Cloning And Functional Analysis Of Putative Bt Recept-Or Genes Belonging To ABCC Transporters Subfamily From Spodoptera Litura

Bacillus thuringiensis(Bt)is a Gram-positive bacterium,which belongs to Bacillus and exists widely in nature.The insecticidal crystal proteins produced by Bt during its sporulation stage are toxic to many insect pests,but are not harmful to animals,plants and human beings.Therefore,Bt as a biopesticide is widely used in the control of pest.However,insect resistance evolves with the broad use of Bt sprays and transgenic plants,which shortens the efficacy in long terms.In fact,it is the changes of Bt receptors that lead to alterations in the binding of the toxins,which is the main reason for illustrating Bt resistance.Spodoptera litura as a pest from Lepidoptera is susceptible to Cry1Ca,insead of Cry 1 Ac.Except for SlAPN having been reported as a receptor of Cry1Ca,it is not clear whether there are any other receptors involved in the susceptibility of Spodoptera litura to Cry1Ca.Therefore,cloning and functional analysis of Cry toxin receptors from Spodoptera litura is very important for insect resistance management.The larvae of Spodoptera litura and Sl-HP cell line derived from its ovary were chosen as materials.Analysis of the transcriptome of S1-HP cells was carried out,then the transcripts of thirteen genes from the ABC transporters superfamily were obtained,which belonged to ABCA,ABCB,ABCC,ABCD and ABCG subfamilies.The phylogenetic analysis based on alignment of amino acid sequences of the selected ABCC proteins revealed that SlABCC3 was more closely related to ABCC3 from H.armigera and Spodoptera exigua.SlABCC1,SlABCCl-like and SlABCC4-like belonged to a different cluster respectively.SlABCC3 ORF from the midgut cDNA of Spodoptera litura was cloned by RT-PCR.The full length of SIABCC3 ORF was 4077 bp,and encoded 1359 amino acids.The predicted molecular mass and the theoretical pI of SlABCC3 were 151.69 kD,8.24,respectively.The fragment of SlABCC3 was expressed by prokaryotic expression system and rabbit polyclonal antibody was made.In order to express SlABCC3 in insect cells,the vector of pie2-EGFP-N1-SlABCC3 was constructed.Analysis of the subcellular localization revealed that most of SlABCC3-GFP was localized in cytoplasm whereas a small amount of SlABCC3-GFP was localized on cell membrane.Over-expression of SlABCC3-GFP could enhance the susceptibility of Sl-HP and TnH5 cells to activated Cry 1 Ac to some degree,but did not increase the susceptibility of TnH5 cells to activated Cry1Ca.It suggested that SlABCC3 was the receptor of Cry 1 Ac,not Cry1Ca.SlABCC4-like ORF from the midgut cDNA of Spodoptera litura was also cloned by RT-PCR.The full length of SlABCC4-like ORF was 4185 bp,and encoded 1395 amino acids.The predicted molecular mass and the theoretical pI of SlABCC4-like were 154.86 kD,8.32 respectively.The vector of pie2-EGFP-N1-SlABCC4-like was also constructed so as to express SlABCC4-like in insect cells.Fluorescence microscopic observation revealed that SlABCC4-like was localized on cell membrane.Over-expression of SlABCC4-like didn't enhance the susceptibility of TnH5 cells to activated Cry 1 Ac,Cry1Fa,CrylAb and Cry2Aa.Knocking down of SlABCC4-like didn't have effects on the susceptibility of Sl-HP cells to activated CrylAc and Cry1Ca.It suggested that SlABCC4-like wasn't the receptor of Cry1Ac,Cry1Ca,Cry1Fa,Cry1Ab and Cry2Aa.In one word,SlABCC3 was the receptor of Cry1 Ac,however,SlABCC4-like wasn't the receptor of CrylAc.These results offered important references for the development of Bt biopesticides and Bt resistance management.