Cloning, Expression And Functional Characteristics Of Cryiac Receptor Aminopeptidase N2in Plutella Xylostella(L.)

The diamondback moth, Plutella xylostella (L.), is one of the world wide agricultural pest of cruciferous vegetables. Bacillus thuringiensis is an aerobic gram-positive bacterium, produces various insecticidal proteins which could kill target insects, but safety to non-target organisms and being friendly to environment, so this bacterium has been used widely in agriculture for pest management. In1990, Tabashnik had reported that the diamondback moth(Plutella xylostella L.) develop resistance to Bt toxin in field, this was the first report and the only insect resistants to Bt toxin in open-field, from now on, six insect(Plutella xylostella, Trichoplusia ni, Helicoverpa zea, Spodoptera frugiperda, Osreinia furnaialis, Pectinophora gossypiella) has developed resistance to Bt toxin or Bt crops in open fields and greenhouses, the control of the resistence to pesticides relies on the identification of the resistance mechanisms, but the molecular mechanisms of the field-and greenhouse-evolved resistance to Bt have not yet been identified. Our research has cloned the the resistance and susceptible strans Plutella xylostella APN2(APN2-S/R) full length genes which may related to the resistance to Bt; Constructed the recombinant vector and expressed in Sf9cells, and then analyzed the function of the APN2-S/R recombinant proteins with Cry1Ac protein. The results were as follows:(1) Cloning of APN2full length gene from Plutella xylostella midgutThe resistance and susceptible strans Plutella xylostella APN2(APN2-S/R) full length genes was amplified according to the gene sequence (accession number:GU479677.1). The results showed that the full length of APN2was2853bp (ORF), coding950amino acids. The protein molecular weight was about107kDa calculated by Compute pI/Mw tool software. The result of BLAST in GenBank indicated that the identity of APN2sequence was98%between Plutella xylostella. There was4amino acids mutation in APN2gene from susceptible strain of Plutella xylostella relative to that of resistant strain, Phe120Val、Glu344Lys、Asp706Glu and Phe709Tyr;(2) Expression of APN2gene from Plutella xylostella in Sf9cellsAPN2genes from susceptible and resistant Plutella xylostella (L.) were cloned into pFastBac HTB vectors, and then expressed in Sf9cells. The results showed that we successfully established the APN2-bacmid using the insect cells Bac-to-Bac baculovirus expression system, and expressed APN2-S/R and GFP protein in Sf9cells. The green fluorescence was observed in Sf9cells infected with the recombinant virus GFP, a107 kDa protein was examined and confirmed by SDS-PAGE and Western blotting;(3) Functional analysis of Plutella xylostella APN2-S/R recombinant proteinsFar-western blot, immunolocalization and the cytotoxin effect of Cry1Ac toxin on Sf9cells analyzed the function of the APN2-S/R recombinant proteins. The results showed that both of Plutella xylostella APN2-S and APN2-R recombinant proteins can bond with Cry1Ac toxin protein which was confirmed by Far-western blot; The green fluorescence was observed in Sf9cells only infected with the recombinant protein APN2-S/R by immunolocalization of Cry1Ac toxin against APN2-S/R proteins; Analyzed the cytotoxin effect of Cry1Ac toxin on Sf9cells-expressing APN2-S/R protein using the trypan blue exclusion assay to quantitate the number of cell deaths, cell mortality of APN2-infected cells was shown to increase significantly in the presence of Cry1Ac toxin protein.