Genetic Modification Of Crystal Proteins From Bacillus Thuringiensis And The Preliminary Characterization Of PlcR Gene

Bacillus thuringiensis firstly came into the world as an insecticide in France early in 1938, and since then it has been successfully used for agricultural pest and medical insect control with its significant benefits based on environmental and safety considerations. However, the deficiency of this pesticide, such as limited spectrum of insecticidal activity, low toxicity to the targets and the inducment of insect resistance, results in the urgent need to exploit new resources of B. thuringiensis or to modify known strains by genetic engineering. Previous reports indicated that domain swapping between different B. thuringiensis toxins might be a possible mechanism for achieving new specificities and more efficient toxins. Besides, the inactivation of some regulator genes of B. thuringiensis is also a useful method for engineering modification of entomopathogenic bacteria. In this study, the genetic modification of crystal proteins from B. thuringiensis by domain swapping and the preliminary characterization of plcR gene were performed as following:(1) Six recombinant B. thuringiensis BT-ACC, BT-AAC, BT-ACA, BT-CAA, BT-CCA and BT-CAC were constructed through domain swapping between crystal protein Cry1Aa and Cry1Ca. SDS-PAGE and Western Blot revealed that only the recombinant BT-CAA and BT-CCA produced a 135 kDa chimeric protein Cry1CAA and Cry1CCA, respectively, but the production level was lower than the native protein Cry1Aa and Cry1Ca. These chimeric crystal proteins could be activated by trypsin, giving a 65 kDa protease-resistant core toxin as the native crystal proteins...