Gene Cloning, Expression And Construction Targeting Vector Of Protein Kinase From Thermophilic Fungi

All living cells must constantly be interfered by all kinds of factors during their life cycle of proliferation, differentiation,development and death. These factors include not only the intercellular hormones but also extracellular light, temperature and water so on. All these request the cell to recognize and receive these signal factors correctly and respond to them as soon as possible to control the courses of progression and cellular activities. In this way, the receivers lie in the membrane of the cell recognizing the external ligand. Then the external signals are transducted into the nucleolus to regulate the gene expression and enzyme activity leading to the cell response. We can conclude that signal transduction plays an important role in the processes of environment cell interaction.Thermomyces lanuginosus is a widely distributed soil-inhibiting fungus which can live normally at about 50. A lot of studies have resulted that kinases and phosphatases are likely to be important mediators of signal transduction. Through phosphrylation and dephosphorylation, the activity and structure of protein changed. External signal are transducted into the cell nucleolus by the signal cascade. Protein kinase mediated phosphorylation regulates protein function, membrane channels and pumps and transcription factors by which changed the gene expression. Indian scientist Ray found in 1994 that three membrane proteins they isolated from Pseudomonas syringae which were phosphorylated in response to temperature changes and resulted that one of these proteins was possibly a histidine kinase.In this study, we cloned the thermophilic fungal protein kinase gene by using the RT-PCR. Structure the pPIC9K/pk and pPIC3.5K/pk expression vector and transformed into Pichia pastoris GS115. We selected the extracellular transformers and the intracellular expression strains, named PS-PK-16 and PS-PK-18. we can no detected the expression producst by SDS-PAGE after fermentation and we think that the detection sensitivity is not enough.The analysis of engineering strain PS-PK-18's fermentation products show that there is a were significantly different protein controled the matched group, the result of the mass spectrometric analysis tell us that it is a ATPase. Plasma membrane ATPase was found to made some contribution to the maintenance of lower membrane permeability of cells under ethanol stress. We conjecture that the over intracellular expression of protein kinase impact the ATPase's excretion. To make the yeast maintain normal growth in the fermentation process and adapt to adverse external environment.We analysis the differential expression of original strains and yeast strains using the FQ-PCR. The highest expression is the first day and it is about five times controled the other six days. This may be in the early stages of growth, the cell's metabolism and is very exuberant and lots of enzymes play an important role in this stage, so requir a large number of protein kinases to regulate the relevant reaction.The th-pk1 gene-disruption vetor were constructed based on the gene homologous combination theory. The gene-disruption vetor were pUCATPB/PK, and Bleomycin resistance gene as the screening label. The th-pk1 gene targeting vector was constructed by using of the homologous fragment was 670 bp and 731 bp. The constrction of the targeting vector is a veryimportent material for the reaserch of the gene's function.