TARGET OF RAPAMYCIN (TOR), a member of the phosphatidylinositol 3-kinase-related family of protein kinases, is encoded by a single, large gene and is evolutionarily conserved in all eukaryotes. TOR plays a role as a master regulator that integrates nutrient, energy, and stress signaling to orchestrate development. TOR was first identified in yeast mutant screens, as its mutants conferred resistance to rapamycin, an antibiotic with immunosuppressive and anticancer activities. In Arabidopsis thaliana, the loss-of-function tor mutant displays embryo lethality, but the precise mechanisms of TOR function are still unknown. Moreover, a lack of reliable molecular and biochemical assay tools limits our ability to explore TOR functions in plants. Here, we produced a polyclonal α-TOR antibody using two truncated variants of TOR (1–200 and 1113–1304 amino acids) as antigens because recombinant full-length TOR is challenging to express in Escherichia coli. Recombinant His-TOR1−200 and His-TOR1113−1304 proteins were individually expressed in E. coli, and a mixture of proteins (at a 1:1 ratio) was used for immunizing rabbits. Antiserum was purified by an antigen-specific purification method, and the purified polyclonal α-TOR antibody successfully detected endogenous TOR proteins in wild-type Arabidopsis and TOR orthologous in major crop plants, including tomato, maize, and alfalfa. Moreover, our α-TOR antibody is useful for coimmunoprecipitation assays. In summary, we generated a polyclonal α-TOR antibody that detects endogenous TOR in various plant species. Our antibody could be used in future studies to determine the precise molecular mechanisms of TOR, which has largely unknown multifunctional roles in plants.

中文翻译：

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抗拟南芥雷帕霉素靶标的多克隆抗体的产生，表征和交叉反应

雷帕霉素的靶标（TOR）是磷脂酰肌醇3激酶相关蛋白激酶家族的成员，由单个大基因编码，并且在所有真核生物中都是进化保守的。TOR发挥主要调节器的作用，将营养，能量和压力信号整合到协调发展中。TOR首先在酵母突变体筛选中被鉴定出来，因为它的突变体赋予了雷帕霉素抗性，雷帕霉素是一种具有免疫抑制和抗癌活性的抗生素。在拟南芥中，功能丧失的tor突变体表现出胚胎致死性，但TOR功能的确切机制仍不清楚。此外，缺乏可靠的分子和生化分析工具也限制了我们探索植物中TOR功能的能力。这里，我们使用两种截短的TOR变体（1–200和1113–1304个氨基酸）作为抗原生产了多克隆α-TOR抗体，因为重组全长TOR在大肠杆菌中难以表达。重组His-TOR1-200和His-TOR1113-1304蛋白在大肠杆菌中单独表达，并使用蛋白混合物（以1：1的比例）免疫兔。通过抗原特异性纯化方法纯化抗血清，并且纯化的多克隆α-TOR抗体成功检测到野生型拟南芥中的内源性TOR蛋白和主要农作物（包括番茄，玉米和苜蓿）中直系同源的TOR。此外，我们的α-TOR抗体可用于免疫共沉淀测定。总之，我们产生了一种多克隆α-TOR抗体，可以检测各种植物物种中的内源性TOR。