Verification For The Interaction Between OsVDAC5 And OsV5IPs In Rice

The voltage-dependent anion channels(VDACs)are known as mitochondrial porins and highly conserved in structure,which play an important role in the process of energy metabolism,cell apoptosis,material transport and signal transduction.Eight putative VDAC genes(the functions of most of them are unknown)were previously identified in rice genome.Also,seven interaction-proteins(OsV5IP1-7)of Os VDAC5 were screened from young panicle cDNA library in rice YTB through membranebased yeast two-hybrid and would be verified through yeast two-hybrid experiment,GST pull-down experiment and bi-molecular fluorescence complementation(Bi FC)experiment.The main results are as follows:1.The full-length cDNA sequence of the seven genes were cloned and linked with pPR3-N vector.These plasmids were co-transformated into yeast strain NMY51 together with pBT3-SUC-OsVDAC5(1-75aa)plasmid and then growthed on SD-2 plate.The monoclonal was selected from SD-2 solid culture medium and cultured in YPAD liquid medium.When the OD546 was 0.6-0.8,bacteria liquid was diluted 10-1 times,10-2 times,10-3 times.2.5 μL culture solution was on the SD-2,SD-4,SD-4(3-AT)solid culture medium by micropipette tips and then placed at 30 ℃for 2-3 d.The results showed that there are interaction between the seven OsV5 IP and OsVDAC5(1-75aa).2.According to the analysis of protein sequences,amino acid sequences of OsV5IP1 and OsV5IP2 were divided into two parts respectively,namely as the OsV5IP1(1-239aa),OsV5IP1(240-364aa),OsV5IP2(1-90aa)and OsV5IP2(91-210aa).cDNA sequence of these four genes were cloned and linked with pET-32 a vector.Then,experiments of protein expression in vitro and pull down were performed.The results showed that there are interaction between OsV5IP1(1-239aa)and OsVDAC5(1-75aa),but there are not interaction between OsV5IP2 and OsVDAC5(1-75aa)in vitro.3.The full-length cDNA sequence of OsVDAC5(1-75aa),OsV5IP1 and OsV5IP2 genes were cloned into pSPYC(N)E vector by Gateway homologous recombination technology.The recombinant plasmids were transformed into agrobacteriuman GV1301-90 G and then infected into N.Benthamiana leaves.The results observed by laser scanning confocal microscopy showed that there are interaction between OsV5IP1,OsV5IP2 and OsVDAC5(1-75aa).