Isolation of the astacin-like metalloprotease coding gene (astl) and assessment of its insecticidal activity / Mervat Ragab Mansour Ali Diab ; Supervied Ebtissam Husseinaly Hussein , Ahmed Mohammed Ahmed Mohammed , Mahmoud Monir Ahmed

Thesis (Ph.D.) - Cairo University - Faculty of Agriculture - Department of Genetics

Biopesticides are considered ecofriendly alternatives to the chemical pesticides for controlling agricultural pests.Spider venoms are a cocktail of thousand peptide toxins, and could have insecticidal activity against a wide range of insect orders. The present investigation was conducted with the main goal of assessing the efficacy of spider-based toxin peptides for pest control and to determine the probability of using these toxins to produce a new biopesticide that is friendly to the environment. Twenty five fragments from genes coding for putative toxins were amplified from five spider species.Sequence analysis revealed that out of the twenty five sequences, only one fragment showed high similarity with the astacin-like metalloprotease toxin gene.Using the barcoding technology, the spider species of this fragment was identified as Hasariusadansoni.The full length of astl cDNA was cloned from thisspider species using the RACE technology. Sequencing of the cloned Ha-astl cDNA proved that its full length includes 802 bp with 714bp open reading frame encoding for 238 amino acids.The predicted molecular weight of the encoded protein is 27.33 kDa with two disulfide bridges between cysteine residues at positions 87-238 and 108-128 and three zinc binding sites at histidine residues at positions 136,140 and 146.A 486bp of the catalytic domain was cloned and expressed by the yeast expression system Pichia pastoris. In an attempt to enhance the insecticidal activity of the toxic protein,the astacin like metalloprotease toxin was fused to the GNA snowdrop lectin in the same frame and expressed in Pichia pastoris. The insecticidal activity of the Ha-astl and the Ha-astl/GNA proteins was determined towards two species of agricultural insects from two different orders, cotton leaf worm, Spodopteralittoralis (Lepidoptera:Noctuidae) and rice weevil, Sitophilus oryzae (Coleoptera: Curculionidae)

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