Siebertz B, Logemann J, Willmitzer L, Schell J
Max-Planck-Institut für Züchtungsforschung, Köln, Federal Republic of Germany.
Plant Cell. 1989 Oct;1(10):961-8. doi: 10.1105/tpc.1.10.961.
The 5' region of the wound-inducible gene wun1, derived from potato, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants. Different 5' deletion fragments were linked to the reporter gene beta-glucuronidase (GUS) as transcriptional fusions, and the expression of these chimeric genes was analyzed in leaf tissue. Sequences 111 base pairs upstream of the transcriptional start site were not able to drive the GUS expression over background levels, whereas sequences between -111 and -571 showed a slightly higher activity with equal levels of transcription in wounded and nonwounded tissue. The addition of further upstream sequences (-571 to -1022) enhanced the level of expression by a factor between 13 and 370. The expression driven by this fragment was inducible by a factor of twofold to ninefold by wounding. Histochemical analysis of different tissue from transgenic plants that contain wun1-GUS fusions demonstrates wound-inducible and cell-specific wun1 promoter activity in plants containing the -1022-base pair fragment. The location of GUS activity appears to be cell-specific, being highest in epidermal cells of leaves and stems and lower in vascular cells. Activity was reduced to levels that could not be detected by histochemical staining in leaves, stems, and roots of plants containing the deleted promoter fragments. Plants that contain the different deletion constructs and plants that carry the -1022-base pair fragment show high expression in anthers and pollen grains that could not be stimulated by wounding.
对来自马铃薯的伤口诱导型基因wun1的5'区域进行了测序，并分析了其中对转基因烟草植株基因表达调控至关重要的顺式作用元件。将不同的5'缺失片段与报告基因β-葡萄糖醛酸酶（GUS）连接作为转录融合体，并在叶片组织中分析这些嵌合基因的表达。转录起始位点上游111个碱基对的序列无法驱动GUS表达超过背景水平，而-111至-571之间的序列在受伤和未受伤组织中具有略高的活性且转录水平相同。进一步上游序列（-571至-1022）的添加使表达水平提高了13至370倍。该片段驱动的表达通过伤口诱导可提高2至9倍。对含有wun1-GUS融合体的转基因植物不同组织进行组织化学分析表明，在含有-1022碱基对片段的植物中，wun1启动子具有伤口诱导性和细胞特异性活性。GUS活性的位置似乎具有细胞特异性，在叶片和茎的表皮细胞中最高，在维管细胞中较低。在含有缺失启动子片段的植物的叶、茎和根中，活性降低到无法通过组织化学染色检测到的水平。含有不同缺失构建体的植物和携带-1022碱基对片段的植物在花药和花粉粒中显示出高表达，且不受伤口刺激。
