Fukuda Y, Shinshi H
Division of Plant Molecular Biology, National Institute of Bioscience and Human Technology, Ibaraki, Japan.
Plant Mol Biol. 1994 Feb;24(3):485-93. doi: 10.1007/BF00024116.
The expression of tobacco class I chitinase gene is effectively induced by a fungal elicitor in suspension-cultured tobacco cells. To identify cis-acting DNA elements that respond to the elicitor, a series of promoter constructs of the chitinase gene CHN50 fused to beta-glucuronidase gene was introduced into tobacco cultured cells. Promoter deletion analysis of the chitinase gene CHN50 in transgenic tobacco calli indicated that the DNA region between positions -788 and -345 from the start site of transcription is required for inducibility by the elicitor. A gel mobility shift assay revealed that nuclear factor(s) specifically interacted with the DNA region between positions -574 and -476. Moreover, this novel DNA-binding activity was present in nuclear extracts prepared from elicitor-treated cultured cells but not in extracts from untreated cells. Competitive binding assays and methylation interference experiments showed that the nuclear factor(s) bound specifically to a sequence of 22 bp that extended from positions -539 to -518 and contained a direct repeat of GTCAG spaced by three nucleotides. This motif is a candidate for a cis-acting elicitor-responsive element (ElRE) that is involved in the transcription of the class I chitinase gene.
在悬浮培养的烟草细胞中，烟草I类几丁质酶基因的表达可被一种真菌激发子有效诱导。为了鉴定对该激发子作出响应的顺式作用DNA元件，将一系列与β-葡萄糖醛酸酶基因融合的几丁质酶基因CHN50的启动子构建体导入烟草培养细胞。对转基因烟草愈伤组织中几丁质酶基因CHN50的启动子缺失分析表明，转录起始位点上游-788至-345位之间的DNA区域是激发子诱导所必需的。凝胶迁移率变动分析显示，核因子与-574至-476位之间的DNA区域特异性相互作用。此外，这种新的DNA结合活性存在于经激发子处理的培养细胞制备的核提取物中，而不存在于未经处理的细胞提取物中。竞争结合试验和甲基化干扰实验表明，核因子特异性结合到一段22bp的序列上，该序列从-539延伸至-518，包含一个由三个核苷酸隔开的GTCAG直接重复序列。该基序是参与I类几丁质酶基因转录的顺式作用激发子响应元件（ElRE）的候选元件。
