A sucrose binding protein homologue from soybean affects sucrose uptake in suspension-cultured transgenic tobacco cells
A sucrose binding protein homologue from soybean affects sucrose uptake in suspension-cultured transgenic tobacco cells
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DOI：
10.1016/s0981-9428(00)00752-x
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发表时间：
2000-05-01
影响因子：
6.5
通讯作者：
Fontes, EPB
中科院分区：
文献类型：
--
作者：
Delú-Filho, N;Pirovani, CP;Fontes, EPB
我们已经获得了以有义或反义方向表达来自大豆（Glycine max L.）的蔗糖结合蛋白（sbp）同源基因（命名为s-64）的烟草转基因细胞系。有义细胞系过度积累S-64蛋白，而反义细胞系内源同源蛋白的水平降低。通过将悬浮培养的烟草细胞与放射性标记的蔗糖在 pH 4.5 或 7.0 下孵育来进行蔗糖吸收实验。将细胞外 pH 值提高到 7.0 会抑制放射性标记的碳吸收效率，这归因于细胞壁转化酶 (EC 3.2.1.26)、H+/己糖转运蛋白和/或 H+/蔗糖同向转运蛋白活性的 pH 敏感性。由于 SEP 介导的蔗糖摄取已被证明对酵母细胞外 pH 值不敏感，因此我们在 pH 7.0 的有义和反义培养细胞中进行了蔗糖摄取实验。在此条件下，SEP同源物的水平与转基因烟草细胞吸收放射性标记的效率相关。此外，S-64 水平的控制以代谢补偿方式改变了蔗糖裂解活性。 S-64 积累的增加导致细胞内蔗糖合酶（裂解，EC 2.4.1.13）活性增加，同时细胞壁转化酶活性下降。这一结果可能反映了有义细胞系由于其以二糖形式直接摄取蔗糖的高效率而引起的代谢调整；相反，反义细胞中细胞壁转化酶活性水平显着增加，有利于转化酶依赖性糖摄取系统。总的来说，这些结果可能在放射性标记流入和 S-64 积累之间建立了功能联系，表明 SEP 影响悬浮培养细胞中的蔗糖摄取。 (C) 2000 年科学与医学版 Elsevier SAS。
We have obtained Nicotiana tabacum transgenic cell lines expressing a sucrose binding protein (sbp) homologue gene from soybean (Glycine max L.), designated s-64, either in the sense or antisense orientation. Sense cell lines over-accumulated the S-64 protein, whereas the antisense cell lines had reduced levels of the endogenous homologue protein. Sucrose uptake experiments were conducted by incubating suspension-cultured tobacco cells with radiolabeled sucrose at pH 4.5 or 7.0. Raising the extracellular pH to 7.0 caused an inhibition of radiolabeled carbon uptake efficiency, which was attributed to the pH-sensitivity of cell-wall invertase (EC 3.2.1.26), H+/hexose transporter and/or H+/sucrose symporter activities. Because SEP-mediated sucrose uptake has been shown to be insensitive to extracellular pH in yeast, we performed the sucrose uptake experiments in sense and antisense cultured cells at pH 7.0. Under this condition, the level of SEP homologue correlated with the efficiency of radiolabeled uptake by the transgenic tobacco cells. Furthermore, manipulation of S-64 levels altered sucrose-cleaving activities in a metabolic compensatory manner. Enhanced accumulation of S-64 caused an increase in intracellular sucrose synthase (cleavage, EC 2.4.1.13) activity with a concomitant decline in cell-wall invertase activity. This result may reflect a metabolic adjustment of the sense cell lines caused by its high efficiency of direct sucrose uptake as disaccharide; In contrast, the level of cell-wall invertase activity was remarkably increased in antisense cells, favoring the invertase-dependent sugar uptake system. Collectively, these results may establish a functional link between radiolabeled influx and S-64 accumulation, suggesting that SEP affects sucrose uptake in suspension-cultured cells. (C) 2000 Editions scientifiques et medicales Elsevier SAS.