Effects Of NO On Expression Of The ETR1 Protein Of Peach Fruit In Prokaryotic System And Oxidative Damage To Mitochondrial Membrane In Harvested Plum Fruit

This paper contains two parts. In the first part, an expression vector was constructed and the optimal conditions for the expression of the recombinant ETR1 protein were determined. In another part of the paper, the effects of NO on oxidative damage to mitochondrial membrane in harvested plum fruit were studied.A ripening related ethylene receptor ETR1 gene in'Feicheng'peach fruits was cloned and the recombination protein of ETR1 was obtained for the further researches on the functions of ETR1 and improving the storage and transport quality of'Feicheng'peach fruits. Using cDNA reverse-transcribed from total RNA in mature'Feicheng'peach fruits as template, the specific PCR product of ETR1 was obtained, and then it was ligated to pEASY-T1 vector. The sequencing data showed that the PCR product was about 2500 bp. After digested by BamHⅠand NdeⅠ, the sequence was ligated to pET15b and transformed to E coli C43(DE3). Recombinant ETR1 was induced by IPTG, and the expression of ETR1 was optimized. Comparison with the cDNA, sequence of AF124527 indicated the homology was 99%,and amino acid identity was 99%.The proper conditions for recombinant ETR1 protein were that the mutant host strains E. coli was C43(DE3). The final concentration of IPTG was 0.5 mol L^-1. The proper temperature was 30 oC and the induction time was 8 h. Recombinant ETR1 was highly expressed under the optimized conditions, which was helpful for studying the functions of E- in vitro. The inhibition by NO on the expression of ETR1 depended on the dose of NO.The second part aimed to study the effects of nitric oxide (NO) on oxidative damage by reactive oxygen species (ROS) to mitochondrial membrane in harvested plum fruit (Prunus salicina Lindl.). The plum fruits were fumigated with 0, 10, 15 and 30μL·L^-1 NO gas and then stored for 13 days at 25 oC. The physiological and biochemical properties, including respiration, contents of hydroperoxide and superoxide radical, activities of superoxidase (SOD), catalase (CAT) and peroxidase (POD), the opening of mitochondrial permeability transition pores, electric potential and cytochrome c/a were determined. Compared with the control and treatment with 10 or 30μL·L^-1 NO, treatment with 15μL·L^-1 NO increased the activities of SOD, CAT and POD, decreased effectively the contents of hydroperoxides and superoxide radical, inhibited the opening of mitochondrial permeability transition pores, delayed the increase of mitochondrial permeability and Cyt c/a, maintained high electric potential. With high concentration of NO, the protection by treatment with 30μL·L^-1 NO reduced. Application of 15μL·L^-1 NO can effectively alleviate oxidative damage by ROS to mitochondrial membrane in harvested plum fruits; protect the integrity of mitochondrial membrane.