Studies On The Characteristics And Function Of NADPH Oxidase Gene NOXA In Pleurotus Ostreatus

The formation and differentiation of the fruit body of Pleurotus ostreatus is a very complex process.Nutrition and external conditions required for this process had been well studied,such as the difference of temperature,light,etc,but studies on the mean of molecular was very few.The NADPH oxidase that can regulate the synthesis of reactive oxygen species exists in filamentous fungi and it plays a key role in the process of differentiation and development of the fruit body.This enzyme had been verified exist in Pleurotus ostreatus,so we deduced that NADPH oxidase played a key role in the process of differentiation and development of the fruit body of Pleurotus ostreatus.The hairpin plasmid NOXA-P8 and plasmid PAN7-1used in this experiment was co-transformated to Pleurotus ostreatus tianda 300 and the positive transformants were screened by hygromycin.The function of the NOXA gene was determined by the traits and molecular analysis.The promoter of alcohol dehydrogenase of Aspergillus nidulans was cloned and the recombinant expression vector p EGFP-C1-alc A was constructed.The vector was transformed to Rhizopus nigricans and Pleurotus ostreatus.By the method of fluorescence microscopy,we verified that the promoter of the alcohol dehydrogenase can start the expression of exogenous gene in Rhizopus nigricans and Pleurotus ostreatus and it can restrain or induce the expression of the exogenous gene.The conclusion of this study were as follows: 1.Hairpin Plasmids NOXA-P8 and plasmids PAN7-1 were conversioned to Pleurotus ostreatus of tianda 300 by the method of protoplast transformation with PEG-Ca Cl2.10 transformants were got after the screening of hygromycin and RCR authentication.The growth rate of the 10 transformants on the plate were lower than the control strain.6 transformants were randomly selected for semi-quantitative PCR,verifying that the NOXA expression amount of 1?3?5 transformants were reduced,decreased by about50%,while the expression levels of the NOXA in the remaining three transformants did not change significantly.The result of the content of H₂O₂,hydroxyl radical,superoxide anion was agree with the result of semi-quantitative PCR.The content of H₂O₂,hydroxyl radical,superoxide anion were significantly reduced compared with the wild strain.Staining transformants and the wild-type strain by NBT also verified the results of the semi-quantitative PCR.The color of the wild-type strain was the same with the effect with the RNAi,while the color of 1?3 transformants were very shallow.Fruit body could be found on the plate of all the wild strains,but none of the 10 transformants formed fruit body except the third transformant.The result verified that the NOXA gene is a key gene for the formation of fruit body of Pleurotus ostreatus.2.The cucumber mosaic virus promoter on the plasmid p EGFP-C1 was replaced by the promoter of the alcohol dehydrogenase of Aspergillus nidulans and the recombinant expression plasmid p EGFP-C1-alc A which was activated by the promoter of the alcohol dehydrogenase was successfully constructed.3.The recombinant expression plasmid p EGFP-C1-ALCA was transformed to Rhizopus nigricans by the method of liposomes conversion.Fluorescent protein was proved to be expressed in Rhizopus nigricans by the method of observation of fluorescence and confirmation of molecules.It was also proved that the promoter of alcohol dehydrogenase could start the expression of the GFP gene on the induction medium and inhibit the expression of the GFP gene on the inhibition medium.4.The recombinant expression plasmid of p EGFP-C1-alc A and the plasmid of p EGFP-C1-PAN7-1 were cotransformed to the Pleurotus ostreatus of tianda 300 by the method of protoplast transformation with PEG-Ca Cl2.The transformants were respectively cultivated on the induction medium and inhibition medium after the screening of hygromycin and molecular authentication.Green fluorescence could be observed in the mycelium cultivated on the induction medium,but it could not be observed in the mycelium cultivated on the inhibition medium.All the result proved that the promoter of the alcohol dehydrogenase can induce or inhibit the expression of the exogenous gene in Pleurotus ostreatus,which laid a foundation for future research of NOXA gene.