Objective: To efficiently construct resistance gene-free Bacillius thuringiensis engineered strain that can stably express heterologous gene.
Methods: We amplified the trigger factor gene located in chromosome of XBU001 strain as homologous arms and constructed an integrative plasmid pKTF12 on the basis of plasmid pKSV7, a temperature sensitive plasmid. We also constructed a recombinant strain KCTF12 containing cry1Ac gene in its chromosome via the integrative plasmid pKTF12.
Results: Site-specific integration of cry1Ac into XBU001 chromosome did not affect its normal growth. The cry1Ac gene could stably express and form bipyramid crystals in KCTF12. When compared with HTX42 harboring a high-copy number plasmid, the recombinant strain KCTF12 has the merit of advanced sporulation and an increase in spore number.
Conclusion: The Site-specific integration proved to be a good approach to construct resistance gene-free Bacillius thuringiensis engineered strain that can stably express the heterologous gene.