Cloning Key Functional Genes And RNAi-based Management Of Striped Flea Beetle, Phyllotreta Striolata (Coleoptera: Chrysomelidae)

Striped flea beetle(SFB),Phyllotreta striolata(Fabricius)(Coleoptera: Chrysomelidae)is one of the most serious insect pests in crucifer crops worldwide.In this thesis,the energy metabolism system and olfactory sense of SFB were exploited to develop a novel strategy for pest control.Phyllotreta striolata Arginine kinase(PsAK) gene and a special olfactory receptor gene(PsOr1)were cloned by RACE technique, respectively.The genes and their expressed-protein sequences were analyzed with bioinformatics methods.According to analysis of the sequences,we designed and constructed double-stranded RNA(dsRNA)based on the functional regions of genes to interfere with the expression of the genes of interest.RNAi technique was applied to develop a novel strategy for insect control,aimed at disturbing the balance of energy metabolism or depriving target-oriented capability of the bettles.The thesis includes five chapters,and is briefed as follows:1.The full-length(1 508 bp)cDNA of PsAK was successfully amplified by RACE technique and comprises of a 51 bp of 5'untranslated region(UTR),1 071 bp of open reading frame(ORF),and 386 bp of 3'UTR.The cDNA sequence has been deposited to GenBank under accession No.EU420057.The ORF encodes a polypeptide of 356 amino acids with most of the residues considered necessary for AK function,which has a theoretical mass of 40.02 KDa and an estimated isoelectric point of 5.86.The highly conserved motif CPTNLGT(position 271-277)around a reactive cysteinyl residue(position 271)constitutes a signature pattern for this family of AK.PsAK also contains a pair of highly conserved amino acids(D62 and R193).The distance between OD₂ atom of D62 and NH₂ atom of R193 is 2.86(?)in our modeled 3-D structure,which can form a strong hydrogen bond stabilizing the closed substrate-bound structure of PsAK.The cDNA-derived amino acid sequence of PsAK shows 66%-91%amino acid sequence identity with other AKs of insects.The conceptual three-dimensional structure of PsAK was generated in silico by the homology-modeling server SWISS-MODEL and protein superposition server SuperPose.The overall structure of PsAK was very similar to that of the transition state analogue complex of L.polyphemus AK(LpAK)(75%sequence identity,root mean square deviation of 0.7(?)for 356 aligned residues),for which the crystal structure had been determined.Additionally,seven conserved residues were predicted to interact with the substrate arginine(S63,G64,V65,Y68,E225,C271 and E314)and five positively charged clusters were predicted to interact with the substrate ADP (R124,R126,R229,R280 and R309)in PsAK,compared with LpAK.The phylogenetic tree was constructed from the amino acid sequence of PsAK and other insect AKs using neighbour-joining(NJ)method.The phylogenetic tree clearly indicated that insect AKs could be separated into two groups.Group 1 is consisted of typical AKs from various insect species,and PsAK also belonged to this group.2.We selected the sequence of 332 bp fragment(nucleotide 790 to 1122)from PsAK cDNA containing the active site motif CPTNLGT to obtain dsRNA.The synchronous adult beetles with a 1:1 sex ratio fed on the cotyledons and leaves of seedlings of Chinese cabbage sprayed with varying doses of dsRNA solutions.LC50 value was determined to 0.80 ng/mL(P＜0.05)by implementing the concentrations of dsRNA with a two-fold serial dilutions.We also tested the effects of dsRNA on the life cycle of striped flea beetle.The results showed that in untreated control(UT)group (dsRNA 0 ng/mL)over 90%of adults survived during the test.Fourty-nine eggs were laid per female and 74%eggs could hatch into larvae.However,there were remarkable differences between feeding dsRNA groups and UT group(P＜0.05).Survival rate was decreased from 85%to 15%for feeding dsRNA groups,which were dramatically lower than UT group.The mean number of egg-laying per female was 7-41 eggs for feeding groups with the 14-84 percentages comparable to those obtained by UT group (P＜0.05).It was also obvious that the significant influence of dsRNA feeding on egg hatching rate.There were less than 13%of the eggs that hatched into larvae in the group fed on dsRNA 1.60 ng/mL.The feeding bioassays indicated that minute quantities of dsRNA can greatly impair beetle's life cycle.RT-PCR analysis and AK activity assay verified RNAi effectiveness at the molecular level.Ingestion of dsRNA not only significantly resulted in adults retarding and mortality,but also severely damaged females' fecundity and fertility,suggesting that RNAi targeting AK can be a potential and attractive tool to control insect pests.3.Host-plants and insects that feast on them have waged war for hundreds of millions of years.The host-plant preference of insect pest relics on olfaction and accordingly mediated via G protein-coupled signal transduction cascades.Here,we cloned and characterized PsOr1,the first candidate odorant receptor from P.striolata. The cDNA sequence has been deposited to GenBank under accession No.EU647221. The ORF(1 437 bp)encodes a 478 amino acid residues and includes seven putative transmembrane domains(TM),which is the typical characteristic of G protein-coupled receptors.However,our predicted PsOr1 membrane topology has an intracellular N-terminus and an extracellular C-terminus,which is contrary to typical N_out-C_in topology of GPCR superfamily.The axis of the helixâ… ,â…¢,â…¤andâ…¦are parallel to that of helixâ…¡,â…£,â…¥,but oppositely directed.The transmembrane helixs consists of 23 AA,except helixâ…£.While conventional odorant receptors were found to be extremely diverse across insect orders,PsOr1 shared rather high primary amino acid conservation with other insect orthologs,particularly for the stretches located in the last third of the polypeptide chain(i.e.in the region which included the putative transmembrane domainsâ…¤-â…¦and the loops connecting them).The last 164 amino acids that comprise the C-terminal region shared an extremely high conservation at levels that ranged between 82%and 95%identity.PsOr1 shared 95%identity and 97% similarity with its most evolutionarily related homolog in the last 164 amino acids from Tribolium castaneum,which reflected a strong negative selection pressure on this part of the protein,and thus suggested a particular functional relevance of this region. Semi-quantitative RT-PCR analysis showed that PsOr1 was highly expressed in antenna of adult beetles,relative low expressed in head(including mouthparts etc.), and undetectable in other tissues of adults,which may play a role in sensing volatile odors as well as sensing liquid chemical stimulus. 4.To test the assumption that the high conserved structure of PsOr1 reflects important biological functions,and that this gene acts as an ortholog of Drosophila melanogaster Or83b(DmOr83b),we micro-injected dsRNA into the last phase of beetle pupae.The sequence of 484 bp fragment of PsOr1 cDNA encoding C-terminal region was chosen to acquire dsRNA and determine PsOr1 function.Compared with null hypothesis(equal time on each side),the wildtype beetles showed significant difference(t₁₄=2.99,p=.010 for allyl isothiocyanate;t₁₃=3.04,p=0.009 for a-pinene,respectively),exhibiting apparent chemotaxis to the odour.However,for RNAi treated beetles,the chemotaxis behavioral response of PsOr1 silencing beetles to attractant odor allyl isothiocyanate or repellent a-pinene were not significantly different from a random distribution.RT-PCR analysis demonstrated the suppression of dsRNA target PsOr1 at the molecular level.Furthermore,RNAi effects on the oviposition preference and feeding preference of P.striolata for 5 species of cruciferous vegetables were tested.For untreated control group,the oviposition preference performed in the same pattern as that of the feeding preference,with Brassica juncea as the most favorable host plant and the Brassica alboglabra as the least favorable host plant.Similar results were achieved when valuating the feeding preference by the feeding area and the number of beedes on the plants,which showed significant correlation with each other(r=0.9472,P＜0.05).In the contrast,after RNAi treatment,the experimental group had significant differences in damage area values and the mean number of beetles on different plants compared with the control group. No correlation was observed between the feeding area and the number of beedes on the plants.The oviposition preference showed no preference among different plants.The data domomstrated that silencing PsOr1 by RNAi apparently impaired host-plant preference in Phyllotreta striolata,indicating the novel insect control approach by RNAi targeting PsOr1 and its orthologs might be effective in blocking host-plant seeking behavior in diverse insect pests.