Construction and Characterization of Novel Bacillus thuringiensis cry1-type Genes with Improved Insecticidal Activities

Abstract

Bacillus thuringiensis (Bt) is a gram-positive, sporulating bacterium that produces a variety of proteins, in the form of large crystalline (Cry) inclusions, which demonstrate toxicity to a variety of insect and nematode pests. Varieties of Cry proteins have been found around the world, which has been widely used as one of the most successful biological control agent. That is regarded as environmentally friendly, with little or no effect on humans, wildlife, pollinators, and most other beneficial insects and are used in Organic farming. Thus, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, to construct of novel Bacillus thuringiensis cry1-type genes for genetically modified crops and to select the most appropriate cry gene for transgenic crops with enhanced insecticidal activity as template. For effective expression in a plant, cry1Ac gene was modified to plant-preferring codon usage by multi site-directed mutagenesis. Through mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Plutella xylostella , Spodoptera exigua and Ostrinia furnacalis were identified. For this mutagenesis, 1857 bp of modified cry1Ac (Mod-cry1Ac) encoding active domain was used as a template and amplified by polymerase chain reaction with 16 mutagenic primers based on cry1Ac sequences. Consequentially, 31 different kinds of mutant cry1Ac genes with improved the insecticidal activity randomly changed 24 amino acid sequences into Cry1Ac were obtained. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. These mutant cry genes encode potent insecticidal proteins in the form of crystalline protoxins of 95 kDa. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella, S. exigua and O. furnacalis were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. In the bioassay of third-instar larvae of P. xylostella, LD50 values of these mutant Cry protein was lower about 5-fold than Mod-cry1A, the control Bt protein. In the bioassay of second-instar larvae of S. exigua, Mod-Cry1Ac showed no insecticidal activity, on the other hand not as effective as Cry1C but 8 Mut-Cry1Ac showed insecticidal activity. Also, LD50 values of mutant Cry protein through quantitative bioassay was lower about 2-fold than Mod-cry1Ac about O. furnacalis. Through quantitative bioassay, several mutant cry genes showing higher insecticidal activity, such as Mut-N04, Mut-N06 and Mut-N16 to P. xylostella and O. furnacalis, might be expected as desirable cry genes for the introduction to genetically modified crops.