MTG-DAPI双染色法观察黄瓜花粉细胞半薄切片中线粒体DNA的研究

The research of detecting the mitochondrial DNA in semi-thin sliced pollen grains of Cucumis sativus L. by MTG-DAPI double-staining method

[目的]MTG(Mito Tracker Green)是一种线粒体特异绿色荧光探针,但是目前还没有研究将MTG应用于细胞的树脂半薄切片中线粒体的荧光染色。分子生物学研究证明黄瓜线粒体是父系遗传但是还没有在细胞学观察中得到进一步证实,因此我们选择MTG和DAPI(4',6-二脒基-2-苯基吲哚)2种染料建立适用于黄瓜花粉细胞的树脂半薄切片荧光双染色技术。[方法]树脂半薄切片厚度为0.3μm,切片首先经20 nmol·L-1的MTG染色液处理30 min,然后用0.1μg·m L-1的DAPI溶液染色6 min,即可在荧光显微镜下清楚地显示出线粒体结构及DNA的存在情况。[结果]观察显示:黄瓜贴壁期花粉细胞中生殖细胞内存在线粒体结构及线粒体DNA,表明黄瓜小孢子第一次有丝分裂时没有将线粒体排除在生殖细胞之外,而是被分配到了生殖细胞中,并且此时的线粒体细胞器携带有基因信息。[结论]MTG-DAPI双染色技术可以有效地对黄瓜花粉细胞中生殖细胞核周围的线粒体进行定位,为探索黄瓜线粒体基因父系遗传现象提供技术支持。同时研究也证实了MTG可以对固定后并进行树脂半薄切片的细胞中的线粒体进行着色,扩展了MTG的应用范围。

[Objectives]MTG（ Mito Tracker Green） is a green fluorescent probe specific for mitochondrial,but there is still no study using MTG as a fluorescent dye for semi-thin sections of resin by now. Mitochondrial genome is paternally inherited in Cucumis sativus L.,but this has not been confirmed by cytological observation. So we explored the double-staining system with MTG and DAPI（ 4＇,6-diamidino-2-phenylindole）,which could be used in cucumber for mitochondrial co-localization. [Methods]The samples were sectioned（ 0.3 μm thick） with a glass knife. And then the Technovit 7100 resin sections were stained with 20 nmol·L^-1MTG for 30 min.They were further stained with an equal volume of 0.1 μg·m L^-1DAPI in Tan Buffer for 6 min. And then we could distinguish the mitochondrial membrane and mitochondrial DNA clearly with the epifluorescence microscope. [Results]We observed the presence of mitochondrial membrane and their DNA in the generative cells in cucumber adherent pollen grains here. The result indicated that mitochondria with its DNA were assigned to the generative cells during the first time mitosis of microspores instead of being excluded totally.[Conclusions]This method provided technical support for further exploration for paternal inheritance of mitochondrial DNA in cucumber. The study here also confirmed the availability of MTG in resin semi-thin sections,which extended the application of MTG.