荔枝果皮的结合态POD及其在果实生长发育过程中的变化

Membrane/wall-bound peroxidases in litchi pericarp and their changes during fruit development

【目的】研究荔枝果皮结合态POD的提取方法及其组分,并监测其在果实生长过程中的变化。【方法】以‘乌叶’荔枝果皮为材料,设置6种不同的结合态POD提取液,对其进行了比较,并采取最优缓冲液提取了果实生长发育不同时期果皮中的结合态POD,观测其活性的变化并同工酶谱的变化。【结果】采用100 mmol·L-1Tris-HCl(pH8.0),1.0 mol·L-1NaCl,10%甘油,0.1%Triton X-100时提取效果最好,在提取液中加入的10%甘油,可以有效地避免结合态POD的失活率;盛花后65 d内,结合态POD活性微弱,在盛花后65 d开始迅速增大。阴极电泳(同工酶)分析表明,盛花后55 d出现2条明显的谱带,相对迁移率分别为0.98和0.99;盛花后75 d开始出现了相对迁移率为0.63、0.69、0.81、0.83和0.94的新谱带。【结论】成熟的荔枝果皮具有高活性的结合态POD,随着果实进入着色和成熟期,其组分明显增多,总活性和各组分活性迅速增强。

【Objective】 The purposes of this study were to explore the extraction methods and major isoforms of the membrane/wall-bound peroxidases(B-PODs) in litchi pericarp and to monitor the changes of these PODs during fruit development. 【Method】 Litchi(Litchi chinensis Sonn.‘Wuye') pericarp was used as materials, and the effects of different extraction solutions were investigated on the activity and zymogram of the B-POD; the activity and the isoform changes of enzymes were observed throughout the whole period of fruit growth and development. 【Result】 The results showed that, the optimal extraction solution for the B-PODs was 100 mmol·L-1Tris-HCl(pH8.0) buffer, containing 1.0 mol·L-1NaCl, 10% glycerol and 0.1% Triton X-100. Addition of 10% glycerol in the solution could prevent enzymes from the activity loss. The activity of B-POD was weak within 65 days after full bloom(DAFB), two clear B-POD bands in alkaline polyacrylamide gel electrophoresis(PAGE), of which the relative migration rate(Rf) values were0.98 and 0.99, respectively were visualized between 55 and 65 DAFB. Then, the B-POD activity increased dramatically with the process of fruit coloring and maturation, and there were other 5 isoforms localized in pericarp of which the Rf values were 0.63, 0.69, 0.81, 0.83 and 0.94, respectively. 【Conclusion】 Mature litchi fruit has high activity of B-POD in pericarp, the B-POD activity, the number and the intensity of isozymatic band increase significantly when fruit start coloring and maturation. Some biochemical properties of the B-PODs were discussed as well.