| 激发子隐地蛋白基因介导的烟草抗病性研究 |
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| 引用本文： | 蒋冬花,郭泽建,陈旭君,程志强,郑重.激发子隐地蛋白基因介导的烟草抗病性研究[J].农业生物技术学报,2003,11(3):299-304. |
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| 作者姓名： | 蒋冬花 郭泽建 陈旭君 程志强 郑重 |
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| 作者单位： | 1. 浙江大学生物技术研究所,杭州,310029;浙江师范大学生命与环境科学学院,金华,321004
2. 浙江大学生物技术研究所,杭州,310029 |
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| 基金项目： | 国家重点基础研究发展规划项目(G2000016203)和浙江省自然科学基金资助项目(301226;302433). |
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| 摘 要： | 摘要：从隐地疫霉（Phytophthora cryptogea）中克隆cryptogein（Crypt）激发子基因。针对病原菌侵染和Crypt基因的特点，选用能在根、茎、叶等的表皮细胞中特异性表达的、弱组成型并受病原菌诱导的水稻(Oryza sativa )苯丙氨酸解氨酶基因（PAL）的启动子；同时为了使表达的cryptogein蛋白能分泌到细胞外，更好地与植物细胞膜受体互作，在Crypt基因前还融合了病程相关蛋白PR1b的信号肽; 然后将此融合基因构建于植物表达载体CHF3中。通过根癌农杆菌(Agrobacterium tumefancens)介导的叶盘转化法转入烟草(Nicotiana tabacum )。经卡那霉素抗性筛选获22株再生植株，PCR检测都为阳性，部分植株的Southern杂交结果表明,Crypt基因已以低拷贝（1～2个）整合到烟草基因组中。抗病性测定结果表明,转化植株对烟草黑胫病菌(P. parasitica var. nicotianae )、赤星病菌(Alternaria alternata )和野火病菌(Pseudomonas syringae pv. tabaci )的抗性均有提高。
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| 关 键 词： | 关键词：激发子 隐地蛋白 载体构建 转化 抗病性 烟草 |
| 修稿时间： | 2002年6月5日 |
Studies on Tobacco Disease Resistances Mediated by Elicitor Gene of Cryptogein fromPhytophthora cryptogea |
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| Abstract: | Abstract: The cryptogein (Crypt ) gene was obtained by PCR amplification of genomic DNA of Phytophthora cryptogea and verified by DNA sequencing. A promoter of a rice (Oryza sativa ) phenylalanine ammonia-lyase (PAL ) gene, which was low-level constitutive induced by pathogen, and specially expressed in epidermal tissues, was selected to control the expression of Crypt gene. This promoter may exhibit a potential to inhibit the infection of pathogen from epidermal tissues. Meanwhile, for functional interaction of cryptogein protein with its outside membrane binding sites, the Crypt gene was fused to signal sequence of the extracellular pathogenesis-related PR1b protein of tobacco(Nicotiana tabacum ), The final construction was subcloned into a binary vector and transformed into tobacco by useing Agrobacterium-mediated transformation method. Twenty-two kanamycin-resistant tobacco plants were obtained by screening in selective medium with 100 mg/L kanamycin. PCR amplification and Southern blot analysis demonstrated that Crypt gene was integrated into tobacco genome. The transgenic plants were challenged separately, with black shank fungi (Phytophthora parasitica var. nicotianae ) , brown spot fungi (Alternaria alternata ), and wild fire bacterium (Pseudomonas syringae pv. tabaci ). More than half of the plants（68.2%）showed that the resistance was strongly enhanced to these pathogens. Results suggested that a low-level constitutive expression of Crypt gene in tobacco should have a potential use to generate a broad-spectrum disease resistant plants. |
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