| The Rhopalosiphum padi(R.padi)is an important pest in wheat,which occurs in all wheat areas in our country.The economic loss caused every year seriously affects the development of Chinese grain industry.At present,the R.padi is mainly dependent on chemical insecticides,but the problem of resistance is becoming more and more serious.Beta-cypermethrin is a common pyrethroid insecticide,which has the advantages of strong insecticidal activity,low toxicity,high persistence and good stability.Its target is sodium channel,and it is widely used in the control of aphids and other stinging insects.The resistance mechanism of insect pests to insecticides is complex and diverse,which is caused by multi-gene and multi-level co-regulation.At present,more researches on the resistance mechanism include target resistance,metabolic resistance and epidermal resistance.In this paper,the sensitivity of R.padi population to beta-cypermethrin and other commonly used insecticides in the field of major wheat areas was systematically monitored in 2021.Then,the metabolic resistance and epidermal resistance mechanisms were analyzed using transcriptional sequencing technology.Finally,cloning and functional verification of UGT single genes with high overexpression ratio were studied.Results are as follows.1.Field population resistance monitoring of R.padiThe monitoring results of the resistance of R.padi to beta-cypermethrin in China showed that the resistance of aphids to beta-cypermethrin in Guiyang,Guizhou Province,Linfen,Shanxi Province,Xinxiang,Henan Province,Hefei,Anhui Province and Kunming,Yunnan Province was at a medium level,while that of Xining,Qinghai province and Xiangyang,Hubei Province was at a low level.Other populations were still sensitive to beta-cypermethrin,and the resistance ratio of Guizhou Guiyang population to beta-cypermethrin was 96.9 times.Risk of developing high levels of resistance;The monitoring results of to sulfoxaflor showed that Xinxiang of Henan had a high level of resistance with a resistance ratio of 145.6 times,Xiangyang of Hubei,Xining of Qinghai,Hefei of Anhui and Guiyang of Guizhou had a medium level of resistance,Linfen of Shanxi,Kunming of Yunnan and Yangling of Shaanxi had a low level of resistance,and other populations were still sensitive to sulfoxaflor.The monitoring results of resistance to imidacloprid showed that Guiyang in Guizhou province had high resistance with a resistance ratio of 159.0 times,Hefei in Anhui Province and Kunming in Yunnan Province had medium resistance,Xinxiang in Henan province and Langfang in Hebei province had low resistance,and other populations were still sensitive to imidacloprid.The monitoring results of abamectin resistance showed that Xiangyang in Hubei Province,Hefei in Anhui Province and Guiyang in Guizhou province had moderate resistance,Langfang in Hebei province,Linfen in Shanxi Province and Kunming in Yunnan province had low resistance,and other populations were still sensitive to abamectin.The monitoring results of chlorpyrifos resistance showed that Langfang(Hebei),Yili(Xinjiang),Xinxiang(Henan)and Guiyang(Guizhou)had moderate resistance,Linfen(Shanxi),Xining(Qinghai),Xiangyang(Hubei)and Yangling(Shaanxi)had low resistance,and other populations were still sensitive to chlorpyrifos.2.Cloning of R.padi sodium channel gene and detection of kdr mutationBy clone the total length of sodium channel of beta-cypermethrin resistant and sensitive strains of R.padi,and compared the two strains of sodium channel except for the mutation of super-kdr M918 L,no other mutation points were found to be involved in the increase of resistance to beta-cypermethrin,and it was found that all the M918 L mutations were homozygous mutations.Then,the mutation point M918 L mutation was detected in 2018,2019 and 2021 all over China,and the resistance degree was significantly correlated with the mutation of sodium channel M918 L by comparing with the results of current virulence tests.3.Screening of R.padi anti-beta-cypermethrin-related detoxification and epidermal protein genesIn this study,we constructed a gene library of R.padi to beta-cypermethrin and assembled the sequenced data.We obtained 17,985,440-25,478,353 clean data from different transcriptomes.Of these,17,183 genes were annotated.These transcripts were then divided into groups for comparison to obtain a more comprehensive picture of resistance-related genes.There were 178-2856 differentially expressed genes(DEGs)in these transcriptomes.DEGs were classified according to the GO and KEGG Pathway databases.In addition,some drug-resistant DEGs are related to keratin and detoxification metabolism.Among them,17 cuticle protein related genes were up-regulated and 20 down-regulated,11 P450 s related genes were up-regulated and 25down-regulated,7 UGTs related genes were up-regulated and 15 down-regulated,2ABC transporter-related genes were up-regulated and 4 down-regulated,and 2 trypsin related genes were up-regulated and 1 down-regulated.Finally,q RT-PCR using DEGs confirmed the trends observed in RNA sequencing expression profiles,and most of the results were consistent with RNA sequencing(RNA-seq).Meanwhile,it was found that the expression level of g4263 was significantly increased in resistant strains.4.Identification and functional verification of aphid detoxification enzyme gene UGT344D31According to NCBI analysis,the g4263 gene highly expressed in the resistant strains was UDP-glucose-transferase gene,which was named UGT344D31(UGTg4263)by the UGT International Nominational Committee.Using nanomaterials combined with microdrip method,the 360 ng ds RNA interfered with UGT344D31 gene,and the interference efficiency reached 60% after 24 h.At the same time,when UGT344D31 was silenced under the same conditions,the mortality rate increased from 25% to 50%at the concentration of 5000mg/L beta-permethrin.Synergist test results showed that the UGT enzyme inhibitor 5-nitrouracil(5-NU)showed synergistic effect on beta-cypermethrin,the synergistic factor of sensitive strain was 3.04,the synergistic factor of resistant strain was 9.86,and the relative synergistic factor of 3.24.The results of UGT344D31 functional verification and UGT enzyme inhibitor tests showed that UGT detoxification enzyme may be involved in the detoxification of pyrethroid insecticides.According to the monitoring results,the R.padi in Guiyang,Guizhou province and Xinxiang,Henan province developed different degrees of resistance to beta-cypermethrin,sulfoxaflor and imidacloprid and other agents.Beta-cypermethrin is still the main agent used in field control of R.padi,so the combination of beta-cypermethrin with sulfoxaflor and neonicotinoid insecticides should be avoided.Moreover,the important mechanism of R.padi ’s high level of resistance to beta-permethrin was significantly related to the mutation of M918 L gene and the significantly increased expression of UGT344D31 gene. |
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