Ramloch-Lorenz K, Knudsen S, Sturm A
Friedrich Miescher-Institut, Basel, Switzerland.
Plant J. 1993 Sep;4(3):545-54. doi: 10.1046/j.1365-313x.1993.04030545.x.
Carrot cell wall beta-fructosidase, previously purified and cloned, is encoded by a single, wound- and pathogen-inducible gene. The developmental regulation of the gene was studied by determining the steady-state mRNA levels in different organs during carrot development: cell wall beta-fructosidase mRNA was detected in roots and leaves of young plants but not during tap root development. A genomic clone was isolated and characterized. The transcription start site was determined by primer extension analysis. Inspection of the promoter sequence (1488 bp) revealed the presence of sequences with high homology to cis-acting elements for the regulation of plant genes by wounding and infection. The 5'-regulatory sequence was fused to the reporter gene beta-glucuronidase (GUS) and tested in a transient expression assay with carrot suspension cells and wounded carrot root tissue (aged disks of carrot roots). The expression of the GUS gene in the transfected cells proved that the isolated promoter was functional. In transgenic tobacco plants containing the cell wall beta-fructosidase promoter fused to GUS, the reporter gene was predominantly expressed in the shoot and root meristems of young seedlings. No GUS expression was detected in mature tobacco plants, showing that the development-specific regulation of the cell wall beta-fructosidase promoter seen in carrot was maintained in tobacco plants. In contrast, expression of the GUS reporter gene in transgenic tobacco was not wound inducible. To analyze the functional organization of the cell wall beta-fructosidase promoter, a 5'-deletion series was generated and tested in a transient expression assay in protoplasts of Nicotiana plumbaginifolia. Two regions containing putative silencer elements were identified. A comparison of these regions with known silencer elements identified in both regions one copy of the negative dominant cis-acting element found in a chalcone synthase promoter of petunia.
胡萝卜细胞壁β-果糖苷酶此前已被纯化和克隆，由一个单一的、受伤口和病原体诱导的基因编码。通过测定胡萝卜发育过程中不同器官中的稳态mRNA水平，研究了该基因的发育调控：在幼嫩植株的根和叶中检测到了细胞壁β-果糖苷酶mRNA，但在主根发育过程中未检测到。分离并鉴定了一个基因组克隆。通过引物延伸分析确定了转录起始位点。对启动子序列（1488 bp）的检查揭示了与伤口和感染调控植物基因的顺式作用元件具有高度同源性的序列的存在。将5'-调控序列与报告基因β-葡萄糖醛酸酶（GUS）融合，并在胡萝卜悬浮细胞和受伤的胡萝卜根组织（胡萝卜根的老化圆盘）的瞬时表达试验中进行测试。转染细胞中GUS基因的表达证明分离的启动子具有功能。在含有与GUS融合的细胞壁β-果糖苷酶启动子的转基因烟草植株中，报告基因主要在幼苗的茎尖和根分生组织中表达。在成熟烟草植株中未检测到GUS表达，表明在胡萝卜中观察到的细胞壁β-果糖苷酶启动子的发育特异性调控在烟草植株中得以维持。相反，转基因烟草中GUS报告基因的表达不受伤口诱导。为了分析细胞壁β-果糖苷酶启动子的功能组织特性，构建了一个5'-缺失系列，并在垂花烟草原生质体的瞬时表达试验中进行测试。鉴定出了两个含有假定沉默子元件的区域。将这些区域与已知的沉默子元件进行比较，在两个区域中均鉴定出矮牵牛查尔酮合酶启动子中发现的负显性顺式作用元件的一个拷贝。
