Fujii N, Yokoyama R, Uchimiya H
Institute of Molecular and Cellular BioSciences, University of Tokyo, Japan.
Plant Physiol. 1994 Apr;104(4):1151-7. doi: 10.1104/pp.104.4.1151.
In cell cultures of carrot (Daucus carota L.), somatic embryogenesis can be induced by transferring cells from a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one devoid of 2,4-D. Previous analysis of transgenic carrot cells containing the 5' non-coding sequence of the Ri plasmid rolC and a structural gene for bacterial beta-glucuronidase (uidA) has shown that the chimeric gene is actively expressed after induction of somatic embryogenesis. In this study, we demonstrate that activation of the rolC promoter is dependent on the process of embryo development but not on the duration of the cell culture in 2,4-D-free medium. We also analyzed the cis region of the rolC promoter that is responsible for somatic embryogenesis-related activation (SERA), namely relatively low beta-glucuronidase (GUS) activity in calli and proembryogenic masses (PEM) and high GUS activity in heart- and torpedo-stage embryos. When the -255-bp region of the rolC gene was used, SERA was retained. Internal deletions within this -255-bp region did not alter SERA by the rolC promoter. Furthermore, when a rolC promoter fragment (-848 to -94 bp) was fused to the cauliflower mosaic virus (CaMV) 35S core region (-90 to +6 bp), it conferred relatively low GUS activity in calli and PEM but high GUS activity in heart and torpedo embryos. When -848 to -255-bp or -255- to -94-bp fragments of the rolC promoter were fused to the same CaMV 35S core region, GUS activity patterns were not related to somatic embryogenesis. These results suggest that the combination of several regulatory regions in the rolC promoter may be required for SERA in carrot cell cultures.
在胡萝卜（Daucus carota L.）的细胞培养中，通过将细胞从含有2,4 - 二氯苯氧乙酸（2,4 - D）的培养基转移至不含2,4 - D的培养基中，可诱导体细胞胚胎发生。先前对含有Ri质粒rolC的5'非编码序列和细菌β - 葡萄糖醛酸酶（uidA）结构基因的转基因胡萝卜细胞的分析表明，嵌合基因在体细胞胚胎发生诱导后被积极表达。在本研究中，我们证明rolC启动子的激活依赖于胚胎发育过程，而非在不含2,4 - D的培养基中的细胞培养持续时间。我们还分析了rolC启动子中负责与体细胞胚胎发生相关激活（SERA）的顺式区域，即在愈伤组织和胚性细胞团（PEM）中相对较低的β - 葡萄糖醛酸酶（GUS）活性以及在心形期和鱼雷期胚胎中较高的GUS活性。当使用rolC基因的 - 255 bp区域时，保留了SERA。该 - 255 bp区域内存在的内部缺失并未改变rolC启动子的SERA。此外，当一个rolC启动子片段（ - 848至 - 94 bp）与花椰菜花叶病毒（CaMV）35S核心区域（ - 90至 + 6 bp）融合时，它在愈伤组织和PEM中赋予相对较低的GUS活性，但在心形期和鱼雷期胚胎中赋予较高的GUS活性。当rolC启动子的 - 848至 - 255 bp或 - 255至 - 94 bp片段与相同的CaMV 35S核心区域融合时，GUS活性模式与体细胞胚胎发生无关。这些结果表明，胡萝卜细胞培养中的SERA可能需要rolC启动子中几个调控区域的组合。
