Construction Of Genetic Transformation System And Transgenic Laccase Producing Strain Of Pleurotus Ostreatus

There were about 600 million tons crop residues produced per year in China. They were rich in lignocelluloses. It was a successful approach to turn crop residues into wealth food using edible fungi. In present study, we constructed an effective genetic transformation system for transformation laccase gene into Pleurotus ostreatus Tianda 300 to enhance its capacity of decomposing the lignocelluloses from crop residues and the biological efficiency. The results were as follows:(1) Cloning of sdi promoter of Pleurotus ostreatus and construction of expression Vector 25Homologous sdi promoters with 1.3 kb of Pleurotus ostreatus tianda 300 was cloned and fused with gene hph by overlapping PCR. The hygromycin resistance expression vector 25 was constructed, which was constituted of sdi promoter, hph gene and CaMV termination in skeleton of pMD19 - T carrie.Protoplasts with regeneration ability of Pleurotus ostreatus tianda 300 were obtained by hydrolysis mycelium using 1.6% lyticase enzyme in 30℃for 3h, and 0.6 mol/L mannitol as osmotic pressure stabilizer. The protoplast yield was about 5.7 X 106/ mL, and regeneration rate on RCM plate was 0.46%.The hygromycin resistance concentration of mycelium and protoplast of Pleurotus ostreatus tianda 300 was determined, and base on it hygromycin concentration of preliminary and counter screening for transformation was set up as 80μg/mL and 100μg/mL, respectively. The expression vector 25 was transformed into mushroom by PEG-CaCl₂ mediated protoplast method, and some transformants grew on RCM plate with 80μg/mL hygromycin. These transformants were all identified as positive ones through the screening test, gene DNA PCR, and mRNA RT-PCR. It was proved that the hph gene integrated into the genome and expressed stability. Thereby, we established a genetic transformation system of Pleurotus ostreatus.(2) Compare between different transformation systems of Pleurotus ostreatusThe transformations frequency of three expression vectors 25 and pAN7-1 into Pleurotus ostreatus tianda 300 were 0.2 transformants/μg plasmid DNA, 0.4 transformants/μg plasmid DNA, respectively, and the lowest of PBHt1. The RNA transcription levels of hph gene of these three expression vectors were similar with their transformations frequency.(3) Cloning and homologous expression of laccase poxc gene of Pleurotus ostreatusThe gDNA and cDNA of laccase poxc gene of Pleurotus ostreatus tianda 300 were cloned. The gDNA length was 3818bp, including 19 introns and 20 exons. The cDNA length was 1607bp, encoding 533 amino acid protein, containing three highly conserved Cu-oxidase domains homologized higher with other fungal laccase protein.Laccase poxc homologous expression vector PIPOXC and PI2POXC of Pleurotus ostreatus were constructed by using gDNA and cDNA of Pleurotus ostreatus tianda 300 replace hph gene of expression vectors 25, and co-transformation with expression vectors 25 into Pleurotus ostreatus protoplast by PEG-CaCl₂ mediated, respectively. 21 hygromycin transformants were obtained. But we found poxc did not fully integrate into the genome by PCR detection, laccase activities did not differ from start strain. There were not any positive transformants obtained.