Construction And Transformation Of Overexpression Vector Of Po.Hyd From Pleurotus Ostreatus

Fungal hydrophobins are a series of small, moderate, secretory proteins with strong surface activity. They are a special class of proteins which is secreted by higher filamentous fungi in a specific period. They can self-assemble at hydrophilic-hydrophobic interfaces and form an amphiphilic membrane. Fungal hydrophobins would be highly expressed in some special growth and development stages of fungi. Beacause of the specific biological function, hydrophobins can be used in food, medical and many other fields. Therefore, the study of hydrophobin gene is very important.In this study, the function of Po.Hyd gene was analyzed by overexpression vector pPHOEx. The results are showed as follows:1. Overexpression vector pPHOEx was successfully constructed and transformed.12 putative transformants were obtained;2. Putative transformants were further identified by PCR.7 of these putative transformants have positive test results. These seven transformants were named G1-G7;3. Southern blot test showed that T-DNA was randomly inserted as single copy;4. RAPD analysis showed that 111 sites had been detected by 13 random primers. Percentage of polymorphic loci was 39.64%, and DNA polymorphism index was 56.63;5. The real time fluorescence quantitative PCR analysis shows that Po.Hyd gene was effectively overexpressed in the transformants;6. Isozyme analysis andicated that there are some differences in esterase and peroxidase in transformants;7. Fluorescence microscopic examination showed that all the transformants can be observed the expression of green fluorescent protein;8. The growth speed of hypha of transformants are different, and there are also some differences about fruiting body morphology;9. The promoter of Po.Hyd gene was cloned by SEFA-PCR.