拟南芥HyPRP蛋白AZI1在转基因烟草中的亚细胞定位及其对灰葡萄孢的抗性  

Subcellular Localization and Resistance to Botrytis cinerea of the Arabidopsis HyPRP Protein AZI1 in Transgenic Tobacco

作　　者：杜改亮[1] 高航[1] 徐子勤[1] 

机构地区：[1]西北大学生命科学学院,陕西省生物技术重点实验室,西部资源与现代生物技术省部共建教育部重点实验室,西安710069

出　　处：《植物生理学报》2012年第8期795-803,共9页Plant Physiology Journal

基　　金：国家自然科学基金(30870194);陕西省重点实验室科研计划(08JZ70和2010JS090);陕西省教育厅科研计划(11JK0612);陕西省科学技术研究发展计划(社发攻关2010K16-04-01)

摘　　要：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。Transgenic tobacco plants harbouring AZll ofArabidopsis thaliana were regenerated by Agrobacterium mediated transformation method. In subsequent experiments, these plants were used in determination of the subcellular localization of AZI1 and identification of the resistance to Botrytis cinerea. A NcoI site and a SpeI site were introduced into the 5＇ end of the forward primer and the reverse primer respectively. The coding sequence of AZll were amplified with Pfu from genomic DNA ofArabidopsis ecotype Col-0. NcoI and SpeI di- gested PCR product and pCAMBIA1302 were ligated with T4 DNA ligase to produce the funsion expression vector ofAZI1-GFP. Agrobacteriurn tumefaciens strain containing the fusion expression vector was used in genetic transformation of tobacco leaves. Transgenic plants were regenerated by hygromycin selection. Observation under laser scanning confocal microscope showed that AZI1 was mainly localized to cell surface. Results of pathogen inoculation indicated that AZ11 could improve the resistance of tobacco plants to B. cinerea effectively. It suggests that the secreted form of AZI1 can repress the infection process of pathogenic fungi to plant tissues.

关 键 词：AZI1基因 '秦烟95' 亚细胞定位 灰葡萄孢 DAB染色 台盼蓝染色 

分 类 号：S572[农业科学—烟草工业]