Enantioselective Metabolism Of Fenpropathrin By TcCCE06 In Tetranychus Cinnabarinus (Boisduval)

The carmine spider mite,Tetranychus cinnabarinus(Boisduval),is a polyphagous mite that is widely distributed in agriculture and difficult to control.Up to now,chemical pesticide is the most effective method to control agricultural mites.The resistance problem of T.cinnabarinus is very serious due to its short generation cycle,strong reproductive ability,small body size and parthenogenesis.Fenpropathrin,a pyrethroid acaricide,often used to control mites.The current commercialized fenpropathrin is racemic,that is,the ratio of R-fenpropathrin to S-fenpropathrin is 1:1,So the result is the combination of two enantiomers.However,the metabolic differences of fenpropathrin enantiomers in T.cinnabarinus have not been reported.In this paper,the metabolic difference of TcCCE06 recombinant protein to fenpropathrin enantiomer was studied,and the following results were obtained:1.Synergist experiment of detoxification enzyme systems in Tetranychus cinnabarinusThe results of detoxification enzyme inhibition experiments showed that Glutathione peroxidase,carboxylesterase and cytochrome P450 enzyme in T.cinnabarinus were inhibited by DEM,TPP and PBO inhibitors,the efficiency ratio was0.87,16.35 and 2.27,respectively.It has been proved that carboxylesterase and cytochrome P450 enzyme are the main enzymes involved in fenpropathrin detoxification metabolism in T.cinnabarinus,and CarEs plays a major role.2.Transcriptome sequencing and quantitative verification after fenpropathrin induced Tetranychus cinnabarinusThe selected 13 CarEs genes and 2 P450 s genes that may be involved in the metabolism of fenpropathrin were analyzed by real-time fluorescence quantitative(RT-qPCR).The m RNA expression profiles of the 15 genes induced by fenpropathrin enantiomers and sensitive strains(SS)were obtained.The results showed that,there was no significant difference in the relative expression levels of 8 genes between S-fenpropathrin and R-fenpropathrin and SS;After R-fenpropathrin induced the relative expression levels of 7 genes were significantly higher than that of SS at the same period,and the up-regulation ratio was 2.03-36.31 times;After S-fenpropathrin induced,the relative expression levels of the 6 genes were significantly higher than that of SS at the same period,and the up-regulation ratio was 3.58-22.96 times;the relative expression levels of 5 genes were significantly different after S-fenpropathrin and R-fenpropathrin induced stress.3.Cloning and prokaryotic expression of fenpropathrin metabolism-related esterase geneAccording to the transcriptome and RT-qPCR quantitative results,the TcCCE06 gene was used as the key gene for metabolizing fenpropathrin enantiomer in T.cinnabarinus.The TcCCE06 esterase gene of T.cinnabarinus was cloned.The open reading frame of this gene is 1737 bp and encodes 578 amino acids.It is named TcCCE06.Bioinformatics analysis showed that TcCCE06 belonged to the J’’ family,and it was found to be homologous to Tetur01g10800 in T.urticae.4.Study on the heterologous expression of TcCCE06 and the difference in the metabolism of fenpropathrin enantiomersThe pCold Ⅱ-TcCCE06 expression vector was constructed,and the TcCCE06 esterase gene was successfully prokaryotic expressed,and the active recombinant protein of TcCCE06 was obtained.In vitro metabolism experiments showed that,TcCCE06 can metabolize fenpropathrin enantiomers,and the metabolic rate of R-fenpropathrin(ineffective body)is faster than S-fenpropathrin(effective body).In 4h,32.1% of R-fenpropathrin(ineffective body)and 13.8% of S-fenpropathrin(effective body)were metabolized,the results showed that the metabolism of fenpropathrin enantiomer was significantly different in TcCCE06 recombinant protein.