Resistance Risk Assessment To Cry1Ab And Ligand Blot Analysis Of Cry1A Toxins To The Midgut BBMV In Chilo Suppressalis

The rice stem borer C.suppressalis(Walker) is one of the most serious pests threaterning rice production in China.Recently,C.suppressalis has become more serious because of cropping system and farming pattern changes,extensive planting of high yield and high quality rice,and suitable weather conditions.Resistance of C.suppressalis to chemical insecticides has obviously increased and let it more difficult to be controlled.Bt transgenic rice offers a new effective and safe weapon to control the rice stem borer.But from the evolutionary biology view,the target pests will inevitably evolve resistance to Bt toxins expressed in the Bt rice after extensive planting of Bt rice cultivars.Therefore, researching the mode of action of Bt toxins to C suppressalis and the potential resistance mechanisms will be important for developing resistance manangement strategy and prolonging the using longevity of Bt rice.In this study,we assessed resistance risk to Cry1Ab,analysized binding of Cry1A toxins to the midgut BBMV of C.suppressalis.The cDNA fragments of four APN genes from the midgut of C.suppressalis were cloned and sequenced.1.Laboratory selection for Cry1Ab resistance and resistance risk assessmentSusceptibility base-lines of activated Cry1Aa,Cry1Ab and Cry1Ac against a laboratory strain(CN) of C.suppressalis were determined using a surface contamination assay.Cry1Ab was more toxic than Cry1Ac,and Cry1Ac was more toxic than Cry1Aa against the third instar larvae of the CN strain.A mixed field population(JAZ) of C. suppressalis collected from Anhui,Jiangsu and Zhejiang provinces during 2006 was selected with activated Cry1Ab for 12 generations,and its susceptibility to Cry1Ab decreased 3.3-fold.The realized heritability(h~2) of Cry1Ab resistance was estimated as 0.068,indicating resistance development potential of Cry1Ab was low.According to the mean realized heritability and the mean slope of LD-P lines,10-fold resistance to Cry1Ab will be developed after 26 generations under selection pressure of 50%,and 8 generations under selection pressure of 95%.2.Ligand blot analysis of Cry1A toxins to midgut BBMV proteinsLigand blot analysis showed that there were 6 major binding proteins(50,70,90,120, 160 and 180 kDa) of Cry1Ac in the midgut brush border membrane vesicles(BBMV) of the CL strain.Binding protein bands of 180,160 and 90 kDa were much darker than the others,indicating their high binding concentration.Homologous competition binding results indicated that 180 kDa and 90 kDa protein bands were of low binding affinity and the other four protein bands(160,120,70 and 50 kDa) were of high binding affinity. Heterologous competition binding assays were conducted to study the binding site cross-reactivity between Cry1Ab and Cry1Ac.Cry1Ab competed for all binding sites recognized by Cry1Ac,with high affinity to 180,120,70 and 50 kDa proteins and low affinity to 160 and 90 kDa proteins.These data suggested that Cry1Ac and Cry1Ab shared several common binding sites in the midgut BBMV of C.suppressalis,but they had different binding affinity for each binding site.Considering the similarity in binding sites, Cry1Ab and Cry1Ac should not be used together in the transgenic Bt rice to control the target pest C.suppressalis.Isolation and identification of the binding proteins of Cry1A toxins in the midgut BBMV of C.suppressalis will facilitate our understanding of mode of action and resistance mechanism of Cry1A toxins.3.Cloning of cDNA fragments of APN genes of C.suppressalisAminopeptidase N is one kind of the important receptors of Bt toxins.A 600kb cDNA fragment of APN genes of C.suppressalis was amplified by RT-PCR with degenerate primers.The fragment was cloned and sequenced,and four cDNA sequences(named as CsAPN1,CsAPN2,CsAPN3 and CsAPN4) of about 600kb bases encoding APN genes were obtained.CsAPN1,CsAPN2,CsAPN3 and CsAPN4 shared 73%,100%,68%and 77% amino acid sequence similarity with Bombyx mori APN(GenBank accession No. BAA32140),C.suppressalis APN(GenBank accession No.ABC69855),Helicoverpa armigera APN2(GenBank accession No.AAP37951),and Ostrinia furnacalis APN (GenBank accession No.ABL01483).