Genome-wide Identification And Expressionprofiles Of ATP-Binding Cassette Transporter Genes In The Oriental Fruit Fly,bactrocera Dorsalis(Hendel) (Diptera:Tephritidae)

The oriental fruit fly,Bactrocera dorsalis?Hendel??Diptera:Tephritidae?,is an important agricultural scratching mouthparts pest in large parts of tropical Asia.Currently,insecticides are the primary compounds used for fruit fly pest management.However,insecticide resistance in B.dorsalis had been reported in China.The ATP-binding cassette?ABC?is the largest transporter gene family and the ABCs are membrane proteins that use ATP to transport substrates across membranes.ABC transporters reduce the concentration of intracellular and exogenous substances,resulting in a decrease in the insect's sensitivity to insecticides.Knowledge of the detoxification process in B.dorsalis mainly involves enzymes of phase I and phase II,such as P450 monooxygenases?P450s?,glutathione S-transferases?GSTs?,and carboxylesterases?CarEs?.However,Less is known about the ABC transporters.In this thesis,according to the transcriptomic and genomic databases of B.dorsalis,we reported a genome-wide identification,phylogenetic,transcriptional expression analysis and RNA interference of the ABC genes in this insect pest.Main results are as follows:We identified 47 BdABCs from the B.dorsalis genome and verified ORFs by different transcriptomic resources into eight subfamilies?A-H?.These included 7ABCAs,7 ABCBs,9 ABCCs,2 ABCDs,1 ABCEs,3 ABCFs,15 ABCGs,and 3 ABCHs,respectively.Different isoforms were found in BdABCB2 and BdABCG1.According to the different transcriptome data sets,29 out of 47 BdABCs contained complete open reading frames?ORFs?.The shortest ORF contained 1830 bp and the longest cantained6705 bp,encoding 610 to 2235 amino acids.Comparative phylogenetic analysis of the ABCs suggested an orthologous relationship between B.dorsalis and Drosophila melanogaster,and the conserved sequence identity was more than 80%,especially in these genes related to pesticide resistance and essential biological processes.Most of BdABCs were highly similar to DmABCs,and the same subfamily proteins were clustered into the same branches.Each subfamily had a distinct functional cluster.Comparison of transcriptome and relative expression patterns of BdABCs via real-time quantitative PCR?RT-qPCR?indicated that BdABCs might have diverse multifunctions within different B.dorsalis tissues.Most of BdABCs genes were highly expressed in Midgut,Malpighian tubules and fat Fat body?transcriptional levels are expressed 5 folds or more to Testis?,which suggested that BdABCs have a key role in insect metabolic detoxification.Among the tested tissues,BdABCB7 was the most highly expressed in the central nervous system.BdABCB7,was the ortholog of DmMDR65 from D.melanogaster,and DmMDR65 was specifically localized at the humoral barrier of D.melanogaster.DmMDR65 and was found to be required for normal chemical protection of the brain,and our results suggested that BdABCB7 might have the similar function in the B.dorsalis.We studied the relative expressions of 18 BdABC transporterss in adult of B.dorsalis after nonlethal exposure to LD₁₀0 or LD₂₀ doses of malathion,beta-cypermethrin,and avermectin.We found that expression levels of most of BdABCs were not significantly different from the control.At relatively low doses,most expressions of BdABCs were increased with increased dose.We also found that many BdABCs had higher expression at the LD₅₀?up-regulate 6 folds or more to control?.Consequently,we added a time-point experiment at LD₅₀ to explore potential functions of the BdABC genes.More than half of BdABC transporters genes were up-regulated to a maximum at 48 h post-treatment and down-regulated at 72 h post-exposure.Additionally,specific ABC transporter was probably responsible for resistance to specific insecticide,such as BdABCB7 may related to resistance of malathion specifically,and BdABCE1 was involved in the detoxification of avermectin and beta cypermethrin and BdABCG1 played a role in the metabolism of malathion and avermectin.RNAi of BdABCB7 was performed to investigate the possible involvements of BdABCB7 in detoxifying malathion.RNAi treatment and RT-qPCR demonstrated that expressions of BdABCB7 were significantly silenced after the adults were injected with single dsRNA and the silence efficiencies were 51%?after 24 h?,and 66%?after 48h?,repectively.Comparing to control flies,RNAi suppression of BdABCB7 significantly increased the toxicity of malathion.The notable difference was for malathion in which mortality was 53%in the control flies and 75%in the BdABCB7 RNAi flies.The death rate of RNAi strains is significantly faster than control.These results may indicated that BdABCB7 was a regulator of the toxicity of malathion and may might be involved in resistance to malathion.In summary,we identified 47 BdABC genes from the B.dorsalis genome and from several tissue specific transcriptome data sets via bioinformatics,molecular biology,RNAi,and biometrics.Based on phylogenetic analysis,the probable relationships were unveiled between these insects via clear orthologs.Most of the BdABC genes were sensitive to exposure to all three insecticides,implying BdABCs may be associated with insecticide resistance.RNAi of BdABCB7 increased the susceptibility of B.dorsalis to malathion.Taken together,this study revealed the molecular mechanisms of BdABCs in mediating insecticide resistance in B.dorsalis,and provide insights into the management of this flie,as well as identification of new insectides targets.