Excavation And Functional Verification Of Tribenuron-methyl Metabolic Resistance Genes In Myosoton Aquaticum L.moench.

China is the world's largest wheat producer and consumer,and wheat is China's second largest food crop.Myosoton aquaticum L.is a common broadleaf weed widely distributed in wheat fields,especially in regions employing wheat-rice rotations.Currently,herbicides used in the field to control M.aquaticum are mainly ALS inhibitor herbicides represented by tribenuron-methyl,and these herbicides have low toxicity,high efficiency and safety to mammals.However,due to the single action site and long-term heavy reuse of this herbicide,M.aquaticum have gradually developed resistance to ALS inhibitors.A recent survey in wheat fields showed more than half the populations were difficult to control using ALS inhibitors during the cropping period,especially in Henan,Anhui,and Jiangsu Provinces.This study aimed to explore the resistance mechanism of HN10 from Henan Province.Meanwhile,focus on the non-target resistance mechanism of HN10 and JS17(coexist of TSR and NTSR in this population)and try to find out metabolic-related resistance genes can provide theoretical guidance for scientific control of M.aquaticum.The main results were as following:1.The HN10 population was treated with tribenuron-methyl(11.25 g a.i.ha^-1)at the recommended rate on the whole plant level.The results showed that the HN10 population has evolved resistance to tribenuron-methyl.Meanwhile,the resistance levels of HN10 field populations were determined.The result showed that HN10 evolved moderate-level resistance to tribenuron-methyl,with resistance index(RI)6.15.2.The gene fragment of ALS gene was amplified and sequenced;the result showed that there were on known ALS resistance mutations in HN10 M.aquaticum population.3.In vitro ALS activity was determined,the total ALS activity of HN10 was lower than that of the sensitive population,but ALS sensitivity to tribenuron-methyl has no significant difference between the two populations.4.A real-time quantification PCR assay was conducted to analyze the relative expression level of ALS gene of HN10 and HN03 populations.The result revealed that there was no difference in expression level of the ALS gene between the two populations before and after herbicide treatment.5.The activity of P450s in resistant populations was investigated by comparing the effective tribenuron-methyl dose causing a 50%growth reduction(GR50)in the absence and presence of P450s inhibitor malathion.The results indicated that malathion significantly decrease the GR50 for tribenuron-methyl in HN10 population.The GR50 value of HN10 and HN03 population decreased 5.53 and 1.69 times respectively.It can be seen that there is higher P450s activity in HN10 population.6.The LC-MS/MS method was used to determine the residual rate of tribenuron-methyl in three populations after 1,3,5,7 and 9 days of application of tribenuron-methyl,in order to determine the metabolic difference of tribenuron-methyl in three populations.The results showed that tribenuron-methyl had the faster metabolic efficiency in the high-resistance population JS17 and moderate-resistant population HN10,followed by the sensitive population HN03.In addition,due to the application of malathion,the residual rate of tribenuron-methyl in three populations was increased,and the residual rate increase ratio from high to low were JS17,HN10 and HN03 compared with no application of malathion.7.The changes of GSTs activity in JS17,HN10 populations and sensitive population HN03 were studied by using CDNB as substrate.The result showed that GST activity could be induced by tribenuron-methyl in three populations.The GST activity of HN10 population was slightly higher than HN03 population at the 2d and 3d after tribenuron-methyl application,but there was no significant difference in GSTs activity between the two populations;The GST activity of JS17 was always higher than that of HN03.8.HN10 population was screened with a range of herbicides with known activity on this species.According to the“R”resistance rating system,HN10 has evolved“RR”resistance to pyrithiobac-sodium,florasulam,flucarbazone-Na and diflufenican,and“R?”(resistance may be developing)to pyroxsulam and imazethapyr,but remained sensitive to MCPA-sodium,fluroxypyr-meptyl,and isoproturon.9.Using the RNA-Sequencing technique,the F2 generation of M.aquaticum was used as a material to screen for differential genes related to the resistance to tribenuron-methyl metabolism.The differential genes were verified by two qRT-PCRs and identified as the final candidate genes.The results showed that there were P450s(3),ABC transporters(3),and peroxidase(2)in MRT_ST and P450s(3),GSTs(2),GTs(2),ABC transporters(4),peroxidase(2),oxidase(1)and hydrolase(1).10.We selected three genes(c36251_g1,c46086_g1 and c40142_g1)in HRT_ST which were annotated to CYP86A2?GST-DHAR2 and Peroxidase 70 for further functional verification.Phenotypic identification of transgenic homozygous Arabidopsis plants showed that transgenic CYP86A2 and GST-DHAR2 Arabidopsis showed higher resistance compared with WT under 0.5?M concentration of tribenuron-methyl;transgenic CYP86A2 Arabidopsis showed higher resistance compared with WT under 0.01?M concentration of florasulam.After treatment with the tribenuron-methyl,the expression of GST-DHAR2 and CYP86A2 was significantly up-regulated in Arabidopsis leaves;the expression of GST-DHAR2 in roots was almost unchanged,and the expression level of CYP86A2 in Arabidopsis roots was relatively low.In summary,this study preliminarily identified the resistance level to tribenuron-methyl and resistance spectrum of HN10,and identified the non-target resistance mechanism of HN10 population.Using RNA-seq,screening for some differential genes related to tribenuron-methyl metabolism,enriching the gene pool of weeds,the preliminary expression of the candidate gene was verified by real-time PCR.The Agrobacterium-mediated transformation technology was used to validate the GST-DHAR2?CYP86A2 and Peroxidase70 genes function.As a result this sutdy provide a theoretical basis for the metabolic genes functional verification and hope to provide scientific guidlines for the control of this specific resistant M.aquaticum.