Study On The Degradation Of Cry1Ac Protein In Transgenic Rice Straw

Bt-transgenic crops is the world's fastest-growing commercialization of genetically modified (GM) crops. Large of plant straw containing Bt insecticidal proteins will be produced after large scale cultivation of Bt-modified crops. When leaving these transgenic crop residues in soil ecosystem, Bt insecticidal protein accumulates and maybe affect the balance of the soil ecosystem. This study attempts to utilize bio-fertilizer fermentation, high temperature alkali method and methane fermentation technology to degradateCry1Ac protein in Bt-transgenic rice straw.Nine Cry1Ac protein-degradating strains was screened from five different soils, and the best Cry1Ac protein-degradating strain was identified. The strain was used for bio-fertilizer and methane fermentation to degradatingCry1Ac protein in Bt-transgenic rice straw. Then the major influential factors of bio-fertilizer fermentation method and high temperature alkali method were analyzed by single factor experiment, then by the orthogonal tests L₉(3⁴) and SAS analysis, the optimum parameter was obtained and the degradation rate has been greatly improved;Cry1Ac protein had been almost completely degradated at the initial stage of methane fermentation.The specific results were as follows:1,Nine Cry1Ac protein-degradating strains was screened from five different soils, 0.1ml each bacterial supernatant was added into 0.9ml Bt protein solution, incubated at 37℃for 4h, after reaction the results were determined by SDS-PAGE, the 130kD strip of each bacteria was disappeared. After repeated trials, strain FJSB3 could degradate Bt protein.Through colony morphology and electron microscope observation, physiological and biochemical tests , 16 SrDNA sequence experiment, the 16SrDNA sequence of strain FJSB3 is most closely related to Stenotrophomonas sp., showed 99%. Taken together, the above results suggest that strain FJSB3 should be identified as Stenotrophomonas sp.2,Fiestly, the effects of incubation temperatures, initial FJSB3 cell concentration, pH value and C/N on the degradation rate of Cry1Ac protein in Bt-transgenic rice straw were evaluated using single factor experimentation, respectively. Then by the orthogonal tests L₉(3⁴) and SAS analysis, the primary relation of the different factors was C/N>initial FJSB3 cell concentration>pH value>incubation temperatures, the optimum parameter was determined:C/N 30/1, initial FJSB3 cell concentration 2.0×10⁶ CFU/ml, pH value 7.0, incubation temperature 33℃. Under the above conditions, the degradation rate of Cry1Ac protein in Bt-transgenic rice straw reached 96.25%.3,Bt-transgenic rice straw and distilled water were mixed with the mass ratio of 1:4, then incubated at 45℃for 1day. Methane fermentation agents, urea solution and FJSB3 bacteria solution were sprayed into transgenic rice straw during methane fermentation, and the temperature maintained 45℃.Cry1Ac protein in Bt-transgenic rice straw reached 95.8% on the 8th. The result showedCry1Ac protein had been almost completely degraded at the initial stage of methane fermentation.4,Fiestly, the effects of temperatures, pH value and treatment time on the degradation rate of Cry1Ac protein in Bt-transgenic rice straw were evaluated using single factor experimentation, respectively. Then by the orthogonal tests L₉(3⁴) and SAS analysis, the primary relation of the different factors was pH>temperature>treatment time, the optimum parameter was determined: pH value 14, temperature 140℃, treatment time1.5h. Under the above conditions, the degradation rate of Cry1Ac protein in Bt-transgenic rice straw reached 100%.