玉米小斑病菌C小种毒素诱导玉米离体根冠细胞凋亡的检测

Detection of Apoptosis in Detached Maize Root Cap Cells Induced by HMC-Toxin

将玉米同核异质体不育细胞质CB37及其正常保持系NB37的离体根冠细胞分为4组,其中3组分别用质量浓度50μg/mL、100μg/mL和150μg/mL的玉米小斑病菌C小种(HMC)毒素诱导,1组用pH6.5PBS诱导作为对照,诱导时间分别为1h、4h和7h。然后采用中性红与伊文思蓝染色、吖啶橙(AO)与溴化乙啶(EB)复染和Hoechst33258染色3种方法检测玉米根冠细胞的凋亡状况。结果表明:HMC毒素诱导玉米根冠细胞发生凋亡,并出现凋亡小体与染色体边集形态特征。中性红与伊文思蓝染色,只能检测出活细胞和死细胞,且在3种毒素浓度、3种处理时间下,CB37的根冠细胞死亡率均高于NB37;采用AO与EB复染方法,用150μg/mLHMC毒素处理7h时,CB37细胞凋亡率达到最高值70.2%,而NB37仅为36.7%;经Hoechst33258染色后,CB37用150μg/mLHMC毒素处理7h时达到最大凋亡率为74.5%,而NB37为30.7%。专效性HMC毒素对C细胞质敏感,在毒素伤害细胞致死过程中,C细胞质的细胞死亡率远高于N细胞质;在诱导细胞凋亡程序中,C细胞质的细胞凋亡率也远高于N细胞质,且其凋亡率随诱导质量浓度和诱导时间的增加而增加;AO、EB复染与Hoechst33258染色两者相比,细胞凋亡率接近,准确度均高,图像清晰,且费用相当,但前者可以区分早期和晚期凋亡细胞,而后者不能区分。

The root cap cells of CB37 and NB37 were divided into 4 groups,in which 3 groups were induced by 50 μg/mL,100 μg/mL,150 μg/mL of Helminthosporium maydis C toxin（HMC-toxin） as the experimental group,and one group induced by pH 6.5 PBS as the control group.The inducing time was 1 h,4 h and 7 h.The apoptosis of root cap cell was examined with 3 methods：neutral red and Evans blue staining,AO and EB staining and Hoechst 33258 staining.In the process of induction with HMC toxin,apoptotic bodies appeared in root cap cells and the chromosome morphology became marginate.Living and death cells could be clearly detected after Neutral red and Evans blue staining and the ratio of death in CB37 cells treated with 150 μg/mL HMC-toxin was higher than that of NB37 with the maximum death rates of 99.2% and 95.5%.Living,necrosis and apoptosis cells could be distinguished clearly by AO and EB complex staining and Hoechst 33258 staining.The ratio of apoptosis in cells of CB37 was much higher than that of NB37,the max ratio of apoptosis cells of CB37 and NB37 were 70.2% and 36.7%.In contrast,the max ratio of apoptosis cells of CB37 and NB37 were 74.5% and 30.7% stained by Hoechst 33258 respectively.The max ratio of apoptosis in CB37 and NB37 occurred in 150 μg/mL HMC-toxin treated for 7 h.Therefore,a conclusion can be made as follows：during the process of the ＂harm＂ by HMC-toxin,apoptosis appeared in the root cap cells,the ratio of apoptosis in C cytoplasm was higher than that in N cytoplasm.Along with the increase of the toxin concentration and treating time,the ratio of apoptosis became higher.In spite of the different staining methods by AO and EB or by Hoechst 33258,the ratio of the cell apoptosis was similar.But the early and late apoptotic cells can be distinguished only by AO and EB complex staining.