Functional Analysis Of Key Regions Of Cry1Ac Toxin Receptor ABCC2 Protein Based On Protein Split Technology

Bacillus thuringiensis(Bt)is a gram-positive bacterium that produces insecticidal protein toxins during spore formation that have been used to control agricultural,medical and forestry pests for more than 60 years.They are widely used,partly as alternatives to chemical pesticides,and have a huge market in organic agriculture and forestry.These markets benefit from the fact that Bt is highly specific to target pests compared to the non-target effects of chemical pesticides.The commercial impact of Bt has increased with the planting of transgenic plants,which are transformed by modified toxin genes to produce protein toxins in the plants themselves.Since it was first commercialized in 1996,more than 200 million hectares of Bt crops have been planted worldwide.Its success is largely due to the high efficiency of insecticidal proteins and their safety to non-target insects.However,the widespread use of Bt insecticide and the increasing planting area of Bt crops,insects have developed a certain level of resistance,which severely limited its long-term use.Among them,the mutation of Bt toxin receptors leads to the change of its ability to bind to toxins,which is one of the key mechanisms for Bt resistance.ABCC2 is considered to be one of the most important receptors of Bt Cryl A toxin so far.The ABC transporter family of insects is also related to pigment transport,uric acid metabolism,development,chemical pesticide and pesticide resistance.Mutations of the ABC transporter family C2(ABCC2)gene in lepidopterans have been identified as one of the main machanisms of high levels of insect resistance to the toxin Cryl A.Six extracellular loops of the ABCC2 transporter have been identified as potential Cryl A toxin candidate binding sites,but have yet to be investigated further.This study indicated that the product of HaABCC2 gene expression in insect cells was mainly localized on the cell membrane.In order to investigate whether or which half transporter of the HaABCC2 protein binds to Cry1Ac toxin,the HaABCC2 transporter was splitted into two half-transpoters by genetic engineering.When transfected into TnH5 cells,both expression products were localized in the cytoplasm.However,when co-transfected into TnH5 cells,expression products was re-localized on the cell membrane.It is speculated that there is an interaction between the two half-transporters,and each of them contains part of the membrane localization signal,or the interaction affects the structure,then the structure affects the location.The EC50 value of Cry1Ac measured after co-transfected was about 10 folds higher than that to TnH5 cells transfected with wild-type HaABCC2 into TnH5 cells,suggesting that the EC50 value may be related to the interaction between the two half-transporters and the lower efficiency of co-transportation to the cell membrane.To further determine the sites of membrane localization signals,two NBD deletion mutants were constructed,which were HaABCC2-D1-del NBD and HaABCC2-D2-del-NBD,respectively.If the NBD-deletion mutant was co-expressed with another half-transpoter(containing NBD),only when the first NBD was absent,partial expressed products were still localized on the cell membrane,and nearly 40%cells were swollen when treated with 40 ng/mL Cry1Ac toxin.When co-expressed the first half-transpoter(containing NBD)and the second NBD deletion half-transpoter,the expressed products can not be localized on the cell membrane,no TnH5 cells were swollen when treated with 40 ng/mL Cry1Ac toxin.This indicated that both NBD could affect the cell membrane localization of HaABCC2,but the second had a greater effect.The results for the second NBD deletion mutant in wild-type HaABCC2 was similar with that just described above.NBD1 and NBD2 of HaABCC2 are the two largest intracellular loops.It was speculated that the membrane localization signal might exist on the two NBDS,so NBD1-HaABCC2-D2 was constructed,which was still localized in the cytoplasm after overexpression in TnH5 cells,indicating that the membrane localization signal was also related to the first transmembrane domain TMD1.When co-transfected it with HaABCC2-D1-del-NBD,re-localized on the cell membrane and was highly sensitive to Cry1Ac toxin(about 10 folds to wild-type).It was speculated that the cell membrane localization of HaABCC2 is related to the integrity of loops.HaABCC4-like protein also localized on the cell membrane,and it can not mediate the toxicity of Cry toxins.It was splitted into two parts and then co-transfected into TnH5 cells,the co-expressed products were also re-localized on the cell membrane,indicating that phenomenons that splitted proteins can be assembled into a whole are common in ABC transporter proteins.The NBDs of the two half-transporters of HaABCC4-like were replaced by the NBDs of HaABCC2,respectively.When co-expressed HaABCC4-like-D1-NBD(from HaABCC2)and HaABCC2-D2-NBD,or HaABCC4-like-D2-NBD(from HaABCC2)and HaABCC2-D1-NBD,the expression products could not be re-localized on the cell membrane,confirming that the two NBDs of HaABCC2 were not enough to make HaABCC2 localize to the cell membrane and the native localization were also related to other parts such as loops.In conclusion,this study showed that the two half transporters of HaABCC2 could interact with each other and co-localized on the cell membrane,and can mediate the toxicity of Cry1Ac toxin.And the localization of HaABCC2 on the cell membrane was related to NBDs and other intracellular loops.These experimental results provided important reference for the study of the binding sites of toxins and receptors,and insect resistance mechanism to Bt toxin.