Study On The Relationship Between TcID1 Gene And Two Acaricides Resistance In Tetranychus Cinnabarinus(Boisduval)

Tetranychus cinnabarinus is one of critical important agricultural mite pests in China.Its control has been and still is largely based on the use of insecticides and acaricides.Abamectin and fenpropathrin are two.However,control failures occurred in the field accompanied with the development of acaricide resistance.Introdial ring-cleavage dioxygenases(ID-RCDs)catalyze the oxygenolytic fission of catecholic substances,allowing bacteria and fungi to degrade aromatic rings,a crucial step in the global carbon cycle.A microarray from our lab revealed ID-RCDs genes were overexpressed in both AbR and FeR strain compared to SS strain in T.cinnabarinus.Thus this thesis mainly investigated a functional link between ID-RCDs metabolism and abamectin/fenpropathrin resistance.The results are as follows.1.Assay of ID-RCDs activityThe crude enzyme activity assay showed ID-RCDs activity in two resistance strains(AbR and Fe R)was significantly higher than that in the reference susceptible strain(SS),with the AbR activity of 1.63 times and FeR of 2.0 times divided by SS.2.TcID1 cDNA cloning and characterizationIn present study,a full length of TcID1 was obtained by PCR and the GenBank under accession number is KF844725.The TcID1 cDNA contains an opening reading frame(ORF)of 774 bp and encodes 257 amino acid residues in T.cinnabarinus.The SignalP 4.1 Server prediction result shows that signal peptide is GCTCAT at nucleotide positions 1-69.Sequence alignment indicate TcID1 share the conserved 2 His and 2 Tyr nonheme iron(III)active site with previously described and functionally characterized ID-RCDs.NCBI blast shows TcID1 has the highest amino acid consistency about 98% with tetur06g00460 gene in T.urticae,then 66% with AFY99039.1 in Tetranychus evansi,while lower than 40% with typical ID-RCDs in fungi and bacteria.Phylogenetic analysis using MEGA 5.0 software further indicates that ID-RCDs share a deeper homology with ID-RCDs in fungi than that in bacteria.3.Expression patterns analysis of TcID1The expressions of TcID1 in different developmental stages,different strains,and upon acaricide induction were investigated using qRT-PCR.Firstly,TcID1 showed a consistent higher expression in larvae and nymphs compared to eggs in SS,and expression levels in a descending order were nymphs,larvae,adults and eggs.TcID1 was over expressed in AbR and FeR compared to SS,conforming to the microarray data in T.cinnabarinus.Induction expression of TcID1 at different time(6h,12 h and 24h)by abamectin/fenpropathrin treatment with corresponding dose of LC30 was detected.When susceptible stains were used as inducing target and induced by abamectin,TcID1 showed a first increased,then decreased expression pattern.However,when it comes to AbR strain,an opposite expression trend was found,TcID1 showed a first decreased,then came back to normal expression pattern.Meanwhile,Induction expression results of TcID1 exposed to fenpropathrin showed a similar trend with abamectin induction.4.RNAi analysisSilencing of TcID1 by feeding TcID1-dsRNA indirectly was conducted respectively in SS,AbR and FeR.The qPCR results showed that the TcID1 expression levels saw a decrease by 60.7% in SS,67.5% in AbR and 47.7% in FeR compared to the control group of DEPC-water or GFP-dsRNA after 48 h feeding.Then ID-RCDs activity of the survival female adults collected after RNA interference showed a significant decrease by 38% in SS,44% in AbR and 40% in FeR.Furthermore,detection of the sensitivity to abamectin and fenpropathrin after TcID1 knockdown was assayed.Silencing of TcID1 via RNAi both in the SS and AbR female adults of T.cinnabarinus significantly increased their susceptibility to abamectin,with an increased mortality.In detail,the mortality in SS saw an increase by 43.9%(LC30 in abamectin)and 38.6%(LC50 in abamectin)respectively.The mortality also increased 19.5%(with dose of abamectin LC30)and 17%(LC50 in abamectin)in AbR.However,the motality of TcID1 sliencing in FeR female adults had no significant changes.5.TcID1 Prokaryotic expression in E.coli Efficient expression was realized based on the elimination of signal peptide sequence of TcID1 gene.A sole and specific band situated between 25 kDa and 35 kDa was observed both in the SDS-PAGE and Western Blot after the process of prokaryotic expression in BL21(DE3)cells transformed with pColdII-TcID1 under IPTG induction and purification using Ni2+-NTA agarose gel column.Which was close to the value for the calculated molecular weight of TcID1 protein(26.86kDa).The specific activity of recombinant TcID1 using protocatechuic acid as substrate was 0.1346 ± 0.0090 nmol/μg.pro/min,and Km was 1.42 ± 4.6 mM and Vmax was 0.1163 ± 0.0153 nmol/μg.pro/min.The recombinant enzyme showed a much higher activity compared with crude enzymes in SS.6.Metabolic studies of recombinant TcID1 with two acaricides Two acaricides were screened for their ability to inhibit recombinant TcID1 mediated protocatechuic acid activity through measurement of IC50.While no inhibition was detected with abamectin or fenpropathrin even in a low micromolar range under assay conditions.What’s more,the HPLC results showed recombinant TcID1 was unreactive against abamectin as there was no apparent substrate depletion observed,despite several efforts including different concentration of abamectin,different protein content as well as different elapsed time were tried.