PROCEDURE FOR WHOLE-MOUNT FLUORESCENCE IN-SITU HYBRIDIZATION OF INTERPHASE NUCLEI ON ARABIDOPSIS-THALIANA
PROCEDURE FOR WHOLE-MOUNT FLUORESCENCE IN-SITU HYBRIDIZATION OF INTERPHASE NUCLEI ON ARABIDOPSIS-THALIANA
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DOI：
10.1046/j.1365-313x.1994.6010123.x
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发表时间：
1994-07-01
期刊：
影响因子：
7.2
通讯作者：
ENGLER, G
中科院分区：
文献类型：
--
作者：
BAUWENS, S;KATSANIS, K;ENGLER, G
报道了一种植物组织整体荧光原位杂交(FISH)方法。用rDNA作为探针，对拟南芥幼苗和花朵进行了直接和间接检测，并进行了标记。结果表明，在1%甲醛溶液中固定效果最好，杂交效果最好。整装FISH和共聚焦扫描激光显微镜的结合使得rDNA基因座在幼苗和花的所有组织中都得到了核定位。直接标记的信噪比最好，尤其是在苗木顶端。利用rDNA和一段重复序列为500bp的拟南芥幼苗进行了双标记实验，进一步说明了该技术的可行性。尽管幼苗所有组织的细胞核都显示了这两种信号，但与rDNA的单一标记实验相比，在致密的顶端区域，这两种信号的杂交效率降低了。
A procedure for whole mount fluorescence in situ hybridization (FISH) on plant tissue is reported. The technique was demonstrated on seedlings and flowers of Arabidopsis thaliana L. with rDNA as a probe, labelled, both for direct and indirect detection. It was found that fixation in 1% formaldehyde yielded the best results with respect to morphology and hybridization efficiency. The combination of whole mount FISH and confocal scanning laser microscopy allowed the nuclear localization of the rDNA loci in all tissues of both seedlings and flowers. Direct labelling yielded the best signal-to-noise ratio, especially in the apical zones of the seedlings. The technique was further illustrated on seedlings of A, thaliana in double labelling experiments with rDNA and a tandemly repeated, 500 bp sequence of A. thaliana. Although nuclei in all tissues in the seedling exhibited both signals, hybridization efficiency for both signals was reduced in the dense, apical zones as compared with single labelling experiments with rDNA.