Study On Methyltransferase Gene OsARM Regulating The Degradation Effect Of Atrazine In Rice

Atrazine is widely used to control broad-leaved weeds and grassy weeds in agriculture.It has a stable structure and high residual capacity in the environment.It is difficult to be naturally degraded.The residue of atrazine causes serious environmental and health problems.Many crops such as rice and wheat have high absorption and accumulation of atrazine in contaminated soil.People began to pay increasing attention to the research on the removal of atrazine residues from environment.Methyltransferases（MTs）are a class of enzymes that transfer methyl groups from S-adenosyl-L-methionine（SAM）to the ends of various secondary metabolites,generating various methylated derivatives and change its molecular activity.Methyltransferases in plants have the functions of regulating plant growth and development,and affecting metabolism.This paper systematically identified an unknown MTs gene（LOC₁1g19840,named Atrazine Responsiveness Methyltransferases,OsARM）that was screened from the rice methylation database and transcriptome database previously produced by our laboratory and studies the effect of its involved in the degradation of atrazine in rice,and provides a scientific basis for reducing atrazine in rice crops and environment and reducing the harm of pesticides to humans.The details are as follows:The effect of atrazine stress on methyltransferase genes was studied.Transcriptome and DNA methylation data under atrazine stress were analyzed by a correlation analysis,based on the 2-fold difference,20 methyltransferase genes were screened for both differential expression and differential methylation.Twelve genes were selected for real-time quantitative PCR verification,and the results showed that they were consistent with sequencing results.Using bioinformatics analysis technology,the 20 methyltransferase genes were analyzed by evolutionary tree,chromosomal localization,and promoter cis-acting element prediction.According to the results of association analysis,a gene（OsARM）whose methylation was hypo-regulated and gene expression was up-regulated under atrazine stress was selected.Detailed bioinformatics analysis was performed.The McrBC enzyme method and chromatin immunoprecipitation method were used to verify that DNA methylation of OsARM was hypo-regulated under atrazine stress,and histone methylation levels and the methylation of DNA in the differentially methylated region of OsARM is specifically regulated by methyltransferase MET1,histone methyltransferase SDG714 and demethyltransferase ROS1.Which in turn co-regulate the expression of OsARM,and then regulates the expression of OsARM.OsARM expression at different density of atrazine stress demonstrated that atrazine could induce OsARM expression.In order to study the effect of OsARM on atrazine degradation and metabolism in rice,an overexpression vector and a CRISPR-Cas9 vector of OsARM gene were constructed by using molecular biology techniques.Three homozygous overexpressing rice lines（OE-1,OE-2,OE-5）and three homozygous CRISPR-Cas9 rice lines（Cas9-1,Cas9-3,Cas9-6）were obtained through genetic transformation,screen and identification.Meanwhile,a heterologous expression vector for the OsARM was constructed to induce the expression of Pichia pastor is X-33 and perform complementary verification of its function.The results showed that under atrazine stress,the growth status of the recombinant yeast strains was significantly superior to that of in blank yeast strains.The degradation rate of the atrazine by the recombinant pichia yeast was 1.55 times of the blank yeast cells.It indicated that OsARM enhanced the resistance of Pichia pastoris to atrazine and promoted the degradation of atrazine by Pichia pastoris.The overexpression and CRISPR-Cas9 rice lines were used as experimental materials,and wild-type rice（WT）was used as a blank control.The OsARMwas systematically studied to enhance the tolerance of atrazine to rice and promote the degradation of atrazine in rice.Under 0.2 mg L-1 atrazine stress,compared with the wild type,the elongation,dry weight and chlorophyll content of OEs rice were higher than that of WT,and the electrolyte permeability was significantly lower than that of WT.However,the growth and physiological and biochemical indicators of CRISPR-Cas9s rice were opposite to those of OEs rice,indicating that OsARM enhanced the tolerance of rice to atrazine.The accumulation of atrazine in OEs rice was lower than that in WT at 0.2 mg L-1 atrazine treatment for 6 d.The accumulation of atrazine in CRISPR-Cas9s rice was higher than that in wild type.Under atrazine stress at 0.2 mg L-1 for 2 d,the amount of removal ranged as OEs>wild type>CRISPR-Cas9s.It showed that OsARM promoted the degradation of atrazine in rice.The accumulation of atrazine in different tissue parts of rice at 30 d,60 d,and 120 d was measured.The results showed that after exposure to 30 d,60 d and 120 d,the accumulation of atrazine in different tissue parts of rice ranged as OEs<WT<CRISPR-Cas9s rice.In the 120-day rice grain,compared with the wild type,the accumulation of atrazine in OE-1 decreased 24.4%,and the content of atrazine in Cas9-1 increased 42.3%.The above results indicate that OsARM can promote the degradation of atrazine in rice and enhance the detoxification function of rice.