Identification And Functional Analysis Of SeABCB3 And SeABCG20 Genes From The Beet Armyworm,Spodoptera Exigua

The beet armyworm,Spodoptera exigua(Hubner)(Lepidoptera:Noctuidae),is a globally distributed omnivorous agricultural pest.It is reported that the insecticidal crystal protein(Cry protein)produced by Bacillus thuringiensis(Bt)is able to bind to the functional receptor,ATP-binding cassette transporter(ABC transporter).Two ABC transporter genes(SeABCB3 and SeABCG20)from S.exigua were identified in this study.The interaction of the two ABC transporters with Bt Cry1Ab35 and Bt Cry1Ca15 proteins was determined by Ligand blot,ELISA,immunofluorescence and cytotoxicity analyses.RNAi technology was used to analyze the functions of SeABCs.The main results are as follows:1.Identification of ABC transporter family genes from S.exigua larvae.After alignment of the S.exigua genome sequence to the known insect ABC transporter sequence in the NCBI database,53 of ABC transporter family genes were discovered to be distributed across its 17 chromosomes.According to the homology of the nucleotide binding domain(NBD)amino acid sequence,it was clustered into 8 subfamilies(ABCA-ABCH).The midgut transcriptome of S.exigua larvae treated with CrylAb protein was analyzed and the differentially expressed genes SeABCB3 and SeABCG20 were identified.The open reading frames of SeABCB3 and SeABCG20 were 3915 bp and 2301 bp,encoding 1304 and 766 amino acids,respectively.The molecular weights of the anticipated proteins were 142.66 kDa and 85.00 kDa.Both SeABCB3 including two TMD(transmember domin,TMD)domains and two NBD domains and SeABCG20 containing one TMD and one NBD domain belong to transmembrane proteins according to SMART and TMHMM software analysis of domains and transmembrane regions.The phylogenetic tree was constructed by MEGA_X software.The results showed that SeABCB3 and SeABCG20 had the highest similarity with ABC transporters of Spodoptera litura,indicating that ABCB3 and ABCG20 had high homology with S.litura.2.The expression analysis of SeABCB3 and SeABCG20 genes in different developmental stages and different tissue structures of S.exigua larvae.The expression level of the SeABCB3 and SeABCG20 gene was found to be highest in the egg stage,according to quantitative reverse transcription PCR data.The Malpighian tubules and midgut of the fifth instar larvae had the highest SeABCB3 and SeABCG20 expression levels,and other tissues expressing these genes to a lesser extent.3.The prokaryotic expression of SeABCB3-OM(1123-1963 bp)and SeABCG20-OM(7-839 bp)genes and their binding analysis with Bt Cry1Ab35 and Bt Cry1Ca15 activated proteins.The prokaryotic expression vector pET30a-SeABCB3-OM/-SeABCG20-OM was constructed.The 38 kDa recombinant protein was successfully expressed in E.coli BL21and the polyclonal antibody was prepared.The binding characteristics of SeABCB3-OM and SeABCG20-OM proteins with activated Cryl Ab3 5 and Cry1Ca15 proteins were identified by ligand blot analysis.The results demonstrated that both SeABCB3 and SeABCG20 had a affinity with Bt Cry proteins.ELISA results revealed that the Kd values of recombinant proteins of SeABCB3-OM and SeABCG20-OM with Bt Cry1Ab35 protein were 29.04±13.83 nM and 5.33±9.017 nM,respectively.The above results demonstrated that SeABCB3 and SeABCG20 were potential functional receptors of Cry1Ab35 and Cry1Ca15 proteins.4.The expression of SeABCG20 gene in Sf9 cells and analysis of its interactions with the activated CrylAb35 and Cry1Cal5 proteins.The Bac-to-Bac insect baculovirus expression system was used to construct the recombinant pFastBacTM HTA-gfp-SeABCG20 expression vector.The recombinant protein with a molecular weight of 120 kDa was efficiently produced in Sf9 cells.The results of immunofluorescence analysis showed that SeABCG20 gene was expressed on the cell membrane and could specifically bind to CrylAb35 and Cry1Cal5 proteins on the cell membrane.The results of cytotoxicity assay showed that the mortality of Sf9 cells expressing SeABCG20 gene increased with the increase of Cry protein concentration compared with the control group.The sensitivity of Sf9 cells to Cry1Ab35 and Cry1Ca15 protein increased with the increasing of SeABCG20 gene expression level,indicating that SeABCG20 is a functional receptor of Cryl Ab35 and Cry1Ca15 proteins.5.Functional analysis of SeABCB3 and SeABCG20 genes.SeABCB3 and SeABCG20 were silenced using RNA interference(RNAi),and the expression levels of the target genes were fallen by 89.07%and 81.46%after 72 hours of injection dsSeABCB3 and dsSeABCG20 to 4th instar larvae,respectively.The corrected mortality of larvae injected with dsgfp,dsSeABCB3 and dsSeABCG20 after 72 hours showed 8.63%,51.82%and 68.73%,respectively.Some of the dsSeABCB3 and dsSeABCG20-injected larvae exhibited a dehydration phenotype.It indicated that SeABCB3 and SeABCG20 genes may be the key genes for the growth and development of S.exigua.The sensitivity of S.exigua larvae after silencing SeABCB3 and SeABCG20 genes to Bt Cry proteins was higher compared with the control group.