【作者】 韩长磊；

【导师】 孟金陵；

【作者基本信息】 华中农业大学 ， 发育生物学， 2008， 硕士

【摘要】 油菜是世界上重要的油料作物之一,是食用油的重要来源。菌核病是油菜中很流行的腐生性病害,它由核盘菌(Sclerotinia sclerotiorum)感染所致,它主要在油菜生长的成熟期发作,严重影响油菜的产量和品质。已有的研究表明,目前暂没有发现抗菌核病的油菜品种,品种之间只有耐性的不同,油菜本身没有抗性的资源,这一局限成为了油菜抗菌核病育种的瓶颈。然而,转基因技术为油菜抗病育种带来了新的契机。针对油菜菌核病的发病特点,本研究采用了在油菜中衰老特异表达病程相关基因—几丁质酶编码基因CH5B和大麦草酸氧化酶HvOxOh,来提高油菜的抗病性。表达载体SAG12:CH5B、SAG12:HvOxOh和35S:CH5B被用来转化甘蓝型油菜品种Westar10,本实验研究了转化过程中影响转化的限制性因素,包括外植体苗龄大小、共培养时间、激素浓度和抗生素浓度。研究表明:4d苗龄的外植体最适合转化;共培养2d时间较为合适;4mg/L的6-BAP影响外植体的分化;500mg/L的羧苄青霉素限制外植体的生长。分别得到了13株转化SAG12:CH5B、5株转化SAG12:HvOxOh和14株转化35S:CH5B的转基因植株。对部分转基因植株进行了Southern杂交分析,结果证明外源基因已经插入到了转基因植株基因组中。RT-PCR和Northern杂交分析了外源基因在RNA水平上的表达,证实了外源基因在转化SAG12:CH5B和SAG12:HvOxOh的转基因植株中衰老时期特异表达。对几丁质酶活性和草酸氧化酶活性的分析结果表明外源基因的表达产物具有活性,同时具有在衰老时期特异表达的特性。对3种不同类型的转基因植株离体叶片进行了菌核病接种,接种受体分别为转化SAG12:CH5B和SAG12:HvOxOh的转基因植株的下部衰老叶片,转化35S:CH5B的转基因植株的幼嫩叶片,结果表明转基因植株在目前的接种胁迫条件下没有明显表现出对核盘菌抗性的增强。更多还原

【Abstract】 Canola is an important oilseed crop in the world. Stem rot is the most devastating disease in Brassica.napus and causes yield loss and quality decline, uaually it infectes the plants in the age of later growth period. No cultivar with high resistance against Sclerotinia sclerotiorum has been developed in B. napus, resulting from no resource resistance gene in B.napus. Therefore, limited germplasm of B. napus could be used on breeding to enhance the resistance against S. sclerotiorum. Along with the development of biotechnology, the method of transformation could be used on the resistance breeding of B. napus.Chitinase encoding gene CH5B and oxalate oxidase encoding gene HvOxOh were used in the research. Three vectors including SAG12 : CH5B, SAG12 : HvOxOh and 35S: CH5B were used in B. napus Westar10 transformation. The results showed that 4d seedling was most suitable for transformation and co-cultivation time with Agrobacteriun for 48 h was better. The medium with 4 mg/L 6-BAP was not benefit to explants regeneration and 500 mg/L Carbenicillin reduced plants growth.Thirty two transgenic plants were obtained, thirteen, five, and fourteen of which were transformed with the vector of SAG12 : CH5B, SAG12 : HvOxOh and 35S : CH5B, respectively. Southern blot analysis evaluated the insertion of transgenes in transgenic plants and the expression of transgenes were further confirmed by Northern blot and RT-PCR. The activity of chitinase and oxalate oxidase was increased in senescening leaves of transgenic plants. The inoculation with mycelium was conducted in the detached leaves from transgenic plants to evaluate the resistance against S. sclerotiorum. No enhancement of resistance in transgenic plants was detected.更多还原