Khunjan Uraiwan, Ekchaweng Kitiya, Panrat Tanate, Tian Miaoying, Churngchow Nunta
Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand.
Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Manoa, HI, United States of America.
PLoS One. 2016 Jun 23;11(6):e0157591. doi: 10.1371/journal.pone.0157591. eCollection 2016.
This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora.
这是首篇报道，呈现了来自经水杨酸处理的橡胶树（巴西橡胶树）的一个推定的碱性HbPR-1蛋白的全长cDNA（命名为HbPR-1）。对其进行了表征，并利用农杆菌介导的瞬时基因表达系统在本氏烟草中进行表达，以研究HbPR-1基因在橡胶树抵抗卵菌病原体棕榈疫霉中的作用，并生产重组HbPR-1蛋白用于微生物抑制试验。HbPR-1 cDNA长647 bp，包含一个492个核苷酸的开放阅读框，编码163个氨基酸残基，预测分子量为17,681 Da，等电点（pI）为8.56，表明HbPR-1蛋白属于碱性PR-1类型。预测的HbPR-1三维结构由四个α螺旋、三个β折叠、七条链和一个连接环组成。利用农杆菌介导的瞬时表达和一步亲和层析成功实现了重组HbPR-1蛋白的表达和纯化。HbPR-1在本氏烟草中的异源表达减少了接种棕榈疫霉游动孢子的坏死面积，表明表达的HbPR-1蛋白在植物对病原体的抗性中发挥了重要作用。与对照相比，纯化的重组HbPR-1蛋白在水琼脂平板上抑制了64%的棕榈疫霉游动孢子萌发，表明它是一种抗棕榈疫霉的抗菌蛋白。
