Study On Agrobacterium Tumefaciens Mediated Transformation Of Laccase Gene From Pleurotus Ostreatus To Tremella Fuciformis

Tremella fuciformis,also named white jelly mushroom,is rich in various ingredients with great nutritional and medicinal value.Basidiospore produce yeast-like spore which is namely the yeast-like cells of T.fuciformis as a budding way under certain conditions.The yeast-like cells of T.fuciformis have the characteristics of being cultured easily,growing fast and possessing great ability of the heterdogous protein sectetion similar to the primary eukaryote yeast,then it is will more secure and economic to express heterdogous protein as a host in food industry.Laccase is an oxygen oxidoreductase with multi copper,containing copper as the cofactor,it has the ability to oxidize phenolic compounds with molecular oxygen as the coculture.Laccase has a non-special molecular structure,extending to a variety of substrates and a high catalytic activity.Therefore,laccase plays an important role in food industry.However,the production of laccase is a low volume in nature,which is difficult to meet the industrial demand,at the same time,the cultivation of the high demand and purification processes increases the cost,so it is a good choice to express heterologous laccase.Pleurotus ostreatus is one of the laccase producing white rot fungi,and the lac3 gene of P.ostreatus has not been reported.The purposes of this study are to establish the highly efficient genetic transformation mediated the yeast-like cells of T.fuciformis by Agrobacterium tumefaciens and construct the heterogeneous expression vector,realizing the heterogeneous expression of laccase gene from P.ostreatus by the means of being transformed into the yeast-like cells of T.fuciformis using A.tumefaciens.The results were as follows:（1）Through antibiotic sensitivity tests,the liquid working concentration of hph is 50μg/m L and cefo is 200μg/m L;The transformation system of A.tumefaciens mediated yeast-like cells of T.fuciformis is as follows:endogenous gpd promoter of T.fuciformis,A.tumefaciens strain EHA105,the liquid working concentration of yeast-like cells of T.fuciformis is 10⁶cfu/m L,the liquid working concentration OD₆₀₀of A.tumefaciens is 0.6,co-cultivation time is three days.（2）To extract total RNA of P.ostreatus and reverse transcribe to c DNA,cloning lac3 gene by designing primers according to the JGI gene library.Constructing the p GEH-lac3 vector of the heterogeneous expression of laccase gene,transformed into A.tumefaciens strain EHA105 by the electroporation method,integrated into the genome of T.fuciformis.The integration and expression of the laccase gene in the T.fuciformis cells were confirmed by PCR,Southern blot,fluorescence microscopy,quantitative real-time PCR and gualacol color plate cultered method.（3）Gainning a strain with high laccase activity which the value is 20.92 U/m L named No.65 through laccase detection with ABTS,and then optimized the fermentation condition like carbon source,nitrogen source and additive amount of Cu²⁺,the optimum results respectively were gulcose,peotone and 60μmol/L.