Isolation Of Pseudomonas Fluorescens And Prokaryotic Expression Of Androctonus Australis Hector Insect Toxin

Pseudomonas fluorescens is plant rhizosphere bacterium or plant-withinendobacterium, which has the functions of both promoting plant root growth andserving as biocontrol agent. Androctonus australis hector insect toxin 1 (AaHIT1)is insect-specific scorpion neurotoxin, uninjurious to mammalian, and allows of newinsecticide. Soil samples were taken from suburb of Shanghai City, from whichrhizosphere bacteria depressing crop pathogenic fungus were isolated through KBmedian and measure of antagonistic ability. The shape of the rhizosphere bacteriawas observed with electron microscope and characterization of these bacteria wasperformed through biochemistry methods. The results showed that the rhizospherebacteria were Pseudomonas fluorescens. The AaHIT1-coding gene sequence wasdesigned according to amino acid sequence of AaHIT1 and preference ofprokaryotic microbe for codon. This gene was synthesized through PCR method.The synthesized gene was sequenced by dideoxynucleotide sequencing and nochanges were found. Then, the AaHIT1-coding sequence was linked with pET32a,resulting in the recombined plamid, pET32a/ AaHIT1, was tentatively expressed inE.coli. Secondly, the AaHIT1-coding sequence was cloned into pSUP106, amultihost expression vector. The resulting vector was introduced into thePseudomonas fluorescens with the activity for depressing crop pathogenic fungus.The expression was induced with IPTG. SDS-PAGE analysis of the expressionproduct showed that the AaHIT1 was successfully expressed in Pseudomonasfluorescens, indicating that an engineering Pseudomonas fluorescens, which mightdepress crop pathogenic fungus and produce insect-specific scorpion neurotoxin,was established. This is the basis for development of polyfunctionalenvironment-friendly biopesticide.