Brinell Beauveria The Establishment Of Genetic Transformation System And The Efficient And Stable Expression Of Pr1 Engineering Strains

Entomopathogenic fungi can be used to control insects and have several advantages over chemical pesticides. They can actively invade hosts and have repeated infection cycles. They can survive in the soil during periods of low host densities and the hosts themselves rarely acquire resistance to them. Entomopathogenic fungi produce extracellular, cuticle-degrading enzymes that include proteases, chitinases and lipases. These degrade the insect cuticle, which is an effective barrier against most microbial infections. An extracellular, subtilisin-like serine protease (Pr1) is the most important factor for the insecticidal activity of entomopathogenic fungi. Previously, the Pr1 genes from Metarhizium anisopliae and Beauveria bassiana have been isolated and analyzed. The former gene was overexpressed in Metarhizium anisoplia, which greatly enhanced the strain virulence.Beauveria brongniartii is a widely employed entomopathogenic fungus all over the world and the Pr1 gene from Beauveria brongniartii had not been cloned or sequenced. So we have explored the mechanism of infection of Beauveria brongniartii at the molecular level. genetics transformation has been recognize as a powerful tool to enhanc the strain virulence of entomopathogenic fungus. However, transformation system for Beauveria brongniarti is far form bing perfect. Therefore, a simple, reliable, and highly efficient transformation system for Beauveria brongniarti need to been found.In this paper, on the base of the Pr1 gene from Beauveria brongniartii had not been cloned and sequenced. The Pr1 protein with a N-terminal fusion to the six-histidine tag was expressed in Escherichia coli as inclusion bodies with the expression vector pBV220, Sodium dodecylsulsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly revealed expressed product. The Pr1 protein was purified, refolded, and had proteolytic activity of 0.288 U mg^-1.According to the fact that Beauveria brongniartii was sensitive to herbicide phosphinothricin, the herbicide resistance gene bar was used as selectable marker gene for Beauveria brongniartii transformation. The PPT concentration for transformant selectable was used to be 60μg/ml. Using PROKII, a commonly used vector for plant transformation, as the backbone, the binary PROBCII-bar, in which the bar gene was under thr promoter PgpdA and the terminater TtrpC, was constructed for Beauveria brongniartii transformation. Mediated by A.tumefaciens strain LBA4404 , T-DNA of PROKII-bar was successful integrated into Beauveria brongniartii genome...