Kehoe D M, Degenhardt J, Winicov I, Tobin E M
Department of Biology, University of California at Los Angeles 90024-1606.
Plant Cell. 1994 Aug;6(8):1123-34. doi: 10.1105/tpc.6.8.1123.
Two small regions of the promoter of an Lhcb gene encoding a light-harvesting chlorophyll a/b protein were identified as essential in conferring phytochrome responsiveness by using a transient expression assay. Initially, 5' deletion analysis of cabAB19, an Lhcb2 gene of Lemna, showed that sequences within the region from -174 to -104 relative to the start of transcription were necessary for phytochrome regulation. Internal deletion and substitution mutants were used to demonstrate that no additional phytochrome-responsive regions exist between -1600 and -174 in this promoter. A 171-bp fragment of the promoter extending from -239 to -69 was sufficient to impart phytochrome responsiveness to a minimal ubiquitin promoter that was not itself regulated by light. Specific binding of Lemna proteins to the region necessary for phytochrome responsiveness was demonstrated using in vitro polyacrylamide gel mobility shift assays and 1,10-phenanthroline copper ion footprinting. Further analysis of the region from -174 to -104 demonstrated that mutations in two separate 10-bp sequences, from -134 to -125 and from -114 to -105, could abolish phytochrome responsiveness; thus, there are two unique regions that are necessary for phytochrome regulation of this gene. One of these regions contains a CCAAT motif and the other a GATA motif. These motifs are conserved in the promoters of many Lhcb genes and may be important elements in the phytochrome responsiveness of this gene family.
通过瞬时表达分析，确定了编码光捕获叶绿素a/b蛋白的Lhcb基因启动子的两个小区域在赋予光敏色素反应性方面至关重要。最初，对浮萍的Lhcb2基因cabAB19进行5'缺失分析表明，相对于转录起始点，-174至-104区域内的序列对于光敏色素调节是必需的。内部缺失和替代突变体用于证明在该启动子的-1600至-174之间不存在其他光敏色素反应区域。从-239延伸至-69的171 bp启动子片段足以赋予对本身不受光调节的最小泛素启动子的光敏色素反应性。使用体外聚丙烯酰胺凝胶迁移率变动分析和1,10-菲咯啉铜离子足迹法证明了浮萍蛋白与光敏色素反应所需区域的特异性结合。对-174至-104区域的进一步分析表明，两个单独的10 bp序列（从-134至-125和从-114至-105）中的突变可消除光敏色素反应性；因此，存在两个对该基因的光敏色素调节必不可少的独特区域。其中一个区域包含一个CCAAT基序，另一个包含一个GATA基序。这些基序在许多Lhcb基因的启动子中保守，可能是该基因家族光敏色素反应性的重要元件。
