【作者】 梁辉；

【导师】 孙东发； 贾旭；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2005， 博士

【摘要】 小麦是中国和世界上最重要的粮食作物之一，但其生产正受到虫害日益严重的威胁。应用基因工程手段培育抗虫品种已成为解决小麦虫害的一种有效途径。豫麦66是高产、优质、抗病小麦新品种，但该品种易感蚜虫，限制了其产量潜力的进一步发挥。植物凝集素是一类具有特异糖结合活性的蛋白，存在于许多植物的种子和营养器官中。基于植物凝集素和糖的特异相互识别的特性，一般认为植物凝集素不仅在植物体内具有一定的功能，如作为氮源、特异的识别因子等，更重要的是它还可能作为防御蛋白发挥作用。已经发现多种植物凝集素具有抵抗农业害虫的特性，特别是对蚜虫等同翅目害虫具有抗杀作用。此外，植物凝集素还可以抵抗植物病原性线虫、真菌及病毒的侵染。雪花莲凝集素是植物凝集素的一种，它属于单子叶甘露糖结合凝集素家族，对具有刺吸式口器的同翅目害虫具有毒杀作用。本研究用基因枪法将雪花莲凝集素基因（GNA14）转入普通春小麦品种中-60634和生产上正在推广的冬小麦高产品种——豫麦66中，分别获得了抗蚜性良好的转基因抗蚜小麦新材料。但实验中发现，基因枪转化法不仅转化频率偏低（平均1％左右），而且使用费用偏高，限制了大规模基因工程分子育种工作的开展。故应用一种适用于普通24孔培养板的便携式、可重复使用的环形电极，将外源基因转入完整的小麦幼胚组织并获得稳定遗传的转基因植株，建立了一种环形电极电激法介导的简便、快速、高效、经济的高频率小麦基因转化系统。 1．应用基因枪转化法将雪花莲凝集素基因（GNA14）转入普通春小麦品种中-60634和生产上正在推广的冬小麦高产品种——豫麦66中，分别获得了转基因小麦植株。对转基因小麦当代及其后代进行PCR及Southern杂交检测，证明外源基因已稳定整合到小麦基因组中。中-60634和豫麦66的外源基因转化频率均为0.37％。 2．抗蚜实验证明，转化gna基因的小麦植株对我国北方冬麦区的主要麦蚜——麦长管蚜和禾谷缢管蚜的抗性效果不尽相同。对禾谷缢管蚜，在接种当代即表现出明显的毒杀作用。对麦长管蚜，则表现为虫体发育减缓并且降低了其所生产的若蚜成活率。在自然放养条件下，gna基因则对这两种麦蚜的取食均起到了一定的抑制作用。 3．获得20个豫麦66抗蚜新材料分别来自由8个独立的转基因T₀代抗蚜植株所获得的73个T₁代转基因单株。T₀代转基因植株与对照相比对蚜虫的抗性表现出极为显著的差异，特别是禾谷缢管蚜在生态盒中的死亡率高达47.1％～71.7％，比对照（16.0％）提高31.1％～55.7％。T₁代植株开放接蚜鉴定，3w后观察发现，转基因植株未找到任何蚜虫，推测可能迁移至对照植株上。2个中-60634抗蚜新材料表现为减缓蚜虫虫体发育并且降低若蚜成活率。更多还原

【Abstract】 Wheat （Triticum aestivum L.） is an important and a major crop not only in China but also in the world. The product loss of wheat due to insects is very high in recent years. Biotechnology is a valid way on insect control comparing with conventional insecticides. Yumai66 is a new commercial variety of wheat with high yield, desirable grain quality and resistance disease, which had been examined and approved by National Crop Variety Approval Committee in 2003. The yield of Yumai66 in each hectare is 10,812 kilogrammes that is equal to 720.8 kilogrammes per Chinese mu, which has achieved the highest yield level of wheat with desirable grain quality in Huang Huai region of China. However, Yumai66 is susceptible to the infection of aphids. Plant lectin is a family of proteins with specific carbohydrate-binding activity distributing in seeds as well as different types of plant vegetative tissues. On the basis of carbohydrate-binding specificity, plant lectins are proposed to not only functions as nitrogen resource or specific recognition factors, but also involved in defense of plant. Plant lectins have deleterious effects on insects, especially against the members of Homoptera. Toxicity of plant lectins towards nematodes, fungi and virus was also reported. Snowdrop lectin （Galanthus nivalis agglutinin; GNA）, as a kind of plant lectins, is belong to monocotyledon mannose-binding lectin that is toxic to sap sucking injurious insects of Homopteran. GNA14 gene has been successfully transferred into a common spring wheat Zhong60634 and a winter wheat "Yumai66" by using the biolistic transformation method in my laboratory. The transgenic plants with good resistance to aphid were obtained either in Zhong60634 or in Yumai66. However, the transformation efficiency by particle bombardment is not only low but expensive, which limits development of molecular breeding. Plasmid DNA was delivered into the tissue of intact wheat immature embryos and transgenic plants with stable integration and inheritance were obtained by a portable and permanent ring electrode which fitted into the wells of 24-well plates in my experiments. A simple, fast, efficient and inexpensive wheat transformation procedure was developed by electroporation with ring electrode-mediated gene transfer, which takes a possibility for molecular breeding via genetic engineering in a large scale. 1. GNA14 gene has been transferred respectively into common spring wheat Zhong60634 and winter wheat Yumai66 by using the biolistic transformation method. Transgenic wheat plants have been obtained in both of the two varieties. PCR and Southern blot analysis confirmed the integration of the foreign gene in the transgenic plants genome. Two transgenic plants （T₀） were obtained from the bombarded 535更多还原