The determination of the environmental fate of Bt insecticidal protein released by Bt rice plants in paddy soils is a key issue in its ecological risk assessment. In this study, the impacts of soil water content, pH, ...The determination of the environmental fate of Bt insecticidal protein released by Bt rice plants in paddy soils is a key issue in its ecological risk assessment. In this study, the impacts of soil water content, pH, and temperature on the degradation of CrylAb protein expressed in the leaves of Bt rice KMD2 were studied in the laboratory. Three types of paddy soils were used, i.e., blue clayey paddy soil, pale paddy soil on quaternary red soil, and marine-fluvigenic yellow loamy paddy soil. Ground powders of KMD2 leaf blades were mixed with each type of soil, and degradation dynamics of Cry lAb were measured using enzyme-linked immunosorbent assay （ELISA）. The degradation rate of CrylAb was high at the early experimental stage, but slowed down steadily at middle and later stages, which could be described by exponential equations, with the half-life period of degradation determined as 1.8-4.0 d. The soil water content, pH, and temperature could affect the degradation of CrylAb, but the effects of soil pH and temperature were relatively greater. In general, CrylAb degradations were slower under lower soil pH and temperature conditions, especially for marine-fluvigenic yellow loamy paddy soil.展开更多
CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and ...CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.展开更多
Two variants of diet composition were prepared to evaluate the susceptibility of ECBs to CrylAb toxin as follows： 1） 38-0600 Stonefly Heliothis Diet mixed with purified CrylAb protein and 2） 38-0600 Stonefly Heliot...Two variants of diet composition were prepared to evaluate the susceptibility of ECBs to CrylAb toxin as follows： 1） 38-0600 Stonefly Heliothis Diet mixed with purified CrylAb protein and 2） 38-0600 Stonefly Heliothis Diet mixed with lyophylized leaves of Bt maize MON 810-YieldGard. A method of sample preparation and extraction of Bt toxin for reproducible ELISA quantification were optimized. The qualitative DAS-ELISA kit from Agdia was optimized for use in quantitative analysis of Cry lAb toxin. The mortality of ECB larvae from the laboratory strain on the diet with CrylAb toxin did not differ significantly from the mortality on the diet with Bt maize leaves with the same rate of Cry lAb toxin. Similarly, the mortality of the ECB larvae from the field population on the diet with Bt maize leaves did not differ significantly from the mortality of ECB larvae from the laboratory strain on the same type of diet. Therefore, the incorporation of Bt maize leaves into the diet did not influence the efficacy of CrylAb toxin against ECBs. Using this method, a susceptibility of one field population of ECBs from the Czech Republic to CrylAb toxin was determined （LC50 of 2.16 μg of Cry 1Ab g^-1 of diet）.展开更多
基金This study was financially supported by the National Natural Science Foundation of China （30070500） to Cheng Jiaan.
文摘The determination of the environmental fate of Bt insecticidal protein released by Bt rice plants in paddy soils is a key issue in its ecological risk assessment. In this study, the impacts of soil water content, pH, and temperature on the degradation of CrylAb protein expressed in the leaves of Bt rice KMD2 were studied in the laboratory. Three types of paddy soils were used, i.e., blue clayey paddy soil, pale paddy soil on quaternary red soil, and marine-fluvigenic yellow loamy paddy soil. Ground powders of KMD2 leaf blades were mixed with each type of soil, and degradation dynamics of Cry lAb were measured using enzyme-linked immunosorbent assay （ELISA）. The degradation rate of CrylAb was high at the early experimental stage, but slowed down steadily at middle and later stages, which could be described by exponential equations, with the half-life period of degradation determined as 1.8-4.0 d. The soil water content, pH, and temperature could affect the degradation of CrylAb, but the effects of soil pH and temperature were relatively greater. In general, CrylAb degradations were slower under lower soil pH and temperature conditions, especially for marine-fluvigenic yellow loamy paddy soil.
基金supported by the Zhejiang Provincial Natural Science Foundation of China (Grant No.Z305650 and No.Y3080361)the Science and Technology Department of Zhejiang Province,China (Grant No.2004C12020)+1 种基金the National Key Technology Research & Development Program,China (Grant No.2006BAD01A01-5 and No.2008ZX08001-001)the 151 Foundation for the Talents of Zhejiang Province,China
文摘CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains （EHA105 and EHA101） contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive （80.94%）, 62 plants were also CrylAb co-transformants （15.05% in total frequency）, and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron.
文摘Two variants of diet composition were prepared to evaluate the susceptibility of ECBs to CrylAb toxin as follows： 1） 38-0600 Stonefly Heliothis Diet mixed with purified CrylAb protein and 2） 38-0600 Stonefly Heliothis Diet mixed with lyophylized leaves of Bt maize MON 810-YieldGard. A method of sample preparation and extraction of Bt toxin for reproducible ELISA quantification were optimized. The qualitative DAS-ELISA kit from Agdia was optimized for use in quantitative analysis of Cry lAb toxin. The mortality of ECB larvae from the laboratory strain on the diet with CrylAb toxin did not differ significantly from the mortality on the diet with Bt maize leaves with the same rate of Cry lAb toxin. Similarly, the mortality of the ECB larvae from the field population on the diet with Bt maize leaves did not differ significantly from the mortality of ECB larvae from the laboratory strain on the same type of diet. Therefore, the incorporation of Bt maize leaves into the diet did not influence the efficacy of CrylAb toxin against ECBs. Using this method, a susceptibility of one field population of ECBs from the Czech Republic to CrylAb toxin was determined （LC50 of 2.16 μg of Cry 1Ab g^-1 of diet）.
