| The striped stem borer, Chilo suppressalis(Walker), is an important rice pest distributed in main rice-growing areas of China. Recently, C. suppressalis population significantly increases with changes in the systematic cultivation of rice, climate warming and insect resistance to chemical pesticide. Transgenic rice expressing Bacillus thuringiensis (Bt) insecticidal proteins has been introduced as an environmentally sound method to control the damage caused by this pest. However, the resistance risk of target pest to Bt rice is one of the key factors to influence its sustainable utilities. Double (multiple) genes pyramided strategy is an important measure to delay insect resistance evolution, namely as transgenic plant containing two dissimilar Bt toxin genes have the potential to significantly delay the evolution of insect resistance. At present, newly developed insect-resistant transgenic rice line with pyramided genes crylAb+cry2Aa has entered environmental safety assessment stage and showed promising prospect for commercial use. In this study, three transgenic rice lines, expressing crylAb, cry2Aa, crylAb and cry2Aa, and their non-transgenic parent commercial cultivars, Minghui63, were used for this research. In vitro insect-feeding bioassay, spatio-temporal expression of insecticidal protein, establishment of selective populations and their continuous screening and binding ability of Bt toxin with midgut BBMV of C. suppressalis were conducted to illuminate the interaction between crylAb and cry2Aa genes in transgenic crylAb and cry2Aa rice line. The results will provide the scientific data and theoretic bases for environmental safety assessment of insect-resistant transgenic crops. The main research content and conclusions are as the following.(1) Control efficacy and expression level of insecticidal proteins in transgenic rice lines:the control efficacy of the transgenic crylAb+cry2Aa rice line and the transgenic crylAb rice line is significantly higher than that of transgenic cry2Aa rice line. Efficacy of transgenic crylAb and cry2Aa rice line against C. suppressalis is equal or even higher than that of transgenic crylAb rice line. In the case of spatio-temporal expression of insecticidal protein among three transgenic rice lines, the expressing level of CrylAb in transgenic crylAb and cry2Aa rice line was significantly lower than that in transgenic crylAb line. However, expressing level of Cry2Aa in transgenic crylAb and cry2Aa rice line was significantly higher than that in transgenic cry2Aa line, which showed that rice line with stacked genes perhaps inhibited the expression of CrylAb, however, no influence was shown on the expression of Cry2Aa in transgenic crylAb and cry2Aa rice line.(2) Measure on the effect of transgenic rice line with pyramided genes on delaying insect resistance evolution:The proportion of CrylAb and Cry2Aa for the screening of CrylAb+Cry2Aa selective population was determined as 1:2. Diagnostic doses obtained based on bioassay data of Fuzhou susceptible population were 5.464 ?g/ml (CrylAb),1900 ?g/?L(Cry2Aa) and 26.15 ?g/mL (CrylAb+Cry2Aa), respectively.Three selective strains, CrylAb, Cry2Aa, CrylAb+Cry2Aa, were established and consecutively selected for 6 generations. Three tolerant strains were tested with their respective diagnostic doses. The survival rates increased with the increase the selective generations of CrylAb-selective and Cry2Aa-selective strains, which indicated that the increase of tolerance of selected strain to Bt toxins. However, the delay of resistance evolution of CrylAb+Cry2Aa strain was not obviously observed due to few selective generations. Therefore, we should conduct long-term selection and observe the differences of resistance evolution rate among different selective strains.(3) The results of saturation binding of CrylAb binding to BBMV from C. suppressalis midgut showed that CrylAb could bind with C. suppressalis midgut BBMV with high and specific binding affinity. The competition binding is under way.Studies on in vitro Insect-feeding bioassay, spatio-temporal expression of insecticidal protein, resistance selection of selective strains and binding ability of Bt toxin with midgut BBMV of C. suppressalis indicated that there was no interaction between crylAb and cry2Aa in transgenic crylAb and cry2Aa rice line. Therefore, this insect-resistant transgenic rice line will play an important role for insect resistance management if this line will be commercially used in future. The results will provide important scientific data and theoretical basis. |
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