Identification Of Hyphantria Cunea Midgut Aminopeptidase N And Its Binding Analysis With Bacillus Thuringiensis Cry Toxins

Hyphantria cunea(Drury) belongs to Lepidoptera, Arctiidae, is a worldwide quarantine pests and cause significant damage to forestry and landscaping in China. Bacillus thuringiensis is one of the bio-control pesticides against H.cunea. Insecticidal Crystal Proteins, the major insecticidal active substances of Bt, bound to the receptor proteins in the midgut of insect and lead to the death of the insect. Aminopeptidase N is one of the most important receptor proteins in the midgut of insect. In order to define the specific receptors of Bt toxins in the midgut of H.cunea and the interaction mechanism, a novel Bt insecticidal protein cry2 Ab gene and the receptor protein Hc APN1 were cloned and expressed in this study. Main results are as follows.1. The lethal and sublethal effect of Bt CYZ-4 strain against Hyphantria cuneaThe experiments were conducted to explore the lethal and sublethal effects of Bt CYZ-4 on 2nd instar larvae of H.cunea. The 96 h LC50 was 2.57 x 105 spores/m L, the pupae and adults of H.cunea were obtained with the larval duration delayed when Bt concentrations were lower than 106 spore/m L. On the contrary, no papae and adults were formed when Bt concentrations were higher than 106 spore/m L, and the development process of larva was prolonged along with the increase of Bt concentration. The mean corrected death rates of 96 h were under 20%, larval stage had a slight delay and pupal weights were significantly higher than control when treated with the sublethal concentration of Bt.2. Cloning and expression of Bt cry2 Ab and preparation the anti-Cry2Ab33 antibodyBt CYZ-4 strain includes the cry2 Ab gene via restriction analysis. The full-length cry2 Ab gene was obtained by PCR. Gen Bank accession number was KP053646 and named cry2Ab33 by International Nomenclature Committee of Bt. The ORF of cry2Ab33 is 1902 bp, encodes 634 amino acid residues, its predicted molecular weight and isoelectric point were 70.67 k Da and 8.36, respectively. We got the EC2Ab33 strain after constructed the recombinant expression vector p ET30a-2Ab33 and transformed into E. Coli BL21, the protein was purified and used to raise anti-Cry2Ab33 antibody in rabbit.3. Cloning and expression of hcapn1RNA was isolated from the midgut of H.cunea, c DNA synthesis was conducted by reverse transcription and used as a template for PCR and RACE-PCR. PCR and RACEPCR products were sequenced assembly, analysis by DNAMAN and BLAST revealed that it belonged to Aminopeptidase N, namaed Hc APN1. The full-length of hcapn1(Gen Bank accession number KP053647) is 3181 bp, open reading frame is 3000 bp, encoding 1000 amino acid residues. Its predicted molecular weight and isoelectric point are 113.14 k Da and 4.85, respectively. Designed a pair of primers SF & SR without signal peptide sequence. The PCR amplified products hcapn1 were cloned into Escherichia coli expression vector p ET30 a to get the recombinant expression vector p ET30a-hcapn1. The Hc APN1 truncated recombinant protein was about 110 k Da induced with IPTG.4. Ligand blot assay of Cry toxins bind to Hc APN1The Cry2Ab33, Cry1 Ac, Cry9Ea6 active proteins were got after the protoxins were treated with the trypsin. The Hc APN1 recombinant protein was separated by SDS-PAGE, and transferred to PVDF and conducted Ligand blot assay with the active proteins, respectively. Ligand blot assay showed Cry9Ea6 active protein bound obviously to Hc APN1 recombinant protein, while either Cry1 Ac or Cry2Ab33 active proteins showed no specifically combination.We inferred that Hc APN1 was the receptor for Cry9Ea6, but not the Cry2Ab33 or Cry1 Ac.5. Binding domain of Hc APN1 with Cry9Ea6In order to further defined the binding domain with Cry9Ea6 in Hc APN1, designed two pairs of domain primers F1&R1, F2&R2. The two domain proteins Hc APN1 G and Hc APN1 E were obtained through cloning and expression and their protein molecular weight were 62 k Daand 48 k Da, respectively. And they located in 34 Asp ~ Asp527 and 563Leu~Gln925, respectively. Vitro binding assay showed binding region with Cry9Ea6 was 563Leu~Gln925 in Hc APN1, the second domain.