الفهرس 
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Abstract
Over 80% of all commercial biological pesticides are based on crystal proteins, produced by the bacterium Bacillus thuringiensis during sporulation.
In order to determine the binding specificities of crystal proteins, the insect receptors should be recognized. However, in Spodoptera littoralis, the toxin receptor(s) has not yet been identified. Therefore, the ultimate goal of this study is to identify, clone, sequence and molecular characterization of S. littoralis midgut Cry1 toxin receptor(s). Because, understanding the mechanism of toxicity for the important lepidopteron insects represents a greet necessity.
In this study the full length cDNA gene of APN receptor gene of Spodoptera littoralis were isolated and cloned in pGEM®-T Easy vector. Clones that contain the desired inserts were subjected to sequencing using the universal primers. Then, the APN gene was fully sequenced by the primer walking method using the specific receptor primers. The putative function of that gene was determined by alignment of the obtained sequence with sequences in the GenBank database using BLASTn. The deduced amino acid sequence was obtained by using the ORF finder function in the NCBI web site and the nucleotide sequence was translated into putative proteins. The homology modeling was used for the prediction of the three-dimensional structure of SlAPN protein. Finally, two different expression systems were performed to express a truncated part of SlAPN gene.
1- Identification of a Spodoptera littoralis BBMVs receptor protein that binds to Cry toxin:
Bioassays were performed to determine the susceptibility of S. littoralis to Cry protoxin of BtkHD1 strain and Cry1C. The pesticidal activities of such toxins were compared and the protoxin of BtkHD1 strain was found 1.3 times less toxic to this insect than CryIC toxin.
2- Binding characteristics of receptor protein on BBMVs of S. littoralis:
BBMVs of S. littoralis were isolated and purified to study the binding ability of Bt. Toxin. To demonstrate which protein of the BBMV is a putative receptor for the binding of Bt toxin, immunoblotting was carried out. A protein band corresponding to 108 kDa was observed on the blots. Hence, a 108 kDa protein of the BBMV was identified as a receptor for the Cry1 type Bt toxin.
3- Cloning, sequencing and characteristics of S. littoralis APN (SlAPN) Cry toxin receptor gene:
A complete cDNA corresponding to 109 Kda protein from midgut of S. littoralis was isolated and cloned into pGEM T-Easy vector. The insert was fully sequenced and the sequence results confirmed that the full-length cDNA clones for SlAPN were obtained. In silico analysis of the present observations showed the primary amino acid sequence identified SlAPN as a member of the aminopeptidase family. Such results depended on the presence of common motifs of aminopeptidase family which included the following conservative regions: zinc-binding/gluzincin motif, gluzincin aminopeptidase motif, GPI anchor aminopeptidase.
4- Homology modeling in predicting the 3-D structure of S. littoralis APN receptor:
Homology modeling was performed to predict the 3-D structure of SlAPN protein. In this model both signal peptide and GPI regions on SlAPN gene were excluded. The present SlAPN model contained four structural domains, which spread from N-terminal domain I to C-terminal domain IV over the regions Asn58-Ile266; Ser267-Gly506; Asn507-Leu581 and Ser582-Ala871, respectively.
5- Expression of S. littoralis APN (SlAPN) Cry toxin receptor gene:
Two attempts have been carried out to express SlAPN proteins in a heterogeneous system. The two attempts include using E. coli and Pichia pastoris expression systems. The current study failed to express the complete S. littoralis APN cDNA in E. coli. On the other hand the expression of APN using Pichia pastories expression system was succeeded. A segment of SlAPN cDNA called F1 SlAPN; from bp 1 to 2185, was successfully expressed in the P. pastoris cell culture.
The cloning of the receptor, characterizing its binding site for the toxin and determining its relationship in binding different toxins, will be of great value in preventing or delaying the development of resistance to bio-insecticides based on Bt.