de Bem Matos Alaide Cristina, Telli Elisa Maria Rodriguez Pazinatto, Camillo Luana Coldebella, da Silva Gustavo Felippe, Gonçalves Mayra Juline, Casa Ricardo, Rufato Leo, de Lourdes Borba Magalhães Maria
Programa Multicêntrico de Pós-Graduação Em Bioquímica E Biologia Molecular, Universidade do Estado de Santa Catarina - UDESC, Lages, Santa Catarina, Brasil.
Biomedicina Universidade Do Planalto Catarinense, Lages, Santa Catarina, Brasil.
Probiotics Antimicrob Proteins. 2024 Sep 7. doi: 10.1007/s12602-024-10333-0.
Plant defensins are small antimicrobial proteins (AMP) that participate in the immune defense of plants through their antibacterial, antiviral and antifungal activities. PgD1 is a defensin from Picea glauca (Canadian Pine) and has antifungal activity against plant pathogens. This activity positions it as an alternative biotechnological agent to pesticides commonly used against these plant fungi diseases. The present study aimed to recombinantly produce PgD1 in Escherichia coli to characterize its in vitro antifungal potential against different phytopathogens. To achieve this, the coding gene was amplified and cloned into pET30a( +). Recombinant plasmid was subsequently introduced into E. coli for the soluble expression of defensin PgD1. To evaluate the antifungal activity of the expressed protein, the growth inhibition test was used in solid and liquid media for approximately 7 days against significant plant pathogens, that cause significant crop damage including: Botrytis cinerea, Colletotrichum gloeosporioides, Colletotrichum musae, Colletotrichum graminicola and Fusarium oxysporum. Additionally, stability assessments included temperature variation experiments and inhibition tests using dithiothreitol (DTT). The results showed that there was significant inhibition of the fungal species tested when in the presence of PgD1. Furthermore, defensin proved to be resistant to temperature variations and demonstrated that part of its stability is due to its primary structure rich in cysteine residues through the denaturation test with dithiothreitol (DTT) where the antifungal activity of PgD1 defensin was inhibited. These data indicate that recombinant PgD1 could be utilized as a plant protection technology in agriculture.
植物防御素是一类小的抗菌蛋白（AMP），通过其抗菌、抗病毒和抗真菌活性参与植物的免疫防御。PgD1是一种来自白云杉（加拿大松）的防御素，对植物病原体具有抗真菌活性。这种活性使其成为一种替代生物技术制剂，可用于对抗这些植物真菌病害常用的农药。本研究旨在在大肠杆菌中重组生产PgD1，以表征其对不同植物病原体的体外抗真菌潜力。为实现这一目标，扩增编码基因并将其克隆到pET30a(+)中。随后将重组质粒导入大肠杆菌，以实现防御素PgD1的可溶性表达。为评估表达蛋白的抗真菌活性，在固体和液体培养基中对引起重大作物损害的重要植物病原体进行了约7天的生长抑制试验，这些病原体包括：灰葡萄孢、胶孢炭疽菌、香蕉炭疽菌、禾谷炭疽菌和尖孢镰刀菌。此外，稳定性评估包括温度变化实验和使用二硫苏糖醇（DTT）的抑制试验。结果表明，当存在PgD1时，受试真菌物种受到显著抑制。此外，防御素被证明对温度变化具有抗性，并且通过用二硫苏糖醇（DTT）进行变性试验表明其部分稳定性归因于其富含半胱氨酸残基的一级结构，在该试验中PgD1防御素的抗真菌活性受到抑制。这些数据表明重组PgD1可作为一种农业植物保护技术加以利用。
