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大豆丛枝菌根共生结构和多聚磷累积双定位方法
引用本文:周佳,张爽,廖红,王秀荣.大豆丛枝菌根共生结构和多聚磷累积双定位方法[J].植物营养与肥料学报,2015,21(1):211-216.
作者姓名:周佳  张爽  廖红  王秀荣
作者单位:华南农业大学, 根系生物学研究中心, 资源环境学院, 广东广州 510642
基金项目:国家自然科学基金项目(31372126)资助。
摘    要:【目的】多聚磷是丛枝菌根内磷的主要贮存形式,定性、定量观察多聚磷对于解析菌根中磷代谢具有重要意义。随着植物体内越来越多的参与菌根真菌与寄主植物之间营养交换过程的基因被鉴定,迫切需要进一步提高根内菌根共生结构和多聚磷累积的染色和定位分析技术。【方法】本研究利用丛枝菌根真菌Glomus mosseae侵染的大豆植株,采集新鲜根样制片,一部分薄根片利用低浓度荧光染料麦胚凝集素,室温染色30 min,在波长488 nm的蓝光激发下使用荧光显微镜观察拍照;另一部分薄根片利用荧光染料4’,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)进行染色,在波长405 nm紫外光激发下观察并拍照;进一步取新鲜制备的薄根片,先后用以上两种荧光染料进行染色,分别在波长405 nm和488 nm的激发光下观察并拍照,完成了菌根共生结构和多聚磷的共定位。【结果】1)使用荧光染料麦胚凝集素,大豆丛枝菌根真菌侵染结构的荧光标记活性染色法,可以清晰地检测到大豆丛枝菌根中所有的共生结构,包括丛枝,泡囊和根内菌丝等。2)在丛枝菌根真菌侵染的根中,各种共生结构都呈现出黄色荧光,为DAPI与多聚磷结合在紫外光激发下的呈色。根段中部分细胞内的蓝白色斑点为DAPI与细胞核中DNA结合的显色结果。在含有成熟丛枝结构的细胞中,也可观察到大部分丛枝呈蓝白色,主要是丛枝膜质结构的呈色。因此,利用荧光染料4’,6-二脒基-2-苯基吲哚二盐酸盐染色法定位多聚磷,能很好地区分多聚磷酸盐、DNA和膜质。3)在以上研究的基础上,通过荧光光路的切换,可以同时观察到菌根共生结构和多聚磷的共定位。处于发育阶段的整个丛枝中多聚磷累积的亮黄色清晰可见。在成熟的丛枝中,由于膜质结构发达,对累积在丛枝结构中的多聚磷的染色观察产生了一定影响,导致仅仅局部的多聚磷累积清晰可见。【结论】本研究建立的大豆菌根共生结构与多聚磷累积的双定位分析系统,能够直观观察植物与丛枝菌根真菌的养分交换,清晰地对丛枝菌根共生结构中多聚磷的累积进行定位分析,可作为从组织和细胞水平研究菌根共生体的重要技术手段。

关 键 词:大豆  丛枝菌根真菌  丛枝菌根共生结构  多聚磷  荧光染料
收稿时间:2015-02-11

Dual localization of arbuscular mycorrhizal structures and polyphosphate accumulation in soybean roots
ZHOU Jia,ZHANG Shuang,LIAO Hong,WANG Xiu-rong.Dual localization of arbuscular mycorrhizal structures and polyphosphate accumulation in soybean roots[J].Plant Nutrition and Fertilizer Science,2015,21(1):211-216.
Authors:ZHOU Jia  ZHANG Shuang  LIAO Hong  WANG Xiu-rong
Institution:Root Biology Center, College of Resources and Environment, South China Agricultural University, Guangzhou, 510642, China
Abstract:【Objectives】 Polyphosphates are major phosphorus forms existing in the structural bodies of arbuscular mycorrhizae. Qualitative and quantitative analyses of polyphosphates are important to understand the phosphorus metabolism in arbuscular mycorrhizae. Many genes have been identified to involve in nutrient exchanges between mycorrhizal fungi and host plants. Therefore, the technology for the staining and localization of polyphosphates in arbuscular mycorrhizae is urgently required. 【Methods】 Fresh roots from Glomus mosseae colonized soybean plants were sampled and made into thin slices. Some of them were stained with low concentration of wheat germ agglutinin for 30 min at room temperature, and photographed using a fluorescence microscope at blue light excitation with wavelength of 488 nm; the others were stained with the fluorescent dye 4, 6-diamidino-2-phenylindole dihydrochloride, and photographed at ultraviolet excitation with wavelength of 405 nm. Thepolyphosphates were thus localized by both the methods. 【Results】1) Using fluorescent active staining method (stained with wheat germ agglutinin), all arbuscular mycorrhizal structures could be clearly detected, including arbuscules, vesicles and intercellular hyphae. 2) In the infected roots, polyphosphates in arbuscular mycorrhizal structures showed yellow fluorescence under excitation of ultraviolet light. DNA in nucleus interacting with 4, 6-diamidino-2-phenylindole dihydrochloride showed blue-white fluorescent dots, and arbuscular membrance of mature arbuscules in the cells exhibited blue-white fluorescence. So, using fluorescent dye 4, 6-diamidino-2-phenylindole dihydrochloride to position the polyphosphates, the pigmentation of polyphosphates, DNA and membrane structure were obviously distinguished. 3) Based on the above results, dual localization of mycorrhizal structures and polyphosphate accumulation was realized by changing the fluorescent channels. Furthermore, polyphosphate accumulation in immature arbuscules showed light yellow fluorescence, and those in mature arbuscules could only partly be observed since highly developed membrane structure affected the staining and appearance of polyphosphates.【Conclusions】The dual staining method using fluorescent dyes wheat germ agglutinin and 4, 6-diamidino-2-phenylindole dihydrochloride is able to localize the mycorrhizal structures and polyphosphate accumulation in soybean roots. Using the method, the nutrient exchanges between arbuscular mycorrhizal fungi and plant roots can be visually observed. Therefore, it is prospective technology for the study of mycorrhizal symbiosis at tissue and cellular level.
Keywords:soybean  arbuscular mycorrhizal fungi  arbuscular mycorrhizal structures  polyphosphates  fluorescent dyes
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