Bioactivity And Purification Of Insecticidal Protein From Xenorhabdus Nematophila CB6

Xenorhabdus and Photorhabdus are entomopathogenic bacteria symbiotically associated with entomopathogenic nematodes, six species included. They are highly virulent to insects. LD50 is generally 1-10 live cells when injected into the hemocoels of insects. Researchers in the world have purified lipopolysaccharide and protein from cells and supernatant of fermentation culture, which had insecticidal activity. The results of bioassay showed that the insecticidal protein was highly virulent against several orders of insects and potential as a biocontroll agent.In this paper, insecticidal activity of eleven strains was examined by means of oral bioassay, and a high virulent strain isolated from the suburb of Beijing was obtained, named Xenorhabdus nematophila CB6. Experiments showed that the substance of insecticidal activity was protein. And the insecticidal protein was isolated and purified from the fermentation culture. The main results gained in the study were as follows:1. Insecticidal activities of fermentation suspension of eleven strains were compared. It was found that the virulence of the eleven strains was different, of which four strains, namely strains CB6, A54,Dan and 4-Ps, were with higher activity of growth inhibition than the others against neonate of H.armigera. However, the mortality of H.armigera was very low after feeding on the diet treated with the fermentation suspension of each strain.2. The bioassay indicated that the substance with growth inhibition activity in X.nematophila CB6 fermentation suspension was protein.This activity didn't depend on the survival of cell, but on the supernatant of either fermentation culture or celllysate solution. Analyzed by enzymatic hydrolysis or heat teatment, the activity of the supernatant decreased greatly. On the concentration of 40 l/g diet, the growth inhibition rates of fermentation supernatant were reduced by 74.51% and 67.01% after treated with 100 C for 10 minites and hydrolyzed with pepsin lor 4 hours, and by 87.13% and 59.14% of cell lysate supernatant. All these properties were proteinous and it colud be concluded that the insecticidal substance was protein.3. The insecticidal protein had high effect on the growth and development of larvae of H.armigera, and had significant antifeedant activity. Compared to the CK, the treatment with insecticidal protein obviously decreased larvae weight, prolonged larvae stage, decreased the rate of pupation and eclosion. In choice antifeedant experiment and no-choice antifeedant experiment, only a little proportion of treated leaves was eaten by insect compared to untreated leaves, and the antifeedant effect was quite notable.4. The insecticidal protein of strain CB6 was isolated and purified. Through centifugation, the cell was removed ,then crude protein was precipitated with ammonium sulfate. After column chromatography on DEAE-Sepharose Fast Flow and Sephadex G-200, several protein were obtained. The bioassay with neonate of H.armigera showed that only one proteir had growth inhibition activity. Analyzed by native PAGE, the molecular weight of the insecticidal protein was more than 670kDa. The protein was sensitive to pepsin and proteinase K, not to trypsin. Toxity of the protein was stable under the temperature of 30 C-60 C and the pH of 5-8. There was only one absorbant peak from 220nm to 400nm, and the maxmun absorbant peak was at 284nm.The experiments indicated that the insecticidal substance in the X.nematophila CB6 culture broth was protein. The insecticidal protein had great effect on feeding, growth and development of H.armigera. The results in the paper provided a base for studying on the properties of the insecticidal protein and the cloning of the insecticidal gene.