Yamamoto Y T, Taylor C G, Acedo G N, Cheng C L, Conkling M A
Department of Genetics, North Carolina State University, Raleigh 27695.
Plant Cell. 1991 Apr;3(4):371-82. doi: 10.1105/tpc.3.4.371.
The expression of the tobacco root-specific gene TobRB7 was characterized. Gel blot hybridizations to RNA isolated from various tobacco tissues demonstrated that steady-state TobRB7 mRNA is not detected in expanded leaf, stem, or shoot apex tissue. To determine the spatial pattern of expression, in situ hybridization to root sections revealed that TobRB7 expression is localized to root meristem and immature central cylinder regions. The 5' flanking region of the gene was studied with respect to its ability to direct root-specific expression. Deletions of 5' flanking sequence were fused to the beta-glucuronidase (GUS) reporter gene and transformed into tobacco. Our data demonstrated that sequences 636 base pairs from the site of transcription initiation are sufficient to direct the root-specific GUS expression in transgenic tobacco, whereas sequences 299 base pairs from the site of transcription initiation fail to direct root-specific expression. A negative regulatory element was apparent between 813 base pairs and 636 base pairs 5' of the transcription initiation site. Histochemical localization of GUS activity in transgenic plants was consistent with in situ hybridization results: GUS activity was localized to the root meristem and central cylinder regions. GUS activity appeared 2 days post-germination in the primary root meristem. In lateral roots, GUS activity was detected from the time of initiation.
对烟草根特异性基因TobRB7的表达进行了表征。与从各种烟草组织中分离的RNA进行的凝胶印迹杂交表明，在展开的叶片、茎或茎尖组织中未检测到稳态TobRB7 mRNA。为了确定表达的空间模式，对根切片进行原位杂交显示，TobRB7表达定位于根分生组织和未成熟的中柱区域。研究了该基因5'侧翼区域指导根特异性表达的能力。将5'侧翼序列的缺失片段与β-葡萄糖醛酸酶（GUS）报告基因融合并转化到烟草中。我们的数据表明，转录起始位点上游636个碱基对的序列足以指导转基因烟草中根特异性GUS的表达，而转录起始位点上游299个碱基对的序列则无法指导根特异性表达。在转录起始位点5'端813个碱基对和636个碱基对之间存在一个负调控元件。转基因植物中GUS活性的组织化学定位与原位杂交结果一致：GUS活性定位于根分生组织和中柱区域。在主根分生组织中，萌发后2天出现GUS活性。在侧根中，从起始时就检测到GUS活性。
