以鞭毛蛋白N端一个保守的22个氨基酸多肽(flg22)为激发子,研究其诱导海带孢子体防御反应的特征,以丰富植物分子病理学理论.采用Evans blue染色方法发现,flg22诱导后2h内,未能观察到海带孢子体细胞死亡现象,诱导后4～10 h,观察到大量死亡的细胞.TUNEL(TdT-mediated dUTP-biotin nick and labeling)检测方法证实,受flg22诱导后海带孢子体有DNA断裂现象,而且3’-OH末端断裂的数量随诱导时间的延长而增多,并有从诱导部位向外扩散的趋势.运用Luminol荧光检测方法检测到受flg22诱导后海带孢子体H2O2释放量迅速增加,至3h时达到峰值,约为20 μmol/L.应用荧光染料2’,7’-二氢二氯荧光黄双乙酸钠(DCFH-DA)进行ROS组织学检测,发现flg22诱导后1h即观察到绿色的荧光信号,信号强度随诱导时间的延长而增加,诱导后3h信号最强,随后绿色荧光信号的强度逐渐减弱.ROS的产生趋势与Luminol荧光检测方法的结果相一致.结果表明,flg22也是诱导海带孢子体防御反应的有效激发子.%Flg22 is an N terminal 22 amino acid peptide of flagellin. Flg22-induced defense responses were investigated in sporophytes of Saccharina japonica in this study. Our results would enrich the theory of plant molecular pathology. The results of Evans blue dye showed that cell death in the sporophytes of S. japonica was not observed within 2 h after flg22 induction, whereas large amounts of cell death were observed from 4 to 10 h. By using TdT-mediated dUTP-biotin nick and labeling (TUNEL), fragments with 3'-OH groups of nuclear DNA were observed. The number of 3'-OH increased gradually with the induction time and spread from the induction sites. The concentration of H2O2 increased rapidly in flg22-induced sporophytes of S. japonica, with a peak of about 20 umol/L at 3 h by using Luminol-dependent fluorescence detection method. Histological observation of radical oxygen species (ROS) production, detected by using the fluorescent dye 2 T- dichlorofluorescein diacetate (DCFH-DA), showed that green DCF fluorescence intensity increased gradually with the induction time, reaching the highest level at 3 h, then decreased from 3 to 5 h. The histological observation showed a consistent result with that of quantitative analysis of H2O2. The results indicate flg22 is an effective elicitor which could induce defense responses in sporophytes of S. japonica.
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