Reduced height-1 dwarfing alleles affect DELLA proteins belonging to a family of putative transcriptional regulators that modulate plant growth and development. The Arabidopsis thaliana genome encodes five DELLA proteins, whereas monocot plants, such as rice, barley, and wheat, each have a single DELLA protein. In wheat, wild-type Rht-B1a and Rht-D1a genes encode DELLA proteins and have many alleles that contain lesions. Among them, Rht-B1b and Rht-D1b are the most common mutant dwarfing alleles, which have played a key part in the creation of high-yielding wheat varieties. Despite their fundamental roles in plant biology, until now, DELLA proteins in wheat have been mainly researched regarding the phenotypic effect of defective Rht mutants on yield-related traits, without studies on the underlying mechanisms. The RHT-1 protein has yet to be detected in wheat tissues, owing to a lack of appropriate molecular tools for characterization of RHT function and protein interactions in signal transduction. This study is focused on the production of a polyclonal antibody to the wheat RHT-D1A protein. To generate the anti-RHT-D1A antibody, we expressed and purified soluble 6xHis-tagged RHT-D1A. The purified recombinant RHT-D1A was injected into New Zealand white rabbits to generate polyclonal antiserum. The polyclonal anti-RHT-D1A antibody was purified by ammonium sulfate precipitation, followed by affinity chromatography on protein A–agarose beads. The purified polyclonal antibody was demonstrated to be effective in immunoblotting, western blot hybridization, and immunoprecipitation. In wheat seedling extracts, the polyclonal antibody recognized a protein with a molecular mass close to the predicted molecular weight of the endogenous RHT-D1A protein. We also demonstrated that RHT-D1A disappears in response to exogenous and endogenous gibberellic acid. The purified polyclonal antibody raised against the recombinant RHT-D1A protein is sufficiently specific and sensitive and could be a useful tool for future insights into upstream and downstream components of DELLA-regulatory mechanisms in wheat plants.

中文翻译：

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针对重组表达的普通小麦RHT-D1A蛋白的多克隆抗体。

高度1矮化的等位基因减少会影响DELLA蛋白质，DELLA蛋白质属于推测的转录调节因子家族，可调节植物的生长和发育。拟南芥基因组编码五个DELLA蛋白，而单子叶植物，例如水稻，大麦和小麦，每个都有一个DELLA蛋白。在小麦中，野生型Rht-B1a和Rht-D1a基因编码DELLA蛋白，并具有包含病灶的许多等位基因。其中，Rht-B1b和Rht-D1b是最常见的突变矮化等位基因，在高产小麦品种的创造中发挥了关键作用。尽管它们在植物生物学中起着基础性的作用，但迄今为止，对小麦中的DELLA蛋白的研究主要是关于有缺陷的Rht突变体对产量相关性状的表型影响的，尚未研究其潜在机理。由于缺乏用于表征信号传导中RHT功能和蛋白质相互作用的分子工具，因此尚未在小麦组织中检测到RHT-1蛋白。这项研究的重点是针对小麦RHT-D1A蛋白的多克隆抗体的生产。为了生成抗RHT-D1A抗体，我们表达并纯化了可溶性6xHis标签的RHT-D1A。将纯化的重组RHT-D1A注射到新西兰白兔中以产生多克隆抗血清。通过硫酸铵沉淀纯化多克隆抗RHT-D1A抗体，然后对蛋白A-琼脂糖珠进行亲和层析。纯化的多克隆抗体被证明在免疫印迹，蛋白质印迹杂交和免疫沉淀中有效。在小麦幼苗提取物中，多克隆抗体识别分子量接近内源RHT-D1A蛋白预测分子量的蛋白。我们还证明，RHT-D1A在响应外源和内源赤霉素时消失了。产生的针对重组RHT-D1A蛋白的纯化多克隆抗体具有足够的特异性和敏感性，并且可能是将来了解小麦植物中DELLA调控机制上游和下游组件的有用工具。