Cloning Of Novel Insecticidal Crystal Protein Gene From Bacillus Thuringiensis And Biosafty Assessment Of Genetically Engineering Strain WG-001

Cloning of new crystal protein genes from Bacillus thuringiensis plays a very important role in controlling the resistance of pests to B. thuringiensis insecticides and studying the function of crystal proteins to B. thuringiensis. In the first section, for the implication of discovering the t(?)xicity to the certain organisms from known "non-toxic" B. thuringiensis, the strains with the special crystal protein profiles but no toxicity to the tested insects were researched, and a novel crystal protein gene, cry7Ba1, from YBT978, the standard strain of B. thuringiensis subsp. huazhongensis (H40), was cloned and analysed. Its crystal protein possesses the high toxicity to the Plutella xylostella. This example indicates a new way of finding new insecticidal crystal protein. In the second section, the safety of genetically engineered B. thuringiensis WG-001 was evaluated. In this biosafety test, the following five categories had been evaluated: the ability of surviving in the fields, spreading and transmitting in the wild environment, influencing the indigenous microorganisms, the ability of mutating, horizontal transferring of the characteristic genes crylAa and crylAc to the indigenous microorganisms. The results testified primarily the safety of the genetically modified B. thuringiensis WG-001 to the ecosystem of fields in the vegetable fields of South ChinaThe results were reported as followed:1. From the stains saved by our laboratory, BMB1518, YBT978, L60, NXP15-04, PBt53, HZ39-04, LT1L-57, NARCBt17, EA15196, EA14696 and T70 were selected. The crystal proteins from BMB1518, YBT978, L60, NXP15-04 and EA15196 had been transferred to PVDF membranes, and the N-terminal first 15 amino acid sequences of 45kD, 130kD and 42kD proteins from BMB1518, YBT978 and EA15196, had been determined respectively.2. The genomic BAC library of YBT978 was constructed. The gene of 130kD crystal protein was cloned, and RBS sequence, promoters of BtI, BtII and the terminor were analysed. The deduced polypeptide is best homologous with Cry7Ab2, and contains the whole of eight conserved regions. This protein had been designated as Cry7Ba1. After transferred into acrystalliferous B. thuringiensis CryB, bipyramidal crystals were formed which were similar with that of YBT978 in shape and dimension, and 130kD protein band was observed according to SDS-PAGE. The bioassay results indicated that Cry7Ba1 was non-toxic to any larvaes of tested insects as the form of crystals, but possessed high toxicity to Plutella xylostella as the form of solution. As the N-half of the crystal proteins...