Cloning Of Pichia Guilliermondii Xylose Reductase Gene And Expression In The Pichia Pastoris GS115

Xylitol has many other physiological functions, such as the inhibition of dental caries, the adjuvant treatment of people with diabetes, liver protection. Its used in many areas such as food, medicine, chemical industry. There are many kinds of microorganisms with a natural xylose-fermenting, high xylitol yields may be difficult to obtain. Genetic engineering methods is used to transform the strain, building high expressing xylose reductase recombinant strain,This can improve the yield of xylitol. In this study, we used the means of genetic engineering, the genome of pichia guilliermondii is as a template, the XYL gene is cloned from the pichia guilliermondii. The XYL gene of pichia guilliermondii was transformed into Pichia pastoris by electroporation, constructed the recombinant strain expressing xylose reductase, this secreted and expressed of xylose reductase excessively, this Laid the foundation for later yielding xylitol by fermentation. The results were as follows:1. The genome of pichia guilliermondii that was screened by our lab was as a template, xylose reductase(XYL) gene was amplified by PCR. The result of the1%of agarose gel electrophoresis showed the amplified fragment was approximately1kb. The sequencing results showed the length of the amplification srip by PCR was1127bp, and the length of the target gene is954bp. Comparising the xylose reductase gene sequences with other mongolia xylose reductase gene sequences of Pichia guilliermondii published in GenBank. we found The homology of XYL gene sequence are respectively92%and96%.2. When the exact sequence of xylose reductase gene of pichia guilliermondii was obtained, after comprehensive analysis of the expression vector restriction site map, EcoRl and Xbal were added into the5’-end of the primers, the primers contain enzyme sites were synthesized again. The PCR amplification product was connected with T vector, then transferred to E. coliDH5α, obtained the Anti-AMP transformants, the transformants had T vector that contain XYL, named T-XYL, The length of T-XYL is about3600bp.3. The xylose reductase gene was connected with pPlCZaA after they were both digested by the same enzymes. Then transferred to E. coliDH5α, constructed yeast expression plasmid containing the xylose reductase pPlCZaA-XYL, It was linearized by restriction endonuclease SacI, the recombinant plasmid pPICZaA-XYL was transformed into Pichia pastoris GS115by electroporation.And the Xylose reductase gene from pichia guilliermondii was integrated into the genome expression host Pichia pastoris GS115,obtained the recombinants that has zeocin resistance.4. The recombinant Pichia pastoris GS115containing xylose reductase gene from pichia guilliermondii was induced by methanol, Containing xylose reductase gene of Pichia guilliermondii was as a control group I.Containing empty plasmid pPlCZaA Pichia pastoris GS115was as control group2, The results displayed when being induced for96h, the supernatant secreted high expression levels of protein, only obtained a kind of protein that the molecular weight is about36KDa, The supernatant of the control group1did not contain any protain stripe, the sample inthe cellof the control group2contained one protain strip that the molecular weight is almost as same as the size of the target protein, but the protein Concentration was far less than the recombinant strain Pichia pastoris GS115-XYL’s. Because the supernatant barely contained hybrid protein that the host strain Pichia pastoris GS115secreted, therefore we can be preliminarily determined that recombinant strain of Pichia pastoris GS115-XYL were induced by methanol after96h, the Xylose reductase was highly expressed.