甘蓝型油菜抗病虫双价基因转化体系的建立  

TRANSFORMATION OF BINARY DISEASE AND INSECT RESISTAN CEGENES IN Brassica napus L.

作　　者：刘宏波 郭翔[1] 崔鹏[1] 刘旦[1] 许玲[1] 唐桂香[1] 周伟军[1] 

机构地区：[1]浙江大学作物科学研究所,浙江杭州310029

出　　处：《核农学报》2011年第1期26-31,共6页Journal of Nuclear Agricultural Sciences

基　　金：国家科技支撑计划(2010BAD01B04);国家自然科学基金(30871652);油菜现代产业技术体系建设专项(nycytx-005);浙江省自然科学基金(R307095)

摘　　要：以甘蓝型油菜(Brassica napus L.)品种浙双758子叶柄外植体为受体,建立农杆菌介导转化体系,研究乙酰丁香酮、羧苄青霉素浓度和潮霉素筛选浓度等对农杆菌遗传转化效果的影响。结果发现,羧苄青霉素对农杆菌的抑制效果以500mg/L最佳,且对子叶柄外植体愈伤组织诱导和分化的影响最小,愈伤组织诱导率和芽再生率分别为75.2%和65.1%。5.0mg/L的潮霉素能完全抑制未转化再生植株的生长,使其最终褐化死亡,在此潮霉素筛选浓度下,获得了32株转化再生植株。共培养时培养基中添加100μmol/L AS的芽再生率为3.9%,显著高于未添加AS的芽再生率(2.0%),说明共培养阶段添加酚类物质乙酰丁香酮有利于转化载体T-DNA的转化。部分转化再生植株经PCR和Southern杂交检测呈阳性,病虫害接种试验表明转化植株对菌核病和小菜蛾有较好的抗性。Effects of acetosyringone,carbenicillin and hygromycin concentrations on the transgenic efficiency were studied to establish the transformation systems using leaf-petiole explants of Brassica napus L.cv.Zheshuang 758.The result showed that the optimum concentration of carbenicillin（500 mg/L） for inhibition of Agrobacterium tumefaciens had little effect on the callus induction and differentiation of leaf-petiole explants.The callus induction and differentiation of leaf-petiole explants were at the ratio of 75.2% and 65.1%,respectively.Hygromycin at a concentration of 5.0mg/L was enough to inhibit the growth of non-transformed regeneration plants,causing them browning to death,whereas 32 regenerated plants were obtained at such lethal concentration.The frequency of shoot regeneration on co-cultivation medium containing 100μmol/L of acetosyringone（AS,） was 3.9% significantly higher than those without AS（2.0%）,indicating that addition of phenolic compounds such as AS to the medium were beneficial to the transformation of T-DNA in co-cultivation.Regenerated plants were confirmed by PCR and Southern blot hybridization,and these transgenic lines were proven to be resistant to Plutella xylostella and Sclerotinia sclerotiorum.

关 键 词：甘蓝型油菜 转化体系 PCR SOUTHERN杂交 病虫害抗性 

分 类 号：S565.4[农业科学—作物学]