The Rules For The Gene Expression Of Tetranychus Cinnabarinus (Boisduval) During The Resistance Development Against Cyflumetofen

The goal of scientific research is to reveal essence of nature.The resistance of pests(mites)to insecticides or acaricides has existed for a long time,among which mites have developed the most prominent resistance problems.As we all know,the expression of a large number of genes is changing during the development of resistance.However,researchers have focused on the relationship between the expression change of a gene or gene family with the development of resistance for a long time,but change rules of all genes in the process of resistance development.Revealing the change rules of differentially expressed gene(DEGs)in the process of resistance development will help to analyze the reasons of development resistance in the molecular level,and then combining the change rules to screen out potential resistance molecular markers,providing theoretical basis and technical support for the management of resistance.Low-cost and high-throughput transcriptome sequencing technology makes it possible to understand the change rules of a large number of genes.Therefore,Tetranychus cinnabarinus and cyflumetofen were taken as the research objects in this study.Transcriptome sequencing technology was used to reveal the change rules of gene expression of T.cinnabarinus to cyflumetofen which was helpful to analyze the molecular mechanism of resistance development and to explore the early and rapid identification method of resistance molecular markers.The main research results are as follows:1.Selection and metabolic resistance mechanism of T.cinnabarinus to cyflumetofenA substrain of T.cinnabarinus,YN-Cy R,was isolated from susceptible strain(YN-S),which was selected for cyflumetofen resistance.The resistance of YN-Cy R was selected for 32 generations,the value of LC₅₀was from 1.15 mg/L increased to 98.96mg/L,and the resistance ratio was 86.05 fold when compared to YN-S strains.According to the classification of resistance levels,the resistance level of YN-Cy R?16,YN-Cy R?25and YN-Cy R?32 reached low,medium and high resistance level respectively,which were abbreviated as L,M and H,respectively.Detoxification enzyme activity and synergism experiment were carried out in YN-S strains and H stage(YN-Cy R high resistance level)of T.cinnabarinus,so as to clarify the resistance mechanism of T.cinnabarinus to cyflumetofen.Compared with YN-S strains,the five SDH subunits of H-stage did not mutate at amino acid level.Compared with YN-S strains,the specific activities of P450s and GSTs crude enzymes in H stage increased significantly to 2.62 fold and 1.20 fold,respectively,while the specific activities of CCEs crude enzymes had no significant change.Synergists PBO and DEM had significant synergistic effect on H-stage resistance,with synergistic ratio of 3.10 fold and1.85 fold respectively,and could eliminate 60.11%and 40.35%of resistance respectively,while synergist DEF had no obvious synergistic effect.The results showed that metabolic resistance was the main mechanism of T.cinnabarinus to cyflumetofen,and P450s was the main enzyme to mediate the metabolic resistance T.cinnabarinus to cyflumetofen.2.Change rules of the gene expression of T.cinnabarinus during the development of cyflumetofen resistanceTranscriptome technique was used to analyze the number,differential expression degree and functional enrichment of differentially expressed genes(DEGs)in the process of resistance development,which could clarify the change rules of gene expression with resistance development.There were 4559 DEGs in L,M and H resistance stages.With the continuous increase of resistance level,the number of DEGs gradually increased.The number of DEGs increased from 2911 to 3359 and then to 3556,when the resistance level developed from L to M and then to H.Among them,there were 226,584 and 657 DEGs unique to L,M and H stages respectively,while there were 2175 DEGs differentially expressed in three stages,accounting for about half of the total DEGs(47.71%).The up-regulation amplitude(median of fold change of up-regulated genes)of up-regulated genes were 4.38,4.11 and 4.26,while the down-regulation amplitude(median of fold change of down-regulated genes)of down-regulated genes were-2.79,-2.68 and-2.73,respectively,which indicated that the expression of DEGs was generally stable during the development of resistance.Of the 2175 DEGs in three stages,1685(77.47%)were up-regulated.Among them,391(23.20%)were up-regulated with the up-regulation amplitude increased from 4.26 in L stage to 5.24 in M stage and then to 6.73 in H stage.The expression of 1148(68.13%)was relatively stable,and up-regulation amplitude was maintained at about 5.24.The expression of 146(8.66%)showed a downward trend,from6.11 to 5.66 and then to 4.32.Functional enrichment analysis showed that the functions of DEGs in L,M and H resistance stages were concentrated in lysosomes,detoxification metabolism,signal transduction and transcription regulation.Up-regulation of detoxification genes was an important factor in the development of metabolic resistance,so the change rules of up-regulation of detoxification genes were analyzed emphatically in three stages of resistance.The results showed that the change rules of four kinds of detoxification genes(P450s,GSTs,CCEs and ABCs)of T.cinnabarinus were consistent with those of the whole genes,with the development of cyflumetofen resistance.For example,most of the up-regulated detoxification enzyme genes(78.86%)were up-regulated in three stages.among them,the most of the up-regulated genes were stably expressed(76.29%),followed by the continuously up-regulated genes(14.43%)and the down-regulated genes(9.28%).It was worth mentioning that P450s gene had the largest number of up-regulated detoxification enzyme genes(40 in total,accounting for 33.06%of the up-regulated detoxification enzyme genes).The number and the fold change of up-regulated detoxification enzyme genes gradually increased with the continuous increase of resistance level.At the same time,the fold change of a P450 gene in three stages(CYP392A1 1,855.23,538.23 and 699.64 times)were much higher than those of other detoxification enzyme genes.Therefore,P450 gene played an important role in the development of T.cinnabarinus resistance to cyflumetofen.To sum up,during the development of the resistance of T.cinnabarinus to cyflumetofen,the changes rules of gene expresssion were as follows:(1)With the development of resistance level,the number of DEGs gradually increases.(2)There were more DEGs in the three resistance stages than in the specific resistance stage.(3)DEGs in the three resistance stages were mainly up-regulated,and most of them were stable in up-regulation expression.Moreover,a few of them increased continuously,and some of them decreased continuously.From this,it could be inferred that genes including detoxification enzyme genes that were stably up-regulated from the stage of low resistance level,which were the main DEGs and constitute the basis of metabolic resistance.During the whole development of resistance,continuously up-regulated genes and newly added up-regulated genes are the main driving forces to promote the continuous increase of resistance.3.Functional verification of resistance genes in T.cinnabarinus to cyflumetofenSeven genes(six detoxification enzymes and one new gene,which were continuously up-regulated in the development of resistance),and CYP392A1(which was the highest fold change and stable up-regulation)and GSTd05 which was stable up-regulation and the highest expression level)were selected for functional verification.The results of RNAi showed that the interference efficiency of 9 candidate genes in the H stage of YN-Cy R ranged from 47.44%to 64.03%.After RNAi,bioassay with LC₅₀ as diagnostic dose showed that the mortality increased by 4.86%to 26.31%,and the order of development mortality was CYP392A1?GSTd05?CCE06?CYP389A1?Tc03g09640?GSTz01?CCE59?CYP389C2 and CYP392E6.These results suggested that 9 genes were involved in the development of metabolic resistance of T.cinnabarinus to cyflumetofen in varying degreesCYP392A1,CCE06 and CYP389A1 were selected as the representative genes(GSTd05 has been studied)for cyflumetofen metabolism experiment after prokaryotic expression.The recombinant CYP392A1,CCE06 and CYP389A1 proteins were obtained by prokaryotic expression,and their catalytic activities on specific substrates were 0.77nmol/mg pro/min,517.14 nmol/mg pro/min and 0.23 nmol/mg pro/min,respectively,and the IC₅₀ of catalytic activities of these three recombinant proteins by cyflumetofen were124.49?M,172.25?M and 218.36?M,respectively.These results indicated that all recombinant proteins had enzyme activity and could bind to cyflumetofen.The results of HPLC showed that the metabolic rates of CYP392A1,CCE06 and CYP389A1,which were 20?g,were 34.61%,17.75%and 12.83%,respectively.The recombinant proteins CYP392A1 and CYP389A1 independently play the role of metabolizing cyflumetofen,and their mixture has no metabolic synergistic effect.4.Exploration on the method of quickly obtaining resistance molecular markersDetoxification enzyme genes were considered as classical resistance genes,so detoxification enzyme genes that was continuously up-regulated and stably up-regulated in the development of resistance could be used as a molecular marker for the resistance of cyflumetofen.Transcriptome sequencing and comparison of DEGs were carried out on YN-S strains that selected at one generation with high dose cyflumetofen(about 80%of mites of YN-S strains were killed by LC₈₀,and F₁ generations of surviving individuals)and YN-S strains induced with different doses cyflumetofen(samples were collected after1.5 h of inducing on YN-S strains with LC₂₀,LC₅₀ and LC₈₀ of cyflumetofen,respectively).The up-regulated detoxification enzyme genes were combined with the resistance molecular markers obtained by long-term resistance selected,so as to explore the method of obtaining resistance molecular markers quickly in early stage.Compared with CK(acetone treatment),the number of DEGs after three doses of cyflumetofen induced treatment was 184,66 and 75 respectively,which was significantly less than the number of DEGs after high-dose selected with cyflumetofen(YN-S-S VS YN-S)(1788).The up-regulation amplitude of up-regulated genes were 1.80,2.16,1.84and 3.14,and the down-regulation amplitude of down-regulated genes were-0.79,-0.77,-0.67 and-1.29,respectively,which indicated that the changes of gene expression after high-dose primary selected were larger than short-term induce.Combined analysis of the over-expressed detoxification enzyme gene under cyflumetofen induced and high-dose cyflumetofen selected with known molecular markers of cyflumetofen resistance showed that only two resistance molecular markers appeared in short-term induction treatment(ABCG11 and GSTm03 were up-regulated after LC₂₀ induced).Sixteen of resistance molecular markers appeared in DEGs which was up-regulated after high-dose screening,among which GSTm04,CYP392A1 and CYP392E6 had been proved to be related to the metabolic resistance of T.cinnabarinus to cyflumetofen.Therefore,it may be an early and rapid method to obtain resistance molecular markers by using the up-regulated detoxification enzyme gene formed after selected with a high dose pesticide.