| At present,the use of pesticides is the most efficient way to ensure crop yield.However,with the expansion of its use,it will cause a series of environmental problems.After the overuse of pesticides on crops,it is easy to leach and runoff in soil and reach groundwater and surface water,resulting in water pollution.Moreover,it is more likely through the food chain(crops)to affect human health,including the skin,eyes,nervous and reproductive system.Bentazone has been widely used in agricultural production owing to its broad spectrum,high effective weeding capability and low resistance.But due to its strong mobility capacity,bentazone is hard to adsorb by soil,and leach easily into water.Thus,the bentazone residues and environmental behavior have attracted the attention of people after application.Rice is one of the most important food crops in the world,which providing a major source of calories for human diet.Meanwhile,the data of rice genetics and genome sequence have been recorded in detail,which makes it be of great practical significance to study the metabolism and degradation mechanism of pesticides in rice.Methyltransferase(MT_S)is a class of enzymes that transfer methyl groups from S-adenosyl-L-methionine(SAM)to the ends of various secondary metabolites and produce various methylated derivatives.The methylation usually can change their molecular activity,regulate plant growth and metabolism.In this work,a methyltransferase gene(LOC＿04g09604,named as Overexpressing Methyltransferase,Os MT1)responding to pesticide stress was screened from the rice methylation database and transcriptome.The bioinformatics analysis of the gene is systematically performed.Os MT1 involved in metabolism and degradation of bentazone in rice was investigated.The results could provide scientific basis for reducing pesticide residues in rice crop and environment.The details of this study are as follows:1.To study the effects of bentazone on the growth of rice,the wild type rice(WT)was treated with 0-1.0 mg/L bentazone for 2-6 d.The results showed that the accumulated concentration of bentazone in rice is increased,the growth of rice was inhibited more obviously,and the degree of cell membrane damage was also more serious.Compared to control,the elongation,dry weight and chlorophyll content of rice was decreased,and the electrolyte permeability and malondialdehyde(MDA)of rice was increased with the increase of treatment concentration under the same exposure time.When the treatment concentration of benzosone is the same,with the extension of the treatment time,its impact on the growth of rice will increase.2.The study of the activities of antioxidant enzymes and detoxifying enzymes in rice under the stress of bentazone was performed.It was found that the activities of antioxidant enzymes(SOD,POD,CAT)and detoxifying enzyme(GST)in the shoots and roots of rice were stimulated under the treatment of bentazone at 0-1.0 mg/L.When rice was exposed at0.8 mg/L bentazone for 6 d,the activities of antioxidant enzymes and detoxifying enzymes reached the maximum value in the shoots and roots.But when the treatment concentration of bentazone was higher than 0.8 mg/L,the activities of antioxidant enzymes and detoxifying enzymes decreased.These results displayed that rice seedlings can resist the oxidative damage caused by bentazone through increasing the activities of antioxidant enzymes and detoxifying enzymes.3.By association analysis of transcriptome and DNA methylation data under pesticide stress,a methyltransferase gene Os MT1 with down-regulated methylation and up-regulated gene expression was selected.Bioinformatics analysis of Os MT1 was performed.The expression profile data of Os MT1 showed that Os MT1 was responsive to bentazone,and showed an up-regulated of expression.In order to explore the regulation of Os MT1 on the metabolism and degradation of bentazone in rice,the Os MT1 overexpression vector and Cas9 expression vector were constructed by using molecular biology techniques.Three homozygous overexpressed rice lines(OE-1,OE-2,OE-3)of T3 generation and three homozygous Cas9 rice lines(Cas9-2,Cas9-3,Cas9-4)of T3 generation were obtained through genetic transformation,screening and identification.4.The enhanced tolerance of Os MT1 to bentazone in rice was confirmed by using overexpressed and Cas9 rice lines as experimental materials and wild-type rice as control.Under the exposure of 0.8 mg/L bentazone,the growth of overexpressed rice was better than that of wild type.The elongation,dry weight and chlorophyll content of the overexpressed rice lines were higher than those of the wild type,and the electrolyte permeability was significantly lower than wild type.While the growth trend of the Cas9rice lines were opposite to those of the overexpressed rice lines under bentazone stress.These results indicated that the participation of Os MT1 could enhance the tolerance of rice to bentazone.After treating bentazone to rice for 2 d,the removal amount of bentzone per plant of overexpressed rice from growth media was greater than that of wild type,and the Cas9 rice were lower than that of wild type.Under the treatment of bentazone for 6 d,the accumulation of bentazone in overexpressed rice was lower than that in wild type,while the accumulation of bentazone in the Cas9 rice was higher than that in wild type.In order to further study the effect of Os MT1 on the metabolism and degradation of bentazone during the whole growth cycle of rice,we also determinated the accumulation amount of bentazone in different rice tissues at 40 d,80 d and 120 d.The results showed that the accumulation amount of bentazone in different tissues of rice was OE<WT and Cas9>WT.In conclusion,Os MT1 can promote the detoxification and metabolism of bentazone in rice.5.To further study the metabolism and degradation pathways of bentazone in rice under the regulation of Os MT1.HRLC-Q-TOF-MS/MS was used to analyze and identify the metabolites and conjugates of bentazone in wild-type,OE-3 and Cas9-4 rice.A total of6 metabolites and 15 conjugates were identified.Metabolism of bentazone in rice is mainly formed by substitution,hydrolysis,reduction,glycosylation,methylation or acetylation.The relative contents of 5 metabolites and 4 conjugates in the shoot and 2 metabolites and12 conjugates in the root of OE-3 were significantly higher than those in the wild-type rice.The relative contents of 3 metabolites and 4 conjugates in the shoot of Cas9-4 and 4metabolites and 8 conjugates in the root of Cas9-4 were significantly lower than wild-type rice.These results indicated that Os MT1 promoted the detoxification and metabolism of bentazone in rice. |
