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通过农杆菌转化法得到了整合有拟南芥AZI1基因的烟草植株,进一步利用转基因烟草分析了AZI1蛋白的亚细胞定位及其对真菌病原体的抗性特征。在上下游引物5’端分别引入NcoI和SpeI酶切位点,采用高保真耐热DNA聚合酶Pfu从拟南芥Col-0生态型基因组DNA扩增AZI1基因的编码序列,用NcoI和SpeI对扩增片段和pCAMBIA1302质粒载体进行双酶切,通过T4DNA连接酶构建产生AZI1-GFP融合表达载体。用包含融合表达载体的农杆菌细胞转化烟草叶片,经潮霉素选择获得了完整的再生植株,并收取了T0代种子。激光共聚焦显微观察发现,AZI1蛋白主要定位于细胞表面。病原体侵染结果显示,AZI1基因能够明显提高烟草对灰葡萄孢的抗性。说明AZI1蛋白通过分泌途径被定位到细胞表面后,能够抑制真菌病原体对植物组织的侵染过程。
Tobacco plants with the Arabidopsis AZI1 gene were obtained by Agrobacterium tumefaciens transformation. The subcellular localization of AZI1 protein and its resistance to fungal pathogens were further analyzed using transgenic tobacco plants. The NcoI and SpeI restriction sites were introduced into the 5 ’end of the upstream and downstream primers respectively. The coding sequence of AZI1 gene was amplified from genomic DNA of Arabidopsis Col-0 genomic DNA using Pfu, a high-fidelity thermostable DNA polymerase. NcoI and SpeI The amplified fragment and pCAMBIA1302 plasmid vector were double-digested to construct AZI1-GFP fusion expression vector by T4 DNA ligase. Tobacco leaves were transformed with Agrobacterium tumefaciens containing the fusion expression vector and intact regenerated plants were selected by hygromycin selection and T0 seeds were collected. Laser confocal microscopy showed that AZI1 protein mainly localized on the cell surface. Pathogen infection results show that AZI1 gene can significantly enhance the resistance of tobacco to Botrytis cinerea. It shows that AZI1 protein can inhibit the infection of plant pathogens by the pathogen after it is localized to the cell surface through the secretory pathway.