Construction And Identification Of Schizosaccharomyces Pombe Secretory Expression Vector Containing CaMV 35S Promoter

Millet wine crack Schizosaccharomyces budding yeast (pombe), is a kind of not only in reproductive and budding produce spores way divide and breeding single-celled eukaryotes, in recent years has been widely used as eukaryotic expression system. It has many and higher eukaryotes similar properties, make its in molecular biology research become a provide information, accurate eukaryotic model, has been successfully applied in cell restructuring, translation, RNA splicing, chromosome structure, mitosis and meiosis, the cell cycle research, is the study cell cycle regulation, chromosome structure, such as the signal transduction sex differentiation, one of the best experimental model has increasingly become the eukaryotic cell biology research and molecular biology of attractive experiment system.However millet wine budding yeast expression vector splitting pESP-2 own promoter Pnmt1 medium vitamin B1 concentration by strict regulation, when medium containing>0.5 VB1, Pnmt1 apms M by strong inhibition, cannot start the purpose gene normal expression. To make Pnmt1 normal exertion startup effect, getting people need exogenous purpose gene expression of the product, cultivating yeasts body must be used when the VB1 does not contain pure substance culture medium, and the medium price is very expensive, don't benefit in fermentation industry of applied to mass production. This paper selects 35S promoter, and its signal peptide with alpha factor pESP-2 connected together with much expression vector, constructing the clone sites containing from VB1 concentration control of crack corn wine promoter budding yeast expression vector secreted type.CaMV 35S as a strong promoter in the expression vector of plant in genetic engineering, GUS often be used to study in transgenic plants 35S promoter the expression distribution as the reports genes. The 35S promoter will not be affected VB1 concentration and we make it inserted into the pESP-2 plasmid, polyclonal sites for GUS gene, through 35S report of budding yeast in millet wine crack plasmid expressing activity in the reorganization of the determination of 35S can succeed, proof of pESP-2 itself replaced promoter.The research also to construct the secretion type, i.e. expression vector to 35S promoters will insert a period downstream signal peptide sequence. In order to validate concatenation signal peptide sequence can be millet wine crack which budding yeast correct identification, and make purpose gene expression, will successfully secretion signal peptide downstream and upstream insert cellulose enzyme terminate the son EGI genes.Because the reason, enzyme cut sites can not be 35S promoter, signal peptide sequence, EGI gene, NOS terminated in the millet wine son even budding yeast expression vector splitting pESP-2 plasmid, so must first building in turn contains the paragraphs gene sequences pCAMBIA1301-35S among carrier ofα-factor. EGI. NOS, then to intermediate carrier for template proposed plasmid, will 35S+α-factor+EGI+NOS as a whole, connected to pESP-2, and plasmid budding yeast corn wine crack transformation. Through the Congo red stain screened positive transformation, the child VB1 carry at the EMM containing liquid medium training, through to the training content qing for DNS cellulose enzyme activity assay, proof contain no medium VB1 concentration control by the strong promoter of budding yeast crack corn wine secreted type expression vector construct success.