New Vector pBUC3 can Express <i>Bt</i> <i>Cry1Ac31</i> during the Vegetative Phase in <i>Bacillus thuringiensis</i>

Abstract

The feature of Bacillus thuringiensis ( B. t .) is the ability to generate parasporal crystal protein during the spore-forming process. In order to realize the expression of the Bt Cry genes independent on spore formation, we attempted to design new vector that could express Bacillus thuringiensis insecticidal crystal protein in the vegetable growth phase in this research. First, we designed a pair of specific primers based on cry 3Aa promoter gene sequence to amplify cry3Aa promoter from B.t. subsp. Tenebrions ; then the DNA fragment with blunt-end was ligated to the cross-species vector pBU4 to build new pBUC3 vector which would be non-dependent sporulation vector expressing in the vegetative growth phase of Bacillus thuringiensis . In order to validate the vector whether express B.t cry gene in the vegetative phase, The B.t cry1Ac31 gene was ligated to the pBUC3 vector to be transformed into B.t non crystal mutant IPS strain by electronic transformation approach. Through the examinations by the PCR, microscopic observation and SDS-PAGE, the results showed that B.t cry1Ac31 was expressed in cell growth vegetable phase. Our study suggested that the feature of cry 3Aa promoter being non-dependent on spore formation might be useful expression element that could achieve the cry gene of Bacillus thuringiensis expressing in vegetative phase.