The Functions Of Laccase On The Mycelium Growth And Fruiting Body Development Of Basidiomycete Hypsizigus Marmoreus

Hypsizigus marmoreus(Peck.)Bigelow(Tricholomataceae),also called bunashimeji and hon-shimeji,is an edible and medicinal fungi.H.marmoreus has become more popular in Japan and China due to its mildly sweet nutty flavor and crunchy texture,and probably the existence of some physiologically beneficial components.H.marmoreus is a drupolar heterothallism basidiomycete and it requires a long culture time(105 d)to allow the fruit body maturation,which is 65 d and 45 d longer than Flammulina velutipes and Pleurotus eryngii respectively,and the efficiency of industrial production is relatively low.The reason for the long culture stage of H.marmoreus is that this mushroom requires a tong spawn-running process(45 d)to allow mycelial maturation,and now the physiological effect of mycelial maturation is still unclear.Because of the lack of genetic background operation method and genomics information,the progress on molecular biology of H.marmoreus is slow.Therefore,this study screening the differential expression genes from four developmental stages of H.marmoreus which are related with the fruiting body development by transcriptome technology,and decided that laccase gene family as the object of our study;Then,we study the functions of laccase in the development of H.marmoreus by adding the inhibitor of laccase;Furthermore,we constructs the Agrobacterium tumefaciens mediated genetic transformation system in H.marmoreus.Finally,we over-express laccase gene lccl to study the functions of laccase gene family in order to shorten the developmental stages of H.marmoreus.1.Screening the differential expression genes related with fruiting body development:In order to study the functions of some crucial genes in the development of H.marmoreus,we firstly sequenced four developmental stages of H.marmoreus by transcriptome technology.After transcriptome sequencing,we screened the differential expression genes by comparing two samples.During these differential expression genes,there are 14 laccase isozyme genes which are named from lcc1 to lcc14.The expression model of this 14 laccase isozyme genes is different from each other during the developmental stages,indicating that laccase gene family might play important roles in the developmental stages of H.marmoreus.Therefore,we decided that laccase gene family as the object to study the fruiting boy development of H.marmoreus.2.The functions of laccase gene family in the fruiting body of H.marmoreus:In this study,we firstly chose the specially inhibitor of laccase Kojic acid(KA).By adding KA to the medium to study the relation between laccase with mycelial growth,primordium initiation and fruit body maturation.Adding the laccase inhibitor can inhibited the laccase activity and mycelial growth markedly,indicating that laccase have relationship with mycelial growth of H.marmoreus.Besides,adding the laccase inhibitor not only can stimulate primordium initiation,but also can induce fruit body maturation.While the mycelium culture time is 60d and the concentration of KA is 10 mM,the output of fruit bodies can be significantly increased;the single bottle production of 60 d is equal with the production of 85 d.From these results,we can indicate that the adding of laccase inhibitor can shorten mycelial culture time 25 days.Besides,we detected the transcription levels of laccase genes family on the effect of KA at the stages of mycelial knot,mycelial pigmentation and primordium,and found that the transcription model of most laccase genes was changed differently.Fromthese results,we know that KA affected the transcription model of laccase gene family and laccase activity to induce the mycelial growth,primordium initiation,fruiting body maturation and shorten mycelial culture time of H.marmoreus.3.The construction the A.tumefaciens mediated genetic transformation system in H.marmoreus:In this study,we constructed the A.tumefaciens mediated genetic transformation system in H.marmoreus,and realized laccase gene over-expression dependent on this system.First,we obtain a bunch of transformants by co-cultured of A.tumefaciens LBA4404 strain and and fungus protoplasts.We then proved that the foreign gene has been successfullyintegrated into the genome of H.marmoreus by PCR amplification and Southern Blot analysis.We optimized this transformation method by adjusting the parameters of the co-culture.Weobtained150 transformants per 105 protoplasts by using A.tumefaciensEHA105,adding 0.25 mM acetosyringone,culturedat 26?for 24 hours based on the bacterium protoplast ratio of 1000:1.Besides,our research applied four fungal promoters and compared the influence to transformation efficiency.The result showed that the plasmid contain a cassettein which gene hph is under the control of promoter of Hm-gpd to get higher transformation efficiency than by using the promoters from Ganoderma lucidum and Lentinus edodes.Our result demonstrated that endogenesis promoter could significantly improve the transformation efficiency.The construction of this system will contribute to study genes functions in H.marmoreus,which will provide technical support for studying the functions of laccase gene family in H.marmoreus.4.Furthermore,we applied over-expression technology in our research in order to study the function of one laccase gene lcc1 in the development of H.marmoreus.We created Hm-lcc1 over-expression vector,and transformed it into H.marmoreus by ATMT.We examined the expression of Hm-lccl gene at the mRNA level in randomly picked five transformants,by using Real-time PCR analysis.The result showed that,the expression level of Hm-lcc1 gene in OE-Lcc1-1 wasover 3 times than that in wild-type strain.And the other four OE-Lcc1,the expression levels of Hm-lcc1 were over 2.0 times than the wild-type strain.To better understand the influence to phenotypic character byover-expression of Hm-lcc1,we detected laccase activity and mycelial biomass.The result showed that the laccase activity and mycelial biomass were increased significantly.In addition,we cultivated the transformants and found that the mycelial growth speed of transformants were quicker and primordium initiation earlier than the wild-type strain.From these results,we found that over-expressing the laccase gene lccl can increase the laccase activity,promote the mycelial growth and induce primordium initiation of H.marmoreus.In conclusions,This study firstly constructed the A.tumefaciens mediated genetic transformation system in H.marmoreus,and provided the technical support for studying the genes functions in the fruit body development of H.marmoreus;By studying the functions of laccase gene family in the development of H.marmoreus,we found that increasing laccase activity could promote mycelial growth,induce primordium initiation and shorten mycelia physiological maturity time.