| Echinochloa glabrescens Munro ex Hook.f.is one of the most common Echinochloa weed species,widely distributed in the middle and lower reaches of the Yangtze River,such as Anhui,Jiangsu,and Shanghai,seriously affecting the quality and yield of rice.The ALS inhibitor herbicide penoxsulam is an important herbicide for controlling weeds in the rice field.In recent years,studies have reported that hardworm has gradually developed resistance to penoxsulam.Cytochrome P450s-mediated metabolic resistance has increasingly become the focus of research by resistant weed institutes.In order to investigate the mechanism of resistance of E.glabrescens to penoxsulam mediated by cytochrome P450s.In our study,the resistant population SHQP-6 and the sensitive population JYJD-3 are taken as the research objects.P450 inhibitors PBO,malathion,and ABT demonstrate the existence of metabolic resistance mediated by cytochrome P450s in SHQP-6.We althrough studied the content and activity of P450s.To investigate the mechanisms of metabolic resistance,transcriptome sequencing analysis was performed to find candidate genes that may confer resistance to penoxsulam in E.glabrescens.27 P450 DEGs were verified by qPCR,then CYP71C2 was selected for functional verification.The following results and conclusions were obtained from the above experiments:1.Study on the relationship between the resistance of E.glabrescens to penoxsulam and cytochrome P450sWe tested the sensitivity of P450s inhibitors PBO,malathion,and ABT pre-treated E.glabrescens to penoxsulam.The results showed that compared with sensitive populations,the cytochrome P450s inhibitors PBO,malathion,and ABT all increased the sensitivity of SHQP-6 to penoxsulam.In this study,we found that the GR50 of the resistant population(SHQP-6)decreased substantially from 25.6 to 5.0 and 6.2 g a.i.·ha-1 after treatment with the P450 inhibitors piperonyl butoxide(PBO)and malathion,respectively.However,P450 inhibitors almost had no effects on the susceptibility of the sensitive population(JYJD-3)to penoxsulam.The result of enzyme content determination showed that after treatment with penoxsulam,the cytochrome P450 content of resistant population SHQP-6 showed an upward trend from 0-3d,and reached a maximum value of 1.2251 nmol·mg-1 pro on the 3rd day,and gradually returned to the unadministered level on 3d-7d.The sensitive population generally showed a downward trend,and the lowest value was 0.6866 nmol·mg-1 pro on the third day,and the values of the resistant population were higher than those of the sensitive population after 1-7d.The resistant population SHQP-6 and the sensitive population JYJD-3 cytochrome b5 content showed an overall upward trend at 0-7d,and reached the highest value at 3d.The resistant population was 0.8994 nmol·mg-lpro,which was significantly higher than(P>0.05)0.3005 nmol·mg-1 pro in sensitive population.The result of enzyme activity determination showed that after treatment with penoxsulam,The NADPH-cytochrome P450 reductase activity of the resistant population and the sensitive population showed an upward trend from 0 to 3d,and the resistance population was 0.1623 nmol·min-1mg-1pro at the highest value on the 3rd day,which was significantly higher than(P>0.05)0.1459 nmol·mg-1pro in sensitive population.The p-nitroanisole-O-demethylase activity(PNOD)of the resistant population showed an upward trend from 0 to 3d,and reached a maximum value of 0.8886 nmol·min-1mg-1pro on the 3rd day,while the sensitive population showed a downward trend after treated with penoxsulam.2.Discovery of cytochrome P450 genes related to penoxsulam.Although the mechanism of metabolic enzymes against resistance E.glabrescens has been studied from the physiological and biochemical level,but the molecular mechanism remains unclear.To investigate the mechanisms of metabolic resistance,transcriptome sequencing analysis was performed to find candidate genes that may confer resistance to penoxsulam in E.glabrescens.A total of 21,077 DEGs(15,114 upregulated,5963 downregulated)were obtained.Among them,233 P450 differentially expressed genes(DEGs)(157 upregulated,76 downregulated)were identified.GO and KEGG analysis revealed that the differential genes were mainly enriched in metabolic processes.Based on the results of gene function annotation of Nr,KO,SwissProt,PFAM,GO,and KOG in 6 large databases,we obtained 27 metabolically related P450 expression differential genes.In order to further explore the expression patterns of these 27 genes,we selected them for qPCR verification at a time point consistent with transcriptome sequencing.The result showed that the relative expression of twenty-three P450 DEGs(eighteen from the CYP72A-71C-74A-96A-734A subfamily;five from CYP81E1-94C1-94B3-714C1-714C2)were upregulated and four P450 DEGs(from CYP724B1-711A1-707A7-97B2)were downregulated.Our study was the first time performed transcriptome sequencing in E.glabrescens,and the P450 genes related to penoxsulam was fully Discovered.3.Study the function of CYP71C2 in penoxsulam-resistant E.glabrescensThis experiment was performed by overexpressing CYP71C2 in Arabidopsis,The constructed pCambia-CYP71C2GFP vector was transformed into Agrobacterium strain GV3101,and then inoculated into Arabidopsis thaliana by inflorescence infection.The transformed Arabidopsis strains were subjected to protein SDS-PAGE electrophoresis and Western blot detection.The result showed that CYP71C2 can be normally expressed in Arabidopsis.qPCR results showed that the relative expression of CYP71C2 in Arabidopsis was up-regulated by more than 22 times compared to the control.After two generations of successfully transformed Arabidopsis strains were cultured for seed bioassay experiments,it was found that the sensitivity of the transgenic Arabidopsis strains to penoxsulam was lower than that of wild-type Arabidopsis strains.At the same time,the subcellular localization of the gene was performed on Tobacco nigra,and observation under a confocal laser scanning microscope revealed that the CYP71C2 gene was mainly localized on the nucleus.In conclusion,one E.glabrescens population(SHQP-R)showing resistance to the ALS herbicide penoxsulam was identified.This population displayed CytP450-induced metabolic resistance.And through a series of experimental,such as discovered of related genes and functional verification,the mechanism of penoxsulam resistance was studied in depth.This study may greatly improve our understanding of herbicide metabolism resistance mechanisms,and also help identify the P450 genes involved in herbicide metabolism.Moreover,these results are potentially beneficial for developing methods to counter herbicide-resistant weeds. |
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