Study On The ARS Structure And Function In Yeast Kluyveromyces

Objective: On the basis of the random mutagenesis library of ARS, we wish to preparation by screening new ARS sequence which not only have higher ARS activity, but also display higher transformation frequency, in order to constructed new gene recombination vector which have higher efficiency in the Nonconventional yeast expression system. Methods: We got purified plasmid 19uA and then amplified the ARS sequence by PCR, by searching the already known sequence and then designing primers. The fragment ARS was got, and used it as template to amplified by PCR random mutagenesis, which created the random mutagenesis libraries of the ARS. The libraties were ligated to a shuttle vector, and the recombinant plasmids were amplified by being transformed into E.coli DH5α. The recombinant plasmids which contained mutations of ARS fragments were extracted and ARS activity was assessed by transforming strain K. fragilis strain HKY039, while transformations frequency was compared with that of vector plasmid free of mutagenesis ARS fragment. Then the yeast plasmids with high frequency were extracted , transformated into E.coli DH5α and sequenced. Then this plasmids were approved mutagenesis and transformated into K. fragilis for confirmed more relativity betweenmutagenesis and transformations efficiency.Results: Successfully constructed the random mutagenesis library forscreen ARS of Kluyveromyces yeast and the new ARS with higherefficiency were screened. Also the new Kluyveromyces vector withhigher efficiency were successfully builded.Conclusion: 1. This work first successfully constructed the randommutagenesis library for screen ARS of Kluyveromyces yeast; 2. The newARS of Kluyveromyces yeast with higher- efficiency were firstlyscreened; 3. It is first research on the relativity between theARSstructure and function;4. The first successfully building of newKluyveromyces vector which have higher efficiency.This study provided great significance to research of the relativity between the structure and function of ARS, DNA replication basic theory, and contributes to the building of new gene recombination vector which have higher efficiency in the Nonconventional yeast expression system.