Preliminary Study On The Function Of Rice OsVDAC4 With Its Interacting Proteins

The voltage-dependent anion channels(VDACs)are pore proteins widely located on the outer membrane of mitochondria and play an important role in the communication between mitochondria and other parts of cells.Our laboratory has identified eight OsVDACs from the rice genome,and screened two proteins OsV4IP1 and OsV4IP2 that can interact with OsVDAC4 by membrane protein yeast two hybrid technology.On this basis,this study preliminarily investigated the functions of OsVDAC4 and its interaction proteins OsV4IP1 and OsV4IP2.The main results are as follows:1.The pM999-OsVDAC4/OsV4IP1/OsV4IP2-GFP transient expression vector was constructed and transformed into the protoplasts of rice to observe the fluorescence signal.Using mitochondrial marker vector mt-rk CD3-991,nuclear marker vector p2300-35S-H2B-mCherry and mitochondrial dye Mito tracker,the results showed that OsVDAC4 and OsV4IP1 were located in mitochondria and cytoplasm,and OsV4IP2 was located in nucleus.2.Using the Gateway homologous recombination technology,the target genes OsVDAC4,OsV4IP1 and OsV4IP2 were cloned into pDONR207.The target gene was finally recombined into pSPYNE/pSPYCE through the specific recombination sites on the vector.The recombinant plasmid was transformed into Agrobacterium tumefaciens,and then injected into tobacco leaf epidermis.The fluorescence was observed by laser confocal microscope,and the interaction between OsVDAC4 and OsV4IP1/OsV4IP2 in living cells was verified.3.The expression levels of OsVDAC4,OsV4IP1 and OsV4IP2 in different tissues of three rice varieties Nip,YTB and YTA were detected by real-time fluorescence quantitative PCR,and their expression patterns were analyzed.The results showed that the expression pattern of OsVDAC4 in different tissues of YTB and YTA was approximate,which was quite different from that in Nip.There was the highest expression of OsVDAC4 in the pollen grain filling and maturation stage of three rice variety,this suggests that OsVDAC4 may be involved in regulating the pollen grain maturation process of rice.The expression patterns of OsV4IP1 in three rice varieties were similar.The expression in roots,stems and leaves at four leaf stage was the highest in leaves and the lowest in stems.The expression of OsV4IP1 was generally low in the four young panicle development stages,and there was little difference in the expression in different stages.OsV4IP2 was expressed differently in different growth and development stages of three rice varieties.Compared with Nip and YTB,the expression level of OsV4IP2 was significantly up-regulated at the pollen grain filling and maturation stage in YTA.It is speculated that OsV4IP2 may be involved in regulating the pollen grain maturation process of YTA,which may affect the fertility of rice.4.The pU1301-OsV4IP1/OsV4IP2 over-expression vector was constructed,and transformed into Nip mediated by Agrobacterium.The over-expression plants of OsV4IP1 and OsV4IP2(OsV4IP1-OE,OsV4IP2-OE)were obtained.Through positive identification and expression detection,3 OsV4IP1-OE plants and 19 OsV4IP2-OE plants with significantly up-regulated expression were obtained.These transgenic plants have harvested seeds,which can provide research materials for subsequent experiments.5.The seeds of OsVDAC4 CRISPR deletion mutant(osvdac4)and over-expression plant(OsVDAC4-OE)were propagated.Through positive identification and expression detection,14 over-expression plants with obvious up-regulation of OsVDAC4 were finally determined.Through the amplification and sequencing comparison of CRISPR editing site sequences,19 homozygous osvdac4 plants and 7 heterozygous osvdac4 plants were finally obtained.All mutant materials have harvested seeds.Comparing the grain size of OsVDAC4-OE,OsV4IP1-OE and OsV4IP2-OE plants,it was found that with the up regulation of their expression,the average single grain weight decreased,and the grain became smaller,and the higher the expression increased,the smaller the grain.It is speculated that OsVDAC4 and OsV4IP1/OsV4IP2 may be involved in the regulation of rice grain development.6.The results of abiotic stress showed that OsVDAC4-OE seedlings have high tolerance to salt stress,while osvdac4 seedlings are more sensitive to salt stress.It is speculated that OsVDAC4 may be involved in the positive regulation of salt tolerance in rice.Under mannitol simulated drought stress,OsVDAC4-OE seedlings were inhibited,while osvdac4 seedlings had better tolerance to drought stress,this indicated that OsVDAC4 may negatively regulate rice drought tolerance.These shows that the phenotypic differences of seedlings under salt stress may be not caused by cell osmotic effect,OsVDAC4 may alleviate the ion imbalance caused by salt stress by regulating the ion transport of rice tissue,and then participate in the regulation of salt tolerance of rice.OsVDAC4-OE seedlings were more sensitive to ABA,while osvdac4 seedlings had better resistance to ABA.It is speculated that OsVDAC4 may be involved in ABA signal transduction.