HMC毒素诱导玉米同核C、N细胞质细胞凋亡的荧光显微观察

doi:10.3864/j.issn.0578-1752.2011.09.008

中国农业科学

HMC毒素诱导玉米同核C、N细胞质细胞凋亡的荧光显微观察

马春红1;李秀丽1;2;董文琦3;张红心4;李运朝1;崔四平1;王立安2;贾银锁1;戴志刚5

1、河北省农林科学院遗传生理研究所，石家庄 050051；
2、河北师范大学生命科学学院，石家庄 050016；
3、河北省农林科学院科技处，石家庄 050051；
4、唐山师范学院，河北唐山 063000；5湖北省土壤肥料工作站，武汉 430064

收稿日期:2010-07-15 出版日期:2011-05-05 发布日期:2010-09-27
通讯作者: 贾银锁，Tel：0311-87652128；E-mail：jiays2005@163.com；王立安，Tel：0311-86268425；E-mail：wlian1965@126.com
作者简介:马春红，Tel：0311-87652128；E-mail：mchdonger@sohu.com
基金资助:
河北省自然科学基金项目（C2011301010，C2006000744）、河北省人才工程培养经费（冀人社字[2010]353号）、科技部国际科技合作项目（2006DFB02480）、河北省科学技术研究与发展计划项目（10393912D，07297162D）、农业部948项目（2008-Z20，2006-G8(5)）

Detection of Apoptosis in Homocaryon with C and N Cytoplasm of Maize Induced by Helminthosporium maydis Race C (HMC) Toxin

MA Chun-hong; LI Xiu-li;DONG Wen-qi; ZHANG Hong-xin; LI Yun-chao; CUI Si-ping; WANG Li-an; JIA Yin-suo; DAI Zhi-gang

1、Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051;
2、College of Life Science, Hebei Normal University, Shijiazhuang 050016;
3、Department of Science and Technology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051;
4、Tangshan Teachers College, Tangshan 063000, Hebei；5Soil and Fertilizer Station of Hubei Province, Wuhan 430064

Received:2010-07-15 Online:2011-05-05 Published:2010-09-27

摘要/Abstract

摘要： 【目的】研究玉米小斑病菌C小种毒素（HMC-toxin）诱导同核异质体玉米的离体根冠细胞发生凋亡的差异性及凋亡规律。【方法】采用吖啶橙（AO）与溴化乙啶（EB）荧光复染以及Hoechst 33258荧光染色的方法检测。【结果】HMC毒素诱导根冠细胞发生凋亡，出现凋亡小体与染色体边集形态特征。在3种毒素浓度、3种处理时间下，Mo17-C的根冠细胞死亡率均高于Mo17-N。采用AO与EB复染方法，在150 μg·mL-1 HMC毒素处理7 h时，Mo17-C细胞凋亡率达最高值，为68.7%，而Mo17-N仅为29%；经Hoechst 33258染色后，在150 μg·mL-1 HMC毒素处理下，Mo17-C也于7 h时达最大凋亡率，为57.3%，而Mo17-N为27.7%。【结论】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细胞质的。凋亡率随诱导浓度和诱导时间的增加而增加。AO、EB复染与Hoechst 33258染色相比，两者细胞凋亡率接近，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。

关键词: 玉米小斑病菌C小种 , 毒素 , 细胞凋亡 , Hoechst 33258 , 吖啶橙 , 溴化乙啶

Abstract: 【Objective】 To study the feature of apoptosis and the change of its ratio induced by HMC toxin in the root cap cells detached from a pair of homokaryon Mo17-C and Mo17-N inbred line of maize. 【Method】AO and EB staining and Hoechst 33258 staining methods were used to detach the apoptosis of root cap cells. In the process of induction, root cap cells appeared apoptotic bodies and marginate of chromosome morphology. Living, necrosis and apoptosis cells could be distinguished clearly by AO and EB complex staining and Hoechst 33258 staining. 【Result】The ratio of apoptosis in Mo17-C cells was sharply higher than that of Mo17-N, and the max ratios of apoptosis of Mo17-C and Mo17-N were 68.7% and 29%. In contrast, the max ratios of apoptosis of Mo17-C and Mo17-N stained by Hoechst 33258 were 57.3% and 27.7%, respectively. After HMC toxin with 150 μg·mL-1 treated for 7 h, the apoptosis rate was the highest. 【Conclusion】 Harmed by HMC toxin, the root cap cells appeared apoptosis. The ratio of apoptosis in C cytoplasm was higher than in N cytoplasm. Along with the increase of 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 could be distinguished only by AO and EB complex staining.

Key words: Helminthosporium maydis (Bipolaris maydis) race C (HMC) , toxin , apoptosis , Hoechst 33258 , AO , EB

马春红1;李秀丽1;2;董文琦3;张红心4;李运朝1;崔四平1;王立安2;贾银锁1;戴志刚5. HMC毒素诱导玉米同核C、N细胞质细胞凋亡的荧光显微观察[J]. 中国农业科学, 2011, 44(9): 1823-1829.

MA Chun-hong; LI Xiu-li;DONG Wen-qi; ZHANG Hong-xin; LI Yun-chao; CUI Si-ping; WANG Li-an; JIA Yin-suo; DAI Zhi-gang. Detection of Apoptosis in Homocaryon with C and N Cytoplasm of Maize Induced by Helminthosporium maydis Race C (HMC) Toxin[J]. Scientia Agricultura Sinica, 2011, 44(9): 1823-1829.

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HMC毒素诱导玉米同核C、N细胞质细胞凋亡的荧光显微观察

Detection of Apoptosis in Homocaryon with C and N Cytoplasm of Maize Induced by Helminthosporium maydis Race C (HMC) Toxin

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