Function Of Laccase Gene In Morphogenesis Of Ganoderma Tsugae

Laccase is a kind of Cu-contained polyphenol oxidase,it has many functions such as degrading lignin,which provides sufficient carbon source for the growth of edible fungi,promoting respiration,improving the quality of the fruit body and restraining hetero fungus growth,so it is an indispensable enzyme in the growth and morphogenesis of edible fungus.Now the studies of laccase were concentrated on enzymatic properties and industrial application,there were quite few literatures about laccase gene.In this study the protoplast monokaryon technique was used to obtain mononuclear strains.The 5'-end and the 3'-end non-coding sequences of laccase gene from Ganoderma tsugae were obtained through the PCR technique.The laccase gene knockout cassette was constructed by the Split Marker strategy and was transformed into the protoplast to obtain the knockout strains.The functions of the laccase gene in the process of growth and development of hyphae and fruit body were carried out by determination of physiological and biochemical indexes in knockout strains and original strains and the fruiting test.Main results were as follows:1 protoplast monokaryon of Ganoderma tsugae and determination of the incompatible factorsIn this study,17 protoplast regenerating mononuclear strains were obtained by protoplast monokaryon technique and the monokaryon rate was 19.50%.Among them,four mononuclear strains of different mating type was determined:the mating type of A1 was A1B?1-?1,the mating type of A5 and A7 was A2B?2-?2,the mating type of A8 was A2B?1-?2,the mating type of A9 and A15 was A1B?2-?2.2 Selection of laccase gene knockout testing strains and detection of hygromycin resistanceThe results of determination of laccase activity in mononuclear strains and dinuclear strains showed that laccase activity in A1,A9 and A19 were high and were respectively 91.0 U/L,76.3 U/L and 64.5 U/L.Combined results of the growth rate and fruiting test,A1,A9 and A19 were selected as laccase gene knockout testing strains.The results of detection of hygromycin resistance in A1,A9 and A19 showed that the growth of A1,A9 and A19 were restrained when hygromycin concentration reached 200 ?g/mL.The plasmid pCB 1003 was transformed to A1 and A19,the transformants were screened by PCR,and two transformants A163 and A1947 were obtained to prove the hygromycin resistance gene could be expressed in Ganoderma tsugae.3 Knockout of laccase gene and identification of transformantsThe 1541 bp 5'-end non-coding sequence of laccase gene was obtained through the SEFA-PCR technique and the 900 bp 3'-end non-coding sequences of laccase gene was obtained through the improved TAIL-PCR technique.The intact laccase gene knockout cassette L1HL-L3HR and the partial laccase gene knockout cassette L3HL-L3HR were constructed by the Split Marker strategy and were transformed into the protoplast of A1 and A9 by PEG.The transformants were screened by PCR,three mononuclear knockout strains ?Lac3-A1,?Lac3-A9 and ?Lac-A9 were obtained,and then they were used to acquire two dinuclear strains ALac3-A19 and ?Lac-A19.4 Functional verification of laccase gene in morphogenesis of Ganoderma tsugaeThe results of determination of growth rate and laccase activity showed that the growth rate and laccase activity in knockout strains were decreased obviously.Scanning electron microscope(SEM)results showed that the hyphae of knockout strains were dry and thin,the surface was rough and had a lot of branch.Around the knockout strains colonies were hackly,the hyphae grew in medium tightly and weren't separated easily.The fruiting test results showed that knockout strains primodium formation need 9-10 d,the color of fruit body was light and lackluster,the stipe was short and the biology efficiency was low.The biology efficiency of A1,A9 and A19 were respectively 11.37%,12.11%and 17.98%,the biology efficiency of ?Lac3-A1,? Lac3-A9,? Lac-A9,? Lac3-A19 and ? Lac-A19 were respectively 2.18%,4.87%,4.86%,2.77%and 3.96%.These results showed that laccase gene was integrallty or partly knocked out,the effects were same,and laccase gene influenced the growth and morphogenesis of mycelium and fruiting body in Ganoderma tsugae.