phenylurea herbicide.The action mode of isoproturon is interfering electrons pass through photosynthesis ?,hindering photosynthesis to control weeds.In this study,the laccase genes from an atrazine-exposed and isoproturon-exposed rice transcriptome were identified through high-throughput sequencing.There were 2 laccase genes under stress of atrazine and 11 laccase genes under stress of isoproturon differentially expressed in rice shoots and roots,and 5 genes were randomly selected to confirm by real-time PCR analysis.Using bioinformatics databases to construct the phylogenetic analysis of laccase gene from atrazine-exposed and isoproturon-exposed rice plants and to predict identification of cis-elements.With the treatment of 0.4 mg·L-1 atrazine or 2 mg·L-1 isoproturon,chlorophyll content of rice were inhibited,the TBARS content was increased in shoots and roots,the electrolyte leakage in rice tissues was induced.On the contrary,the laccase activity was significantly increased by 0.4 mg·L-1 atrazine or 2 mg·L-1 isoproturon exposure.It was showed that the herbicide could stimulate the defense mechanism of rice seedlings.In order to study the function of laccase gene,we successfully generated transgenic yeast cells(Pichia pastoris X-33)expressing two laccase genes(LOC_Os12g15680 and LOC_Os01g63190)of rice.The LOC_Os12g15680-tranformatant were found to accumulate less atrazine and produced more atrazine degraded products,atrazine degradation rate increased 1.56 times.The LOC_Os01g63190-tranformatant were found to accumulate less isoproturon and produced more isoproturon degraded products,isoproturon degradation rate increased 1.83 times.The atrazine and isoproturon degraded products in the transformed yeast cells were analyzed by ultra performance liquid chromatography mass spectrometry(UPLC/MS/MS).The major metabolite of atrazine chemically modified by LOC_Os12g15680-tranformatant was hydroxylation of atrazine(HDHA).The major metabolite of isoproturon chemically modified by LOC_Os01g63190-tranformatant was 2-OH-isopropyl-IPU.In summary,the modified laccase genes were overexpressed in Pichia pastoris and the recombinant laccase exhibited highly expression.In addition,the recombinant laccase had the degradation ability to atrazine and isoproturon.These results demonstrated that laccase was a promising candidate for promising application in the generation of atrazine-resistant and isoproturon-resistant plants.The significance of the study may in order to provide basis for the degradation and detoxification of toxic herbicide residues.