Preliminary Analysis Of The Subcellular Localization And Function Of OsVDAC3 And Its Interaction Protein In Rice

Mitochondria is the center of cell energy metabolism and death regulation.Voltage-dependent anion channels?VDACs?are a class of highly conserved proteins found on the outer membranes of all eukaryotic mitochondria.In our laboratory,8 kinds of VDAC protein OsVDAC1⁸ were previously identified in rice,and 3 OsVDAC3 interacted proteins,OsV3IP1³,were screened.In this experiment,the subcellular localization and function of OsVDAC3 and OsV3IP1³ were further studied,and the results are as follows:1.Bioinformatics and qRT-PCR were used to analyze the OsVDAC3 and OsV3IP1-3 expression patterns of young spikes in different developmental stages,roots,leaf sheaths and leaves of the fourth leaf stage in YTA,YTB and NIP.The results showed that the expression patterns of OsV3IP2 and OsV3IP3 were similar in YTB and NIP,but were significantly different in YTA,and OsVDAC3 was expressed at a very high level in 1.5-4.0 cm young spike of YTB.It is suggested that the differential expressions of those genes between NIP?a regular variety?,YTB?male fertile line?and YTA?male sterile line?might be in relations to their differential male fertility,which is worth studying further with these isonuclear allocytoplasmic lines of YTB and YTA.2.The pM999-OsVDAC3/OsV3IP1³/N/C-GFP transient expression vectors were constructed,and transformed into rice protoplasts,and the fluorescence was observed with laser scanning confocal microscope.The results showed that OsVDAC3-N,OsVDAC3,OsV3IP1-C,OsV3IP1 were located in mitochondria;OsVDAC3-C,OsV3IP1-N,OsV3IP2-N,OsV3IP2-C1,OsV3IP2-C2,OsV3IP2,OsV3IP3-N,OsV3IP3-C and OsV3IP3 were located in the cytoplasm.The signal peptide of OsVDAC3 is located at the N-terminus,OsV3IP1 is located at the C-terminus,and there are no signal peptide in OsV3IP2 and OsV3IP3.These results laid the theoretical foundation for the future study of the structure and function of OsVDAC3.3.The results of BiFC showed that there were interaction between OsVDAC3with OsV3IP3,and OsV3IP3-N-terminal domains,while no interaction between OsVDAC3 with OsV3IP3-C-terminal domain.This indicated that OsVDAC3interacts with OsV3IP3 through OsV3IP3-N-terminal domain.4.The positive plants of T₀ OE-OsV3IP1²,T₀ and T₁ CRISPR edited-OsVDAC3 and-OsV3IP2 were obtained through genotype identification.There was a larger difference in the fertility and setting rate within T₀ OE-OsV3IP1²transgenic plants,but no distribution mode could be found.The fertility of positive T₀CRISPR edited-OsVDAC3 and-OsV3IP2 plants were significantly lower than the negative plants.The pollen fertility of T₁ CRISPR edited-OsVDAC3 and-OsV3IP2was significantly lower than that of T₀,and the number of seeds was also significantly lower than that of T₀.Whether the function of OsVDAC3 and OsV3IP2 was related to the fertility of rice needs to be further demonstrated.