| Transgenic cotton plants,expressing insecticidal proteins from Bacillus thuringiensis（Bt）,have been effectively used to control the major target pests since Bt cotton commercially introduced in 1996.However,the population of secondary pests,such as mirid bug Apolygus lucorum（Hemiptera:Miridae）has increased in China,as the amount of insecticide used in cotton fields decreased after the transgenic cotton has planted.It is especially attributed by the fact that current available Bt crops showed little to no toxicity against sap sucking pest.Recently,Cry51Aa1 and Cry51Aa2 proteins from B thuringiensis,belonging to aerolysin-typeβpore-forming protein family,have been reported to have potential activity against stink bugs and plant bugs.A Cry51Aa2 mutant,Cry51Aa2.834＿16,modified based on Cry51Aa1gene sequence have shown high toxicity against plant bugs,Lygus hesperus and Lygus lineolaris.In this study,we investigated the insecticidal activity of Cry51Aa1 and Cry51Aa2 against A.lucorum,and the action mode was also studied.Firstly,we conducted bioassays to test the toxicity of Cry51Aa1 and Cry51Aa2 protoxins against A.lucorum nymphs.The proteolytic activations of protoxins by A.lucorum midgut-homogenate or-juice or trypsin/chymotrypsin were then studied in vitro.ELISA assays were used to detect the binding interactions of midgut proteins with toxin proteins.We discovered the midgut receptors binding to both protoxins and activated toxins by Ligand blot analysis,and then identified the probable receptor proteins by pulldown assays and LC-MS/MS analysis.The main findings are as followed:Cry51Aa1 protoxin was produced from the recombinant Bt strain BMB171 hosting full-length gene.Protoxin solubilized from Bt spores was purified by ion exchange chromatography.The purified protoxin appeared at around 35 k Da.Cry51Aa2 protoxin was produced as recombinant His-tag protein using BL21（DE3）cells transformed with p ET30a＿51Aa2 plasmid.His-tag region in the N-terminal of the recombinant Cry51Aa2 protein was removed by the digestion with enterokinase enzyme and purified again with ion exchange chromatography.The purified Cry51Aa2 protoxin showed at around 35 k Da.1.Toxin-diet incorporated bioassays results showed A.lucorum nymphs were susceptible to both Cry51Aa1 and Cry51Aa2 protoxins.Toxicity was higher when feeding with Cry51Aa1 protoxin(LC50=11.87μgml-1),than with Cry51Aa2 protoxin(LC50=61.34μgml-1).2.Cry51Aa1 and Cry51Aa2 protoxins were activated by trypsin/chymotrypsin complex in vitro. The activated forms of both Cry51Aa1 and Cry51Aa2 toxins appeared at the same size just about25 k Da.The consistent results were observed when protoxins were incubated with midgut homogenate and midgut juices of A.lucorum and detected by western blot analysis.In addition,we detected the self-dimers of both Cry51Aa1 and Cry51Aa2 toxins appearing at 60 k Da.For in vivo activation,nymphs were fed with sublethal concentrations of protoxins for 1 hour.Midgut samples taken at different time points after 1-hour-feeding were analyzed by western blot using Cry51Aa1 and Cry51Aa2 antibodies.In 3 h and 6 h after toxin-feeding,both Cry51Aa1 and Cry51Aa2 toxins were partially activated,having protoxin,pre-activated,and activated forms in three separate bands.In samples of 12 h,24 h and 48 h after toxin-feeding,only a single band representing activated forms were detected.3.ELISA assays showed midgut proteins bind with both Cry51Aa1 and Cry51Aa2 toxins and the binding affinities were concentration dependent.When using 100 n M and 200 n M of Cry51Aa1, bindings were increased with increasing midgut concentrations and reached the highest binding in 20μg midgut proteins.But low bindings were observed when using 25 n M and 50 n M of Cry51Aa1.Bindings of all tested concentrations of Cry51Aa2,25,50,100,and 200 n M,were increased with increasing midgut proteins and highest bindings were also in 20μg and 30μg midguts.Dissociation constants（Kd）of the specific bindings to the fixed midgut proteins（20 μg）were 248.6±47.4 n M in Cry51Aa1 and 113.8±25.4 n M in Cry51Aa2.4.Ligand blot analysis showed both protoxin and activated Cry51Aa1 mainly bind with a protein, molecular size approximately around 25 k Da,in the midgut proteins.Binding with Cry51Aa2 activated and protoxin also showed the same band,while there was an additional receptor band approximately at 50 k Da in binding with activated Cry51Aa2.5.Midgut proteins binding to Cry51Aa1 and Cry51Aa2 protoxins were obtained by pull-down assay.The binding bands resolved in SDS-PAGE were excised and identified by the LC-MS/MS analysis.Among all the identified proteins,15 plasma membrane proteins were common bound to both Cry51Aa1 and Cry51Aa2 toxins.They included ABCA3,ABCB6,ABCC7,ABCD1, ABCG1,ABCG4,ABCF1 and one aminopeptidase protein.Twenty-four plasma membrane proteins were identified only bind with Cry51Aa1 or Cry51Aa2,respectively.Two aminopeptidase and ABCF2 proteins were found binding with Cry51Aa1.ABCB10,ABCD3,ABCG4,and ABCF3 proteins were found binding with Cry51Aa2. |
