Sels Jan, Delauré Stijn L, Aerts An M, Proost Paul, Cammue Bruno P A, De Bolle Miguel F C
Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, Heverlee, Belgium.
Transgenic Res. 2007 Aug;16(4):531-8. doi: 10.1007/s11248-006-9057-8. Epub 2006 Dec 19.
Plant defensins, exhibiting various levels of inhibitory activity against fungal pathogens, are potent candidates for pharmaceutical or agricultural antimycotics. Study of the plant defensins from the model plant Arabidopsis thaliana requires the purification of these peptides. However, heterologous production of defensins for large-scale in vitro bioactivity assays is often experienced as a major problem. In this study we describe the transgenic expression of a previously identified seed-specific and a so far uncharacterized plant defensin gene in their host A. thaliana using a formerly developed plant expression system. Therefore, both genes were cloned in a matrix attachment region (MAR) based plant transformation vector and expressed in post-transcriptional gene silencing (PTGS) impaired A. thaliana plants. The peptides were purified to homogeneity and were correctly processed, as confirmed by mass spectrometry analysis. Finally, they were assessed for their in vitro antifungal activity and mode of antifungal action. Our results indicate that the PTGS-MAR expression system can be applied to obtain significant amounts of bioactive, rightly processed plant peptides from leaves of first generation transgenic plants.
植物防御素对真菌病原体具有不同程度的抑制活性，是制药或农业抗真菌剂的有力候选物。对模式植物拟南芥中的植物防御素进行研究需要对这些肽进行纯化。然而，用于大规模体外生物活性测定的防御素的异源生产常常是一个主要问题。在本研究中，我们描述了使用先前开发的植物表达系统，在其宿主拟南芥中对先前鉴定的种子特异性和迄今未表征的植物防御素基因进行转基因表达。因此，将这两个基因克隆到基于基质附着区域（MAR）的植物转化载体中，并在转录后基因沉默（PTGS）受损的拟南芥植物中表达。通过质谱分析证实，这些肽被纯化至同质且加工正确。最后，评估了它们的体外抗真菌活性和抗真菌作用模式。我们的结果表明，PTGS-MAR表达系统可用于从第一代转基因植物的叶片中获得大量具有生物活性、加工正确的植物肽。
