Itzhaki H, Maxson J M, Woodson W R
Department of Horticulture, Purdue University, West Lafayette, IN 47907-1165.
Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8925-9. doi: 10.1073/pnas.91.19.8925.
The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene.
康乃馨花瓣衰老过程中乙烯产量的增加调节了编码谷胱甘肽 - S - 转移酶一个亚基的GST1基因的转录。我们通过研究参与GST1基因表达的顺式元件和反式作用因子，来探究这种乙烯响应转录的分子基础。将与β - 葡萄糖醛酸酶报告基因融合的GST1 5'侧翼DNA导入后进行瞬时表达分析，以功能鉴定负责乙烯响应表达的序列。对GST1 5'侧翼序列的缺失分析确定了在 - 667至 - 470之间一个197 bp的单一正向调控元件，它是乙烯响应表达所必需的。这个乙烯响应区域内的序列进一步定位到 - 596至 - 470之间的126 bp。该区域内的乙烯响应元件（ERE）以方向独立的方式赋予最小花椰菜花叶病毒 - 35S TATA框启动子乙烯调控的表达。凝胶电泳迁移率变动分析和DNase I足迹法用于鉴定与ERE内序列结合的蛋白质。结果表明，康乃馨花瓣中的核蛋白与126 bp的ERE特异性相互作用，并且这些蛋白质的存在和结合与乙烯或花瓣衰老无关。DNase I足迹法确定了ERE内 - 510至 - 488之间被结合蛋白特异性保护的DNA序列。受保护区域内的一个8 bp序列（ATTTCAAA）与番茄果实成熟E4基因乙烯响应所需的启动子序列具有显著同源性。
