Cloning And Characterization Of NADPH Oxidase Gene (noxa) In Pleurotus Ostreatus

Like other basidiomycetes,the mushroom Pleurotus ostreatus’s fruiting body differentiation and development is a very complex and advanced physiological process. Many researches contribute to nutritional requirements and external environmental conditions of the of this process, yet aspects of molecular mechanism is relative little. NADPH oxidase （NOXA） in filamentous fungi, regulates reactive oxygen species （ROS） synthesis, which plays a key role in induction of differentiation and development of fruiting bodies. Also existence similar physiological process in Pleurotus ostreatus, suggesting that maybe NOXA genes control the differentiation of Pleurotus fruiting body development. In this study, we had been cloned Pleurotus ostreatus NOXA gene and then constructed RNA inference expression vector. The plasmids was transformed into mushroom by the methods of protoplast. We have obtained positive transformants via hygromycin resistance screening and molecular detecting with special primers. The preliminary study of the NOXA gene regulatory for fruit body development have been carried ou by analyzing the traits of transformants. The main conclusions as follows .1. Pleurotus ostreatus NOXA gene cDNA partial core sequence was cloned from total RNA extracted from Tianda 300 mycelia by PCR with the primers that ware designed according to other creatures reported. Sequencing shows that the length of the NOXA gene is 679 bp and could encodes 226 amino acids polypeptide. Bioinformation analysis indicates that the Pleurotus ostreatus NOXA gene cDNA shared 54.87%,40.71% and 73.89% homology with A.nidulans,N.crassa and L.bicolor respectively in amino acid sequences of protein conservative structure.2. We have cloned forward and reverse hairpin sequences which were used to construct hairpin structure on the basis of part NOXA sequence already cloned. The hairpin structure connected to the expression vector PAN7-1 to replaced part of the hph gene was and to build RNAi expression vector NOXA-P8.3. Plasmid PAN7-1and NOXA-P8 were transformed into Pleurotus ostreatus protoplast, using PEG-CaCl₂ co-transformation method. By means of hygromycin resistance screening and molecular detection after protoplast regeneration,each sample can detect hygromycin resistance gene and there is only one sample can not detect hpRNA in seventeen random choosen transformants. These sixteen transformants growth rate were significantly lower than the original strain in the PDA plate, but with no difference in cotton seed medium in test tube, the reasons needed to be further study.We have been cloned the NADPH oxidase gene noxA from higher Basidiomycetes for the first time. ROS content and the growth rate was lower than in the original strain in noxA-RNAi transformants indicate that the content of ROS is closely relate to the function of noxA and further studies may reveal the relationship between noxA gene and the formation of Pleurotus ostreatus fruiting bodies.