Regulation Of Nitric Oxide On Mitochondrial Phosphate Carrier And Mitochondrial Membrane Permeability Transition Pore

Membrane permeability transition pore(MPTP) plays a very important role on cell mitochondria-mediated apoptosis, it has a huge influence on mitochondria and cell. As an important part of MPTP, the role of mitochondrial phosphate carrier(PiC) in the mitochondrial permeability transition is not clear. Nitric oxide(NO) is a kind of small biologically active molecules. Existing studies of between NO and mitochondrial function are mainly concentrated in the NO and mitochondrial respiration, mitochondrial protein oxidative damage, and so on, but it is rarely reported on postharvest fruit cell mitochondria MPTP opening and NO regulation function of mitochondria MPTP. So conducting research on the effects of exogenous NO on PiC protein and MPTP is helpful to clear the role of PiC in mitochondrial permeability transition pore, and is of great significance to maintain good fruit quality and prolong fruit postharvest storage period.In this sthdy, primer was designed according to the other plants PiC gene on GenBank, the full length of Feicheng peach fruit PiC gene cDNA was cloned by touchdown PCR, rapid amplification of cDNA ends(race) method and nested PCR. The full length of gene was 1769 bp, total of 1116 bp open reading frame encoding 372 amino acids. The login number in GenBank is KP122948. Hydrophobic prediction of PiC as a hydrophobic protein; transmembrane prediction of PiC as a transmembrane protein. By amino acid sequence comparison, it is found that the amino acid sequence of peach fruit PiC protein was high similar with other plant PiC protein homologous; phylogenetic tree shows, Feicheng peach fruit PiC protein amino acid sequence with the apple, plum has the closest relationship. Introduced the plasmid pET-30a-PiC into Escherichia coli Transetta(DE3) to express the recombinant protein, the electrophoresis showed that the protein appeared in the 39 kDa band. Using nickel column to purifie recombinant protein and the recombinant protein was refolded by gradient dialysis method. Fluorescence quantitative PCR results showed that NO treatment significantly increased the expression of PiC gene during storage. NO protein can react with PiC, formation of 1:1 complexes. The SPR method showed that NO can make PiC protein S-nitrosylation. Artificial simulation of MPTP bilayers in vitro using phospholipid compounds, and its components were studied, when MO and DPPC ratio of the 1:1 concentration of 0.1 mg·m L-1 had the best effect. The yeast two hybrid experiments show that the reaction of PiC protein and CypD protein.