Construction and characteristic of a constitutive expression chi of engineered Bt strains

Abstract:

[Objective] We constructed Bti engineering strains that can highly express chitinase genes constitutively, so that the strains can inhibit the growth of fungi more effectively. [Methods] The full length and series of deletion promoter fragments of chitinase gene chiB from Bacillus thuringiensis were cloned into a shuttle promoter-probe vector pCB, and all of the constructed plasmids were transformed into Bti75 strains. A 190 bp-length high constitutive expression promoter was identificated based on the results of β-galactosidase activity and real time-PCR. By using this promoter, we constructed Bti75 engineering strains that express chitinase gene chiMY of Bacillus licheniformis and chiA gene of Bti75 itself. SDS-PAGE and zymogram analysis were used to examine chitinase expression. In order to evaluate the antifungal activity assay of engineering strains against 3 plant pathogenic fungi, mycelium growth and spore germination inhibition assay were done. [Results] Without induction, β-galactosidase activity and mRNA contents of β-galactosidase of Bti75(pBPΔ7) was about 7-fold and 2.5-fold high than that in Bti75(pBP), respectively. Without chintin induction, the chitinase activity of Bti75(pDA) and Bti75(pDM) was up to 3.5-fold higher compared with the parent strain. The results of SDS-PAGE and zymogram analysis confirmed that chitinase could be highly expressed in engineered strains. The results of antifungal activity assay indicated that the two engineered strains achieved a significant improvement in inhibiting three plant pathogenic fungi species. [Conclusion] The 190 bp deletion mutant promoter could drive the expression of different chitinase genes constitutively and efficiently. Without induction, the two engineered strains with the deletion promoter exhibited high antifungal activity.

Key words:Bacillus thuringiensis, Chitinase, Deletion mutant promoter, Constitutive expression, Antifungal activity