The Recombinant Expression And Functional Analysis Of ABCC2 And ABCC3 Genes From Four Different Insect Species

Bacillus thuringiensi(Bt),as a microbiological pesticide,is produced in a huge scale and widely used.It has very good economic value.The produced crystal proteins(ICPs)could directly kill pests,and the genes encoding ICPs are used as target genes for transgenic crops.Therefore,this microorganism has great practical significance.During the process of insecticidal activity,the important keys are the recognition and binding between the ICPs and the receptors on insect midgut epithelial cells.So identification of the BT toxin receptors is very important for further elucidation the model of action.Four types of receptors of Bt toxins were found in the insect midgut:alkaline phosphatase(ALP),aminopeptidase N(APN),cadherin(Cadherin)and glycolipids.In recent years,various ABC family proteins of Lepidopteran insects have been reported to serve as BT toxin receptors and may be associated with resistance to Bt toxin.So far,whether the ABCC proteins of Spodoptera litura,Helicoverpa armigera,Pectinophora gassypiella and Mythimna separata can mediate toxicity of BT toxin to insect cell lines is not clear.In this study,firstly,RNAi-mediated knockdown targeting SlABCC3 significantly decreased the susceptibility of TH cells to the activated Cry 1 Ac(p<0.05).Then,the constructed eukaryotic plasmids pEE2-EGFP-N1-SlABCC3-GFPwere transfected into TH and TnH5 cells,respectively.Over-expression of the gene strongly increased the susceptibility of both the two different cells to the CrylAc toxin(p<0.05).Comparing to the Hacadherin of Helicoverpa armigera,the SlABCC3 was more toxic to the TnH5 cells.In addition,SlABCC3 and Hacadherin had a strong synergistic effect on cytotoxicity of the activated Cry1Ac(p<0.05).These results suggested that the SlABCC3 served as BT toxin receptor and had a strong synergistic effect on cytotoxicity of the activated Cry1Ac with Hacadherin.Besides,the HaABCC2-GFP({Helicoverpa armigera),PgABCC2-GFP(Pectinophora gassypiella),MsABCC2-GFP and MsABCC3-GFP(Mythimna separate)were localized on the cell membrane and in the cytoplasm of TnHs cells;and all these proteins can mediate the cytotoxicity of the activated Cry1Ac to TnH5 cells.It showed that the ABCC2 and ABCC3 proteins of these Lepidopteran insects are potential BT toxin receptors.We infer that both ABCC2 and ABCC3 in many insects may be common BT toxin receptors.