Safety Evaluation Of Bt Toxin On Rice Planthopper And Screening Active Strain

Rice planthoppers are the most important pests on rice, including Nilaparvata lugens (Stal), Sogatella furcifera (Horvath) and Laodelphax striatellus (Fallen), and cause big loss in rice production. Due to its safety, high efficiency and specificity against the target pests, Bacillus thuringiensis (Bt) products gradually became biological pesticides that were studied most deeply and used most widely. Currently, research on Bt focused on the control effects on the Lepidopteran, Coleopteran and other target pests. Bt biological pesticides pose good economic and ecological benefits to control target pests, but with no reports against rice planthoppers until now.In order to evaluate the influence of Bt rice on rice planthoppers, we established the Tier-1toxicity test model to determinate the potential effects of insecticidal compounds, with L. striatellus as test insects. The model has been successfully used to assess risk of Bt toxins on non-target pests planthopper, so that we can predict possible trends of L. striatellus development and damage in insect-resistant transgenic rice fields. Bt toxin binding to receptors in mid-gut BBMV is a key step in its toxic effects. We preliminarily studied the putative Bt receptor alkaline phosphatase (ALP) with N. lugens as test insects and cloned two ALP genes. Finally, we conducted the bioassay for a large number of standard strains of Bt and screened active and specific Bt strains on planthoppers, which may provide a theoretical basis for the subsequent development of transgenic Bt rice to control rice planthoppers.1. Risk assessment of Bt toxins on non-target insect pests rice planthoppersFirstly, we select a suitable artificial diet to develop a toxicity test model to evaluate the potential effects of the pure insecticidal protein on L. striatellus, Tier-1test model. The sensitivity of Tier-1test model was verified with E-64and PA, which were rendered as a model compound, and the results showed that the Tier-1toxicity test model posed the high sensitivity. Based on this model, we assess the effects of insecticidal protein Cry2Aa on L. striatellus survival rate, nymph development period and the fresh weight of eclosion adults. The results showed that the Cry2Aa had no toxic effects on L. striatellus. In addition, the concentration, stability and biological activity of insecticidal compounds were entirely monitored by ELISA and sensitive bioassay.2. Biochemistry and molecular biology preliminary studies on two alkaline phosphatasesIn gene clone and enzyme activity assay, the whole body of the brown planthopper was used as the object of study. We separated and purified soluble and membrane-bound alkaline phosphatases by ultracentrifugation and the activity of both alkaline phosphatases were determinated under the optimal pH environments. The full-length cDNAs encoding two forms of alkaline phosphatases in BPH were obtained by RT-PCR and RACE technology, which was firstly identified in the Hemipteran insects. Both N1-ALP1and N1-ALP2show approximately the same degree of sequence identity (40-50%) to other insect soluble and membrane-bound forms of alkaline phosphatase. Analysis of the deduced amino acid sequences of Nl-ALP1and N1-ALP2indicated the presence of one putative alkaline phosphatase signature and all13functionally important amino acids for alkaline phosphatases. In addition, N1-ALP2contains a putative GPI-anchor site, which is absent from N1-ALP1. Western blotting experiments provided direct proof that the two genes encode the soluble and membrane-bound alkaline phosphatase respectively, so that the two genes were named as Nl-sALP and Nl-mALP.3. Screening the active Bt strains against planthoppersWe used the artificial fiet for L. striatellus to screen a large number of Bt strains, including68standard Bt strains. Among them, the crystal protein and soluble small molecules of Bt strain T42001posed higher toxicity on L. striatellus. The bioassay results showed that the LC50value of crystal protein is6.58μl/ml onZ,. striatellus, which indicated the strain had a high toxicity on L. striatellus. The electrophoresis of the crystal protein had three bands,90kDa,60kDa and31kDa, which were different from that found in other reported Bt strains.This study reveals the possibility of Bt toxin in controlling rice planthoppers at different levels and provides a theoretical basis for successful usage of Bt toxins on rice planthoppers, including the development of transgenic Bt rices.