The Functional Analysis Of DsPLC From Dunaliella Salina

Dunaliella salina is one of the most salt tolerant eukaryotes so far, but the molecular mechanism and the signal pathway of tolerence salt stress is not clear yet. Phospholipase C can hydrolyze phosphatidyl inositol phosphate into two important second messenger. The phosphatidyl inositol signal pathway take part in many cellular signal transduction andphospholipase C is a key enzyme. So it has important scientific significance to study the biological characteristics of the phospholipase C. The methods and results are as follows:1. The open reading frame sequence of DsPLC was amplified by RT-PCR technique, and the prokaryotic expression vector pGS21a-DsPLC was constructed. Then the recombinant plasmid was introduced into the E. coli BL21(DE3) competent cells, and the fusion proteinwas expressed successfully. 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 result showed that there was a single protein band about 96 KD, indicating that the fusion protein was purified effectively. The western blot result showed that the obvious hybridizing bands was about 96 KD, and it proved that the purified protein was thephospholipase C with His tag.2. The open reading frame sequence of DsPLC was connected with the expression vector pMDCG which has green fluorescent protein gene, and structured theubcellular loalization recombinant vector pMDCG-DsPLC successfully.The pMDCG-DsPLC was transformed into Agrobacterium GV3101 competent cell byfreeze-thaw method, the positive monoclonal was transfected in onion epidermal cell by Agrobacterium tumefaciens-mediated method. The target gene in onion epidermal cells expressed successfully, so we could observed that the phospholipase C was mainly expressed in intracellular membranes systems by fluorescence microscope. 3. The bait expression vector was connected with the open reading frame of the DsPLCand yeast two-hybrid expression vector pGBKT7-BD was constructed, the bait vectorpGBKT7-DsPLC was transformed into the yeast Y2 H Gold by heat shock method. The yeast Y2 H Gold had a hybridization experiment with yeast Y187 from cDNA library, and analysised by bioinformatics/information biology to make sure the biological function of interactions protein.