Function Analysis Of OsVDAC3 And Screening Of Its Interacrion Protein In Rice

Voltage dependent anion channel(VDAC) proteins are mitochondrial outer membrane pore proteins and have many important physiological functions. Our laboratory have found 8 osvdac genes in the genome of Japonica rice, but the functions of most of them are unknown.In this study, to focus on fuctions of osvdac3, we constructed a osvdac3 RNAi vector, and obtained positive transgenic plants; and the previously transgenic positive over-expression(OE) families with pCAMBIA1302-osvdac3 were treated with abiotic stress; in addition, we constructed bait vectors for membrane yeast-two hybrid and screened the cDNA library to select the proteins which are interacted with OsVDAC3. The main results were as follows:1. RNAi primers were designed according to ORF of osvdac3 and the target fragments was cloned into the dual vector pMCG161 to construct the pMCG161-osvdac3 RNA interference vector, and transformed it into japonica rice mediated by agrobacterium. The results showed that 6 positive RNAi plants were obtained by detection with primers PMCG3 F / 3R and PMCG2 F / 2R. RT-PCR results showed that the osvdac3 gene expression in 4 transgenic positive families had reduced 20-50% compared with the negative ones.2. The expression level of osvdac3 in the T1 seedlings with OE- osvdac3 was confirmed by RT-PCR. And the positive T1 families were treated with NaCl and mannitol. The results showed that over-expression of osvdac3 could increase the length of root in NaCl, as well as seedling and root in mannitol. It is suggested that osvdac3 could increase tolerance to salt and drought stress.3. Two bait vectors pBT3-N-osvdac3 ORF and pBT3-SUC- osvdac3 ORF? were constructed. The result from the membrane yeast two-hybrid analysis showed that the N-terminal of OsVDAC3 was in the cytoplasm while the C-terminal was out. So pBT3-N-osvdac3 ORF can be used as the bait vector. Through the 3-AT titration experiment the culture medium was optimized and the optimum 3-AT concentration was 7.5 mmol/L. By screening the dualmembrane yeast two-hybrid cDNA library and Colony-lift Filter Assay, 3 potential proteins interacted with OsVDAC3 were obtained. They are DNA-binding protein S1 FA, signal recognition particle receptor and ribulose bisphosphate carboxylase large chain precursor.