Proteomic Analysis Of Up-accumulated Proteins During Autumn Olive Fruit (Elaeagnus Umbellata Thunb.) Ripening And Cloning Of IPI Gene

Autumn olive (Elaeagnus umbellata Thunb) fruit is a rich source of many human health-related nutrients, such as amino acids, minerals, vitamins, essential fatty acids, flavonoids, lycopene and other substances. It has a great development potential of natural antioxidants and nutritional supplements. The sweet-tart fruit can be eaten fresh, or used as a substitute for tomato products. In addition, the extracts can be used as anti-cancer or other drugs. This nutritional fruit may have a broad prospect in healthcare food and clinical application.Autumn olive as a wild plant is not yet widely cultivated, and little is known about the development and ripening processes of the fruit. In this case, the fruit protein extraction method and 2-DE electrophoresis conditions with high-resolution and good reproducibility were optimized. This study was carried out by using differential proteomic analysis combined with the organic compound determination during autumn olive fruit ripening. In addition, the full-length IPI was also cloned. All of these studies will help to understand the fruit ripening mechanism. The results are as follows:1. High resolution and good reproducibility of 2-DE maps could be obtained by the phenol extraction method, appropriately extending the equilibration time and timely replacing the equilibration buffer. The improved procedures significantly decreased interference of rich pigments, polyphenols and their oxides on the stability and clearness of 2-DE gels, which may be helpful for the downstream manipulation. This approach was suitable for proteomic analysis of fruits.2. Fruit ripening is highly coordinated, genetically programmed, and an irreversible phenomenon involving a complex series of physiological and biochemical events, which generally include conversion of starch to sugar, accumulation of carotenoid and organic acid metabolism. In the full-ripe autumn olive fruit, glucose and fructose were the principal sugars, and malic acid was the most abundant organic acid besides that lycopene concentration was extremely high. The proteomic analysis was used to identify up-accumulated proteins induced by the ripening. The expression of three selected proteins was confirmed by Western blot. The 63 up-accumulated protein spots were composed of 42 known proteins, one unknown protein and 20 unidentified ones. Thirty-eight proteins of known function were involved in sugar metabolism, citric acid cycle, isoprenoid metabolism, oxidative stress, defense response, fatty acid synthesis, and protein synthesis and fate. The possible roles of these induced proteins in the above physiological processes and autumn olive fruit quality were discussed.3. The full-length cDNA sequence of IPI was shown to be 1185 bp long and contained an ORF of 705 bp encoding a polypeptide of 235 amino acids. IPI identified in this case was strongly up-regulated over three-fold during the autumn olive fruit ripening. This suggested that IPI could be a key enzyme that made lycopene accumulation in the full-ripe fruit.