Study On The Relationship Between Short Chain Dehydrogenase And The Fenpropathrin Resistance In Tetranychus Cinnabarinus (Boisduval)

The carmine spider mite,Tetranychus cinnabarinus,is an important pest that endangers many economic and food crops.At present,the control of the mites priority relies on chemical pesticides.Mites,r-biological countermeasures,was controlled by single pesticide for a long time,which caused that resistance level rose sharply and most of the popular acaricides had been resistant to mites,finally,which caused irreversible losses in agriculture and economics.Fenpropathrin is a kind of pyrethroid insecticide.At present,the mites in many regional of the world had developed a certain degree of resistance to fenpropathrin,and the research of resistance mechanism had made some progress.Short chain dehydrogenases/reductases?SDR?,an oldly kind of superfamily enzymes,widely distributed in eucaryote and prokaryote.Many studies showed that SDR mainly participated in the growth and a variety of endogenous and exogenous metabolism of substances?drugs?of the organism.Previous studies showed that the the gene expression level of SDR was significantly higher in FeR strain,suggesting that SDR might be involved in the resistance to fenpropathrin.At present,there was little research between SDR and resistance of mites,so studying the relationship between SDR and resistance of T.cinnabarinus not only enrich the mechanism of metabolic resistance of T.cinnabarinus and but also provide theoretical support for the prevention and control of the mites.Therefore,this research based on the laboratory transcriptome data and DNA microarray data,and used the analysis of the expression pattern?RNA interference?RNAi?and prokaryotic expression to study the difference between fenpropathrin resistant strain and susceptible strain in the laboratory,and then,this paper could clear the mechanism of SDR mediated resistance.The main results of this research are as follows:1.Toxicity and SDR enzyme assay to FeR and SS of T.cinnabarinusThe bioassay results showed that the LC₅₀ of fenpropathrin to FeR strain and SS strain of T.cinnabarinus were 64621.39 mg/L and 583.72 mg/L,respectively,which showed that the fenpropathrin resistance ratio of FeR strain was 110-fold.The activity of SDR enzyme to FeR strain and SS strain of T.cinnabarinus were0.1041±0.0197 nmol/mg pro.min^-1 and 0.0451±0.0109 nmol/mg pro.min^-1,respectively,which showed that the SDR activity of FeR is 2.31 times of SS.The results of enzyme activity showed that the activity of SDR was significantly higher in FeR strain than SS strain,suggesting that SDR might be involved in the resistance to fenpropathrin.2.Identification,cloning and expression patterns analysis of SDR genes and its function related gene involved in fenpropathrin resistance in T.cinnabarinusAnalysising the transcriptome data and DNA microarray data,we found two overexpression SDR Unigenes?UN9502 and UN13129?in the FeR strain of T.cinnabarinus.Futher qPCR resules showed both of two genes were higher expressed in FeR strain compared to SS starin upregulated 3.13-fold to 6.27-fold,respectively.The full length of UN9502 gene was cloned and sequenced by reverse transcription PCR technology,and UN9502 was named as TcSDR112C1.The length of complete open reading frame?ORF?of TcSDR112C1,867bp,encoded deduced peptide of 288 amino acid residues which was predicted that the molecular weights was 31.6 kDa,which GenBank accession numbers was MG595714.The fragment nucleotide sequence of UN13129 gene was 336 bp,encoded deduced peptide of 112 amino acid residues.And then,the bioinformatic analysis of TcSDR112C1 showed that it contains the SDR Classical family character sequence:the NADPH binding regions of TGxxxGxG near the N-terminal and substrate catalytic area of YxxxK in the downstream and the phylogenetic tree analysis showed that it belongs to SDR112C subfamily.Detected the mRNA expression levels of SDR genes of the difference development stages in FeR strain and SS strain of T.cinnabarinus showed that two SDR genes were expressed highest in adult.Detected the mRNA expression levels of SDR genes after fenpropathrin induction indicated that two SDR genes were dynamically changed in 24hours that UN13129 in 6 hours and TcSDR112C1 in 12 hours up-regulated and then down-regulated in FeR,except the TcSDR112C1 gene was upregulated in 6 hours in SS strain,the mRNA expression levels of UN13129 was signigicantly downregulated at 24hours,but no change in 6 and 12 hours compared to control.These results suggested that these two SDR gene were responsive to the induction of fenpropathrin,and the response was more sensitive in FeR strain.3.The RNA interference research of the SDR genes of T.cinnabarinusThe RNAi method was the leaf disc feeding,after feeding the target genes'dsRNA,the silence efficiency of TcSDR112C1 and UN13129 were 53.19%and 62.89%in FeR strain,respectively,causing the specific activities of SDR enzymes decreased significantly 44.96%and 24.87%compared to the control?water?.Furthermore,after feeding of SDR dsRNAs,assessed the susceptibility of FeR mites to fenpropathrin(three concentration of fenpropathrin LC₁₀?LC₃₀ and LC₅₀)showed that the mortalities increased significantly 16.31%,17.21%,21.76%and 14.62%15.96%,16.46%compared to the control?water?,respectively.The silent efficiency of TcSDR112C1 and UN13129 were 48.80%and 57.17%in SS strain,however,the susceptibility of SS mites to fenpropathrin was no significant difference.Surprisingly,after feeding the mix of these two genes'dsRNAs,the silence efficiency of TcSDR112C1 and UN13129 were48.43%and 54.11%,and the specific activities of individual SDR enzymes decreased significantly 59.74%,compared to the control?water?.After feeding the mix dsRNAs,the susceptibility of FeR mites to fenpropathrin increased significantly 20.49%?26.26%and 30.08%,respectively.These results of RNAi showed that SDR genes participated in the resistance of T.cinnabarinus to fenpropathrin and two SDR genes may collaboratively involve in fenpropathrin detoxification.4.Prokaryotic expression of TcSDR112C1 gene in T.cinnabarinusRecombining and expressing TcSDR112C1 protein in vitro by the Escherichia coli prokaryotic expression system obtained the recombint protein and its function analysis.Recombining and expressing TcSDR112C1 protein in vitro was successful,unfortunarely,there was no souble protein and the recombint protein all existed in the form of inclusion body.After inclusion body renaturation,although the protein became soluble,the souble protein lost SDR enzymes activity which was caused by the restitution change the structure of protein.Changing the expression system may be a good choice to recombine and express TcSDR112C1 protein in vitro,and then,studying the interaction between TcSDR112C1 protein and fenpropathrin.In summary,the SDR enzyme assay and fenpropathrin induction indicated that SDR was related to the formation of fenpropathrin resistance,and RNAi further proved the correlation.