Stotz H U, Long S R
Howard Hughes Medical Institute, Department of Biological Sciences, Stanford University, CA 94305-5020, USA.
Plant Mol Biol. 1999 Nov;41(5):601-14. doi: 10.1023/a:1006338401808.
Microtubules are thought to be major determinants of plant morphogenesis, through effects on planes of cell division and on directions of differential cell expansion. In differentiation and redifferentiation processes, tubulin expression may prove a useful early indicator of cell activity. We examined the expression and localization of the pea alpha-tubulin gene TubA1 in situ and in transgenic alfalfa (Medicago sativa) to explore its use as a probe for plant development, and as a test case for correct developmental expression between two legume species commonly compared for studies of symbiosis with Rhizobium. The TubA1 mRNA was more abundant in root tips and immature leaves than in other tissues of pea. The promoter of TubA1 was fused to beta-glucuronidase (GUS) to analyze alpha-tubulin expression in transgenic alfalfa. Transient assays indicated that the TubA1 gene is transcribed at moderate levels compared to the cauliflower mosaic virus (CaMV) 35S promoter. Histochemical staining for GUS activity confirmed a correlation between TubA1 expression and cell division in nodules, roots and leaves. TubA1 promoter activity was first detected in the inner cortex of the root between 18 h and 24 h after spot inoculation with Rhizobium meliloti. Expression of a c-myc epitope fused to the carboxy-terminus of TubA1 resulted in an incorporation into the microtubular cytoskeleton, demonstrating the effectiveness of at least one epitope tag in creating functional tubulin fusions.
微管被认为是植物形态发生的主要决定因素，通过影响细胞分裂平面和细胞差异扩展方向来实现。在分化和再分化过程中，微管蛋白的表达可能是细胞活性的一个有用的早期指标。我们研究了豌豆α-微管蛋白基因TubA1在原位和转基因苜蓿（紫花苜蓿）中的表达和定位，以探索其作为植物发育探针的用途，并作为比较与根瘤菌共生研究的两种豆科植物之间正确发育表达的测试案例。TubA1 mRNA在豌豆根尖和未成熟叶片中比在其他组织中更丰富。将TubA1的启动子与β-葡萄糖醛酸酶（GUS）融合，以分析转基因苜蓿中α-微管蛋白的表达。瞬时分析表明，与花椰菜花叶病毒（CaMV）35S启动子相比，TubA1基因以中等水平转录。GUS活性的组织化学染色证实了TubA1表达与根瘤、根和叶中的细胞分裂之间的相关性。在用苜蓿根瘤菌点接种后18小时至24小时之间，首次在根的内皮层中检测到TubA1启动子活性。与TubA1羧基末端融合的c-myc表位的表达导致其整合到微管细胞骨架中，证明了至少一种表位标签在创建功能性微管蛋白融合体方面的有效性。
