Evaluation Of Brauveria Bassiana Transformation Efficiency Mediated By Ustilago Maydis Heat Shock Protein Promoter

Beauveria bassiana is a kind of entomopathogenic fungus which is highlypathogenic, has strong compatibility and wide-ranging hosts.During the research offunctional genomics and gene engineering breeding to Beauveria bassiana,promoter isone of the most important elements for the efficient of foreign or endogenous genetransfection.This studyintent to replace the Beauveria bassiana promoter with the heatshock protein promoter hsp70which obtained from Ustilago Maydis,aimed to enhancethe level of transcription and translation of the foreign genes which transfected intoBeauveria bassiana,and to lay a certain foundation for functional gene modification ofBeauveria bassiana using genetic engineering.The PHD3-hsp70plasmid which contains gene ofhsp70promoter was gained usingcarboxin as a selection marker from Ustilago maydis. The PF plasmid was gained bytaking the steps of enzyme digestion, PCR and transformation. The protoplast wasgained by optimizing the conditions, and genetic transformation of Beauveria bassianaprotoplast was conducted via PEG. To get requested transformants, then filtered them onthe Czapek medium which contain PPT herbicide. The transformation efficiency of PFand hsp70-F were compared with the plate counting method and PCR detection.Thesuccessive transfer culture of transformants were conducted for threegenerations toobserve if the biologicalcharacteristics of recipientbacterium was changed, and if theyhad inheritable stability after transformation. The third stage larvae of Asiatic cornBorerv was chosen as objects, to test the toxicities of Beauveria bassiana aftergenetictransformation.In this study, the heat shock protein promoter hsp70of Ustilago maydis was firstlyused for mediating genetictransformation of Beauveria bassiana. The results showedthat, the transformationefficiencymediated by hsp70promoterwas60%higher toPgpdApromoter. The positive rate of PCR was achieved to90%through molecularbiology and fluorescent microscope analysis. The green fluorescence was detectedwithin hypha and spore in the fluorescent microscope test. The evidence aboveindicated that the selectable marker gene of hsp70and carboxin had been integrated intothe genomes of Beauveria bassiana, and expressed well. A genetic transformation system of Beauveria bassiana mediated by hsp70promoter was been successfullyestablished. Through the comparison and analysis of biological characteristics oftransformants and recipients which named D1-5, it indicated that thetransformationprocess did not affect the biological characteristics of transformants.Meanwhile the results of insecticide toxicity tests on Asian corn Borerv indicated thetoxicity was not affected by this transformation process.