Subcellular Localization Of Four OsVDAC Proteins And The Effects On Rice Growth By Over-expression Osvdac3

Voltage-dependent anion channel (VDAC) widely exists in from fungi to theplant and animal organisms, and depends on the voltage to adjust its selectivity of ionof the anion and cation. Research shows that the VDAC is encoded by a small genefamily, not only exists in the mitochondrial outer membrane, but also exists in othercellular components. Generally plants have more VDAC isomers than mammals.Our lab identified eight rice vdac genes by bioinformatics and expressionanalysis. Compared with three vdac genes in mammalian, we know little about theirbiological functions, subcellular localization and higher structure of eight vdac genesin rice.In this study, four vdac genes, including osvdac3and osvdac5, which have ahigher expression level during the period of development of the rice pollen, andosvdac1and osvdac4were selected. the subcellular localization of the four vdac geneswere investigated. In addition, the effects of overexpression osvdac3on the growth oftransgenic rice plants were also investigated. The main results are as follows:1. The ORFs of vdac3and vdac5, obtained from the cloned plasmids ofpET-30a-osvdac3and pET-30a-osvdac5by PCR with the specific primers, werecloned into pCAMBIA1302-rfp vector to contruct CaMV35S∷osvdac3∷rfp andCaMV35S∷osvdac5∷rfp, respectively. By Agrobacterium-mediated transformation,the positive transgenic YTB plants with CaMV35S∷osvdac3∷rfp were obtained.RT-PCR analysis comfirmed that the expression level of osvdac3in positivetransgenic plants was higher than that in both YTB and the negative transgenic plants.According to phenotypic observation, the height of T0transgenic positive plants waslower and the pollen fertility declined, compared with the negative transgenic plantsas well as YTB. Moreover, it was also observed that there were lower height andshorter root in positive T1plants than those in both negative T1and YTB plants asthey were treated with NaCl and Mannitol.2. The four ORFs of osvdac1, osvdac3, osvdac4, osvdac5, and the C-terminaland N-terminal sequences of both osvdac3and osvdac5were obtained from the plasmid of pET-30a-osvdac3and pET-30a-osvdac5and cDNA of YTB by PCR withthe specific primers, and then cloned into the transient expression vector pM999-gfp,respectively. Transient expressions in the rice protoplast showed that OsVDAC1protein pesented in the cytoplasm, OsVDAC4in both cytoplasm and mitochondria,OsVDAC3in all parts of cell except the nucleus and mitochondria; both C-terminaland N-terminal of OsVDAC3located in the cytoplasm and plasma membrane,N-terminal of OsVDAC5presented in the cytoplasm and plasma membrane; furthermore, OsVDAC5proteins were distributed around the nucleus as the crimpled shape,indicating it may be in the ER.3. OsVADC3and OsVADC5were expressed in E.coli by using the constructedprokaryotic expression vector pET-30a-osvdac3and pET-30a-osvdac5And therenatured proteins were preparated by the method of urea gradient treatment ofinclusion body protein. CD analysis revealed that there exist the dominant β secondstrcuture in OsVDAC3and none in OsVDAC5.