Molecular Cloning And Expression Analysis Of Lipoxygenase Gene From Whangkeumbae

China, where is the country of origin of pear, has the most different varieties pears in the world. Whangkeumbae is the new variety of Pyrus pyrifolia‘Nakai’in Japan and South Korea. This kind of pear is named Whangkeumbae because its rounded fruit and golden yellow peel. Whangkeumbae is known for excellent flavor of the tender, juicy, sweet and crisp,Therefore, Whangkeumbae is very popular in domestic and foreign markets, and the sell price is higher than other kinds of pears in the market of Southeast Asia and Europe currently. Whereas, the shelf life of Whangkeumbae fruit was very short, it easy to lose water and rot, and thus lose the value of the goods. Lipoxygenase (EC 1·13·11·1,LOX) is a kind of enzyme, which is relative with the ripening and senescence of fruits. In some certain stress conditions, LOX and its metabolites can replace the ACO and catalyze ACC to generation ethylene.It was one of the key factors of fruit ripening, softening, senescence. In this study, the Whangkeumbae pear LOX gene fragment was made the cloning and sequence analysis, and the activities of LOX were determinated in the different development period of fruit. Expression quantity of the gene in the different process of fruit development was researched by using fluorescence real-time quantitative PCR. It is aimed to provide the theoretical basis for the study of fruit ripening and senescence mechanism by physiological and molecular biology means, and provide a new approach for the effective regulation of fruit ripening and senescence. further more, Thus lay a solid theoretical foundation and technical support for the further studies of LOX gene in the pear mature and aging mechanism and genetic engineering breeding of resistant storage. The main results in this study are as follows:1. Gene merger primers was derived from Nakai LOX gene(GenBank Accession No. EF215448.1) in GenBank. High purity RNA was extracted from Whangkeumbae pear and used as a template for cloning the Lipoxygenase gene cDNA by RT-PCR. A cDNA fragment which contains about 570bp was cloned.2. Sequence alignment and amino acid analysis on the Whangkeumbae pear LOX gene sequences by using application software showed that the homology of the gene shared 99% identity with Nakai (GenBank Accession No. EF215448.1)、apricot(GenBank Accession No.EU439430.1)�'�peach(GenBank Accession No.EF568783.1), which proved that the cloned cDNA fragment in this experiment was the LOX gene in Whangkeumbae pear fruit, encoding 193 amino acid residues.3. The test in the activity of lipoxygenase showed that the activity of LOX during fruit development in general exhibited a increased trend, indicating that the enzyme may be associated with the cell division and fruit swollen reaction during fruit development.4. Q-PCR detection showed that LOX gene in fruit growth and development, maturation and aging process were expressed in the fruit of growth, mature and aging process were expressed, indicating that the gene was involved in fruit growth development, ripening and senescence process.