疫霉31kDa激发子的分离纯化及其诱导烟草细胞死亡机理的初步研究

Purification of a 31 kDa elicitor produced by Phytophthora and its mechanisms of inducing tobacco cell death

通过沸水浴、硫酸铵沉淀、非变性电泳、割胶电洗脱等方法,从一种疫霉的培养液中分离得到分子量约31kDa的蛋白类激发子。该激发子在低浓度下即可诱发烟草叶片发生过敏反应,处理烟草悬浮细胞12h能导致24.6%的细胞死亡,处理24h后细胞的死亡率达67.8%。用该激发子处理烟草悬浮细胞24h后细胞基因组受到明显降解,但无DNAlad-dering现象。用该激发子处理1h即可显著降低烟草悬浮细胞中的ATP水平,而用氧化磷酸化的解偶联剂CCCP处理在降低胞内ATP水平的同时也能显著诱导烟草悬浮细胞死亡,因此抑制胞内ATP合成可能是该激发子诱导烟草细胞死亡的重要机制之一。

A proteinaceous elicitor was extracted and purified from the culture filtrate of a Phytophthora isolate through the procedures of boiling-water bath, ammonium sulfate precipitation, non-denaturing polyacrylamide gel electrophoresis （PAGE）, corresponding gel cut and electroelution. It showed an apparent molecular weight of about 31 kDa in SDS-PAGE gel. Inoculation with this elicitor caused necrosis in tobacco leaves. Treating suspension-cultured tobacco cells with the elicitor resulted in 24.6% cell death after 12 h, and 67.8% after 24 h. Obvious genome degradation occurred in suspension-cultured tobacco cells after treatment with the elicitor for 24 h, but no DNA laddering was observed. A significant decrease of ATP level was found in sus- pension-cultured tobacco cells after treatment with the elicitor for 1 h. As treating the suspension-cultured tobacco cells with carbonyl cyanide 3-chlorophenylhydrazone （CCCP）, an oxidative phosphorylation uncoupler, resulted in both obvious intracellular ATP level decrease and cell death, it is inferred that inhibition of ATP synthesis is one of the important mechanisms of inducing tobacco cell death by this elicitor.