Molecular Cloning And Function Study Of Detoxifying Genes From Cydia Pomonella

The codling moth Cydia pomonella (L.), an economically important fruit pest, iswidespread in the majority of pome fruit cultivated regions worldwide. This pest haswide-ranging hosts and fruit damages by C. pomonella include boring tunnels to the center offruit to feed on seeds, and causing fruit abscission, enormous economic losses to global fruitproduction are caused each year. Effective management of the codling moth mainly dependson chemical insecticides worldwide. However, a major problem associated with chemicalcontrol of the C. pomonella frequently is its development of resistance to groups ofinsecticides in many countries. Previous researches have revealed that activities elevation ofdetoxifying enzyme including the cytochrome P450monooxygenases (P450), glutathioneS-transferase (GST) and carboxylesterase (CarE) are the main resistance mechanisms incodling moth. However, very little is known in the molecular cloning and functional analysisof detoxifying genes in C. pomonella. Therefore, investigation of the resistance status of C.pomonella populations from northwestern China are urgently required and of guidingsignificance; moreover, study on the function of detoxifying genes in term with molecular andbiochemical methodology is also a good avenue to reveal the resistance mechanism in C.pomonella.To investigate the resistance status of C. pomonella populations from northwestern Chinaand the role of detoxifying genes in insecticide metabolism and potential insecticideresistance, the susceptibility of four insecticides to C. pomonella populations fromnorthwestern China was investigated by applying bioassays. Meanwhile, RT-PCR and RACEstrategies were adopted to clone the three detoxifying enzyme genes, namely CYP9A61,CpCE-1and CpGSTd1. The spatiotemporal and insecticide-induced expression profiles ofthese genes were analysed by RT-qPCR. The effect of insecticide-exposure on enzymeactivities of three detoxifying enzymes in larvae was also determined. Beside what mentionedabove, these detoxifying enzyme genes were expressed using prokaryotic expression system,and the enzymatic hydrolysis characteristics of these enzymes toward model substrates,organophosphorus and pyrethroid insecticides were determined both on molecular andbiochemical level; The3D models were constructed and the site-directed mutagenesistechnique, combined with the computational molecular simulation methods were effectivelyutilized to study the effect of key amino acid mutation on the hydrolysis of the model substrate and insecticide was structurally studied. The results are as follows:1. Investigation of insecticide-resistance status of C. pomonella populations in NorthwestChinaThe insecticide-resistance status of four field populations from northwestern China tolambda-cyhalothrin, chlorpyrifos-ethyl, carbaryl, and imidacloprid was investigated byapplying bioassays and the result suggested that field populations were less susceptible tochlorpyrifos-ethyl and carbaryl than laboratory strain. Insensitive strains displayed anelevated glutathione transferases (GST) activity and the three strains---Zha, Wuw andLan---showed reduced carboxylesterase (CarE) activity. Reduced acetylcholinesterase activitywas observed only in the Wuw strain. Neither the known-resistance mutation F399V in theacetylcholinesterase (AChE) gene, ace1, nor mutations in carboxylesterase gene CpCE-1were found in adult individuals from our field strains. Native-PAGE revealed that variousCarE isozymes and AChE insensitivity were occurring among these strains.2. Molecular cloning and expression profiles of cytochrome P450cDNA from C.pomonellaThe first P450gene, designated as CYP9A61, was cloned from C. pomonella. Thefull-length cDNA sequence of CYP9A61is2071bp long and its open reading frame (ORF)encodes538amino acids. Sequence analysis shows that CYP9A61shares51%–60%identitywith other known CYP9s and contains the highly conserved substrate recognition site SRS1,SRS4and SRS5. Quantitative real-time PCR showed that CYP9A61were67-fold higher inthe fifth instar larvae than in the first instar, and more abundant in the silk gland and fat bodythan other tissues. Exposure of the3rd instar larvae to12.5mg L1of chlorpyrifos-ethyl for60h and0.19mg L1of lambda-cyhalothrin for36h resulted in2.20-and3.47-fold inductionof CYP9A61, respectively. Moreover, exposure of the3rd instar larvae to these twoinsecticides also significantly enhanced the total P450activity. The results suggested thatCYP9A61is an insecticide-detoxifying P450.3. Molecular cloning, expression analysis, and5’-flanking sequence of CpCE-1from C.pomonellaA type-B carboxylesterase gene CpCE-1cDNA was cloned from C. pomonella. Thefull-length cDNA sequence of CpCE-1is1978bp long and its ORF encodes545amino acids.Sequence analysis shows that CpCE-1shares high identity with other knowncarboxylesterases and contains some highly conserved motifs, such as the catalytic triad andGxSxG pentapeptide. Absolute quantitative real-time PCR showed that CpCE-1was expressed predominantly in pupae and adults and differs between sexes, with a highertranscript in females than males. CpCE-1was primarily expressed in cuticle, midgut and head.Exposure of3rd instar larvae to chlorpyrifos-ethyl and lambda-cyhalothrin resulted ininduction of CpCE-1expressions and inhibition of total carboxylesterases activity. The5’-flanking sequence of CpCE-1was isolated by thermal asymmetric interlaced PCR(TAIL-PCR). This promoter sequence contains several transcription factor binding sites suchas heat-shock factors, deformed motifs, ternary complex factors, broad-complex Z4,hunchback, TATA-box, CAAT-box, GATA-box, zinc finger protein, TFIID, tramtrack69andsome elements involved in development and morphogenesis.4. Expression of CpCE-1and its functionin metabolism of insecticidesThe fusion CpCE-1protein was expressed in E. coli BL21(DE3) at18℃with0.6mMsorbitol in the growth medium. The fusion protein was purified from the lysate supernatantusing a Ni2+-NTA agarose gel column. The enzyme exhibited a higher affinity and substratespecificity for ɑ-naphthyl acetate (ɑ-NA), with kcat/Kmof100s-1μM-1for ɑ-NA, and thevalue is29.78s-1μM-1for β-NA. The Vmaxand Kmwere also determined to be12.9μmol/min/mg protein and13.4μM using substrate ɑ-NA. The optimum pH was7.0andtemperature was25℃. An enzyme inhibition assay shows that protease inhibitor PMSF andDEPC, as well as insecticide cypermethrin, methomyl, and acephate strongly inhibit theenzyme activity, while the metal ions Cu2+and Mg2+significantly activated the activity.Molecular dynamics (MD) simulation and computational alanine scanning (CAS) indicatethat Asn232in CpCE-1constitutes an approximate binding hot-spot with a binding freeenergy difference (ΔΔGbind-exp) value of3.66kcal/mol. The N232A and other mutants wereobtained using the site-directed mutagenesis technique. The catalytic efficiency (kcat/Km) ofN232A declined dramatically, only with a value of17.91s-1μM-1. The half inhibitoryconcentrations (IC50) value of acephate against wild CpCE-1was42.18μM, whereas thevalue was more than10000μM in N232A mutant. The calculated binding free energydifference (ΔΔGbind-cal) value was3.17kcal/mol, which is close to the predicated value(ΔΔGbind-exp). The Metabolism assay in vitro reveals that the acephate could be metabolizedby wild CpCE-1, whereas N232A mutation is unable to metabolize the acephate, suggests thatthe hot-spot Asn232is a crucial residue for acephate metabolism.5. Expression of CpGSTd1and its functionin metabolism of insecticidesA delta class GST gene CpGSTd1was cloned from C. pomonella, the648bp long ORFencodes a protein with215amino acids. Real-time quantitative PCR shows that CpGSTd1was up-regulated with aging, and the mRNA level of CpGSTd1was higher in the fat body and silk glands than in other tissues. The expression level of CpGSTd1exposure to insecticidesuggests that CpGSTd1is up-regulated after chlorpyrifos-methyl and lambda-cyhalothrintreatments. Both lambda-cyhalothrin and chlorpyrifos-methyl altered GST activity in vivo.The fusion CpGSTd1protein was expressed in E. coli BL21(DE3) and was purified using aNi2+-NTA agarose gel column. The catalytic constant kcat and catalytic efficiencies (kcat/Km)of the purified CpGSTd1protein were3.66×104min-1and1.37×105min-1mM-1respectively. The CpGSTd1exhibited IC50values of0.18and0.26mM forlambda-cyhalothrin and chlorpyrifos-methyl respectively in vitro. Metabolism assays indicatethat lambda-cyhalothrin was significantly metabolized while chlorpyrifos-methyl was notmetabolized by CpGSTd1. The bindling models and molecular docking indicated thatlambda-cyhalothrin and chlopyrifos-ethyl occupied the H-and G-site, respectively. Bindingfree energy analysis suggested that CpGSTd1binding is tighter with lambda-cyhalothrin thanwith chlorpyrifos-methyl, with the ΔGbindvalue of-28.26kJ/mol for lambda-cyhalothrin and-16.08kJ/mol for chlorpyrifos-methyl. Our study suggests that CpGSTd1plays a key role inthe metabolism of insecticides in C. pomonella.