| Bacillus thuringiensis (Bt) is an insecticidal microorganism widely commercialized in the world. Its activity is attributed largely to insecticidal crystal proteins (ICP), while, the application of Bt in agricultural production in the future will be limited due to some flaw, such as narrow insecticidal spectrum, low toxicity and resistant risk of insects against Cry toxin. Novel cry gene cloned from natural strains is more and more difficult because of increasing new cry genes yearly, .therefore, improvement of known Cry protein by molecular design and protein engineering is imperative and feasible.The study includes a series of aspects: a new approach of producing random mutation library with prospective base substitution rate, directed evolution of Cry1Ie. The results ara shown below:The promoter of cry1Ac gene and full length cry1Ie were fused by overlapping primer PCR to result in a expression vector in E. coli.In the process of constructed the random mutation library, calculated the required cycles of PCR according to the intrinsic error rate rate of Taq DNA polymerase and the required number of Cry toxins mutation. At the same time, this study used real time PCR to monitor the process of PCR amplification to control the times of polymerization.In this experiment, we used cry1Ie gene which has toxic to Plutella xylostella, Ostrinia furnacalis, as amodel to constructed three libraries with the prospect that the libraries of base-substitutions in DNA molecules were 1 (Library I), 2 (Library II) and 3 (Library III) respectively. 100 random clones from each library were selected for subsequence analysis. The result shown that the number of mutated clone of Library I, Library II and Library III was 59, 93 and 98 respectively; the number of nonsense mutation of the three library was 36, 43, 50 respectively, the number of missense mutation of the three library was 13, 41, 38 respectively; the average number of nucleotide substitutions of the three library was 0.86, 2.1, 2.79 respectively. In general, this method can control the rate of mutation of mutation library and mutation of two bases is the most suitable for directed evolution of Cry toxins.Furthermore, the toxicity of the 92 missense mutants of Cry1Ie against Plutella xylostella was assayed, and intensions of 11 mutants were increased comparable with Cry1Ie which LC50 is 1.96252μg/ml. The LC50 of the 11 mutataions were 0.20470,0.81601,0.15063,0.44387,0.61911,0.49740,0.29259,0.51284,0.58315,0.29723,0.67218μg/ml respectively and the changes of amino acid were Y162H&E181K,N214S,V236A,K237R,Y239H&A307V,N258Y,I285T&R506G,P320H&Y599N,Y422H,I507V,A522V respectively. This mutation site were distribution in the three domain of Cry1Ie, and the most obvious of which increased activity is located in domain II of the double mutant Y239H & A307V, increased 13-fold.The results show that the directed evolution of this research is appropriate for Cry toxins and develop of a simple approach to constructed random mutationlibrary with prospective base subtationrate. This study was used cry1Ie gene that with self-owned intellectual property as experiment material, and acquired 11 mutants with increased toxic to P. xylostella which provides a new material for the development of transgenic Bt plants...
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