ACC Deaminase Of Pseudomonas Putida Expression In Agricus Bisporus And Mechanism Studies

The involvement of Pseudomonas putida UW4 in the process of basidiome initiation of the cultivated mushroom Agaricus bisporus was investigated,which can significantly improve the growth of mycelium of Agaricus bisporus,induce fruiting body formation and development,but its mechanism has not been reported.This study mainly use clonal ACC deaminase gene from Pseudomonas putida,and build the ACC deaminase gene(acd S)engineering bacterium of Agaricus bisporus,investigation ACC deaminase to the influnce of Agaricus biosporus fruiting 。The results are as follows:(1)The gene encoding ACC deaminase(acd S)of Pseudomonas putida was cloned according to the published DNA sequence.The GPD promoter of A.biosporus was used to construct the ACC deaminase gene basing on PBHG vector.building the binary expression vector of ACC deaminase PBHG-acd S.(2)Transferred the constructed binary expression vector PBHG-acd S into Agrobacterium EHA105,then this new constructed vector,PBHG-acd S,wastransformed into wild-type AS2796 through Agrobacterium tumefacien-mediated transformation.Successfully screening to transgenic engine-ering strain of Agaricus bisporus,which obtain the ACC deaminase gene of Pseudomonas putida.(3)Hygromycin resistance,molecular testing,Determination of the ACC deaminase enzyme activity,Determination of the mycelial growth and fruiting,the results showed that the transformants can grow on PDA plate with hygromycin concentration up to 100ug/ml,indicating that exogenous gene was integrated stably into the genome of AS2796,that obtain ACC deaminase gene and ACC deaminase activity,also can promote the groth of hyphae,reaching extremely significant level.After covered the wermiculite of sterilization,The formation time of Agricus bisporus primordium of the transformants is earlier than the ck.In summary,these results indicate that the exogenous gene exogenous gene was integrated stably into the genome of AS2796 through Agrobacterium tumefacien-mediated transformation.it is possible to carry out transcription and translation under endogenous promoter starting.The high concentrations of Hygromycin resistance,indicating exogenous gene was integrated stably into the genome of AS2796 without the gene lose due to vegetative propagation.ACC deaminase enzyme testing results also showed that exogenous ACC deaminase gene expression in AS2796.Fruiting experiment shows that ACC deaminase can degrade procrent ACC of Agaricus bisporus mycelium,thereby reducing the amount of mycelium produced ethylene,thereby removing inhibition of the ethylene for the primordium formation and development of fruiting body of A.bisporus,promoting the growth of Agaricus bisporus.