Construction Of Transgenic Laccase Producing Strain Of Pleurotus Ostreatus

ABSTRACT There were multitudinous crop residues produced per year in China, such as wheat straw and maize straw. They were rich in lignocelluloses. It was a successful approach to turn crop residues into wealth food using edible fungi. In order to enhance the capacity of edible fungi decomposing the lignocelluloses from crop residues and its biological efficiency, we constructed an effective genetic transformation system for transformation laccase gene into Pleurotus ostreatus Tianda 300 to. The results were as follows:1. The expression of laccase poxc gene of Pleurotus ostreatus in Pichia pastoris. The cDNA that code mature laccase protein was constructed to pPIC9K, then the carrier was transformed to Pichia pastoris by using electrotransformation method, alpha-factor from Saccharomyces cerevisiae as signal peptide. After G418 screening, we got some transformants. But when we tested their activity on MM plates that contain ABTS as substrate, we couldn’t get a normal expression of laccase transformant.2. The construction of eukaryotic expression vector. Replacing the whole hph gene on the PAN7-1 expression vector by laccase poxc gene of Pleurotus ostreatus, we got the modifacated eukaryotic expression vector pAN7-1’-cDNA.3. Plasmid pAN7-1, pAN7-1’-cDNA was cotransformrd in Pleurotus ostreatus protoplas through PEG-CaC12 method. After protoplast regenerating, transformants were detected by hygromycin screening and molecular means. It is founded that all the transplants can be detected by exogenous hygromycin resistance hph gene, of which 15 transformants were detected by exogenous laccase gene POXC.The. comparion of the growth rate between the 15 transformants and the start strain in PDA plate did not differ significantly, presumably because PDA medium does not contain lignin, in which the function-of laccase was not obvious. Analysis of variance of the tube experiment results showed that the growth rate of the transformant did not differ significantly compared with the control, presumably because the exogenous gene did not really play a role.Determine the enzyme activity of transformants under different culture conditions, the results were not same. The enzymatic activity of the transformants in tube were lower than the start strain, presumably because the amount of Pleurotus ostreatus hyphae tested is too low. Flat mycelium and shaking the measured enzyme activity in the extracellular intracellular enzyme activity of several transformants was significantly higher than the start strain. However, by further RNA expression level of verification, we couldn’t get the corresponding results, suggesting that it mightoccur co-suppression, which needed further validation to determine.