Molecular Cloning and Expression of Polyphenoloxidase Genes from the Mushroom,Agaricus bisporus

摘要

The polyphenoloxidase(PPO) is the key enzyme considered to be responsible for mushroom browning.By using homology cloning and rapid amplification of cDNA ends(RACE),two new PPO genes and the corresponding cDNA were identified from the fruit bodies of Agaricus bisporus(AbPPO3 and AbPPO4,GenBank accession nos.GU936494 and GU936493,respectively).The genomic DNA sequences of AbPPO3 and AbPPO4 are 2 080 and 2 189 bp in length,respectively,encoding putative polypeptides of approximately 66 and 68 kDa.The deduced amino acid sequences show characteristic features of two copper-binding domains conserved in the type III copper proteins including fungal polyphenol oxidases.Sequence comparisons indicate that AbPPO3 and AbPPO4 present 55.3% similarity to each other(48% identity).We also obtained more than 1.5-kb long sequences upstream of the start codon of the AbPPO3 and AbPPO4 and recognized them as their respective putative promoters.Analyses of the two PPO promoter regions show that they contain abundant cis-acting elements which are probably responsible for anaerobic induction,light,wound,stress,and auxin response.Semi-quantitative RT-PCR results indicate that AbPPO3 and AbPPO4 were highly expressed in the mature fruit bodies and up-regulated after 2-d storage of mushroom.These results suggest that AbPPO3 and AbPPO4 may play roles in A.bisporus browning and pigmentation during development and postharvest storage and the elements in promoters may act as regulatory elements for the inducible expression of AbPPO3 and AbPPO4.The successful cloning and expression analysis of AbPPO3 and AbPPO4 warrant a further investigation on the structure and function of A.bisporus PPO which points to the possible targets for genetic manipulation.