高效抗虫基因PinⅡ转化水稻的研究

Studies on transforming high efficiency insect-resistant gene Pin Ⅱ into rice

利用农杆菌介导的转化方法对1个籼稻品种和3个粳稻品种进行了转化。所用质粒为pCAMBI1300,其上带有马铃薯蛋白酶抑制剂基因PinⅡ和高效启动子ACTⅠ以及调节子Intron。通过添加有利于转化的物质乙酰丁香酮及影响转化的各因素(农杆菌的培养方法、共培养天数、愈伤组织诱导方法及继代次数)进行综合优化后,建立了农杆菌介导的水稻高效转化体系。将成熟胚用pCAMBI1300/LBA4404浸染后,筛选出抗性愈伤组织并获得转化植株。其中抗性愈伤组织产生率最高达51 8%(粳稻)和31 5%(籼稻),转化植株再生率最高达67 6%(粳稻)和47 5%(籼稻)。初筛苗中PCR阳性苗得率最高达33 3%(粳稻)和27 4%(籼稻)。PCR结果初步表明PinⅡ基因已整合到水稻基因组中。田间抗虫试验初步表明,阳性苗虫害率低于其受体亲本虫害率。本研究还在转基因水稻中发现了性状明显变异的植株,是进行水稻遗传背景分析和优良性状利用的宝贵材料。

One japonica variety and three indica varieties were applied in the genetic transformation of rice by using Agrobacterium tumefaciens LBA4404 harboring binary vector containing Pin Ⅱ and high efficiency promoter ACT I and ACT I intron.The system of high efficiency transformation was established by optimizing many factors involving in tranformation effect,which were methods of callus induction,days of co-culture and adding Ace to medium and etc.Callus derived from mature embryos of rice were infected and co-cultured with Agrobacterium,and many transgenic rice plants were obtained.The highest transformation frequency was 51.8?%(japonica) and 31.5?% (indica),respectively.The ratio of transgenic plants regenerated from selected calli was 67.6?% (japonica) and 47.5?%(indica).The amplified PCR results indicated that insect-resistant gene was integrated into rice genome and the percentage of positive plants was 33.3?% (japonica) and 27.4?% (indica). The putative transformed plants were conformed by insect-resistant assay in field.Pin Ⅱ had effect on transgenic plants because the frequency of destroyed plants by insects was lower than that of its original no-transgenic plants.Many plants,of which agricultural characters were obviously changed,were obtained,and were useful materials for studying its genetic backgrounds and rice breeding.