Flg22诱导的海带(Saccharina japonica)孢子体防御反应的初步研究

Preliminary study on flg22-induced defense responses in sporophytes of Saccharina japonica (Phaeophyta)

  • 摘要: 细菌鞭毛蛋白及其N端一个保守的22个氨基酸的多肽(flg22)是高等植物的有效外源激发子。低等藻类与高等植物在防御反应途径上具有保守性。本研究以flg22为激发子,研究其诱导海带孢子体防御反应的特征。运用Evans blue染色方法发现,flg22诱导后2 - 6 h,未能观察到海带孢子体细胞死亡现象,诱导后8 - 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3 h时达到峰值,约为20 μM。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1 h 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3 h 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。我们的研究结果显示,flg22也是诱导海带孢子体防御反应的有效激发子。

     

    Abstract: Flagellin and its N terminal 22 amino acids peptide (flg22) function as an effective elicitor, which can induce defense responses in higher plants. Marine algae and higher plants share some conserved characteristics on defense response pathways. Flg22-induced defense responses were investigated in sporophytes of Saccharina japonica in this study. The results of Evans blue dye showed that cell death in the sporophytes of S. japonica was not observed within 2 to 6 h after flg22 induction, whereas large amounts of cell death were observed from 8 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 site. The concentration of H2O2 increased rapidly in flg22-induced sporophytes of S. japonica, with a peak of about 20 μM 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’,7’- 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. According to our data, flg22 is an effective elicitor which could induce defense responses in sporophytes of S. japonica.

     

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