Specific Binding Protein ABCC1 Is Associated With Cry2Ab,Cry1Ac Toxicity In Helicoverpa Armigera

The binding of specifical receptor proteins on the brush border membrane vesicles of insect midgut epithelium with Bacillus thuringiensis insecticidal proteins is a key step in the mode of action of Bt toxicity.Mutations or altered expressions of receptor proteins maybe make insect resistance to Bt proteins.The previous studies indicate that ATP-Binding Cassette(ABC)transporter family protein is a kind of Cry1 A receptors,and the mutation of ABC transporter family protein relate with the insect resistance to Bt.In this study,based on the full-length sequence of ABCC1 coding gene in Helicoverpa armigera was found,we further tested the expression levels of ABCC1 in different tissues and life stages of H.armigera,the binding characters of ABCC1 with Cry2 Ab and Cry1 Ac in vitro,and the functional roles of ABCC1 in the toxicity of Cry2 Ab and Cry1 Ac separately.The results can provide the theoretical basis for indicating the role of ABCC1 in Cry2 Ab and Cry1 Ac insecticidal mechanism,and rational application of Cry2 Ab and Cry1 Ac to delay the resistance evolution in cotton bollworm.The main results were as followed:1.The full length Ha ABCC1 gene was cloned using PCR and RACE-PCR,and it was analyzed by bioinformation tolls.The open reading frame of Ha ABCC1 was 4545 bp(Gen Bank:KY796050)and the translated protein encoding with 1515 amino acids.The predicted molecular weight was 169.75 k Da and isoelectric point was 6.68.The predicted protein structure consisted of two large extracellular regions,14 transmembrane regions,two ABC transporter integral membrane type-1 fused domains(TMD1 and TMD2)and two ABC transporter-type domains(NBD2),14 dispersed N-glycosylation sites,and 16 concentrated O-glycosylation sites,but no signal peptide was predicted.Phylogenetic analysis of ABCC1,ABCC2 and ABCC3 genes of different Lepidopteran insects revealed that Ha ABCC1 was most closely related to Slit ABCC1,followed by Atra ABCC1,Ppol ABCC1,and Pxut ABCC1.The Ha ABCC1 gene expression was found in all the developmental stages and different tissues.The highest expression was observed in fourth-instar and fifth-instar larvae and Malpighian tubules,but the lowest expression was found in adult and foregut tissues.2.Two c DNA fragments baring TMD1 and TMD2 domains respectively were cloned and expressed as the recombinant peptides in the prokaryotic expression system.Following purification,ligand blot analysis was conducted and the results showed that both Ha ABCC1 fragments TMD1 and TMD2 could specifically bind to activated Cry1 Ac and Cry2 Ab toxin separately.3.Ha ABCC1 gene was knockdown by RNAi,the gene expression level was significantly decreased after 48 hours.In the diet overlay bioassay with Cry1 Ac and Cry2 Ab toxin,the mortality rate of the larvae preinjected with si ABCC1 obviously reduced than that of injected with DEPC water and si EGFP.Sf9 cells were transfected with p Ac-ABCC1 plasmid construct and the success of transfection was confirmed by PCR.The cell mortality rate was increased after being treated with Cry1 Ac and Cry2 Ab protein,which is significantly higher than that of the control transfected with empty p Ac plasmid.4.The ABCC1 gene was amplified and sequenced in the susceptible strain(96s)and the Cry1Ac-resistant strain(Bt R)of H.armigera respectively.The results showed that there was no difference in the amino acid sequence of the coding region of the ABCC1 gene.However,the expression of ABCC1 significantly reduced in Bt R resistant strain compared with the sensitive strain 96 S,and the expression level in the sensitive H.armigera was about 2.5 times higher than that of in resistant strain.5.Altogether current results suggested that ABCC1 of H.armigera was the specific binding protein for Cry2 Ab and Cry1 Ac,they affected the toxicity of Cry2 Ab and Cry1 Ac,maybe they were functional receptors for Cry2 ab and Cry1 Ac respectively and maybe the reduced ABCC1 expression was related with Cry1Ac-resistance.