Studies On The Relationship Between The Midgut Cadherin-like Protein, Alkaline Phosphatase And The Evolution Of Cry1Ac Resistance In Ostrinia Furnacalis

Asian corn borer (ACB), Ostrinia furnacalis (Guenee), is a major insect pest that attacks cornand other cereals in China. The transgenic Bt corn, which could protect the plants from the insectsby expressing Cry toxins, will be threatened by the evolution of insect resistance to Cry toxins.Recently, the resistance in lepidoptaran pests to Bt corn was reported in the field, and alsolaboratory selected colonies of ACB exhibited resistance to Cry1Ab and Cry1Ac. These indicatethat the ACB has potential to evolve resistance to Bt Cry toxins.“Multiple genes” strategy is oneof the important tactics in the IRM (Insect Resistant Management), which demands for thedifferent toxins with limited cross-resistance distinguished by mode of actions. In addition, thisstrategy is based on the understanding of toxins binding receptors on the insect gut. Furthermore,to investigate the relationship between the receptors and the evolution of resistance will providescientific basis for establishing the resistance detection and monitoring program. The objective ofthis study is to understand the action of ofCAD and ofALP on the resistance to Cry1Ac in the ACBCry1Ac resistance strain.The ofCAD was truncated expressed in Escherichia coli and the recombinant proteins bind toCry1Ac/Cry1Ab. The Cry1A binding region was located between the amino acid1347and1562,which were within the membrane proximal region (MPR). Screening the MPR of survivors withhigh resistance to Cry1Ac at200μg/g (protein/diet). Three mutant alleles were derived, i.e.MPR1-r1and MPR-r3with2or3amino acids substitutions, and MPR-r2, both deletion andsubstitution of amino acids were observed. Within these three alleles, the substitution1458T�'Swas occurred uniformly. The recombinant expressed MPR-r2showed lower Cry1Ac bindingability in the Co-IP compared to the MPR, and lost the synergistic effect to Cry1Ac presented inMPR. qPCR analysis showed a significantly down-regulation of ofcad transcription level in3of5instar during the larvae development.Beyond the ofcad, we cloned another putative Cry1Ac receptor gene ofalp (accession no.JN392445), which encoding542amino acid residues. The ofALP was mainly located on the apicaltip of microvilli (AMv) and basal lamina (BL) in the posterior region of the8-day-old larvalmidgut, using a McAb and immunofluorescence, was characterized as a Cry1Ac receptor by Co-IP.The binding between the ofALP and Cry1Ac could be dissociated by GalNAc. A turncated E.coliexpressed ofALP fragement with the Cry1Ac binding ability served as synergist of Cry1Ac in thebioassay. No significant transcription level differences of ofalp were detected during the larvaedevelopment between susceptive-and resistance-strain in the qPCR analysis.This is the first report that the down-regulation transcription level of ofcad and the MPRmutants may be responsible for the Cry1Ac resistance in the ACB, and the MPR sequence may bedevelop into a molecular marker incorporate in the field resistance monitoring.