Functional Characterization Of 2-CYS PRX In Pleurotus Ostreatus

Pleurotus ostreatus which not only contains an abundance of essential amino acids and vitamins, but also appropriate quantities of carbohydrates and minerals, has medicinal properties and ecological and economic values. Oxidative stress has a negative impact upon P. ostreatus exposed to environmental stresses, therefore, P. ostreatus has developed a series of antioxidant mechanisms to remove reactive oxygen species(ROS). 2-cys peroxiredoxin(2-Cys Prx), an antioxidative enzyme, can scavenge peroxides and protect cells against oxidative stress. In this study, the gene function of 2-Cys Prx of P. ostreatus was analyzed tentatively by overexpression and RNA interference technology,the results were as follows:1. A 929 bp open reading frame of 2-Cys Prx gene contained five introns and six exons, and encoded a protein of 218 amino acids corresponding to the c DNA of 651 bp in length. The predicted molecular weight and isoelectric point was 23.9 k Da and 5.4, respectively. The deduced amino acid sequence showed the structural feature of typical 2-Cys Prx which contained two conserved cysteine regions(Cys48 and Cys170) and two conserved motifs GGLG(Gly-Gly-Leu-Gly) and YF(Tyr-Phe) acted as a signal transmitter through sensitively recognizing H2O2 changes. The result of phylogenetic analysis revealed 2-Cys Prx from P. ostreatus had the closest evolutionary relationship with Agaricus bisporus.2. The overexpression vector p CAMBIA1302-OE and RNAi vector p CAMBIA1302-RNAi-1 and p CAMBIA1302-RNAi-2 were constructed and successfully transformed into P. ostreatus by Agrobacterium tumefaciens-mediated transformation(ATMT). In order to confirm stable integration of the target gene into the genome DNA, PCR and fluorescence observation were carried out. 23 overexpression transformants, 28 R1 silencing transformants and 18 R2 silencing transformants were obtained. The transformation efficiency was about 62 %, 67 % and 60 %, respectively. q RT-PCR was used to determine the expression levels of the 2-Cys Prx gene in different transformants, The maximum expression level in the overexpression transformants was about 3.5-fold higher than the wild-type strain. The silencing efficiency of R1 and R2 was average 58 % and 34 %.3. Results showed that silencing or overexpression transformants had no apparent effects on morphology of mycelia and colony. Abiotic stress resistance assays showed, overexpression of 2-Cys Prx in transgenic P. ostreatus conferred obvious resistance to high peroxide concentration, high temperature and high osmotic stress. While silencing transformants were insensitive to oxidative stress in comparison to WT strain. This suggested that 2-Cys Prx acted as regulators of stress-responsive signaling transduction pathways.All these data indicated that 2-Cys prx was involved in protecting the cellular proteins against oxidative damage.