Study Of POD Characterization And Cloning POD During Stone Cell Development Of Pyrus Bretschneideri

As one of Pyrus bretshneider Rehd, Pyrus bretschneideri cv. is one of the largest varieties of pears in our China. In recent years, due to the factors such as deterioration of variety and poor field management, stone cell content gets increased gradually, moreover, the texture becomes rougher with more pomace.The changes have adversely affected pear flavor and quality. Stone cells result from the formation of secondary cell wall thickening and lignin deposition. Therefore, the formation and development of stone cells is closely relevant to lignin biosynthesis, lignin transportation and lignin deposition. As a key enzyme, Peroxidase （POD） involves in the last step reaction of lignin biosynthesis. Three monomers, namely coumaric alcohol, coniferyl alcohol and erucic alcohol, were formated in the phenylpropanoid metabolism, then dehydrogenated to form lignin under the role of POD. However the types of POD, involving in lignin biosynthesis, still keep unkown in the plants, and the enzyme substrate has not yet been identified. With the materials of the fruit cultivar Pyrus bretschneideri at different development stages, the types of POD were investigated to clarify the POD’s role of lignin metabolic pathway in the formation of stone cells, in order to provide the theoretical basis for controlling the contents of stone cells, inhibiting lignin biosynthesis and monomer polymerization by mean of genetic engineering.The major findings were as follows:1. During the fruit development, POD located in the cell wall of stone cells as well as the neighboring regions was involved in the procedure of lignin deposition in the cell wall of stone cells.2.In15-23days post anthesis, POD isozyme spectrums were mainly divided into three groups of PODⅠ, PODⅡand PODⅢ, of which the Rf values were0.13～0.23, 0.44and0.63～0.96respectively. Moreover, PODⅡwith the Rf value of0.44was the base spectrum. PPOD was the main type of the key enzyme POD in lignin metabolic pathway, in which it was involved in the polymerization of lignin and promoted the development of stone cells during fruit development.3. During the fruit development, GSH-PX activity peaked at day27after flowering (611.76U g^-1 min^-1), three peaks of POD activity respectively occurred at day31, day43and day55after flowering (184.5U g^-1 min^-1,221.4U g^-1 min^-1a nd242.9U g^-1 min^-1) APX activity peaked at day47after flowering (35.48U g^-1 min^-1), stone cell content peaked at day51after flowering （17.82%） lignin content peaked at day47and63day after flowering （6.47%,7.00%）4. The optimum conditions of PPOD activity were established, namely, pH value:6.5, temperature:35℃and substrate concentration:0.02mol L^-1, Km4.4×10-2m mol L^-1.5. A cDNA sequence of POD gene was amplified by RT-PCR and3’RACE at fruits. The results of sequence analysis show that the length of POD gene is833bp, containing a3’ untranslated region of41bp and translated region of792bp, and encoding264amino acids. The nucleotide acid sequence was60%-81%identical to Gossypium hirsutum, exhibiting the closest relationship with Gossypium hirsutum, and the farthest relationship with Spinacia oleracea.