Abstract
ABSTRACTTo expand the usage of endophytes in agriculture and in forestry, the insecticidal gene cry218 of Bacillus thuringiensis was introduced into a poplar bacterial endophyte Burkholderia pyrrocinia JK-SH007. The cry218 gene was cloned by polymerase chain reaction (PCR) and was inserted into a PHKT2 expression vector that was introduced into the bacterial endophyte JK-SH007. By using sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE) and western blotting, we confirmed that the engineered bacterial endophyte was successfully constructed, and it harboured insecticidal function after the bioassay in planta. The toxicity of the expressed insecticidal protein was analysed on second instar silkworm. The regression equation showed that the median lethal concentration (LC50) of the insecticidal protein was 0.77 (0.57–1.04) g/L at 72 h. The insecticidal bacteria genetically modified in this study have laid the foundation for further exploitation of biocontrol bacteria.
Highlights
Bacillus thuringiensis can produce insecticidal Crystal proteins (ICP) with specific virulence on pests which severely damage crops and trees
The Cry218 expression vector was constructed by reverse polymerase chain reaction (PCR) and in vivo homologous recombination
The results showed that 78.3% of the silkwarms were dead in 72 h after treatment with 3.16 g/L of recombinant bacterial lysate
Summary
Bacillus thuringiensis can produce insecticidal Crystal proteins (ICP) with specific virulence on pests which severely damage crops and trees. By constructing BT engineered bacteria, focus has been laid on a few aspects, like expanding the insecticidal spectrum and extending the effective duration of the toxicity. In 1983, Klier et al [2] transferred plasmid pBt42-1 carrying an ICP gene into Bacillus subtilis, and transferred this plasmid to B. thuringiensis. They gained engineered bacteria which were insecticidal to both Ephestia kuehniella and Anopheles stephensi [2]. Rang et al [3] cloned a CIP gene encoding a 34 kDa crystal protein from B. thuringiensis subsp.
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