Expression And Functional Analysis Of Ds64PHR In Pichia Pastoris

UV radiation (UV-B, UV-C) can induced serious damage in organism. The major damage on the nucleic acid level is DNA biologic oxidation damage: formation of pyrimidine dimmers. This kind of dimmers is lethal to organism. But the organism has evolved many systems to this damage, and photoreact-ivation that can reapair this damage through (6-4) photolyase is a very important repair mode.Dunaliella salina is a unicellular eukaryotic organism (alga), it not only resist high density salt, but also be able to bear strong light. So the (6-4) photolyase (Ds64PHR) is cloned from Dunaliella salina by our colleague, so we design experiments to research if (6-4) photolyase from Dunaliella salina can highly expressed in Pichia pastoris and if the engineering strains can resist stronger UV than wild type strains. The main research contents in this experiment are:1. The pulse voltage, the harvest period that be used for preparing competent cells, the density of transformed plasmid DNA, carrier DNA of used or not, and the restoring of transformed mixture which can affect the transformation efficiencies of Pichia pastoris are studied. We found that the highest transfer m-ation efficiency can be obtained when the pulse voltage is 1500V/cm, the cell density is OD600=1.3, and the total amounts of plasmid DNA is from 500ng to 1ug , and the density of plasmid DNA is 100ng/ uL. Besides, when the carrier DNA is added, and after pulsed, the transformed mixture is restored some times (from 1 hour to 4 hours) before spread onto the YPD/G418 plates, the transformation efficiencies increase to some extent.2. The Ds64PHR cDNA is inserted in the constitutive type expression vector pPGAPZB purchased from Invitrogen, this construct is called pGAPZB-Ds64PHR, and the molecular biological test is performed.3. The construct pGAPZB-Ds64PHR is transformed into Pichia pastor is and integrated into the genome DNA of Pichia pastoris. As above, the molecular biological test is also performed.4. The expression ability of engineering strain GS115-PHR is analysed through SDS-PAGE, and the relationship between the level of resistant to UV with the extent of expression heterogous proteins (Ds64PHR protein) of GS115-Ds64PHR is also studied, we found that the relationship is not fixed.