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富含亮氨酸重复序列的受体样激酶(leucine-rich repeat receptor-like kinase, LRR-RLK)是一类富含亮氨酸的单跨膜类受体蛋白激酶，也是植物类受体蛋白激酶家族中最大的亚家族。本研究通过分段克隆LRR-RLK家族的氧化应激相关蛋白基因(oxidative-stress related protein kinase 1,AtORPK1)编码序列构建原核表达载体，转化至大肠杆菌BL21,经IPTG诱导获得重组蛋白，并纯化出可溶性的AtORPK1蛋白C端。经免疫兔子获得了多克隆抗体，通过免疫荧光和western blot实验验证多克隆抗体与目的蛋白识别的特异性。本研究成功利用大肠杆菌表达了AtORPK1蛋白C端，并获得特异性良好的多克隆抗体，为快速制备植物富含亮氨酸单跨膜类受体蛋白的抗体提供了有效方法；获得的多克隆抗体可用于检测并鉴定目的蛋白在植物中的表达水平，还可用于免疫荧光分析，快速定位目的蛋白在植物细胞和组织中的分布，为研究植物类受体蛋白激酶信号通路的分子机理奠定基础，也为黄河流域盐碱地抗逆植物的分子育种提供参考依据。
Abstract：LRR-RLK is a class of leucine-rich single transmembrane receptors, which is also the largest subfamily of RLKs in plants.In this study, prokaryotic expression vectors were constructed by cloning the coding sequence of the oxidative stress-related protein gene AtORPK1 of LRR-RLK.The recombinant plasmids were transformed into E.coli BL21,and the cAtORPK1 recombinant proteins were obtained with IPTG induction, then C-terminus of the protein was purified using affinity chromatography and quantified.Subsequently, rabbits were immunized to produce polyclonal antibodies, and the specificity of polyclonal antibodies binding with target protein was verified by immunofluorescence and western blot assay.Here, the cAtORPK1 was successfully expressed in E.coli,and its polyclonal antibodies with good specificity were obtained, providing a method for rapid preparation of plant LRR-RLK.The obtained polyclonal antibodies could be used to detect and identify the expression level of target protein in plants, and could also be used for immunofluorescence to analyze target protein distribution in plant cells and tissues, which laid the foundation for the study of the molecular mechanism of LRR-RLK signaling pathway to cultivate new varieties with drought resistant and water-saving, cold resistance, and salt tolerance by molecular breeding, improving the vegetation coverage and land utilization efficiency of saline soil in the Yellow River basin, and strengthening ecological protection of the Yellow River basin.
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基本信息:
DOI：10.20062/j.cnki.CN37-1453/N.2025.04.009
中图分类号:Q943.2
引用信息:
[1]盛卓然,周厚君,张洪霞.拟南芥类受体蛋白激酶AtORPK1的原核表达与多克隆抗体制备[J].鲁东大学学报(自然科学版),2025,41(04):361-370.DOI:10.20062/j.cnki.CN37-1453/N.2025.04.009.
基金信息:
国家自然科学基金(32001330)