酸碱滴定法优化香蕉磷脂酶A活性测定及炭疽病胁迫下其活性变化研究

Optimization on Activity Assay of Banana Phospholipase A by Acid-base Titration and Change of PLA Activity under Anthracnose Stress

植物磷脂酶A(phospholipase A,PLA)是细胞膜磷脂代谢中的关键酶,可以催化磷脂分子上多个功能基团发生裂解生成信号分子,参与调节植物生长发育以及环境和生物胁迫,具有重要研究价值。本研究主要考察底物浓度、反应温度、缓冲溶液pH、缓冲溶液体积、反应时间等多种因素对香蕉PLA催化卵磷脂水解活性的影响,从而优化磷脂酶A活性测定方法,进一步根据最佳测定条件研究炭疽病胁迫下香蕉PLA活性的变化趋势。结果表明,酸碱滴定法测定香蕉PLA活性的优化条件:反应体系pH 9,底物浓度为4 g/L,反应温度为55℃,底物为15 mL,酶粗提液为3 mL,提取缓冲液体积与果皮重量的比值(mL/g)为3,反应时间为7 h。在最优条件下对炭疽病胁迫下的香蕉果皮组织进行PLA活性测定,发现果皮的病害程度与PLA活力呈正相关,即随着病害程度加深,PLA活性增大,且远大于对照组。表明PLA活性大小可能与果实的抗性有关,随着果实病害程度加深,PLA活性增大,膜磷脂降解,细胞膜的完整性被破坏,从而导致果实腐烂。本研究明确了一种方便可行的香蕉PLA活性测定方法,为深入研究香蕉PLA的催化作用机制奠定基础。

Plant phospholipase A(PLA)is a key enzyme in cell membrane phospholipid metabolism.It can catalyze the cleavage of multiple functional groups on phospholipids to generate signaling molecules,which are involved in regu-lating plant growth and development as well as environmental and biological stresses.PLA possesses important research value.In this study,the effects of various factors such as substrate concentration,reaction temperature,pH of buffer solution,buffer solution volume,and reaction time on catalytic activities of banana PLA for phospholipid hydrolysis were investigated,so as to optimize PLA activity assay method.According to the optimal assay conditions,the change of banana PLA activity under anthracnose stress was further studied.The results showed that the optimal conditions for the determination of banana PLA activity by acid-base titration method were as follows:pH 9,reaction temperature 55℃,substrate concentration 4 g/L,15 mL substrate,3 mL crude enzyme extract,the ratio of extraction buffer volume to peel weight(mL/g)3,and reaction time 7 h.According to the optimal conditions,the PLA activity of banana peel tissue under anthracnose stress was determined.It was found that the degree of disease in peel tissue was positively correlated with PLA activity,indicating that PLA activity increased with the deepening of disease.This suggested that the level of PLA activity was probably related to fruit resistance.As disease degree deepened,PLA activity increased,membrane phospholipids degraded,and cell membrane integrity disrupted,leading to fruit decay.In this article,a con-venient and feasible method for the determination of banana PLA activity was established,which laid a foundation for further study of the catalytic mechanism of banana phospholipase A.