Cloning And Expression Analysis Of ACC Synthase Gene Of The Whangkeumbae Pear

Whangkeumbae pear (Pyrus pyrifolia‘Whangkeumbae’) is one of the main cultivars in China, which is a variety of Pyrus pyrifolia Nakai. It is known for excellent flavor of the tender, juicy, sweet and crisp. However, it is difficult to be storaged and transported. Whatever in room temperature or low temperature during storage, it is easy for fruits to have water loss, fruits decay and core browing. It will cause many problems such as losing the value of goods because of these things. So if the factors of impacting the mature of fruits can be controlled during the mature period, it is benefit of improing the storability, and extending the shelf life of pears.In this paper, the fruit of different periods of Pyrus pyrifolia‘Whangkeumbae’were collected as the trial materials, getting the sequence of ACC synthase gene through cloning and analyzeing it. Using fluorescence real-time quantitative PCR, expression quantity of the gene in the different process of fruit development was detected for further researching the ethylene synthesis process from the molecular level. It is useful to regulate and control the ethylene synthesis process better. Above all the purpose is to extend the storage and shelf life. The main findings are as follows:1. According to the characteristics of the high level of polyphenol, polysaccharides and other secondary metabolities of Pyrus pyrifolia‘Whangkeumbae’, three methods were used to find out which was the best for extracting totai RNA. They are modified 3% CTAB method, TAKARA plant tissue method and TIANGEN RNAplant plus Reagent method. The results show that TIANGEN RNAplant plus Reagent method is better than others, which can effectively remove the influence of polyphenol, polysaccharides and other secondary metabolities. The electrophoresis of RNA was showed less degradation and higher purity.2. According to the amino acid sequence of Pyrus pyrifolia‘Zaoshengxinshui’, a pair of primers was designed to obtaine the sequence in Pyrus pyrifolia‘Whangkeumbae’. The analysis showed that the sequence was 99% homology to that of Pyrus pyrifolia and Pyrus bretschneider and more than 85% homology to that of other rosaceous plant.3. Real-time quantitative fluorescence PCR detection showed that ACS gene in fruit growth and development, before fruit ripening the expression level was increased slowly until the day of 150 after flowering. During that period the expression level was increased rapidly and reached the highest, after that it was followed by a sharp decline.