Expression Analysis And RNA Interference Of Arginine Kinase Genes In Chilo Suppressalis And Frankliniella Occidentalis

The rice stem borer,Chilo suppressalis(Lepidoptera:Pyralidae),is one of the most serious rice pests in China,which causes large crop losses through feeding on the stems of rice.The western flower thrips(WFT),Frankliniella occidentalis(Thysanoptera:Thripidae),is one of the most economically important insect pests worldwide,which causes extensive damages to vegetables,fruit and ornamental crops directly through feeding and indirectly by vectoring of tospoviruses.Arginine kinase(AK)is a phosphotransferase,which catalyzes the reversible phosphorylation of arginine through MgATP to form MgADP and phosphoarginine.Since it is absent in vertebrates and plays an important role in insect energy metabolism and growth and development,AK has become an important target for the development of highly selective insecticides and RNAi-based control of insect pests.In this paper,C.suppressalis CsAK and F.occidentalis FoAK were functionally expressed in vitro,and the enzymatic activities of the purified products were determined,and the mRNA levels of CsAK and FoAK in response to stresses were analyzed Furthermore,knockdown efficiency of CsAK and FoAK using different RNA interference(RNAi)methods were systematically evaluated.The research results will lay foundation for further study on the function of CsAK and FoAK as well as for the development of RNAi-based control strategy for C.suppressalis and F.occidentalisRT-PCR and RACE were used to clone the full-length cDNA sequence of CsAK gene.The CsAK open reading frame is 1068 bp long and encodes 355 amino acid residues.Multiple sequence alignment analysis revealed that the amino acid sequence identity of CsAK with other species AK is more than 73%,of which the identity with Helicoverpa armigera HaAK is as high as 98.0%.Genomic structure analysis of CsAK,FoAK and other representative insects AK showed that AKs from Lepidopteran insects including CsAK,HaAK,and BmAK of Bombyx mori contain only one intron,FoAK contains five exons connected by four introns,while no introns were identified in two Drosophila melanogaster AK genes,DmAK1 and DmAK2.Analysis of temporal-spatial expression patterns revealed that CsAK had the lowest and highest expression level in pre-pupae and 12h-old adults,respectively,and the transcript level of CsAK in the hindgut was significantly higher than other tissues.The 1-day-old prepupae exhibited the lowest mRNA level of FoAK,and relatively constant expression levels of FoAK were observed throughout adult stages.These results indicate the occurrence of frequent loss or gain of introns during the evolution of insect AKs,and provide evidence that CsAK and FoAK might be transcriptionally regulated by the ecdysone signaling pathwayThe 3rd instar larvae of C.suppressalis were used for CsAK expression analysis under various environmental stresses inliuding high temperature(42?),low temperature(4?)and paraquat treatments.While no significant change of mRNA expression level of CsAK was observed after high temperature treatment,transcript levels of CsAK increased by 4.65± 0.31 fold at 4h after low temperature treatment,and 2.10 ±0.09 and 4.74±0.24 fold,respectively,at 3h and 9h after paraquat treatment.Two-day-old adults of F.occidentalis thrips were treated with high temperature of 39?,low temperature of-8? and paraquat,respectively.At 1 h after high temperature treatment,the expression level of FoAK increased significantly by 3.72±0.40 fold compared with control.FoAK mRNA expression level did not change significantly at 1h and 2h after low temperature treatment,but increased significantly by 1.70±0.12 fold at 12h after low temperature treatment.Paraquat treatment also significantly increased the expression of FoAK by 3.43±0.25 and 4.69±0.54 fold at 8h and 12h after treatment.These results indicated that CsAK and FoAK might be involved in adaptation to stressesImmunofluorescence and western blot were used to determine the subcellular localization of CsAK.The results showed that most of CsAK was localized in the cytoplasm,but not in the nucleus,and a few were localized in the mitochondria Heterologous expression of CsAK and FoAK and determination of enzymatic activity in the purified product showed that the KmArg and Kcat values of CsAK were 1.27 ± 0.07 mM and 18.23±0.34 s-1,respectively,and those of FoAK were 1.79 ± 0.05 mM and 6.74±0.12 s-1,respectively.The investigation of substrate specificity with potential arginine substrate analogs showed a high specificity of CsAK and FoAK for L-arginine over D-arginine.Among the other potential guanidino substrates investigated,CsAK and FoAK showed an appreciable reaction rate of 29.3%and 52.4%of that obtained with L-arginine for L-canavanine.The median inhibitory concentration of quercetin and luteolin on the prokaryotically expressed CsAK and FoAK were determined.The results showed that the median inhibitory concentrations of quercetin on CsAK and FoAK were 21.5?M and 6.74?M,respectively,while luteolin did not show any inhibitory effect within the gradient concentration range of 200?M.These results lay foundation for further screening of CsAK and FoAK inhibitorsKnockdown efficiency of CsAK and FoAK using different RNAi methods were evaluated.In the case of CsAK,the injection of synthetic dsCsAK resulted in 47.35%silencing efficiency at 72 h after treatment,the feeding of synthetic dsCsAK decreased the expression of CsAK by 51.72%at 24h after treatment,the topical application of nanoparticle-encapsulated dsCsAK resulted in 42.65%silencing efficiency at 48h after treatment,the feeding of synthetic dsCsAK after pretreatment with ds-dsRNase decreased the expression of CsAK by 54.22%at 96 h after treatment,the feeding of bacteria-expressed dsCsAK after pretreatment with ds-dsRNase resulted in 64.15%and 62.20%silencing efficiency at 48 h and 72 h after treatment,respectively.However,the treatments by feeding nanoparticle-encapsulated dsCsAK and bacteria-expressed dsCsAK or injection of synthetic dsCsAK after pretreatment with ds-dsRNase did not achieve significant knockdown effects AS for RNAi of FoAK,the feeding of synthetic dsFoAK resulted in 45.92%silencing efficiency at 96 h after treatment,the topical application of nanoparticle-encapsulated dsFoAK decreased the expression of FoAK by 29.54%and 42.62%at 24 h and 48 h after treatment,the feeding of bacteria-expressed dsFoAK resulted in 53.83%silencing efficiency at 48h after nymph treatment and 79.65%at 72 h after adult treatment.However,the feeding of nanoparticle-encapsulated dsFoAK did not decrease the expression of FoAK.These results indicate that different RNAi methods will affect the silencing efficiency.