【作者】 陈浩；

【导师】 张启发； 林拥军；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 水稻是主要粮食作物之一，全世界有超过20亿的人口以水稻为主食。虫害是造成水稻减产的主要原因之一。在现代农业中，使用化学杀虫剂为减少虫害损失起到了巨大作用。但是，化学杀虫剂的使用会不可避免的造成环境污染以及威胁人类健康。 转基因抗虫作物是近些年兴起的一种新的害虫防治技术，它从1996年开始在世界上大规模种植。这种转基因作物可以表达一种细菌Bacillus thuringiensis(Bt)来源的杀虫蛋白。这种细菌作为一种生物杀虫剂在农业上已经使用了60多年了。Bt作物在农业上的使用可以大幅度的减少杀虫剂的使用量，这不仅直接降低了种植者的种植成本，而且对保护人类的健康和生态环境都有积极的意义。 本研究通过农杆菌介导的方法，将3个人工合成的Bt基因(cry1Ac、cry2A~*和cry9C~*)分别转入优良的水稻恢复系明恢63中，并获得了一批抗虫性优良的转基因家系。本研究获得的主要研究结果如下： 1．证实了cry2A~*和cry9C~*这两个由本实验室设计合成的Bt基因能够在转基因水稻中正常表达，转基因植株表现出了良好的抗虫性。因此，这两个基因的人工改造是成功的。 2．获得了11个外源基因单拷贝插入、抗虫性优良且农艺性状较好的纯合转基因明恢家系，其中Cry1Ac家系2个(命名为TAc-1和TAc-2)，Cry2A~*家系4个(命名为T2A-1、T2A-2、T2A-3和T2A-4)，Cry9C~*家系5个(命名为T9C-1、T9C-2、T9C-3、T9C-4和T9C-5)。这些转基因家系的选择标准为：高抗虫性，没有明显农艺性状的改变，单拷贝插入和后代分离符合孟德尔遗传规律。这些抗性家系不仅可以用来作为新的抗性育种资源，也可以用于发展双价Bt水稻。 3．通过室内的抗虫性生物检测，所有的Bt转基因水稻家系均表现出对两种主要的水稻钻蛀害虫三化螟(Tryporyza incertulas walker)和二化螟(Chilo suppressalis walker)的高度抗性。结果显示，所有转基因家系可在5天内彻底杀死一龄三化螟幼虫；彻底或者近似彻底地杀死一龄二化螟幼虫。 4．研究了二化螟幼虫对Bt水稻茎杆取食的选择性。结果表明，在同时喂饲Bt转基因水稻茎杆和原品种明恢63茎杆时，二化螟幼虫可以辨识Bt茎杆和非Bt茎杆。在仅有3种Bt转基因水稻茎杆(含Cry1Ac，Cry2A~*和Cry9C~*)被喂饲时，二化螟幼虫更多还原

【Abstract】 Rice is one of the most important crops, and the staple food for over two billion people in the world. Insect damage is one the major cause of yield loss. Synthesized pesticides have significantly contributed to decreasing yield losses caused by the pests in modern agriculture. However, the long-term employment of synthesized pesticide has inevitably led to the environmental pollution and human health problems.As a novel pest control technology, genetic modified (GM) insect-resistant crops began to grow on a large scale in 1996. These insect-resistant crops can express the insecticidal proteins from Bacillus thuringiensis (Bt) that has been used in agricultural production as a biological insecticide for more than 60 years. Commercialization of Bt crops has significantly reduced the use of synthetic insecticides. It not only cut down the cost of the growers, but also is friendly to environment and human health.In this study, three modified-synthesized Bt genes (cry1Ac, cry2A~* and cry9C~*) were introduced into an elite rice CMS restorer line Minghui 63 respectively, and an array of high insect-resistant transgenic lines were obtained. The main results in this study are as follows:1. It was confirmed that the two Bt genes (cry2A~* and cry9C~*), which were designed and synthesized in our lab, could express effectively in the transgenic rice plants. And the transgenic plants exhibited excellent insect resistance. Thus, the artificial modification of the two Bt genes is successful.2. Eleven homozygous transgenic lines, including two CrylAc lines (designated as TAc-1 and TAc-2), four Cry2A~* lines (designated as T2A-1, T2A-2, T2A-3 and T2A-4) and five Cry9C~* lines (designated as T9C-1, T9C-2, T9C-3, T9C-4 and T9C-5), were obtained in this study. The selection of these transgenic lines followed the same criteria: high insect-resistance, no significant phenotypic changes, single-copy insertion, and Mendelian segregation. These insect-resistant transgenic lines can be used to produce insect-resistant hybrids and serve as a resistant source for development of two-toxin Bt rice.3. All transgenic Bt lines exhibited highly resistant against two main rice stem bores, yellow stem borer (YSB, Tryporyza incertulas walker) and striped stem borer (SSB,更多还原