Mutagenesis, Screening Of Auxotroph In Tremella Fuciformis And Cloning, Expression Of The Related Gene

In this study,high-energy pulsed ultraviolet was used to induce mutation on the yeast-like-conidia(YLC) of Tremella fuciformis.This is a new way for the induce mutation of edible fungi.Myo-inositol-1-phosphate synthase(MIPS) gene is the key enzyme in synthesizing inositol.MIPS gene was cloned from T.fuciformis prototrophic stain by Molecular biology technique and expressed in E.coli BL21(DE3) sequentially.Simultaneously,enhanced green fluorescent protein(EGFP) gene expression vector of T.fuciformis was constructed and transformed to T.fuciformis by electroporation.And then the transformants were verified.Finally,the MIPS gene expression vector of T.fuciformis was constructed from the vector pCB-BEGFP and transformed to T.fuciformis AU811 strain by electroporation.These results were following.1.High-energy pulsed ultraviolet mutation on the YLC of T.fuciformisInduced effects on the spores of T.fuciformis by traditional ultraviolet mutation and high-energy pulsed ultraviolet mutation were compared.70 auxotrophic strains from traditional ultraviolet mutation were obtained,but the mutants were not steady.90 auxotrophic strains were obtained from high-energy pulsed ultraviolet mutation and one was steady auxotroph.It was inositol-defect and pyridoxine-defect mutant by identification.Lag phase of growth of Tau813 was four days more than wild-type strains.2.Cloning and expression of the cDNA of MIPS gene from T.fuciformisThe full sequence of MIPS cDNA from T.fuciformis was cloned and sequenced by molecular biology technique as TAIL-PCR、3’-RACE.Primers were designed, and the MIPS gene was amplified by RT-PCR from the total RNA.The cDNA full length of 1695 bp encodes a protein of 564 amino acids.The cDNA fragment was inserted into expression vector pET28a(+) and the resulting plasmid was expressed in E.coli BL21(DE3) by IPTG induction.The results of SDS PAGE analysis indicated that the MIPS gene from T.fuciformis was expressed.The expression level raised along with the time.3.Construction of EGFP gene expression vector of T.fuciformisThe DNA fragment including Agaricus bisparus GPD promoter,Tnos terminator and EGFP gene was excised from plasmid pEGFP with HindⅢand EcoRⅠenzymes and then was inserted into plasmid pCAMBIA1300.The recombinant plasmid was named as pCB-BEGFP.The expression vector pCB-BEGFP with EGFP gene was transformed to T.fuciformis by electroporation.Many transformants were gained and verified by PCR of EGFP gene and Tnos sequence.The results demonstrated that the EGFP gene had been integrated into the genome of transformants.The transformants emited green fluorescence under the fluorescence microscope induced by blue-ray.It showed that the EGFP gene was expressed in T.fuciformis.4.Transformation of MIPS gene to T.fuciformis AU811 strainThe MIPS gene was inserted into plasmid pCB-BEGFP instead of the EGFP gene.The recombinant plasmid was named as pCB-MIPS.The expression vector pCB-MIPS with MIPS gene was transformed to the inositol-defect strain of T. fuciformis by electroporation.Many transformants were gained and verified by PCR of 35S promoter,hpt gene and Tnos sequence.The results demonstrated that the MIPS gene had been integrated into the genome of transformants.The growth situation of the transformants on the minimal medium was tested.The result indicates that the inositol-defect strains didn’t recover to prototroph.