Preliminary Study On Tremella Fuciformis Bioreactor

Tremella fuciformis was used as material for researches as follows:selection and determination of T.fuciformis strains for transformation;optimization of electroporation parameters;construction of expression vectors pCB-BEGFP with Agaricus bisparus gpd promoter and detection expression of egfp gene;cloning of gpd promoter of T.fueiformis and study its function using green fluorescent protein gene;construction of expression vectors pCB-TBCA400 with gpd T.fuciformis promoter and transformation.The results were following.1 Selection of T.fuciformis recipient strainsFourteen strains of T.fuciformis were analyzed with two molecular markers ISSR and RAPD.The fourteen T.fuciformis strains were divided into five clusters.Cluster V included 9 strains which had no differences at DNA level.The result showed some strains were the same strains with different names.Five different strains were selected based on dendrogram of cluster.Growing speed and production yield of every strain were determined.The results showed growing speed and production yield of the strain T0006 was best, strains T0001 and T0026 followed.T0006 had resistance to kanamycin and hygromycin,but T0001 was sensitive to hygromycin.The strain T0001 was used to recipient for transformation.2 Construction of high efficiency Strain T0001 electroporation transformation systemStrain T0001 was used to recipient for transformation.The optimum parameters for electroporation were studied.The results showed yeast-like-conidia of strain T0001 was needed pretreatment with 2%lywallzyme for 1h.The optimal conditions were voltage 300 V,electric resistance200Ω,capacitance 25μF.In addition PEG4000 was added to electroporation buffer; transformation efficiency can be improved and had 9.25×10~6 transformants perμg plasmid DNA. The concentration of PEG4000 was 7%.3 Construction of T.fuciformis expressed vectors and preliminary study on expression of egfp gene under control of promoter from Agaricus bisporusT.fuciformis expressed vectors pCB-BEGFP was constructed with Agaricus bisparus gpd promoter,Tnos terminator and egfp gene.The expression vector pCB-BEGFP with egfp gene was transformed to strain T0001 by electroporation.These transformants were 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 emitted green fluorescence under the fluorescence microscope induced by blue-ray.SDS-PAGE analysis showed that the molecular weight of expressed EGFP protein in YLCs was identical to predicted EGFP protein.These showed that the enhanced green fluorescent protein gene was expressed in strain T0001.The promoter of A. bisparus gpd promoter was sufficient to confer EGFP gene transcript.4 Cloning and analysis of gpd gene and its promoterglyceraldehyde-3-phosphate dehydrogenase cDNA was cloned by RT-PCR with total RNA of strain T0001,using a pair of degeneracy primers.The length of cDNA was 1008 bp and encoded protein contained 336 amino acid residues.Multiple sequence alignment revealed high similarities of the amino acid sequences and exhibited 81.52%of identity to other glyceraldehyde-3-phosphate dehydrogenases of nine fungi in GeneBank.A 500bp promoter region from strain T0001 genomic DNA was isolated by thermal asymmetric interlaced PCR(Tail-PCR).Sequencing analysis showed that the cloned fragment only contained TATAAA motif located upstream of transcription start site 'A'.Many motifs of conventional eukaryotic promoters were found in this promoter sequence.Expression vector TGP1301 was constructed with strain T0001 gpd promoter and was transformed into strain T0001 YLCs by electroporation.Five transformants were selected at random for determination GUS activity.Four transformants changed to light blue.The function of T.fuciformis promoter was preliminarily identified.5 Studies on the activity of promoter of strain T0001 using green fluorescent protein geneThe cloned promoter was fused to 5'-upstream of enhanced green fluorescent protein gene to construct T.fuciformis expression vector pCB-TEGFP.Electroporation was performed to transfer plasmid DNA of pCB-TEGFP into yeast-like conidia of strain T0001.PCR analysis indicated egfp gene had been integrated into the genome of transformants.By fluorescence microscopy,distinct green fluorescence could be observed from the colonies.Maximal excitation wavelengths(Ex)of two transformants were 492 nm and 486 nm respectively.Two transformants had same maximal emission wavelengths(Em)of 510 nm.SDS-PAGE analysis showed that the molecular weight of expressed EGFP protein in YLCs was identical to that of the positive control.The molecular weight of EGFP protein is predicted to be ca.27 kDa.As expected,a dark band of the protein was visualized at 27 kDa.The results indicated that egfp was expressed in YLCs of strain T0001.This promoter can be used to carry out regulated expression of heterologous gene products in strain T0001.6 Transformation of human insulin gene to strain T0001 and its molecular evidencesExpression vector pCB-TBCA400 with human insulin gene was transformed into strain T0001 by electroporation and gained many antibiotic resistant transformants.These transformants contained human insulin gene,gus gene by PCR determination.Expression of human insulin gene will be researched in future.