砀山酥梨石细胞发育过程中PPO酶学特性及其cDNA片段克隆  

PPO Characterization and a PPO cDNA Coloning during the Development of Stone Cell in Pyrus bretschneideri cv.

作　　者：李玲[1] 于娟娟[1] 金青[1] 蔡永萍[1] 林毅[1] 吕芮宏[1] 

机构地区：[1]安徽农业大学生命科学学院,安徽合肥230036

出　　处：《激光生物学报》2011年第3期353-359,共7页Acta Laser Biology Sinica

基　　金：国家自然科学基金(30771483)

摘　　要：以砀山酥梨果实为材料,研究了影响石细胞形成的木质素合成酶-多酚氧化酶(polyphenol oxidase,PPO)的酶学特性,并对PPO基因进行了克隆。结果表明,在砀山酥梨果实发育的过程中,PPO活性在花后27d和47d出现高峰,早于木质素含量高峰(花后47d、61d)和石细胞团含量高峰(花后51d),且PPO活性变化与两者极显著相关。砀山酥梨果实PPO最适反应条件为pH值7.0,温度40℃左右,底物浓度0.16mol/L。酶促褐变反应动力学符合米氏方程,动力学参数V_(max)为454.5455U/g,K_m为0.1364mol/L。克隆得到了558bp的PPO基因的cDNA片段,该序列已登录GenBank,登陆号为HQ329065。经序列分析及同源性比对发现,该基因与其它植物的同类基因有较高同源性。The enzymatic characteristics of Polyphenol oxidase （PPO） affected the formation of stone cell were investigated and the PPO gene was cloned with the material of fruit of Pyrus bretschneideri ev. The results showed that during the development of Pyrus bretschneideri cv. , PPO activity reached peaks in 27th and 47th days after anthesis （DAA） respectively, which appeared earlier than the peaks of contents of lignin （ in 47 DAA and 61 DAA） and stone cell （ in 51 DAA） respeetively, meanwhile, the variation of PPO activites had extremely significant correlation for both of them. The optimum conditions of PPO activity were established, namely, pH value 7.0, temperature 40 ℃ and substrate concentration 0.16 moL/L. The kinetics of enzymatic browning reaction in this experiment was conformed to Michaelis equation and the kinetic parameters Km and Vmax were 0. 1364 tool/1, and 454. 5455 U/g respectively. The fragment of 558 bp cDNA of the PPO gene was cloned from the fruit organ of Pyrus bretschneideri cv. and its sequence was submitted to the GenBank database with accession No. HQ329065. Sequence analysis and homology comparison revealed that the fragment of PPO gene from Pyrus bretschneiderl cv. had a high correlation with other plant species.

关 键 词：砀山酥梨 多酚氧化酶 木质素 石细胞 酶学特性 RT—PCR 

分 类 号：Q502[生物学—生物化学] Q785