Engineered Bacillus thuringiensis GO33A with broad insecticidal activity against lepidopteran and coleopteran pests

Abstract

A recombinant plasmid pSTK-3A containing cry3Aa7 gene encoding a coleopteran-specific insecticidal protein was constructed and introduced into wild Bacillus thuringiensis subsp. aizawai G03, which contained cry1Aa, cry1Ac, cry1Ca, and cry2Ab genes and was highly toxic to lepidopteran insect pests. The genetically engineered strain were named G033A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis demonstrated that the cry3Aa7 gene was expressed normally and produced a 67 kDa protein in G033A, and the flat rectangular crystals of Cry3Aa7 toxin protein was observed under scanning electron microscope. The recombinant plasmid was maintained in bacteria cultured for 180 generations in culture media containing no antibiotics. Synthesis of the Cry3Aa7 toxin conferred high and broad toxicity to the recombinant strain G033A against coleopteran order, elm leaf beetle (Pyrrhalta aenescens) (LC(50) 0.35 mg/ml), for which the parental strain G03 was not toxic. Both the parental strain G03 and recombinant strain G033A showed strong insecticidal activity to lepidopteran pests, beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella), and cotton bollworm (Helicoverpa amigera), respectively. The lethal concentration 50% (LC(50)) of G033A against S. exigua, P. xylostella, and H. amigera was 4.26, 0.86, and 1.76 microg/ml, respectively.

Structure and Regulation of thegabGene Cluster, Involved in the γ-Aminobutyric Acid Shunt, Are Controlled by a σ54Factor inBacillus thuringiensis

“…The bacterial strains and plasmids used in this study are listed in Table 1. E. coli JM110 was used for cloning, while SCS110 was used to produce nonmethylated plasmid DNA for B. thuringiensis transformation (45).…”

Transcriptional Regulation and Characteristics of a Novel N -Acetylmuramoyl- l -Alanine Amidase Gene Involved in Bacillus thuringiensis Mother Cell Lysis

“…The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli TG1 was used as the host for the cloning experiments, while SCS110 was used to generate unmethylated plasmid DNA for B. thuringiensis transformations (23,24). B. thuringiensis HD73 was used as the recipient strain to monitor promoter activity and clone the target gene (25,26).…”

Lectins and Their Roles in Pests Control

“…Currently, the two major groups of plant derived genes used to confer insect resistance on crops are lectins and inhibitors of digestive enzymes (proteases and amylase inhibitors). Lectins have been introduced into crops genomes and are now being tested in field conditions Hilder et al, 1987;Hilder et al, 1999;Carlini et al, 2002;Schuler et al, 1998;Ranjeker et al, 2003;Schnepf & whitely, 1981;Smith & Boyko, 2006;Christou et al, 2006;Wang, 2006;Zhao, 2006;Ferry, 2006). Also, for the first time Jjanhong et al (2003) reported that transgenic tobacco expressing Pinellia ternata agglutinin (pta) gene induced enhance level of resistance to M. persicae.…”