高压静电场对采后葡萄果实总黄烷-3-醇及隐色花色素还原酶表达的影响

Effects of High Voltage Electrostatic Field on the Content of Total Flavan-3-ols and Expression of Its Related Synthetic Enzyme, Leucoanthocyanidin Reductase(LAR) in Harvest Grape Berries

目的：阐明葡萄果实采后用高压静电场（HVEF）处理对总黄烷-3-醇含量和隐色花色素还原酶（LAR）表达的影响。方法：赤霞珠葡萄成熟果实经-2 kV/cm高压静电场处理,分别于30,60,120,240 min取样,采用分光光度计法、蛋白质印迹法和实时荧光定量PCR对果实中超氧化物歧化酶（SOD）活性、丙二醛（MDA）、总酚、黄烷醇类多酚、总黄烷-3-醇含量及其LAR表达进行分析,从底物、酶活性、酶蛋白含量及基因转录水平揭示HVEF对Vv lar1、Vv lar2表达的影响。结果：HVEF诱导葡萄果实中SOD活性增强,抑制果实中MDA的积累;诱导果实中总黄烷-3-醇含量增加,而对总酚、黄烷醇类多酚含量无明显作用。HVEF处理60 min后,LAR酶活性明显增强。蛋白质印迹法结果表明,HVEF明显诱导LAR1、LAR2新蛋白的合成,特别是在处理后30 min和60 min。实时荧光定量PCR结果表明,HVEF处理后30~60 min,Vv lar1、Vv lar2表达明显增强。结论：HVEF诱导Vv lar1、Vv lar2表达,LAR1、LAR2新蛋白合成,LAR酶活性增强,从而导致葡萄果实中总黄烷-3-醇积累。

Objective： The main purpose of this paper was to study the effects of high voltage electrostatic field （HVEF） on the content of total flavan-3-ols and expression of its synthetic related enzyme, leucoanthocyanidin reductase （LAR）, in harvest grape berries. Method： The harvest grape （Vitis vinifera L. Cabernet Sauvignon） berries were treated with HVEF of -2 kV/cm in strength for 30 min, 60 rain, 120 rain and 240 rain in a parallel plate electrode system, and the content of malondialdehyde （MDA）, total phenolics, and total flavan-3-ols, as well as the transcriptional and trans- lational level of Vv htrl and Vv /ar2 were determined by spectrophotometer, Real time PCR, and Western blot. Results： An increasing activity of superoxide dismutase （SOD） induced by HVEF, which resulted in an obvious inhibition in ac- cumulation of MDA in harvest grape berry was observed. HVEF induced the accumulation of total flavan-3-ols in grape berry, whereas there no obvious influences on the concentration of total phenols and flavanols were found. The activity of LAR was increased significantly by HVEF treatment at 60 rain, followed by an accumulation of total flavan-3-ols in grape berry. The results of Western blot and Real time PCR suggested that the translational and transcriptional level of Vv larl and Vv /ar2 were induced by HVEF treatment for 30-60 rain. Conclusion：All the results obtained in this experiment indicated that the HVEF treatment could induced the mRNA accumulation of Vv /arl and Vv /ar2, the synthesis of LAR1 and LAR2 new protein, the increased of LAR enzyme activity, and resulted in the increasing accumulation of total flavan-3-ols in harvest grape berries.