Ukrainian National Forestry University, Chuprynka St., 103, Lviv, Ukraine.
Appl Microbiol Biotechnol. 2011 Feb;89(4):1093-101. doi: 10.1007/s00253-010-2935-2. Epub 2010 Oct 19.
Plants produce a variety of molecules to defend themselves from fungal pathogens. Defensins belong to the family of antimicrobial peptides that play a central role in innate immunity in all species of plants. We have previously reported the purification of antimicrobial peptides from Scots pine seedlings and the identification of some of them, including defensin, by mass spectrometry. In this study, we extend our original study on molecular cloning of Pinus sylvestris defensin 1 (PsDef1) by presenting the expression and affinity purification of recombinant defensin 1 (rPsDef1). The full-length coding sequence of PsDef1 has an open reading frame capable to encode a protein of 83 amino residues, including a signal peptide of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5,601.6 Da. We have employed pET system to express mature form of PsDef1 fussed to GST. As GST-PsDef1 fusion protein was not biologically active, we removed GST moiety from the mature defensin 1 peptide by proteolytic cleavage with Factor Xa. The resulting rPsDef1 protein exhibited strong antifungal activity against a panel of pathogenic fungi which is comparable to that of endogenous Scots pine defensin 1. In addition, rPsDef1 was used to produce specific polyclonal antibodies. Using generated antibodies, we found that the level of PsDef1 is significantly increased in Scots pine seedlings during germination and in their response to pathogenic infection with Heterobasidion annosum.
植物会产生多种分子来抵御真菌病原体。防御素属于抗菌肽家族，在所有植物物种的先天免疫中发挥核心作用。我们之前曾报道过从苏格兰松幼苗中纯化抗菌肽，并通过质谱鉴定了其中一些，包括防御素。在这项研究中，我们通过展示重组防御素 1（rPsDef1）的表达和亲和纯化，扩展了我们之前对苏格兰松防御素 1（PsDef1）的分子克隆的原始研究。PsDef1 的全长编码序列具有一个开放阅读框，能够编码一个 83 个氨基酸的蛋白质，其中包括 33 个氨基酸的信号肽，其后是代表其活性形式的 50 个氨基酸的特征防御素结构域。PsDef1 成熟形式的计算分子量为 5601.6 Da。我们使用 pET 系统表达融合到 GST 的成熟形式的 PsDef1。由于 GST-PsDef1 融合蛋白没有生物活性，我们通过用 Factor Xa 进行蛋白水解切割从成熟防御素 1 肽中去除 GST 部分。所得的 rPsDef1 蛋白对一系列病原真菌表现出强烈的抗真菌活性，与内源性苏格兰松防御素 1 相当。此外，rPsDef1 被用于产生特异性的多克隆抗体。使用生成的抗体，我们发现 PsDef1 的水平在苏格兰松幼苗的萌发过程中以及对 Heterobasidion annosum 病原感染的反应中显著增加。
