Expression Regulation Of The Cytochrome P450 Genes Associated With Deltamethrin Resistance In Small Brown Planthopper,Laodelphax Striatellus(Fallen)

The small brown planthopper,Laodelphax striatellus(Fallen)(Hemiptera:Delphacidae)(hereafter referred to as SBPH)is one of the most destructive pests of agriculture in Asia.Deltamethrin is a pyrethroid insecticide acted in the voltage gate sodium channel,which is used for controlling wheat field L.striatellus population.Previous work in our laboratory showed that strong resistance to deltamethrin was due to enhanced detoxification rather than target site insensitivity,and that five monooxygenase genes and one esterase gene were over-expressed in the resistant strain(CYP6AY3v2 24.0-fold,CYP6FU1 16.0-fold,CYP353D1v2 5.3-fold,CYP314A1v2 2.3-fold,CYP439A1v3 4.6-fold,and LSCE1211.1-fold).Constitutive gene over-expression can arise from gene amplification or up-regulation of expression.Some cis-acting factors and trans-acting factors have been implicated.Cis-acting regulation of resistance-conferring monooxygenases can be achieved through indels or mutations in their promoter region,such as a transposable element insertion in the 5'-flanking region.This work focused on cloning of 5'-flanking region and 3'UTR of these genes from both resistant and susceptible strains to determine the change of polymorphism under insecticide stress.We also tested the function of 5'-flanking region and 3'UTR by luminescent reporter assay and site mutation recovery.This work provided a theoretical basis to clarify the evolution mechanism of resistance to insecticides and promoted the development of more efficient resistance management strategies.1.Molecular cloning and polymorphism analysis of 5'-flanking regions for P450 genes associated with deltamethrin resistance in L.striatellusPrevious work found that five P450 genes(CYP6AY3v2,CYP6FU1,CYP353D1v2,CYP314A1v2 and CYP439A1v3)were overexpressed in the resistant strain.The 5'-flanking regions of these genes were cloned by genome walking technique in resistant and susceptible strains.The obtained 5'-flanking region of CYP6AY3v2,CYP6FU1,CYP353D1v2,CYP314A1v2 and CYP439A1v3 was 2183bp,1515bp,1771bp,1870bp and 1924bp,respectively.By analyzing 5'-flanking sequences from individuals of both resistant and susceptible strains,nucleotide diversity of CYP6AY3v2,CYP6FU1?CYP353D1v2 and CYP439A1v3 in resistant strain was significantly lower than that in susceptible strain.Percent similarity from pairwise comparisons and phylogenetic analysis of the cloned sequences showed that different genes had several types in 5-flanking region.CYP6AY3v2,CYP6FU1 and CYP353D1v2 had 2,5 and 2 types,respectively.Type distribution in 5'flanking region of CYP6AY3v2,CYP6FU1 and CYP353D1v2 had significant difference between two strains.For CYP6FU1,only one type was found in the resistant strain,and all the 5 types were found in the susceptible strain.These results demonstrated that regulation region of these P450 genes showed pronounced footprint of selection.2.Analysis of cis-acting factors in 5'-flangking regions of P450 genes associated with deltamethrin resistance in L.striatellusTranscriptional regulation is the most important step for gene expression regulation in eukaryotes.Regulation factors mainly act with 5'-flangking regions.Here we analysis the regulatory region of these gene.First,the 5'UTR of mRNA were cloned from resistant and susceptible strains by 5'RACE,which showed different types in these genes(CYP6AY3v2 4 types,CYP6FU1 6 types,CYP353D1v2 6 types,CYP314A1v2 2 types,CYP439A1v3 1 type).Then,the prediction of transcription factor(TF)binding boxes showed that the 5-flanking region of these genes abounded with functional elements,including multiple transcription start sites.Finally,some TF binding boxes and transcription start sites in different type were influenced by polymorphism sites.These results provided an important basis for investigating expression regulation of cytochrome P450 genes.3.Promoter activity of 5'-flanking regions of P450 genesCYP6AY3v2,CYP6FU1,CYP353D1v2 and CYP314A1v2 had a different type distribution of 5-flanking region between resistant and susceptible strains.The function of 5'-flanking region was tested by luminescent reporter assay in S2 cell line for over-expressed P450 genes.Result showed that all types of CYP6AY3v2 and CYP353D1v2 were not significantly different in promoter activity;three 5'-flanking regions of CYP6FU1 had significant promoter activity,while the expression of the reporter gene driven by the type 1 flanking sequence from the resistant strain was significantly higher(2.4 and 3.7-fold,respectively)than that driven by Type 2 and 3 cloned from the susceptible strain;three of CYP314A1v2 also had significant promoter activity,DNA5'-D1 from the resistant strain had significantly higher(1.5 and 1.6-fold,respectively)promoter activity than DNA5'-S3 and S5 cloned from the susceptible strain.This finding indicated that some over-expression of P450 genes(CYP6FU1 and CYP314A1v2)in the resistant strain resulted from an enhanced promoter activity,4.Transposable element affected expression of CYP6FU1In addition to arising from single point mutations in binding sites or detoxifying enzymes,it is likely that the mechanisms of insecticide resistance are frequently under the control of multiple genetic factors resulting in resistance.However,empirical evidence for the latter is still rare.Luminescent reporter assay showed that the entire flanking region of CYP6FU1 from resistant strain had higher promoter activities.Here,progressive 5'deletions and site mutation recovery in the fragments with higher promoter activity were tested.Results showed four mutations made significant contributions to up-regulation.Of these,two mutations led to new TF binding boxes XBP-1 and D1 at-1368 and-1292/-1283 positions,another changed an existing TF binding box Dfd to a new one BR-C at-352/-342 positions,and the fourth added a unique transcription start site at 100 bp fragment insertion(between-294 and-194 positions),where the major CYP6FU1 mRNA was transcribed in the resistant strain.Other elements appeared to make minor contribution.These results demonstrated that multiple elements were involved in up-regulation of CYP6FU1,revealed the base for insecticide resistance being inherited as a quantitative trait,which is important for understanding how resistance traits accumulate in pest insect populations,and should promote the development of more efficient resistance management strategies.5.Molecular cloning and polymorphism analysis of 3'UTR for P450 genes associated with deltamethrin resistance in L.striatellusThe 3'UTR regulation can impact mRNA stability and translational efficiency in post-transcriptional regulation.Here,we analyzed the post-transcriptional regulation of up-regulated monooxygenase genes implicated in resistance to pyrethroids in L.striatellus.3'UTR regions of these P450 genes were cloned from individuals of both resistant and susceptible strains.Sequence analysis showed that 3,UTR regions of five P450 genes had a large diversity of SNP sites and insertion-deletion polymorphic sites.Based on the distribution of these polymorphism sites,two types of 3'UTR region were differentiated for CYP6FU1 and CYP353D1v2,respectively.3'UTR types of these genes had different frequency in resistant and susceptible strains.However,the polymorphic sites in 3'UTR region of CYP6AY3v2 and CYP439A1v3 were random.These results demonstrated that selection appeared in 3'UTR region of some genes(CYP6FU1 and CYP353D1v2)when the insecticide was used continuously.6.Function assay of 3'UTR region of P450 genes associated with deltamethrin resistanceLuminescent reporter assay,site mutation recovery and mRNA stability testing were used to investigate the function of 3'UTR for CYP6FU1,CYP353D1v2 and CYP314A1v2.Results of CYP6FU1 indicated that the expression of the reporter gene affected by type A 3'UTR from the resistant strain was significantly higher(1.61-fold)than Type B from the susceptible strain.Type B reduced the stability of the corresponding mRNA.The prediction of miRNA indicated that three mutations located Type B led to two miRNA binding sites(miR-306 and miR-275).Results of CYP353D1v2 showed that the expression of the reporter gene affected by 3'UTR D1 from resistant strain was significantly higher(2.1-fold)than S1 from the susceptible strain,and D1 increased the stability of the corresponding mRNA.Site mutation recovery at D1 indicated that a fragment insert(+51/+73)resulted in increase of mRNA stability.Interestingly,the stem loop structure was found in the fragment insert(+51/+73).These results showed that 3'UTR might contribute to up-regulation of over-expressed P450 genes(CYP6FU1and CYP353D1v)in insecticide-resistant strain.