Interactor Screen And Gene RNA Interference Of OsVDAC5 In Rice

The voltage-dependent anion channels(VDAC) are known as mitochondrial porins in mitochondrial outer membrane. VDAC forms complexes with other mitochondrial membrane proteins to transport metabolites between cytoplasm and mitochondria. Eight putative VDAC genes(OsVDAC1-8) were previously identified in rice genome, while the functions of most of them are unknown. In this study, interaction-protein screening of OsVDAC5 was performed through membrane-based yeast two-hybrid in cDNA library from young panicle in rice YTB. Meanwhile, the vectors for over-expression and RNA interference of OsVDAC5 are constructed and introduced into rice for analysis of its function. The main results are as follows:1. Prediction of bioinformatics analyses showed that OsVDAC5 might contain twelve or eighteen transmembrane helices and its N-terminus and C-terminus in intra-mitochondria. Seven bait vectors with mutiple different fragments of OsVDAC5 were constructed for analysis of the transmembrane regions and their orientations by yeast split-ubiquitin system. The results showed that the bait vector pBT3-SUC-OsVDAC5 75 with N-terminus 75 amino acids could be utilized for subsequent interaction-protein screening of OsVDAC5. Seven proteins interacted with OsVDAC5 were then screened from young panicle cDNA library in rice YTB, and the reality of the interactions would be further validated.2. Specific cDNA fragment of OsVDAC5 was cloned into vector pMCG161 for RNA interference, and then introduced into rice Nipponbare mediated by Agrobacterium. The result of real-time PCR showed that the expression of OsVDAC5 was suspressed in most T0 plants.3. Two induced-expression vectors were constructed by cloning full-length OsVDAC5 gene in the downstream of two inducible promoters HVA1 and pGTPB, respectively, and transformed into Nipponbare mediated by Agrobacterium. However, we could not get transgenic plant because of failure of re-gerneration.