Metabolic And Detoxification Mechanism Of Isoproturon In Rice Involved By Cytochrome P450

Pesticides have been mainly used to prevent and control diseases,pests,weeds and protect crops.Application of pesticides is one of the effective ways to improve crop quality and yield.But the excessive and non-standard use of pesticide,not only causes certain effect on ecological environment,but also has a potential hazard to human health,and then it will has drawn great attention of people.Isoproturon(IPU)is one of the herbicides used in China today.Because of long use time,difficult degradation and accumulation in the ecological environment,IPU is easy to cause environmental pollution and irreversible damage.Cytochrome P450,a monooxygenase encoded by the supergene family,involves in the metabolism and degradation of a variety of pesticides in plants.In this study,a differentially up-regulated cytochrome P450 gene(LOC＿Os08g39730,this is called CYP76C6)induced by IPU was screened to study its role in the detoxification and metabolism of IPU in rice.The specific contents are as follows:Firstly,the effect of IPU on cytochrome P450 was studied.By analysis of the transcriptome data under the stress of IPU,62 differentially expressed genes were screened out,including 28 in shoots and 34 in roots.Six genes which showing a trend of up-regulation of expression levels were shared by both shoots and roots.The differentially expressed genes were analyzed by heat map,evolutionary tree,chromosome localization and promoter cis-acting element prediction.The variation trend of expression levels of selected 12 genes was consistent with the sequencing data by verification of q RT-PCR analysis.According to the results of transcriptome data analysis,a cytochrome P450 gene(CYP76C6)(five-fold differential expression and up-regulated expression level)was selected and performed the bioinformatics analysis.When the concentration of IPU was 2 mg/L,the expression level of CYP76C6 in shoots of rice was 32.56 times of control group,and that in roots was 2.25 times of control group.Under 4 mg/L IPU treatment,the expression level of CYP76C6 was 36.76 times compare with control group in shoots,and 10.14 times in roots.These results indicated that CYP76C6 can be induced under IPU stress.In order to study the detoxification and metabolism function of CYP76C6 on IPU in rice.The vector of CYP76C6 overexpression and the CRISPR-Cas9 were constructed using molecular biology techniques.Through genetic transformation,screening and identification,three homozygous overexpressed lines(OE-1,OE-2 and OE-3)and two homozygous Cas9 lines(Cas9-1 and Cas9-2)of rice T3 were obtained.The effect of cytochrome P450 gene CYP76C6 on the detoxification and metabolism of IPU in rice were studied using OEs and Cas9 s as experimental materials,and wild-type(WT)rice as control.At 2 mg/L of IPU treatment,compared with WT rice,OEs growth were better,and the elongation,dry weight and chlorophyll content were significantly improved,the degree of membrane damage is reduced.The accumulation of IPU is decreased in OEs rice tissues,and removing rate of IPU from the growth media of OEs rice is increased.However,the phenomenon of Cas9 s rice lines was completely opposite to the OEs lines.These results indicated that the CYP76C6 enhanced the tolerance of rice to IPU and accelerated the metabolism and degradation of IPU in rice.To further explore the metabolism and degradation effect of CYP76C6 to IPU in rice,WT,OEs and Cas9 s were exposure respectively to 0.02 mg/L of IPU for a long time.The accumulation of IPU in different tissues of rice at 30 d,90 d and 120 d were detected respectively.The results showed that IPU accumulation in different tissue was following the trend: OEs(27)WT(27)Cas9s.In 120 d mature grains,the accumulation of IPU in the OEs lines was 0.58～0.81 times that of WT,and Cas9 s 1.38～1.54 times of WT.These results indicated that CYP76C6 could promote the detoxification and metabolism of IPU in rice.Finally,HRLC-Q-TOF-MS/MS was used to detect the metabolites of OEs,Cas9 s and WT.Fifty metabolites and 9 conjugates of IPU were detected and indentified.The possible metabolic pathway of IPU was proposed.According to the relative quantitative analysis of metabolites and conjugates,the relative contents of most of the metabolites and conjugates in OEs were higher significantly than those in WT,while those in Cas9 s were lower significantly than WT.The above results further elucidated that the cytochrome P450 enhanced the metabolism of IPU in rice.CYP76C6 involved in the metabolism and detoxification mechanism of pesticides in rice was further proved.