Cloning And Expression Of The Polyphenol Oxidase From Agricus Bisporus

Polyphenol oxidase is recognized as the major enzyme responsible for the browning, browning is one of the key factors that influence the shelf-life and product quality of the cultivated Agricus bisporus. Agaricus bisporus browning cloning-related gene, is targeted to achieve mushroom breeding of anti-browning the basis of an important research work. This study used 2796 as Agaricus bisporus samples using RT-PCR, RACE cloning from the fruiting bodies of Agaricus bisporus PPO browning-related genes, and its expression in E.coli. Homology of its gene sequence comparison and analysis. Paper includes two parts of the experiment:(1) Agaricus bisporus PPO gene cloning and amino acid sequencing:Using primers designed on the basis of sequence homologies in the copper-binding domains for a number of plant and fungal tyrosinases, and in accordance with the sequence information obtained by rapid amplification of cDNA ends (RACE) method, two tyrosinase encoding cDNAs were cloned from an Agaricus bisporus. The sequences PPO1 and PPO2 were, respectively,1.88kb and 2.023kb in size and encoded amino acids of approximataly 576 and 611. Homology analysis showed that, PPO1 encoding amino acid sequence homology with other fungal PPO protein similarity is about 21-50%; PPO2 with other fungal PPO protein is about 20-63% similarity. Phylogenetic analysis showed that, PPO1 and PPO2 are close evolutionary relationship with AbPPO2 cloned by Agaricus bisporus.(2) Polyphenol oxidase gene expression in the semi-quantitative RT-PCR analysis:Using semi-quantitative RT-PCR method, with 18s rRNA gene as internal control, study the browning of Agaricus bisporus PPO mRNA-related gene expression levels.Analysis of results showed that the fruiting bodies of Agaricus bisporus in the different developmental stages, PPO1 expression in the needles is very low period in the cup to express a view in both Tai higher line period. Period in PPO2 needles to large amount of line to express a view to gradually. Agaricus bisporus at 4℃storage, PPO1 and PPO2 days in paragraph 1 to the micro-expression detection,2 to 4 days the expression of both the stronger the 7th day after the expression to decrease.(3) The Polyphenol oxidase was expressed in Escherichia coli:The ppo was inserted into the expression vector of pET28a(+) and transformed into E.coli BL21(DE3) which was expressed successfully in Escherichia coli by inducing with IPTG.The product was analyzed by SDS-PAGE, the result showed that a specific band about 67kDa was obtained.