Study On The Expression System Of Foreign Gene In Tremella Fuciformis Spores

T.fuciformis spores have the characteristics as a good expression host for heterologous gene.But the low expression level of heterologous gene limits the application of T.fuciformis spores as bioreactor.The study on the expression systems of foreign gene in T.fuciformis spores has not yet been reported home and abroad.A series of different expression vectors were constructed,and then transformed into T.fuciformis spores.High expression systems will be confirmed by detecting the expression level of heterologous gene.And the adjustment of membrance permeability of spores by different reagents were studied to optimize expression of heterologous genes in T.fuciformis spores.The results were as follows:1.Construction of eight different eukaryotic expression vectorsEight eukaryotic expression vectors were successfully constructed by PCR,restriction enzyme digestion and connections from plasmid pET-28a-rfp,pA0815-lacZ and pMD18-TAT-survivin(T34A).The vectors contained rfp gene,lacZ gene,Laccase gene and survivin(T34A)gene,were respectively named pTE11-rfp,pTE11-lacZ,pTE11-Laccase,pTE111-survivin(T34A),pTE11-co-survivin(T34A),pCAMBI A1301-survivin(T34A),pCAMBIA 1301-s-survivin(T34A)and pCAMBIA1301-co-survivin(T34A).The target genes were under the control of Magnaporthe grisea strong promoter RP27 in the expression vectors of pTE11-rfp,pTEll-lacZ,pTE11-Laccase,pTE11-survivin(T34A),pTE11-co-survivin(T34A).The signal peptide was added in front of survivin(T34A)gene in pCAMBIA1301-s-survivin(T34A)vector.The codons of survivin(T34A)genes were optimized in pCAMBIA 1301-co-survivin(T34A)under the control of the cauliflower mosaic virus CaMV 35S promoter.2.Transformation and expression analysis of expression vectorsThe Pichia pastoris expression vector pPICZa A-Laccase,pA0815-lacZ and the eight vectors were transformed into T.fuciformis spores and 41 transformants were screened by antibiotic resistance.The distinct red fluorescence can be observed from pTE11-rfp transformants by fluorescence microscope.It means that rfp gene was expressed in T.fuciformis spores.The pA0815-lacZ and pTE11-lacZ transformants were induced on the CM/Amp/IPTG/X-Gal plates and were showed in blue.The β-galactosidase activity were 0.40 U/L and 0.27 U/L respectively by ONPG method determination and proved LacZ gene were expressed in T.fuciformis spores,the laccase activities were 14.08 U/L and 7.55 U/L respectively,while the control group did not detect any activity.In conclusion,Yeast expression vectors had higher efficiency of exogenous gene in T.fuciformis spores comparing with the pTE11 vectors.3.Effects of different treatments on T.fuciformis spores cell membrane permeabilityThe cell membrane permeability of T.fuciformis spores was researched by adding organic solvents,surfactants,inorganic membrane surface effects of antibiotics and substances.The results showed that the cell membrane permeability was improved by DMSO,TritonX-100,Tween 80,Amphotericin B and Potassium chloride.But some severe surfactantsand organic solvents is not conducive to the synthesis and secretion of laccase in T.fuciformis spores,which may due to its strong role in the destruction of T.fuciformis spores cell,which lead to large number of deaths and affect the synthesis of Laccase.