Resistance Mechanism Of Empoasca Onukii To Thiamethoxam

Empoasca onukii(Matsuda)is one of the most important insect pests in tea-growing areas,it does great harm to the production of tea due to widely host,strong adaptability and many generations annually,chemical control is the main method for the management of this pest.The resistance of E.onukii has become more obvious caused by chemicals abuse recently.With its excellent contact,systemic and stomach toxicity activities,high-efficiency,low toxicity and broad spectrum of insecticidal properties,thiamethoxam has become one of the main nicotine insecticides for chemical control to E.onukii.Previous studies have shown that the resistance of E.onukii to thiamethoxam has increased year by year by year.The evaluation of the resistance mechanism of the leafhopper to thiamethoxam is an important basis for the resistance management strategy and development of the pesticides.Therefore,the biochemical and molecular resistance mechanism of E.onukii to thiamethoxam was studied from the aspects of life table,screening of resistant and sensitive strains,enzyme activity determination,transcriptomics,amplification and prokaryotic expression of resistance genes based on the resistant and sensitive strains which were acquired from indoor domestication,and the main results are as follows:1.The Life Table of E.onukii in Yunnan-Guizhou Plateau was Constructed and the Resistant Strain and Sensitive Strain were ScreenedThe results showed that the adult has longest age which was 18.90 days,next was eggs which was 9.02 days,and L1-L5 was 1.28,1.92,2.12,2.78,1.37,respectively.There is an significant overlap between the curves of the age-stage survival rate(S_xj)of different instar at 25°C.With the time increased,the age-specific survival rate(l_x)of E.onukii began to decline at 31 days,and all individuals died at 44days;the curves of female age-stage specific fecundity(f_x)and age-specific fecundity(m_x)increased with the time gradually,and then began to decline a round 31 days;the curves of life expectancy(e_xy)gradually decreased with the increase of age;the reproductive value(v_xj)of a newborn egg of the female of E.onukii was 1.065,and the reproductive value increased with the development stages and the increasing age,the reproductive value of the female reaches the peak at the tenth day.The toxicity regression equations of sensitive strains of E.onukii at 24 h,48 h,72 h was y=4.2952+1.6306x,y=4.6528+1.9017x,y=5.0957+2.0957x,respectively;the correlation coefficients was:0.9982,0.9705,0.9362,respectively,indicating that the linear relationship of these equations was good;the LC₅₀ was 2.7055 mg/L,1.5226 mg/L,0.8971 mg/L,respectively.After 15 generations continuous screening,LC₅₀ of resistant strain reached to 52.4806 mg/L which was 19.40 times of the initial LC₅₀,the E.onukii becomes moderately resistant to Thiamethoxam.2.The Biochemical Mechanism of Resistance of E.onukii to Thiamethoxam was ExploredThe synergistic ratios of Piperonyl butoxide(PBO),Diethyl maleate(DEM),Triphenyl phosphate(TPP)on sensitive strain of E.onukii was 1.4115,1.2251,0.8911,respectively;while the resistant strain was 3.0153,2.8508,1.010,respectively.The above results showed that the synergism of PBO and DEM to thiamethoxam were good,while the synergism of TPP was general.The mixed-function oxidase activity of the sensitive strain and resistant strain of E.onukii was 0.2977mmol/pro(mg)/30 min and 1.9707mmol/pro(mg)/30 min,respectively.The difference was significant between the sensitive and resistant strains of E.onukii,and the mixed-function oxidase activity of the resistant strain was 6.62times than that of the sensitive strain.The glutathione S-transferase activity of the sensitive strain and resistant strain of E.onukii was 33.3337 U/mg prot and 182.2680 U/mg prot,respectively.The one-way ANOVA showed that the difference was significant between two groups,the glutathione S-transferase activity of the resistant strain was 5.47 times than that of the sensitive strain.The acetylcholinesterase activity of the sensitive strain and resistant strain of E.onukii was 0.2934 nmol/min/mg prot and 0.3002 nmol/min/mg prot,respectively,and the difference was not significant between two groups throught the one-way ANOVA,the acetylcholinesterase activity of the resistant strain was only 1.02 times than that of the sensitive strain.3.This Research Expounds the Molecular Mechanism of Resistance of E.onukii to ThiamethoxamThrought the analyzing of the transcriptome of the sensitive strain and the resistant strain of E.onukii,a total of 222,059 unigenes were obtained,these unigenes were annotated through the Nr,NT,KO,Swiss Prot,PFAM,GO,KOG databases;7844 unigenes were successfully annotated in all of the databases;107653 unigenes were successfully annotated in at least one database;and 114406 unigenes were not successfully annotated,the role and function of which need to be further explored and analyzed.Through the alignment of Blastx,a total of 67224 nucleic acid sequences(CDS)that encode the protein were obtained;a total of 56437 sequences encoding the nucleic acid and amino acid(CDS)were obtained by estscan prediction.The differential unigenes between the treatment group and the control group were screened based on the threshold pval<0.01,fold change?2,3131 differential unigenes were obtained,of which 169 were significant up-regulated and 1423 were significant down-regulated.Of the 3131 differential unigenes,a total of 1598 were successful annotated to thirty functional groups of three categories of the GO database;in the category of biological process,the function group of proteolysis and oxidation-reduction process have more unigenes,the number of unigenes of them was138 and 139,respectively;in the category of molecular function,the function group of peptidase activity that acting on L-amino acid peptides,peptidase activity,endopeptidase activity,oxidoreductase activity and hydrolase activity have more unigenes,the number of unigenes of them was 142,144,102,137 and 296,respectively;the results showed that the resistance of E.onukii to thiamethoxam in biology was reflected as the different metabolic activities of cells in vivo,which was mainly related to the binding or catalytic activity in cells.A total of 11 unigenes in differentially expressed unigenes were related to pesticide resistance;these unigenes included cytochrome P450,glutathione S-transferase,Cytochrome b,ATP synthase,and the log2Fold Change of P450 and glutathione S-transferase was higher which indicated that metabolic resistance may be the main resistance of E.onukii to thiamethoxam.4.The Full-length c DNA of Resistance Gene Eo GSTs1 was Cloned and Bioinformatics Characteristics was Analyzed.The full-length c DNA of Eo GSTs1 gene(Cluster-166.0)was successfully amplified throught cloning,its length is 841 bp.Its coding region is 624-bp long,encoding a total of 207 amino acids including the start codon ATG and the stop codon TGA.The noncoding region at the 5'end is 99 bp in length,and the 3'end noncoding region is 118 bp in length followed by a 16-bp poly(A)tail;the molecular weight is23.68932 k Da,with a calculated isoelectric point(IEP)of 6.00.The numbers of negatively and positively charged residues are 30 and 29,respectively.Its N-terminus begins with a methionine and the half-life of the protein is 30 h.The instability index of the protein in solution is 31.87,the fat coefficient is 78.26,and the total average hydrophobicity index is-0.376.The protein contains a total of 3331 atoms,including1078 carbon atoms,1662 hydrogen atoms,276 nitrogen atoms,305 oxygen atoms and10 sulfur atoms.It contains 20 different types of amino acids,among which the top three are alanine(Ala or A),lysine(Lys or K),and glutamic acid(Glu or E),with numbers of 19(9.2%),19(9.2%),and 16(7.7%),respectively;the number of amino acids of numbers Cys(C)and His(H)was low,with 2(1%)and 1(0.5%),respectively.There is no signal peptide and no transmembrane helix region in the Eo GSTs1 gene protein.In addition,the catalytic domain of the protein contains 69amino acid residues.Comparison of the protein sequences between EoGSTs1 and the GSTs of four other insects in the Hemiptera order using Clustal Omega revealed that multiple conserved domains exist.The amino acid sequence of Eo GST was highly homologous to GSTs in Sub psaltriayangi,Locusta migratoria,Daktulosphaira vitifoliae and Leptinotarsa decemlineata,with a similarity of 53%,52%,52%,and 50%,respectively.The phylogenetic tree of the full-length EoGSTs1 protein sequence and GST sequences of insects from 5 other orders,including Hemiptera,Orthoptera,Coleoptera,Odonata and Hymenoptera,were constructed using BI phylogenetic analysis and an ML-based method.The topological structures of the phylogenetic trees generated using either ML or BI displayed substantial similarities.In the cluster analysis,the Eo GSTs1 gene appears to cluster with the known insect GST in the Hemiptera order,but it did not cluster with GSTs from insects in the Odonata or Hymenoptera order.These results suggest that E.onukii Matsuda is very closely related to insects in the Hemiptera order and has a relatively close genetic association with the order Orthoptera but is only remotely related to insects in the orders of Hymenoptera,Coleoptera,and Odonata.A cluster analysis of Eo GST gene and 54 other GST sequences belonging to 6GST families was performed using the adjacency method.The cluster analysis demonstrated that the Eo GST gene clustered with the Sigma subfamily of GSTs with a high bootstrap value of 99%of a close genetic association.However,it did not cluster with GSTs in the Delta or Epsilon subfamilies,indicating a distant phylogenetic relationship between Eo GST with these two subfamilies of GSTs.The Eo GSTs1 gene encodes a protein with 207 amino acids and contains 9?-helices and 2?-sheets.The Eo GSTs1 gene protein belongs to the superfamily of GST and shares sequence similarities with GSTs in other insects.In addition,the protein sequence of EoGSTs1 contains 2 Asn-Xaa-Ser/Thr motifs,one of which is an N-glycosylation site.The protein sequence also contains 8 other potential phosphorylation sites:4 serine phosphorylation sites located at positions 95,118,152,and 166 in the peptide chain;2 threonine phosphorylation sites located at positions 47and 125 in the peptide chain;and 2 tyrosine phosphorylation sites located at positions148 and 188 in the peptide chain.The C-score of the protein structural comparison is 1.21,indicating that it is very similar to 4Q5R in protein folding and secondary structure.The TM-score,representing the structural similarity between the target sequence(EoGSTs1)and the template protein sequence(Bg GST),is 0.88±0.07.5.EoGSTs1 Has been Successfully Obtained,and Its Enzymatic Characteristics was Preliminarily AscertainedThe target protein EoGSTs1 was obtained by prokaryotic expression and Ni affinity chromatography.The results of SDS-PAGE electrophoresis showed that the recombinant enzyme was mainly presented in an soluble form in the protein gel.Western blot results showed that the target protein EoGSTs1 was consistent with the predicted protein molecular weight of 23.68932 k Da.The results of enzyme activity assay showed that recombinant EoGSTs1(r EoGSTs1)protein had catalytic activity for substrate CDNB,and had the highest activity at p H 7 and 25?.The Michaelis constant Km of r EoGSTs1 was 0.07782±0.01990 mmol/L,and the maximum reaction rate Vmax was 12.15±1.673?mol/min·mg.Our study clarified the taxonomic identity of Eo GST of E.onukii and revealed some properties of the gene and its encoded protein sequence;according to the catalytic activity of its r EoGSTs1 enzyme to the model substrate CDNB,we inferred that it has the function of degradation of exogenous substances.