Site-specific Integration Of Heterologous Gene Into Bacillus Thuringiensis Chromosome And Its Expression

During construction of high-efficiency and broad-spectrum Bacillius thuringiensis engineered strain, the introduction of high-copy number plasmid resulted in a significant decrease in the spore number and retardance of sporulation, and as a consequence, negatively affected the toxicity of Bacillius thuringiensis. It needs plentiful energy during sporulation of B. thuringiensis. It would consume a mass of energy to replicate and maintain higy copy number plasmid. So the sporulation of B. thuringiensis is retarded. Besides, unstable single-stranded DNA(ssDNA) always formed in the course of Bacillius plasmid replication, which will lead to removal of the plasmid and heterologous gene. The object of this project is to integrate heterologous gene into the chromosome of B. thuringiensis to solve these problems and construct high-efficiency Bt engineered strain that can stably express heterologous gene.The trigger factor gene located on the chromosome of B. thuringiensis was chosen as the integration site. The Trigger Factor protein has been characterized in Bacillius thuringiensis aerystalliferous strain XBU001 via proteomics methods in our laboratory. By analyzing the homology of trigger factor gene sequence from several strains of B. thuringiensis, two pairs of primers TF1-F/TF1-R and TF2-F/TF2-R based on conservative region were synthesized. And the 5' end and 3' end of trigger factor gene were amplified as the homologous arms. An integrative plasmid pKTF12 was constructed by cloning the the homologous arms onto plasmid pKSV7, a temperature sensitive shuttle vector. An engineered strain KCTF12 containing cry1Ac gene in its chromosome was constructed via this integrative plasmid pKTF12.Several characteristics of KCTF12 has been compared with recombinant strain HTX42 harboring a high-copy number plasmid and XBU001, including growth curve, stability of heterologous gene, morphology of crystals and cells, spore number, express of Cry1Ac protein and insecticidal activity. The results demonstrated that site-specific integration of cry1Ac in the chromosome did not affect the normal growth of XBU001. The KCTF12 could stably express cry1Ac gene and produce bipyramid crystals. When compared with HTX42, KCTF12 has the merit of advanced sporulation and an increase in spore number. Cry1Ac began to express after 12 hours of incubation and reached its maximum after 20 hours of incubation in KCTF12 strain, while in HTX42 strain Cry1Ac began to express after 18 hours of incubation and reached its maximum after 28 hours of incubation. The insecticidal activity of Cry1Ac protein produced by KCTF12 and HTX42 to the second instar lavea of Helicoverpa armigera did not show significant difference.The site-specific integration proved to be a good approach to construct resistance gene-free B. thuringiensis engineered strain that can stably express the heterologous gene. And resistance gene in B. thuringiensis engineering strain can be eliminated by the temperature sensitive plasmid pKSVT, which can avoid the risk of environmental release.