Identification Of The PPO Gene Family And Analysis Of Gene Function In Agaricus Bisporus

With unique flavor, delicious taste, high nutritional value and pharmacological effects, Agaricusbisporus is the edible fungi having the largest cultivation scale and consumption in the world andalso having the largest export in China. The fruiting body of A. bisporus easily get browning afterpostharvest, reducing the commodity of fresh mushroom and affecting the development ofmushroom industry. According to the researchs, Polyphenol oxidase (PPO) catalyzed phenolicsubstrates to aggregate into quinones and formed melanin, which is the leading factor of A. bisporusbrowning. Firstly, this study identified the PPO gene family of A. bisporus and then analyzed theexpression of PPOs at the different stages of fruit-body developing. And that of at15℃during thestorage time, finally constructing expression vectors of Agaricus bisporus. The results are asfollows:A. bisporus genome identified5members of PPO gene family. All the PPO proteins contains aconserved CuA and CuB domains, and there are many conserved residues in the entire Cu-bingdingdomain. Phylogenetic tree analysis showed that AbPPO1,AbPPO2, AbPPO3and AbPPO5hadhigher homology, but AbPPO4had the close evolutionary relationship with Agaricus blazei,Laccaria bicolor, and Lentinus edodes.Semi-quantitative RT-PCR analysis showed that at different development stages of A. bisporusfruiting body, AbPPO1had lower expression level at the pin head stage and the button stage,increased gradually from closed cup stage to flat2stage, and reached the maximum at the flat2stage.AbPPO2, AbPPO3and AbPPO5had lower expression at pin head stage, while reached higherexpression from cup stage to flat2stage. AbPPO4had the same expression from pin head stage toflat2stage, which is the constitutive expression. At the storage of15℃, AbPPO1had higherexpression level at the first day, and then gradually decreased until the fifth day. AbPPO2hadstrong expression at the fourth and fifth day. AbPPO3had higher expression at the first day, whileAbPPO5had the highest expression in the storage of five days. The expression of AbPPO4wasunchanged during the storage.Replaced CaMV35S promoter in pCambia1301with gpd and PPO3promoter respectively,constructed pCghG1and pCghP3expression vector, then transformed into the mycelia of A. bisporus through agrobacterium-mediated method and realized the expression of exogenous gene inA. bisporus. After GUS histochemical staining, hypha with pCghG1vector was stained blue, whilehypha with pCghP3vector was not changed, which indicates that PPO3is the inducible promoter.