The Study Of PAL Characterization And Cloning PAL Gene In Pyrus Bretschneideri Cv.

Pyrus bretschneideri cv.originates in Dangshan, Anhui Province, which is one of the pear products for main cultivating and exporting. However, due to poor management, species degradation and many other factors in recent years, the stone cell content is significantly increased in the pear. What's more, the flesh becomes thicker, more residue and taste variation than before, which severely decline the quality of Pyrus bretschneideri cv. and economic value. At the present time, reducing the stone cell content by utilizing biotechnology to improve the quality and enhance the market competitiveness of Pyrus bretschneideri cv. become an urgent question. In this experiment, we use Pyrus bretschneideri cv. fruit as research sample. We try to analyze the fluctuation of stone cell content, lignin content and the PAL activity, a critical enzyme to produce lignin, in the process of fruit development; and dissect the relationship between fruit stone cell development and PAL activity. In addition, in order to understand the nature of PAL, the characterization of the PAL was studied; and PAL gene was cloned at the same time. All of these provided a theoretical foundation for regulating the stone cell development from the level of physiological, biochemical and molecular biology. The main research results are as follows:1. Slices observation of five types of pear fruit about the shone cell group and instensive situation of structure, the order is: Pyrus bretschneideri cv., Fung pear, Korla fragrant pear, Gong pear and apple pear. Among these pears, Pyrus bretschneideri cv. showed the largest stone cell group and the most densely distributed, Gong pear and Fung pear were secondary, Korla fragrant pear show the smaller stone cell group and distribute comparably scattered, and Apple pear show the smallest stone cell group and the most scattered distributed.2. In the whole process of Pyrus bretschneideri cv. fruit development, stone cell content peaked at the stage of DPA51, the peak value is 17.83%; lignin content appeared two peak at the stage of DPA47 and DPA63, the peak value are 6.47% and 7.00% respectively; PAL activity peaked at the stage of DPA27, the value is 85.53U/g·h. The emergences of these three peaks showed some sequential relationship. PAL activity was first to reach the peak, then lignin content, the final one was stone cell content. Their trends were basically same, generally was first increased then declined. Besides, all of them showed significantly positive correlation.3. The most suitable pH for Pyrus bretschneideri cv. fruit PAL activity was 9.2. The PAL activity was comparably stable at around pH9.0. The PAL activity depressed remarkably both at pH4.0 and pH10.0 buffer due to the conformational destruction. The most suitable reaction temperature was about 60℃.The PAL activity and reaction time present linear relationship when the reaction time within 180min. In addition, PAL has a high specificity on L-phenylalanine, and also shows some activity on L-Tyrosine. The most suitable substrate concentration of PAL is 0.0004~0.0012 mol/L. Dangshan pear fruit PAL has two Km values, Km1 is 0.7500mmol/L and Km2 is 1.0690mmol/L.4. The PAL gene was cloned from the fruit of Pyrus bretschneideri cv.by RT-PCR. The sequence of the cDNA fragment was 582 base pairs and encoded a 195 amino acid protein. And it was found to have 90% homology with those of other plants PAL genes via multiple alignments. The catalytic active sites in PAL protein of other plants were also found in the deduced PAL protein. Phylogenetic tree analysis revealed that the PAL amino acid of the Pyrus bretschneideri cv. had closer relationship with the PAL amino acid of the Pyrunus avium. than other plants.