Subcellular Localization Of Four OsVDAC Proteins

Voltage-dependent anion channel(VDAC) lies in mitochondrial outer membrane,which is the channel of small molecular substances(such as ATP, ADP, DNA, tRNA)to move in and out of mitochondria. VDACs in plasma membrane, sarcoplasmic reticulum and chloroplast, etc are also observed.In the previous study, our laboratory identified 8 VDAC in rice genome using bioinformatic analysis and expression confirmation. In this study, four VDAC genes,including Os VDAC2, OsVDAC3, OsVDAC5 and OsVDAC7 were selected to research the subcellular localization by methods of protoplast transformation and particle bombardment transformation. The main results are as follows:1. The ORFs of OsVDAC2 and OsVDAC7 were amplified from Nipponbare cDNA using the specific primers, and then cloned into the transient expression vector pM999-GFP. It was showed that pM999-OsVDAC2-GFP and pM999-OsVDAC7-GFP fusion expression vectors were successfully constructed.2. Optimal observation time for transient expression in the rice protoplast was investigated by former constructed fusion expression vector pM999-OsVDAC3-GFP and pM999-OsVDAC5-GFP. Subcellular localization of OsVDAC3 and OsVDAC5 was conducted by transforming expression vectors with full-length, C-terminal and N-terminal sequences of OsVDAC3 and OsVDAC5, respectively, and with different cell organelle markers in the rice protoplast system. The results showed that the optimal observation time was 16 h after transformation in rice protoplast, while protein aggregation was observed after 20 h. OsVDAC3 and OsVDAC5 were localized in the nucleus and cell membrane, respectively, but the locating signals of those OsVDACs were unable to determine. OsVDAC2 and OsVDAC7 were localized in the cytoplasm using the same method.3. Different expression vectors were transformed into onion epiderm by particle bombardment, and were used for subcellular localization by the optimal conditions. It was showed that the highest transformation efficiency of particle bombardment could be obtained at the plasmid concentration of 1 ?g/?L, distance positioning of 6 cm and the pressure of 1100 psi. OsVDAC3 in the nucleus and the cytoplasm membrane, as well as in the vesicles was observed, and its signal peptide might lie at the N-terminal.Meamwhile, OsVDAC5 in the nucleus, the cytoplasm membrane and the Golgi vesicles was observed, and its signal peptide might lie at the C-terminal.