Cloning And Expression Analysis Of Protein Kinase C Gene From Dunaliella Salina

Dunaliella salina is a unicellular eukaryotic green alga which can grow in hypersaline environments. And it is an important model organism to reveal thesalt resistance physiological and molecular mechanism. Salt-tolerant genes and proteins were studied by many scholars and some progress had been made. But the resistance of salt algae is related to a series of genes and proteins, the molecular mechanism is very complicated. So far, the salt-tolerant of salt alga’s molecular mechanism and signal pathway is not clear yet.Many substrates, such as the key enzymes of metabolic pathways, ion channels and transcription factors, can be phosphorylated by PKC. It plays an important role in regulation of cell cycle, growth, differentiation and gene expression. So it has important scientific significance to explore the function of PKC and clarify the molecular mechanism of salt stress. Experimental methods and results of this study are as follows:1. DsPKC was amplified by PCR, the prokaryotic expression vector pET32a(+)-PKC and eukaryotic expression vector pMDCG-PKC and pRI 101-PKC were respectively constructed successfully. The open reading frame of DsPKC was sequenced rightly.2. The prokaryotic expression vector pET32a(+)-PKC was introduced into E. coli. BL21(DE3), and the fusion protein was expressed successfully by IPTG induction. The fusion protein were presented as the supernatant proteins and inclusion body proteins. It was purified by His column and detected by SDS-PAGE. The high purity of fusion protein were obtained from the soluble protein. Western blot results showed that the purified protein was protein kinase C with His tag. Activity analysis results showed that the protein kinase C had no dependence of protein kinase Ca2+ and diglyceride cofactor, so it was proved that it was atypical PKC subtypes.3. The eukaryotic expression vector pMDCG-PKC was introduced into Agrobacterium GV3101 competent cells, onion epidermal cells were impregnated through the method of agrobacterium, the DsPKC was successfully expressed in onion epidermal cells. The PKC was expressed in the cell membrane and cytoplasm by fluorescence microscope. The PKC was transfered from cytoplasm to the cell membrane under 0.5 M NaCl.4. The eukaryotic expression vector pRI 101-PKC was introduced into agrobacterium GV3101 by the freezed and thawied, using leaf disc with Agrobcterium infection of tobacco leaves, transgenic plants were obtained. The agrobacterium was transfected in tobacco leaf by leaf discs and the transgenic plants was obtained. The tobacco leaf total DNA as a template for PCR, the 2000 bp specific amplification bands was obtained. It preliminary proved that PKC was transferred into tobacco.