导入双价基因的转基因杂交油菜亲本及其对菌核病抗性的研究  

Transgenic Hybrid Parents in Brassica napus Transformed with Bivalent Genes for Resistance to Sclerotinia sclerotiorum

作　　者：王新发[1] 王汉中[1] 刘贵华[1] 胡赞民[2] 郑元本[1] 

机构地区：[1]中国农业科学院油料作物研究所 [2]中国科学院遗传与发育生物学研究所,北京100101

出　　处：《植物学通报》2005年第3期292-301,共10页Chinese Bulletin of Botany

基　　金：国家863计划项目(2001AA241101)

摘　　要：比较了乙酰丁香酮、pH、共培养温度及不同激素配比对根癌农杆菌转化油菜(Brassicanapus)的影响,建立了油菜高效转化体系。按该体系,将组成型表达几丁质酶和β-1,3-葡聚糖酶基因转化甘蓝型双低杂交油菜亲本恢复系和保持系,获得了转基因恢复系和保持系植株。对再生植株进行PCR和SouthernBlot检测,结果表明外源基因已整合到油菜基因组中。连续3代的PCR检测及转基因植株抗病性在自交后代中得到保持,证实外源基因已遗传到T3代。对转基因植株T1代离体叶进行菌核病菌丝体接种和T1及T2代大田自然侵染鉴定,结果表明,转基因恢复系棚40-12、棚40-32和保持系96B-2对菌核病的抗性比受体对照大幅度提高,大田鉴定其发病率连续2年均比受体对照减少78%以上,比抗病品种‘中油821’减少75%以上,病情指数比受体对照和‘中油821’减少均达显著水平,其抗病性能在后代稳定遗传,获得了高抗菌核病的转基因育种材料。After careful study of factors that may affect transformation such as acetosyringone, pH, temperature of co-cultivation with Agrobacterium and hormones, we established an efficient and reproducible transformation method for Brassica napus. Using this protocol, we introduced β-1,3- glucanase and chitinase genes into pol-CMS hybrid parents (restorers and maintainers). Transgenic plants of restorers and maintainers were successfully obtained. PCR and Southern blot analysis revealed that both β-1,3-glucanase and chitinase gene were introduced into the genome of B. napus, and that they were inherited stably to the T3 generation. Inoculation of detached leaves with mycelia and field assessment revealed that the resistance to Sclerotinia sclerotiorum of the T1 and T2 transgenic plants was stronger than that of the control line. In a 2003-2004 field assay, the disease incidence and the index of disease of the transgenic lines Peng40-12, Peng40-32 and 96B-2 was lower than that of the receptor line and resistant ‘Zhongyou821’ by 78% and 75%, respectively, was significantly lower than that of the receptor line and ‘Zhongyou821’.

关 键 词：双价基因 亲本 甘蓝型双低杂交油菜 Southern 转基因恢复系 中油821 抗性 导入 3-葡聚糖酶 外源基因 乙酰丁香酮 农杆菌转化 共培养温度 组成型表达 植株抗病性 PCR检测 转基因植株 保持系 激素配比 转化体系 几丁质酶 

分 类 号：S565.4[农业科学—作物学]