Purification And Gene Clone Of Insecticidal Proteins From Xenorhabdus Nematophila HB310

Xenorhabdus and Photorhabdus spp. are harbored as symbionts in the intestine of the infective juvenile stage of nematodes belong to the families Steinernematidae and Heterorhabditidae respectively. The bacteria are carried into the hemolymph of the host insects by the nematodes and are subsequently released into the insect hemolymph. The nematodes and the bacteria together rapidly kill the insect larva, but in most cases the bacteria alone are highly virulent. The insecticidal protein toxins and insecticidal toxin genes have been found to be elaborated from symbiotic bacteria. The bacteria may be new weapon for insect pest control or be a viable alternative to Bt. In this study, the symbiotic bacteria strains with highly virulence were screened. Three insecticidal toxins were isolated. The cosmid genomic DNA library was constructed successfully in order to clone the insecticide toxin genes.The 719 soil samples were collected randomly from Hebei province. 28 of the samples (3.89%) contained the entomopathogenic nematodes (EPNs). Twenty-eight symbionts were isolated, including 25 Photorhabdus and 3 Xenorhabdus spp.Partial 16S rDNA genes sequences of 28 isolates and 4 reference strains of the genera Xenorhabdus and Photorhabdus were determined by direct sequencing of PCR products. Aligned sequences against those from described species were subjected to phylogenetic analysis by DNAstar software. The results showed that HB310 and HBml belonged to X. nematophila; HB89 and HBgy belonged to P. luminescens laumondii. The gwl5 was likely to be new species of genus Xenorhabdus; HBxhl, HBxh5, HB3, HB140, HB8, HB06, HB36, HB73, HB141, HB202, HB231, HB237, HB287, HB288, HB301 and HB352 were likely to be new species or new subspecies of P. luminescens. The homology of 16S rDNA between phase I and Phase II from HB310 and HB202 isolates was.all 100%.The oral toxicity of culture broths from 28 strains of genera Xenorhabdus andPhotorhabdus against Helicoverpa armigera neonates was tested. The bioassay indicated the most bacteria strains had oral insecticidal activity, which also showed distinct differences between two genera, or species and strains. The all strains of X. nematophila displayed more highly oral toxicity than other species. Plutella xylostella and Pieris rapae larvae were sensitive to the most symbiotic bacteria, especially Photorhabdus spp.The virulence of X nematophila HB310 was studied in detail. The culture broth from the bacterium showed oral insecticidal activity against 10 species of insects in Lepidoptera and Coleoptera, especially with highly oral insecticidal activity against P. rapae, Pontia daplidice, P. xylostella and Hencsepilachna Vigintiocyomaculata. The bacteria killed 100% P. daplidice neonates (LC50 6.4998 X 105 cells/mL), P. xylostella second-instar larvae (LC50 1.3614 X 106 cells/mL) and H. Vigintiocyomaculata neonates within 72 h, killed 100% P. rapae neonates (LC50 2.7418 X 106 cells/mL) and P. daplidice fourth-instar larvae within 96 h. The growth inhibiting rates of HB310 broth to H. armigera Spodoptera exigua, Ostrinia furnacalis and Mythimna separata were 98.74%, 97.43%, 75.80% and 68.85% respectively at 120h. The equal volume of the culture broth, supernatant and cells all showed similar high oral toxicity. The bioassay results indicated that the bacterium had produced oral insectcidal toxins from early log period and had very high toxicity in middle log period.The bioassay showed that the both extracellular and intracellular protein crude extracts from X. nematophila HB310 have highly oral and injectable toxicity. Two kinds of oral toxins, toxin I and toxin II, and one hemocoel toxin, toxin III, were isolated and pured by native-PAGE from both intracellular and extracellular protein crude extracts. All of the purified toxins exhibited single band on native-PAGE, but four peptides of 55~170 kDa, three peptides of 55-250 kDa, and one peptide of 65 kDa respectively on SDS-PAGE. The oral insecticidal activity of toxin II was higher than one of toxin I to H. armigera neonates. The hemoc...