F-box proteins play important roles in the regulation of various developmental processes in plants. Approximately 1796 F-box genes have been identified in the wheat genome, but details of their functions remain unknown. Moreover, not much was known about the roles of kelch repeat domain-containing F-box genes (TaKFBs) in wheat. In the present study, we isolated five TaKFBs to investigate the roles of KFBs at different stages of colored wheat grain development. The cDNAs encoding TaKFB1, TaKFB2, TaKFB3, TaKFB4, and TaKFB5 contained 363, 449, 353, 382, and 456 bp open reading frames, respectively. All deduced TaKFBs contained an F-box domain (IPR001810) and a kelch repeat type 1 domain (IPR006652), except TaKFB2. Expression of TaKFBs was elevated during the pigmentation stages of grain development. To clarify how TaKFB and SKP interact in wheat, we investigated whether five TaKFB proteins showed specificity for six SKP proteins using a yeast two-hybrid (Y2H) assay. An Y2H screen was performed to search for proteins capable of binding the TaKFBs and interaction was identified between TaKFB1 and aquaporin PIP1. To examine the subcellular localization of TaKFBs, we transiently expressed TaKFB-green fluorescent protein (GFP) fusions in tobacco leaves; the TaKFB-GFP fusions were detected in the nucleus and the cytoplasm. Y2H and bimolecular fluorescence complementation (BiFC) assays revealed that TaKFB1 specifically interacts with aquaporin PIP1. These results will provide useful information for further functional studies on wheat F-box proteins and their possible roles.

中文翻译：

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从有色小麦中分离和鉴定含有海带重复序列的F-box蛋白。

F-box蛋白在调节植物各种发育过程中起重要作用。在小麦基因组中已鉴定出约1796个F-box基因，但其功能细节尚不清楚。此外，关于小麦中含有海带重复结构域的F-box基因（TaKFBs）的作用的了解还很少。在本研究中，我们分离了五个TaKFB，以研究KFB在有色小麦籽粒发育的不同阶段的作用。TaKFB1，TaKFB2，TaKFB3，TaKFB4和TaKFB5编码的cDNA分别包含363、449、353、382和456 bp开放阅读框。除TaKFB2外，所有推导的TaKFB都包含一个F-box域（IPR001810）和一个海带重复1型域（IPR006652）。TaKFBs的表达在谷物发育的色素沉着阶段升高。为了阐明TaKFB和SKP在小麦中如何相互作用，我们使用酵母双杂交（Y2H）分析研究了五种TaKFB蛋白是否对六种SKP蛋白具有特异性。进行了Y2H筛选以搜索能够结合TaKFB的蛋白质，并确定了TaKFB1和水通道蛋白PIP1之间的相互作用。为了检查TaKFBs的亚细胞定位，我们在烟草叶片中瞬时表达了TaKFB-绿色荧光蛋白（GFP）融合蛋白。在细胞核和细胞质中检测到TaKFB-GFP融合蛋白。Y2H和双分子荧光互补（BiFC）分析表明TaKFB1与水通道蛋白PIP1特异性相互作用。这些结果将为进一步研究小麦F-box蛋白及其可能作用提供有用的信息。进行了Y2H筛选以搜索能够结合TaKFB的蛋白质，并确定了TaKFB1和水通道蛋白PIP1之间的相互作用。为了检查TaKFBs的亚细胞定位，我们在烟草叶片中瞬时表达了TaKFB-绿色荧光蛋白（GFP）融合蛋白。在细胞核和细胞质中检测到TaKFB-GFP融合蛋白。Y2H和双分子荧光互补（BiFC）分析表明TaKFB1与水通道蛋白PIP1特异性相互作用。这些结果将为进一步研究小麦F-box蛋白及其可能作用提供有用的信息。进行了Y2H筛选以搜索能够结合TaKFB的蛋白质，并确定了TaKFB1和水通道蛋白PIP1之间的相互作用。为了检查TaKFBs的亚细胞定位，我们在烟草叶片中瞬时表达了TaKFB-绿色荧光蛋白（GFP）融合蛋白。在细胞核和细胞质中检测到TaKFB-GFP融合蛋白。Y2H和双分子荧光互补（BiFC）分析表明TaKFB1与水通道蛋白PIP1特异性相互作用。这些结果将为进一步研究小麦F-box蛋白及其可能作用提供有用的信息。我们在烟草叶片中瞬时表达TaKFB-绿色荧光蛋白（GFP）融合蛋白；在细胞核和细胞质中检测到TaKFB-GFP融合蛋白。Y2H和双分子荧光互补（BiFC）分析表明TaKFB1与水通道蛋白PIP1特异性相互作用。这些结果将为进一步研究小麦F-box蛋白及其可能作用提供有用的信息。我们在烟草叶片中瞬时表达TaKFB-绿色荧光蛋白（GFP）融合蛋白；在细胞核和细胞质中检测到TaKFB-GFP融合蛋白。Y2H和双分子荧光互补（BiFC）分析表明TaKFB1与水通道蛋白PIP1特异性相互作用。这些结果将为进一步研究小麦F-box蛋白及其可能作用提供有用的信息。