| During the biodegradation of lignin,lignin peroxidase,manganese-dependent peroxidase and laccase are 3 main kinds of lignin degrading enzymes,among these microbial which can produce these enzymes,white rot fungus is the main microorganism,and mushroom is one of the important groups.Mushroom under their own secretion of extracellular enzyme degrades macromolecular substances in the matrix into small molecule and translates them into their own nutrition for growth.Besides,the type and activity of its extracellular enzyme are closely related to the decomposition and utilization of nutrients.Therefore,to a certain extent the activity of these enzymes determines hyphae's biological conversion rate of medium.Research on the Agaricus bisporus casing fruiting Mechanism shows that agaricus bisporus has the synthesis of methionine by ethylene through ACC,and ACC oxidase(ACO)is a key enzyme in the ethylene biosynthetic pathway,which directly catalyzes the shift from ACC to ethylene.Preliminary study focuses on the role of ACO gene on growth of sporocarp.The study results are as follows:1.Build P.ostreatus laccase poxc gene segments for homologous recombination.Firstly,use gpd promoter homologous to swapping laccase gene poxc promote of P.ostreatusr.Then utilize e.coli hygromycin B resistance gene as marker gene,build P.ostreatusr laccase poxc gene segments for homologous recombination.2.Use tender P.ostreatusr mycelium as experimental material and liposomesTM2000 as the carrier of the transformation,transport the homologous recombination of P.ostreatusr laccase poxc gene fragments into hypha cells.By doing this we can obtain a series of transformation in hygromycin RCM medium(80?g/m L).By hygromycin selection and molecular methods and on PDA plate containing the substrate ABTS color validation,and under the condition of different training methods such as enzyme activity,RNA expression quantity verification,finally we can get a group of normal expression of laccase enzyme activity to transformation.3.The cultivation test found out that in spawn runing phase,the transformation strain of hyphae fulfill the bag about 2 days faster than starting strains;After fulfilling the bag,hyphae was induced via 16 ? ? low temperature for 2 days,proportion of conversion of strain retort stand-up pouches into primordium is about 50%-75%,and that of the original strain is 30%.After the ?mushroom cultivation material 2 stubble with fann(Van Soest)washing cotton seed shellis fiber analysis test cultivation of cellulose,hemicellulose,and lignin content in the material.Results for genetically modified strains of oyster mushroom cultivation material for 29.3%,content of cellulose in the hemicellulose content is about 22.45%,and the content of lignin was 16.61%;Starting strains cultivated material was 32.0%,content of cellulose in the content of hemicellulose was 26.9%,the content of lignin was 17.86%.Transgenic mushroom strains increased to 30% of the cotton seed shellis for effective utilization of the above,the yield increased by more than 20%.4.We designed a pair of PCR-specific primers according to the conserved sequence of the Gene Bank fungi ACO gene,took the extracted total RNA from the bisporus As2796 mycelium as a template,obtained a product of 630 bp in size by RT-PCR,based on the obtained ACO sequence cloned and got what was used to construct a hairpin structure forward and reverse sequences.The forward and reverse sequences were connected to bisporus GPD promoter and T35 S terminator together,respectively.The hairpin structure was built on this basis,which was connected to the plasmid p BHg expressing of RNAi-hairpin structure to build the vector the PBHG – ds ACO.5.Took bisporus young gill tissue as experimental materials and liposomes TM 2000 the carrier of the transformation,transferred the ACO gene RNAi fragments to bisporus mycelium cells,through hygromycin and molecular means to detect transformants,we found that as the co-culture time of the liposome-DNA increases gill germination rate is getting higher and higher.The best co-culture time for liposome-mediated transformation is 100 min.In all of the 22 randomly selected transformants,the exogenous hygromycin resistance hph gene and hairpin RNA(hp RNA)could be detected.6.After culturing the transformed strain of bisporus agaricus for 20 days in PDA liquid medium,the amount of ethylene and ACC synthetized in bisporus cultivation were measured by using gas chromatography and ion chromatography.The amount of ethylene is 6.9?l / L and 4.13?l / L,respectively,and they have a significant difference(p <0.05).Then add the ACC solution,giving a final concentration of 5 mmol / L,after incubated,determining the enzyme activity(nmol/mg·h).The enzymatic assays results show that starting strain and genetically modified strains of ACC synthase activity(nmol / mg·h)are 20.43 and 4.97,respectively,and they have a significant difference(p <0.05). |
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