The novel cry1Ai gene that cloned from Bacillus thuringiensis strain SC6H8 encoded a protein exhibiting strong toxicity against Plutella xylostella and Chilo suppressalis in our previous study. Using the available information for the active fragments of other Cry toxins, eight truncated fragments were constructed to identify the minimal active fragment of Cry1Ai. All truncated fragments were expressed in Escherichia coli strain BL21(DE3), and the insecticidal activity against 2ndinstar P. xylostella larvae was assessed using full-length Cry1Ai as a positive control. The results indicate that the minimal active fragment of the Cry1Ai toxin against P. xylostella is located between amino acid residues 36I and 605I, which is smaller than the regions previously reported for Cry1A. The fi rst two amino acids(34T and 35P) on helix α-1 and whole helix α-2 of domain I and sheet β-32 of domain III are necessary for Cry1Ai toxin to keep its toxicity against P. xylostella.
ZHOU Zi-shanLIN Hui-yanLI YingSHU Chang-longSONG Fu-pingZHANG Jie
Bacillus thuringiensis is one of the most widely used bioinsecticides, and cry gene is the major insecticidal gene.Because Cry1 Ac protein shows strong toxicity against many lepidopteran species, it has been applied widely in spraying products and transgenic Bt-crops.The preparation of Cry protoxin is the first step in the very important processes of understanding the insecticidal mechanism, resistance screening, and biosafety assessments.The media for crystal production and the method for Cry protoxin preparation were varied, however, it was not clear which was better for preparing a larger amount of Cry protoxin.In this paper, three media for crystal production and the method for Cry1 Ac protoxin preparation from HD73 strain were compared to find an efficacious way to prepare a large number of Cry1 Ac protoxin.The results showed that the 1/2 LB(Luria-Bertani) medium was the ideal medium for crystal production, because the total yield of Cry1 Ac protoxin in 300 m L 1/2 LB medium was(112.38±5.64) mg, the highest one among three media; the repeated crystal solubilization method was better for the preparation of the Cry protoxin comparing with the continuous crystal solubilization method.It will be a reference for other Cry protoxin preparation, especially for larger number.
ZHOU Zi-shanYANG Su-juanSHU Chang-longSONG Fu-pingZHOU Xue-pingZHANG Jie
Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1 Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alaninesubstituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly373 and Asn375 caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.
LIU Yu-xiaoZHOU Zi-shanLIANG Ge-meiSONG Fu-pingZHANG Jie
