Study On Highly Efficient Expression System About Exogenous Gene In The Yeast-like Conidium Cells Of T·fuciformis And Its Application

The basidiospore of Tremella fuciformis(T·fuciformis)is the yeast-like conidia reproduced by its basidiospore,the yeast-like conidium cells of T·fuciformis breed by budding under conditions of the absence of xianghui strains,and it is fast reproducing and easy to culture by submerged fermentation,so it is a good bioreactor for exogenous gene expression.Studies showed that there were multiple genes which could be expressed in the yeast-like conidium cells of T·fuciformis.However,their expression levels have been reported to be low and lack of safe and effective selection markers.Therefor,the expression system was not perfect yet.At present,we had successfully proved that rfp gene can be expressed in the yeast-like conidium cells of T·fuciformis,but the HIV-TAT-survivin(T34A)mutant gene(TAT-survivin(T34A))has not been expressed successfully.To improve those problems,in this study,we used the PNP that transformed by the yeast expression vector pPICZ A as an expression vector and lacZ gene as a reporter gene to constract foreign gene expression vectors of the yeast-like conidia which with different promoters.The expression capacity of promoters were compared by the level of ?-galactosidase enzyme activity.Then we constructed a fusion expression vector containing the genes rfp and HIV-TAT-survivin(T34A)mutant gene and transformed the yeast-like conidia of T·fuci-formis to express the HIV-TAT-survivin(T34A)mutant.1.Studies on the selection of exogenous Genes Construction and selection marker and transformation methodIn this study,we compared the transformation effectiveness of pCAMBIA 1301 and PNP expression vector as well as screening effect of hygromycin b and zeocinTM.The results showed that,the spores were sensitive to zeocinTM,and the lowest sensitive concentration was 25?g·mL-1,and the PNP expression plasmid was suitable for foreign gene expression in the yeast-like conidia of T·fuciformis.We chose PNP,which has no exogenous gene expression promoter and transformed by the yeast expression vector pPICZ A.And the TEF1-EM7(the promoter of zeocin resistance gene on PNP plasmid)was amplified successfully as well as lacZ gene.The expression vector PNP-lacZ without exogenous gene expression promoter and the pPZ-lacZ expression plasmid with the promoter of TEF1-EM7 promoter were successfully constructed,and then transformed into yeast-like conidia.We got a certain amount of positive clones successfully.The lacZ gene showed normal expression in some clone of the conidia which transferred by Recombinant Plasmid pPZ-lacZ.Nevertheless,the lacZ gene couldn't express in any clone of the conidia which was transferred by Recombinant Plasmid PNP-lacZ.These results demonstrated that the PNP vector could be used to express foreign gene in yeast-like conidia of T·fuciformis,where there's no promoter in lacZ gene.And The expression system which using PNP as an expression vector and lacZ gene as a reporter gene can be used for screening of exogenous gene expression promoter.2.Construction of exogenous genes construction containing different promoters and lacZ geneWe successfully obtained seven promoter with PCR amplification and restriction enzyme digestion.The seven promoters as follows:CaMV35s,CaMV35s2,gpd ?,RP27,gpd-Tf,AOX I and GAP.Construction of expression vectors contained the above promoter and lacZ gene using PNP,and designated as p35s-lacZ,p35s2-lacZ,pgpd ?-lacZ,pRP27-lacZ,pgTf-lacZ,pAOX ?-lacZ and pGAP-lacZ.After,those plasmids were identified by PCR,and confirmed by double restriction enzyme digestion and sequencing.3.Transformation and expression verification of lacZ gene in yeast-like conidia of T·fuciformisTransferred those plasmid constructed in the chapter 2 into the yeast-like conidia of T·fuciformis by electroporation,all transformants were obtained by zeocinTM resistance screening,and positive trans-formants were identified by the phenotypes of inactivaton of lacZ gene,then PCR identify the expression promoter,target gene as well as resistant gene promoter of the transforments.The result showed that all plasmids have been successfully transformed with a conversion rate of 700/?g plasmid DNA.The colour reaction result showed that lacz gene expressed normally in the conodia which transformed by plasmid p35s-lacZ,pRP27-lacZ,pAOX ?-lacZ and pgpd ?-lacZ.And 17,23,8 and 3 transformants were obtained respectively.The expression rate was only 0.3%.The expression of lacz gene of the transformant cotaining pgpd ?-lacZ plasmid is slow and the expression level is low.The P-galactosidase activity of three carrier transformants of p35s-lacZ,pRP27-lacZ and pAOX ?-lacZ was determined by ONPG colorimetric method,in order to compare the expression of exogenous genes in yeast-like conidia between the three promoters of CaMV35s,RP27,AOX ?(Because the expression of the transformant of the pgpd ?-lacz carrier was extremely low and slow,so it was not included in the comparison).After comparison,it was found that the expression effect of the two promoters RP27?AOX ? was obviously stronger than that of CaMV35s promoter.There was no significant difference in the expression betweenthe the two promoters,RP27 and AOX ?.AOX ? promoter was slightly stronger than RP27 promoter.4.contruction and expression of TAT-survivin(T34A)gene with rfp geneThe fusion gene of rfp and TAT-survivn(T34A)gene was designed and respectively cloned into expression vectors PNP-RP27 and PNP-AOX ?,and recombinant fusion vectors of pRP27-RS and pAOX ??-RS were constructed and transformed into yeast-like conidia of Tfuciformis by electroporation.Conidia transfected with pRP27-RS?pAOX ?-RS vector can be observed with red fluorescence.And PCR experiment showed the objective bands.A interesting band about 44 KDa was visible in the result of SDS-PAGE,which was consistent with expected size of recombinate protein.The results showed that,fusion gene was integrated into yeast-like conidia of T·fuciformis genome and was expressed successfully.