Scutt Charles P, Jenkins Tom, Furuya Masaki, Gilmartin Philip M
Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, U.K.
Plant Cell Physiol. 2002 May;43(5):563-72. doi: 10.1093/pcp/pcf069.
Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.
荧光差异显示（FDD）已被用于筛选在雌雄异株植物宽叶蝇子草雄花中差异上调的cDNA，该植物中存在XY染色体性别决定系统。为使FDD适用于大量差异cDNA的克隆，已设计出一种确认这些差异表达的新方法。将FDD凝胶进行Southern电印迹，并用来自不同FDD产物连接反应的单个cDNA克隆混合物进行杂交检测。然后将这些Southern印迹洗脱，并再次用单个克隆的FDD产物的进一步混合物进行杂交检测，以鉴定携带真正差异扩增产物的重组克隆的最大数量。通过FDD鉴定出的135条差异条带中，有56个差异扩增产物得到确认；通过虚拟Northern分析确定，这些代表23个独特的差异表达基因，以及两个在虚拟Northern分析检测水平或以下表达的基因。这两个低表达基因在基因组Southern印迹上显示出雄性植物特有的杂交条带，表明它们源自Y染色体基因或与Y染色体基因密切相关。
