Kawaoka A, Kawamoto T, Sekine M, Yoshida K, Takano M, Shinmyo A
Department of Biotechnology, Faculty of Engineering, Osaka University, Suita, Japan.
Plant J. 1994 Jul;6(1):87-97. doi: 10.1046/j.1365-313x.1994.6010087.x.
The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5'-deleted promoters showed that a positive element involved in the response to wounding was located between -307 and -99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from -296 to -283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5' end to -289 with a disrupted Box 1 was fused to a reporter gene for beta-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2 (-289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from -296 to -283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix-loop-helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2(-529)/GUS.
已对控制辣根过氧化物酶（HRP）的prxC2基因伤口诱导表达的机制进行了研究。对5'端缺失启动子的调控特性分析表明，参与伤口反应的一个正向元件位于距翻译起始位点-307至-99 bp之间。在烟草核蛋白与prxC2启动子DNA片段的体外结合试验中，结合位点是包含CACGTG基序的-296至-283处的Box 1。为了确定Box 1的功能作用，将从5'端消化至-289且Box 1被破坏的prxC2启动子与β-葡萄糖醛酸酶（GUS）报告基因融合。在具有prxC2（-289）/GUS构建体的转基因烟草植株中未观察到GUS活性的诱导。这些数据表明，prxC2对伤口的反应表达需要-296至-283处的Box 1序列。此外，筛选了烟草cDNA表达文库，并鉴定出一个与Box 1序列特异性结合的蛋白质的cDNA克隆，命名为TFHP-1。推测的TFHP-1蛋白包含一个碱性区域和亮氨酸拉链（bZip）基序以及一个螺旋-环-螺旋（HLH）基序。TFHP-1的mRNA在根和茎中丰富，并且在叶片中不受伤口诱导。在烟草原生质体中，反义TFHP-1抑制了prxC2（-529）/GUS的表达。
