| Pesticides are used to prevent diseases,insects and weeds in modern agriculture and make an important contribution to the improvement of crop yield and quality.However,due to the longevity of their residual period and nonstandard utilization,pesticide pollution and residue have been becoming an essential issue concerning about animals,plants and human health.Isoproturon is a widely used substituted urea pesticide in the world.The use of isoproturon for long-term causes not only the pollution to the natural environment,but also the enrichment of the food chain threatening human health.Acetochlor is one of the herbicides most commonly used in China,which not only is toxic to mammals,but also has a certain impact on soil microorganisms and crops.Besides,it causes potential risks in surface water and underground water contamination.Therefore it has attracted people's attention all over the world.Glycosyltransferases(GTs)is a group of enzymes universally existing in living organism,transfer sugar molecules from donors to receptors to form glycosidic bonds.Specifically,GTs in plants play an important role in synthesis of polysaccharides of cell wall and plant glycoproteins,as well as the regulation of metabolism.This thesis mainly focus on the function of GTs genes in detoxification and metabolic of isoproturon and acetocholor in rice for reducing the potential risks of pesticides to the ecological environment,crops,animals and human.In this research,high-throughput sequencing results of rice seedling transcriptome under isoproturon stress were analyzed.Rice GTs genes induced by isoproturon were screened.Tobacco subcellular localization and bioinformatics of a GTs gene with significantly up-regulated differential expression were studied.The rice of overexpressing isoproturon-responsive glycosyltransferase gene(here referred as OsIRGT1)was obtained by molecular cloning.This gene can alleviate the toxicity of isoproturon to rice and accelerate the degradation of isoproturon in rice.The toxicity of acetochlor and its accumulation were studied for rice.The metabolism and detoxification of acetochlor by this gene in rice were also studied.We generated transgenic pichia pastoris expressing OsIRGT1 by genetic engineering technology to further verify its functionality.It was testified that the rice GTs gene was an effective way to remove and degrade herbicide.Specific content as follows:1.High-throughput sequencing results of rice transcriptome under isoproturon stress were analyzed.145 differentially expressed GTs genes were screened.Among them,18 GTs genes were expressed differently two times.12 GTs genes were randomly selected which analyzed and verified by qRT-PCR.The result of verification was consistent with the trend of regulation.Based on the bioinformatics databases,we presented phylogenetic assay of these 18 GTs genes,as well as their cis-elements prediction,chromosome location and domain analysis.The activity of GTs and the accumulation of isoproturon in rice were analyzed.2.High responsive level OsIRGT1 to isoproturon was selected as the research object from two times of differential expression in GTs genes.Molecular biological information of OsIRGT1 was systematically analyzed and the subcellular localization of OsIRGTl in tobacco was recorded where located in ER.Studies on the expression profile of the gene have shown that it's not only responded to the induction of isoproturon,but also responsive to another kind of herbicide acetochlor.Three OsIRGT1 homozygous overexpression rice lines(OE-1,OE-3 and OE-5)were obtained by the molecular biology technology to study the regulatory function of this gene on the degradation of two pesticides in rice.The OsIRGT1 expression levels in the three overexpression rice lines were examined,as well as the corresponding GTs activity.OsIRGT1 gene can be expressed stably in all three lines.3.To investigate the effect of OsIRGT1 on detoxification and degradation of isoproturon in rice,three homozygous overexpression lines(OEs)and mutant were selected as the research objects,and wild-type rice as the blank control.Compared with the wild type,the elongation,dry weight and chlorophyll content of OEs were significantly increased,and the damage of membrane and the content of malondialdehyde(MDA)were decreased,after 4 days treatment of 2 mg/L isoproturon.On the other hand,the growth of mutant was worse than that of wild-type rice,with the electrical conductivity and the content of MDA significantly increased,indicating that OsIRGT1 gene helped detoxification of isoproturon in rice.The accumulation of isoproturon in OEs rice was lower than that in the wild type,while the accumulation in the wild type was lower than that in the mutant.This result suggested that OsIRGT1 promoted the degradation of isoproturon in rice.Meanwhile,the residual amount of isoproturon in the nutrient solution planted OEs rice was significantly lower than that of the wild type,while the residual amount of isoproturon in the nutrient solution planted mutant rice was significantly higher than that of the wild type.To further investigate the degradation effect of OsIRGT1 on isoproturon in the environment,the concentration of 0.05 mg/L isoproturon was selected for long-term treatment of wild-type,with OEs and mutant rice.It was found that the contents of isoproturon in shoots and roots of the OEs were significantly lower than that of the wild-type,while the mutant was higher than that of wild-type rice after 30,45,60 and 90 days exposure to isoproturon.After 100 and 120 days of exposure,the accumulation of isoproturon in OEs green and mature grain was reduced by 30.8-40.7%,which is lower than that in wild type while that of mutant was higher than wild-type.To explore the detoxification pathway of isoproturon by OsIRGT1 further.The metabolites and conjugates of isoproturon in the line OE-3 of overexpression rice and wild-type rice was detected by HPLC-Q-TOF-MS/MS.Sixteen metabolites and nine conjugates of isoproturon were detected,in which two metabolites(1-Methyl-3-phenyl-urea,1,1-Dimethyl-3-phenyl-urea),five glycosylation conjugates(1-Isopropenyl-IPU-N-malonylglucoside,2-Methoxyl-IPU-N-acetylglucoside,2-IPU-N-acetylglucoside,1-OH-Isopropyl-benzene-O-glucoside,2-Methoxyl-IPU-N-glucoside)and two amino acids conjugates(4-Amino-4-[4-(1-hydroxy-1-methyl-ethyl)-phenylcarbamoyl]-butyric acid,2-Amino-N-(4-vinyl-phenyl)-acetamide)were found in plant for the first time.The contents of twelve metabolites and seven conjugates in OE-3 shoots,and the contents of four metabolites and three conjugates in roots were higher than that in wild type.Finally,the possible metabolic pathways of isoproturon degradation in rice were speculated.The aforementioned results confirmed that OsIRGT1 promoted the detoxification and degradation of isoproturon in rice.4.For investigating the toxicity of acetochlor to rice,a series of acetochlor concentrations(0,0.05,0.1,0.2,0.4 and 0.8 mg/L)were selected to treat rice seedlings.The results showed that the content of malondialdehyde in rice firstly increased and then decreased with the increase of acetochlor concentration,and reached the maximum at a concentration of 0.1 mg/L for 6 days,indicating the most severe impair of rice cell membrane.Acetochlor also inhibited the biosynthesis of chlorophyll of rice.The GTs activity of rice was induced to improve under exposure to different concentrations of acetochlor for 6 days.It was found that with the increment of treatment time and concentration of acetochlor,the accumulation of acetochlor in rice gradually increased,as well as its toxity.5.The degradation and detoxification ability of glycosyltransferase OsIRGT1 gene was further verified through treatment with acetochlor,a different type of pesticide compared to isoproturon.Rice seedlings were treated with acetochlor at 0.1 mg/L for 6 days.The results were similar to those with isoproturon.The growth of OEs rice was apparently better than that of wild type,and the chlorophyll content was significantly higher than that of wild type.The content of malondialdehyde was lower than that of wild-type rice seedlings.The electrolyte permeability of the OEs shoot and root was significantly lower than that of the wild-type.The results of mutants were contrary to those of OEs.Compared with the wild-type,the accumulation amount of acetochlor in the OEs and in the nutrient solution were both significantly reduced.Meanwhile,the concentration of acetochlor in the mutant plant tissues and the nutrient solution were distinctly increased compared with the wild-type.The data analysis of the content of acetochlor in rice and nutrient solution demonstrated that the OEs rice had a stronger accumulation ability and upward conduction effect on acetochlor,while the mutant's were relatively weaker.Under long-term exposure,the content of acetochlor in shoots and roots of OEs was lower than that of wild-type rice,and the mutant was higher.After 120 days of the exposure,the content of acetochlor in mature grains decreased by 42.6-48.4%in OEs compared with the wild-type,while increased in mutant.The metabolites and conjugates of acetochlor in wild-type and OE-3 rice were identified by HPLC-Q-TOF-MS/MS.Nineteen metabolites and thirteen conjugates of acetochlor were detected and identified.The relative amount of eleven metabolites and eight conjugates in OE-3 shoots,and nine metabolites and six conjugates in roots were significantly higher than the wild-type.These results confirmed that OsIRGTl gene has the detoxification and degradation effect in rice not only on isoproturon,but also on acetochlor.6.To further verify the ability of OsIRGT1 on degradation and detoxification of isoproturon and acetochlor,OsIRGT1 was transferred into Pichia pastoris X-33 for heterogenous expression OsIRGT1 and functional analysis.To study the toxicity resistance of recombinant yeast to isoproturon and acetochlor,of isoproturon and acetochlor 0,0.5,2 and 8 mg/L isoproturon and 0,0.02,0.1 and 0.5 mg/L acetochlor were added into the YPD medium,respectively.Compared with blank yeast cells,the recombinant one showed no difference in the control group,while grew better under pesticide treatment,especially at the level of 8 mg/L isoproturon or 0.5 mg/L acetochlor.Subsequently,recombinant yeast and blank yeast cells were inoculated with 2 mg/L isoproturon and 0.1 mg/L acetochlor in YPD liquid medium,respectively.For 24,48 and 72 hours,results showed the degradation rate of recombinant yeast was significantly higher than that in blank yeast cells.At 72 hours,the degradation rate of two pesticides in recombinant yeast strains was by 2.4 and 2.6 times,respectively,which is higher than that of blank yeast strains.The above results futher validated that OsIRGT1 could enhance the degradation and detoxification of pesticides. |
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