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首页> 外文期刊>Plant and Cell Physiology >Molecular Cloning, Functional Expression and Subcellular Localization of Two Putative Vacuolar Voltage-gated Chloride Channels in Rice (Oryza sativa L.)
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Molecular Cloning, Functional Expression and Subcellular Localization of Two Putative Vacuolar Voltage-gated Chloride Channels in Rice (Oryza sativa L.)

机译：水稻中两个推测的液泡电压门控氯化物通道的分子克隆，功能表达和亚细胞定位

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摘要

We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.
机译：我们从水稻（Oryza sativa L. ssp。japonica，cv。Nipponbare）中分离了两个与烟草CLC-Nt1同源的cDNA克隆（OsCLC-1和OsCLC-2），它编码一个电压门控的氯离子通道。推导的氨基酸序列是高度保守的（彼此的87.9％同一性）。水稻基因组DNA的Southern印迹分析表明，OsCLC-1和OsCLC-2分别是染色体4和2的单拷贝基因。 OsCLC-1在大多数组织中表达，而OsCLC-2仅在根，节，节间和叶鞘中表达。用NaCl处理可提高OsCLC-1的表达水平，但不会提高OsCLC-2的表达水平。通过在离子胁迫下恢复生长，两个基因都可以部分替代GEF1，GEF1编码酵母中的唯一氯离子通道。这些结果表明两个基因都是氯通道基因。在液泡膜级分中免疫化学检测了来自这两个基因的蛋白质。标记的合成绿色荧光蛋白，融合到液泡膜中的OsCLC-1或OsCLC-2。这些结果表明蛋白质可能在氯离子跨液泡膜的运输中起作用。我们从在外显子区域插入反转录转座子Tos17产生的一组水稻突变体中分离了这两个基因的功能缺失突变体，发现在所有生命阶段均具有生长抑制作用。

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