Construction Of A Fusing Chitinase And Protease Gene And Improvement Of Mycoinsecticide In Beauveria Bassiana

Mycoinsecticides are important biocontrol agents. Entomopathogenic fungi penetrate the insect cuticle by combination of mechanical pressure and cuticle-degrading enzymes including proteases, chitinases, carboxylesterase and lipases. During this process, a subtilisin-like protease and a chitinase were proved to play important roles. Overexpressing the protease gene PrlA or the chitinase gene Bbchitl can enhance the virulence of entomopathogenic fungi. However, the interaction between cuticle-degrading enzymes remains unrevealed. In this study, we investigated the interaction between chitinases and protease.Protease gene CDEP-1 and chitinase gene Bbchitl were firstly expressed in yeast. The mixture of CDEP-1 and Bbchitl can degrade, observed by scanning electron microscope, aphid cuticle to a greater extent than CDEP-1 or Bbchitl. Based on this result, vectors, overexpressing a protease gene CDEP-1, a chitinase gene Bbchitl from Beauveria bassiana and fusion gene CDEP-1::Bbchitl, were constructed to increase the virulence of B.bassiana. Vectors, overexpressing a chitinase gene Machitl from Metarhizium anisopliae and fusion gene CDEP-1::Machit1, were also constructed and transformed into B.beauveria. The insertions of these vectors into the genome of B.bassiana were confirmed by PCR. The overtranscription and overexpression of CDEP-1, Bbchitl, Machitl and the fusion genes CDEP-1::Bbchit1, CDEP-1::Machitl were detected by Northen blotting and Western blotting.Insect bioassays revealed that, overproduction of CDEP-1, Bbchitl as well as fusion genes CDEP-1::Bbchitl and CDEP-1::Machitl all can enhance the virulence of B.bassiana for Myzus persicae. Transformants overexpressing CDEP-l::Bbchitl, however, have higher toxicity. At 1×10⁷ conidia/ml, compared to wild type strain Bb0062-15, the average lethal middle time (LT₅₀) of transformants overexpressing CDEP-1 and Bbchitl was reduced by 8.84% and 9.92%, respectively. The average LT₅₀ of transformants overexpressing CDEP-l::Bbchitl was reduced by 21.12%. At 108h and 120h after inoculation, relative to wild type strain, the lethal middle concentration (LC50) of a transformant with CDEP-1::Bbchitl was reduced by 67.42% and 60.50%, respectively. The reduction of LC₅₀ in transformant with CDEP-1::Bbchit1 was 2.22 (108h) or 1.65 (120h) times bigger than the sum of reduction in LC₅₀ of transformants with CDEP-1 and Bbchitl.