Study On Characteristics And Function Of Laccase Gene Of Ganoderma Tsugae

Laccase(benzenediol:oxygen oxidoreductases,EC 1.10.3.2)are blue multicopper oxidases.Laccases have been ascribed diverse biological functions in different organisms.In fungi,laccases have been assigned a variety of physiological roles in their life cycle,such as lignin degradation,detoxification of phenolic compounds inhibitory to fungal growth and lignocellulolytic enzyme activity,and sporophore development.However,only a few of these functions have been experimentally proven.The laccase were separated and purified by ammonium sulfate fractional precipitation,dialysis and DEAE sepharose anion exchange chromatography,and then the characteristics of laccase were determinated.The function of laccase gene were be explored by determination of physiological and biochemical indexes of gene knockout,complementation and over-expression strains during mycelial growth and fruit body development.The results were as follows.1 Purification and characterization of laccase of Ganoderma tsugae_The laccase from Ganoderma tsugae we purified only have one isoenzyme which molecular weight is about 52 kDa.The optimum reaction temperature of the laccase we purified is 40? and it is very stable at the temperature of 4?,at this temperature its enzymatic activity almost has no lost even being hold 12 hours.When the temperature become higher,the laccase become less stable.The optimum reaction pH value of it is 4 and its stability is about 4.However,when the pH value is 3 the laccase may lose its activity easily.As concentration of different metal ions was 0.05mmol/L,K+ and Fe3+ had the most effective actiavation on laccase while Fe2+ can inhibit enzyme activity completely.2 Construction of laccase gene complementation and overexpression strains of Ganoderma tsugaeIn this study,the pTE11-laccase vector was constructed which contained cDNA sequence of the laccase gene from G.tsugae.The recombinant plasmid was transformed into the laccase gene knockout strain ?Lac-A 19 and the original strain Gpsl to construct laccase gene complementation strain ? Lac-A 19/pTE 11-laccase and overexpression strain Gpsl/pTE11-laccase.By the same time the plasmid pPICZa-laccase which contained the Cerrena unicolor laccase gene was transformated into ?Lac-A19 and Gpsl to construct laccase gene complementation strain ? Lac-A19/pPICZ?-laccase and overexpression strain Gps1/PICZa-laccase.The expression quantity of laccase gene from high to low were respectively Gps1/pPICZ?-laccase,Gps1/pTE11-laccase,? Lac-A19/pPICZ?-laccase,Gps1,?Lac-A19/pTE11-laccase,?Lac-A19 at transcriptional level by qRT-PCR detection.The laccase activities of the strains Gps1/pPICZ?-laccase,Gps1/pTE11-laccase,?Lac-A19/pPICZ?-laccasep,Gps1 had high activity,but the laccase activity of the strain ?Lac-A19 could not determinated.3 Effect of Laccase gene on growth and development G.tsugaeThe results of mycelial grow rate and characters observation of above strains showed that the growth rate of the strains with high laccase activity was fast and the mycelia were very thick and density.The growth rate of strain ?Lac-A 19 was slow and the mycelia were very sparse and brittle.The mycelia of the strains with high laccase activity had lots of small new branches by SEM.The results of G.tsugae cultivation showed the strains with high laccase activity had high lignin degradation ability,fruit body yield,biology efficiency and good quality.But strain ?Lac-A19 could hardly degrade the lignin of the cultivation material and had a little fruit body yield and bad quality.These results showed that laccase ge ne played an important function with the mycelial growth and morphogenesis in G.tsugae.