琼胶寡糖诱导坛紫菜活性氧爆发

Oxidative burst in Porphyra haitanensis (Rhodophyta)

研究了琼胶寡糖诱导坛紫菜氧爆发的响应及其活性氧产生的位点。用琼胶寡糖诱导坛紫菜,以微呼吸测量系统检测坛紫菜氧消耗的变化;以对羟基苯乙酸(POHPAA)化学发光法检测坛紫菜的H2O2释放量;利用DCFH-DA染色观察活性氧在细胞上的定位;并利用实时定量PCR检测坛紫菜NADPH氧化酶基因Phrboh的差异表达;通过2-AMAC标记琼胶寡糖,观察其在坛紫菜的结合部位。结果显示,100μg/mL琼胶寡糖处理坛紫菜,出现两次呼吸爆发,分别在4min和10min左右,强度分别是基础呼吸的4倍和14.1倍;5min内出现H2O2的积累,15min达到峰值,比对照组高出近10倍。10μmol/LDPI可部分抑制H2O2的产生。3min内Phrboh的表达已上调,并持续近10min。DCFH-DA染色显示,活性氧主要产生在膜系统上。荧光标记琼胶寡糖的定位发现,坛紫菜细胞膜上存在琼胶寡糖的结合位点。综上所述,琼胶寡糖能识别细胞膜上的结合位点,并诱导坛紫菜膜系统上的H2O2爆发,H2O2的产生与NADPH氧化酶相关。

Oligosaccharides could induce some physiological responses in algae to defend attacks from pathogens. One of the earliest responses induced by oligosaccharides was so-called oxidative burst. This study has surveyed the recognition and oxidative burst response of Porphyra haitanenesis to agaro-oligosaccharide. Oxygen consumption was detected by an oxygen electrode. H2O2 concentrations in the medium were determined by measuring the dimerization of （p-hydroxyphenyl） acetic acid. The elicited oxidative burst was observed by loading the P. haitanenesis with redox-sensitive fluorescent probe dichlorohydrofluorescin （DCFH-DA）. Gene expression level of NADPH oxidase （named Phrboh） was detected by real-time PCR. 2-AMAC labeled agaro-oligosaccharides were used to research the binding of agaro-oligosaccharide on algae. Results showed that two respiration peaks were observed after addition of 100 μg/mL agaro-oligosaccharide in P. haitanensis. Fourfold increase occurred in the first respiration peak 4 minutes after the addition, and fourteen times higher in the second response 10 minutes after the exposure to agaro-oligosaccharide. The accumulation of H2O2 in the culture medium was detected 5 minutes after challenged with agaro-oligosaccharide. The concentration of H2O2 reached its peak after 15 minutes, which was nearly ten times higher than control. The gene expression level of NADPH oxidase in P. haitanensis was up-regulated 3 minutes after the addition of agaro-oligosaccharide, suggesting that the synthesis of H2O2 was closely related to the activation of Phrboh. Based on the analyses of the fluorescence of redox-sensitive dye, activated oxygen species mainly accumulated around the plasma membrane. Under the fluorescent microscope, specific binding of 2-AMAC labeled agaro-oligosaccharide on cell plasma membrane was also observed, which indicated that there was recognition site of agaro-oligosaccharide on P. haitanensis plasma membrane. In summary, P. haitanensis could recognize agaro-oligosaccharide and respond with oxidative burst which was associated with the activation of NADPH oxidase.