Analysis Of PPO Gene Expression And Construction Of Efficient Expression Vector Of Agaricus Bisporus

Polyphenol oxidase (PPO) is the key enzyme leading to browning oi Agaricus bisporus. In order to further study PPO gene activity, we discussed the expression of PPO under different stages and conditions. Constructing proper transformation method of A. bisporus and improving efficiency of transformation will provide a powerful tool for studying PPO gene and other functional genes of Agaricus bisporus at molecular level, and are meaningful for genetic breeding of Agaricus bisporus as well. This article discussed the expression activity of PPO under different stages and conditions, constructed an efficient transformation method and a PPO gene promoter expression vector for Agaricus bisporus. Tissues of fruiting bodies of Agaricus bisporusat different stages were sampled and stored under4℃, total RNA of Agaricus bisporus’was extracted and tested by half real-time fluorescent quantitative PCR useing18s rRNA as a reference to analyse PPO gene mRNA expression in samples of Agaricus bisporus under different conditions. The four PPO genes in Agaricus bisporus are AbPPO1、 AbPPO2、 AbPPO3and AbPPO4. Under different stages of fruiting bodies of Agaricus bisporus, AbPPO1expressed nearly equally in all stages, AbPPO2was higher in middle stage, AbPPO3was higher in ripe stage, and AbPPO4expressed gradually higher step by step. Stored under4℃, the expression of AbPPOl and AbPPO2weren’t changed obviously, but the expression of AbPPO3and AbPPO4increased first and then decreased.Gpd promoter was cloned form Agaricus Bisporus and inserted to the plant expression vector pCambia130l to take place the35S promoter of cauliflower mosaic virus, then expression vector pCghg was construct, which is suitable for transformation of Agaricus Bisporus, and different transformation conditions were discussed to establish an efficient transgenic system. The optimized transgenic system was as following: OD600nm of Agrobacterium tumufaciens cells was0.05, infection for30min, the concentration of Acetosyringone was200μmol in pre-culture, cocultivating for one day, and the efficiency of transformation achieved to70%at least.We designed specific primers according to the PPO3sequence of Agaricus Bisporus to clone promoter of PPO3, inserted the promoter to the expression vector pCambial301to take place the35S promoter of cauliflower mosaic virus, and got vector pCghP3. Then, pCghP3was transferd into A. Bisporus on the basis of the established transgenic system of pCghg to achieve an efficient exogenous gene transformation system for Agaricus Bisporus, providing a powerful tool for studying the expression activity of PPO gene.