| Genetic transformation has been playing an important role not only in basic research of entomopathogenesis investigation but also in genetic modificaiton in many organisms. However, transformation system for Beauveria bassiana is far from being perfect. In this report, optimal medium screening for growth and spore production, selection marker gene screeing, protoplast preparation conditions, PEG-mediated protoplast transformation were investigated. Results revealed that: (1) The myceliums of B. bassiana grown fast in the L-broth medium. (2) The optimal medium for B. bassiana spores production was PDA. (3) The optimal strain age for protoplast production was 24 hours culture, 2.25×1010 cells/g mycelium. (4) The optimal concentration of β-mercaptoethanol was 0.01mol/L for protoplast production (99%) and regeneration. (5) The best enzymes was fungus cell wall degradation enzyme and the optimal concentration, treatment time were 6.0 mg/ml and 1.5 hours. (6) The optimal osmotic stabilizer was 0.7 mol/L NaCl for protoplasts production. (7) The optimal osmotic pressure stabilizer was 0.7 mol/L glucose. (8) The optimal agar concentration was 0.75 % for protoplasts regeneration to a high level of 57%. (9) The growth of protoplasts and spores of B. bassiana were fully inhibited in the L-broth medium that containing 25 mg/L or 150 mg/L herbicide (phosphinothricin, PPT) respectively, while homomycin B and other antibiotics used in this paper had no inhibition. The result indicates that bar gene should be used as a selection marker gene in genetic transformation of B. bassiana. (10) Plasmid PTF102 containing bar gene was introduced into B. bassiana protoplast. The putative transformants were selected after 5 generations and characterized by PCR. Penetration of host cuticle is one of the most important steps fungal pathogenesis. In common with phytopathogenic fungi, entry of B. bassiana into the host involves both mechnical pressure and enzymatic degradation. The complex refractory nature of insect cuticle suggests that penetration would require the synergistic action of several enzymes including chitinase, protease and lipase. Therefore, cloning of these related genes are very important for investigation of entomopathogenicity molecular basis. In this report, conserved primers were designed according to previous reported chitinase gene, sublitisin-like protease gene and cyclophilinA gene. The homology genes were isolated by PCR from the local B. bassiana isolate from Ostrinia funacalis in Gongzhuling. Sequencing and bioinformatics analysis indicated that: (1) A chitinase gene BbChitl was cloned from Beauveria bassiana isolated from Ostrinia furnacalis. Sequence analysis revealed a cDNA sequence of 1047 bp long and encoded a protein consisting of 348 amino acids. The deduced amino acid sequence exhibited 99% or 82% identities to that of Cordyceps bassiana chitinase and Trichoderma harzianum chitinase, respectively. (2) A subtilisin-like cuticle degrading protease gene was also isolated by PCR from the local B. bassiana isolate. Sequence analysis revealed a gene of 1760 bp long containing three introns between 331～405, 586～646, 1157～1223 bp. The deduced protein consist of 377 amino acids and exhibited 98.9% or 98.7% identities to the previous reported sublitisin-like enzymes of B. bassiana. (3) A cyclophilinA gene was isolated by PCR from the local B. bassiana isolate. Sequence analysis revealed a gene of 606 bp long containing two introns between 215～269, 376～413 bp. The deduced protein consist of 164 amino acids and exhibited 99.4% identity to previous reported cyclophilin A protein of B. bassiana. |
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