Cloning,Expressing And Fanction Testification Of Receptor Aminopeptidase N1in The Midgut Of Helicoverpa Armigera

Harmless to human and animals, and no pollution to environment, Bacillus thuringiensis has been uesd as microbiol insecticide for60years, the cry gene has also become the most widely used bioinsecticide over the world. Cry toxins show special toxicity against some insects, and the specificity is decided by the interaction between toxins and the special receptors in the brush border membrane of insect midgut, as a result, the research on the construction and function of receptors in the midgut of pests not only provide meterial for the interaction between toxins and receptors, but also build the foundation of mechanism of action of toxins. Aminopeptidase N is an accepted receptor of Cry toxins, being studied by most people. It belongs to exopeptidase. In the lepidopteran insects, APN can hydrolyze amino acid of protein and polypeptide.The cry1Ah is a novel insecticidal gene which was cloned from natrual strain BT8by our lab with independent intellectual property rights.The size of Cry1Ah protein is about133kDa, and the toxin shows high toxicity against Helicoverpa armigera, Ostrinia furnacalis and Chil suppressalis, even better than the commercial gene cry1Ac.Homology comparing results indicate that crylAh gene has a84percent similarity with cry1Ac gene, at the same time, the two toxins show the same bioassay that is toxic to H a but are safe to B m.By pull-down platform, Professor Zhou found that there are different binding proteins in the BBMV of H a with Cry1Ah and Cry1Ac proteins, it is interesting that there is one special binding protein band in the BBMV of H a, however no one in the B m, the MS result indicates this protein is aminopeptidase N. So we guess that this protein is the reason that makes CrylAh and CrylAc toxins kill Ha.In this report by desiging full-length primers, we cloned Ha midgut receptor gene apn1and expressed in prokaryotic system, at last testified the expressed APN1could bind to CrylAh and CrylAc toxin using western blotting. Bioinformatics make clear that the expressed APN1has characters of APN including conserved region GAMEN and HEXXH motif. There are also some potential N-or O-linked glycosylation sites. In order to demenstrate the binding ability of this expressed receptor in prokaryotic system to toxins, Western blotting is experimented. We found that APN1fragments of100kD and70kDa could also bind to Cry1Ah and Cry1Ac proteins, the results of MS indicate they are degraded from APN1.To furthur comfirm the binding region of H.a APN1and CrylAh protein, according to the characteristic of APN1, we cloned and expressed four fragments of APN1, then made these fragments bind to CrylAh protein, except H4fragment, other three fragments all expressed in prokaryotic successfully. Binding experiment showed that only H3could bind to CrylAh protein.In the study, the expressing system not only establish basis on the other receptors in cloning and expressing, but also provide thinking for the research on interaction machenism of Cry toxins and receptors. Binding experiment in vitro amd western blotting can serve as a fessible plan, initially comfirming relationship between receptors and Cry toxins. The follow-up study will focus on the purification of H3and bindind to CrylAh again, finally determing binding region of H a APN1and Cry1Ah.