The Study On The ACC Oxidase Genes Clone Of Pear Fruit

The genus Pyrus, a member of Pomoideae of Rosaceae, is one kind of important economic fruit tree in China, which has a longtime history to be cultivated. There is a difficulty problem in pear manufacture, which is how to stove the fruit for much longer. It had been proved that the ethylene play a major maturative role in fruit ripening by more and more investigations. Based on the studies of ACC oxidase which is the key enzyme in ethylene biosynthesizing, this research is to clone the ACC oxidase genes of pear fruits and analyze the gene structures. This work will do great help to made a solid foundation for which the research of building anti-sense expression vector of ACC oxidase genes in genus Pyrus.Main results of this research are as follows:1. It'd been built a method, which was suit to the extraction of the total RNA of Whangkeumbae (Pyrus pyrifolia Nakai.), Xuehua (Pyrus bretschneideri Rehd.) and Clapp's Favorite (Pyrus communis Linn.), that was improved CTAB method. On the basis of making comparison as to the effect of extracting total RNA by four methods, that were 1. Hot borate method, 2. Guanidine isoisothiocyanate method, 3. CTAB-LiCl method, 4. Improved CTAB method, due to high levels of polyphenol content and polysaccharides in genus Pyrus fruit tissues, the special stage of getting rid of polyphenol content and polysaccharides was added so as to improve the conventional CTAB method in isolating RNA. The results showed that the improved CTAB method was better than others.2. Obtained the cDNA fragments of ACC oxidase genes in pear fruits. Chose the fruits of Whangkeumbae (Pyrus pyrifolia Nakai.), Xuehua (Pyrus bretschneideri Rehd.) and Clapp's Favorite (Pyrus communis Linn.) to be materials, extracted and purified the total RNA, then the ds-cDNA was synthesed by reverse transciptase. The double degenerate primiers were designed by the homologous sequence of the ACC oxidase genes. Using PCR, three genes fragments of three pears had been cloned.3. Compared the homology analysis of the three pears with the other pears ACC oxidase genes from Gene Bank, and developed bioinformatics analysis, the homology were all above 98%. The homology analysis result had proved that the three genes fragments had been gotten were ACC oxidase genes.