转无毒基因马铃薯抗晚疫病的研究

晚疫病菌(Phytophthora infestans)引起的马铃薯晚疫病是危害全球马铃薯生产的严重病害.通过基因工程方法把外源基因导入植物体内以增强抗病性被证明是一条行之有效的途径.应用植物基因工程技术将具有能激活植物自身防御系统的无毒基因与适合于植物背景、非专一性的病原物诱导启动子组合成嵌合基因构建到植物表达载体中.通过农杆菌或基因枪的介导转化植物,可筛选出高效广谱的抗真菌和细菌病害的转基因植株.本研究从病原细菌Pseudomonas syringae PV.tomato中获得的无毒基因avrD(0*93kb)和从病原真菌Phytophthora parasitica中获得的无毒基因Elicitin(0.294kb)分别与非专一性病原物诱导启动子Pill和BG组成含2个嵌合基因(Pill-avrD,BG-Elicitin)的植物表达载体pYH144和pYHEt.通过农杆菌LBA4404介导转化马铃薯,其中用pYH144载体转化2个品种(克新1号,2号),用pYHEt载体转化3个品种(Desiree,克新2号,4号),通过组织培养分别获得潮霉素(HygromycinB)标记的转基因马铃薯试管苗.将转基因试管苗扩繁,应用马铃薯脱毒微型种薯生产技术获得转无毒基因微型薯,在温室(15～25℃和湿度高)条件下,观察转无毒基因马铃薯植株中对晚疫病菌自然感染的抗性.1998年和1999年(每年的3-5月)的温室试验初步表明用avrD和elicitin基因分别转化的转无毒基因马铃薯植株都具有较明显的对晚疫病菌侵染的抗性,大部分转基因植株不表现或表现轻微的感病症状,对照植株(未转化)则表现明显的感病症状.转基因植株生长正常,且在感染后期(恢复期)生长良好.对照植株在恢复期生长弱和缓慢.在获得较多数量的转无毒基因马铃薯微型种薯的时候,将进行人工接种晚疫病菌和田间种植试验,从中筛选出抗真菌病和细菌病的转基因马铃薯株系.

Study on resistance of potato with transferred avirulent genes to Phytophthora infestans

Potato late blight caused by Phytophthora infestans is a serious and destructive disease worldwide. To transform exogenous genes to plant and enhance its resistance to diseases by genetic engineering methods has been proved to be an effective way. Using plant genetic engineering techniques, a chimeric gene is constructed by combining an avirulent gene which can activate plant autologous defensive system with a non-specific pathogen induced promoter which is suitable to plant background, and inserted into a plant expressive vector. Then it can be transformed to plant by Agrobacterium tumefaciens or microparticle bombardment. Transgenic plant with effective and extensive resistance can be screened. In this study, an avirulent gene avrD (0.93 kb) isolated from a pathogenic bacteria Pseudomonas syringae PV. tomato and another avirulent gene Elicitin (0.294 kb) isolated from a pathomycete Phytophthora parasitica were ligated respectively with the non-specific pathogen induced promoters Pill and BG to construct plant expressive vectors pYH144 and pYHEt containing the two chimeric genes (Pill-avrD, BG-Elicitin). They were then transformed to potatoes by Agrobacterium tumefaciens LBA 4404. The vector pYH144 was transferred into two potato varieties (Kexin No.1 and No.2), and the vector pYHEt was transferred into three potato varieties (Desiree, Kexin No.2 and No.4). Transgenic potato tube seedlings labelled with Hygromycin B resistance were obtained by tissue culture. Then the transformed seedlings were proliferated. Transgenic potato minitubers were produced by a method of producing virus free potato minitubers. The resistance of the transgenic potato plant naturally infected P. infestans was recorded in greenhouse conditions (15～20℃, with high humidity). The greenhouse experiments in 1998 and 1999 (from March to May every year) primarily showed that the potato seedlings transformed with avrD gene or Elicitin gene have significant resistance to potato late blight, and most of them have no or slight symptoms of the disease, while the control (non-transgenic) potato seedlings have manifest symptom of the disease. The transgenic potato seedlings grow normal, and grow better in the late phase (the recovery phase), while the control potato seedlings grow weaker and slower in the recovery phase. When we have got enough number of the trans-avirulent gene minitubers, we will inoculate P. infestans to the transformed potato plant in the field, and select the transgenic potato plant with high resistance to fungal and bacterial diseases.