The Cloning And The Expression In Pichia.pasteoris Of Resveratrol Synthase Gene From Grape

Resveratrol, a kind of time livings to metabolize the outcome and a kind of plant endotoxin, has the anti- oxidize, anti- tumor, anti- blood platelets coagulates, keeps low density in human body LDP from oxidizing and increases muscle immunity dint and so on. Res is extensive in seed plant and it is low in plant, so it is difficulty to only use traditionally withdraw method to get plentiful Res. Research shows that resveratrol synthase is the main element to influence the content of Res. In this paper, we use the method of gene ungenerous to establish Pichia.pasteoris expression system.In order to establish the cloning vector of RS, I amplify the RS gene from grapvive leaf total DNA by PCR. Then it was inserted into pUC19 .The recombinant vector was verified with restriction analysis. The results showed that the RS gene was cloned correctly into pUC19. It's concluded that the vector was constructed successfully. Then the recombinant plasmid was turn into the E.coli to make the RS gene large amplified.The cloning vector was amplified by primer with kozak sequence, then RS gene was gotten after electorphoresis. The expression vector pPIC3.5K and RS gene were cutted with the same restrict enzyms. The RS gene and pPIC3.5K wre ligated together. The restriction analysis results showed that the RS gene was ligated correctly into pPIC3.5K.With the electricity dash the recombinant plasmid was switch to the Pichia.pasteoris strain GS115. The strain with recombinant plasmid was chosen by PCR. Then the strain express the RS in the culture medium using methyl alcoholas the carbolic rescource.