【作者】 张晶；

【导师】 朱延明；

【作者基本信息】 东北农业大学 ， 植物学， 2003， 硕士

【摘要】 水稻是世界上最重要的粮食作物之一，但容易受到多种真菌病害的侵染，使其产量和品质受到严重的威胁。实践证明，最经济有效的、也是最根本的防治方法就是培育抗病品种。常规育种由于品种选育年限长、抗源基因匮乏及随病源生理小种的变化抗病性消失等原因，远不能满足选育抗病品种的需求。利用基因工程技术进行抗病育种是一条崭新而有效的途径。但水稻抗真菌病基因遗传转化中仍存在单个抗病基因的抑茵谱窄等问题；另外，现有的水稻遗传转化体系还存在着转化频率低等因素，难以获得大量的转基因植株。本研究针对以上问题，构建菜豆几丁质酶基因与大麦核糖体失活蛋白基因双价植物表达载体，通过农杆菌介导法和基因枪法转化水稻(选用东北地区4个主栽水稻品种)，系统优化植株再生条件和遗传转化条件，建立高效的植株再生体系和遗传转化体系。为水稻以及其它禾本科植物抗病分子育种奠定基础，并为有性杂交育种提供基因资源。主要研究结果如下：(1) 构建单价和双价植物表达载体共构建了5个载体，其中包括一个中间克隆载体pUchB，1个植物载体卡盒p33EG，2个单基因植物表达载体p33EC、p33ECB，1个双价植物表达载体p33ECR。(2) 优化水稻再生体系干燥处理可显著提高水稻愈伤组织的分化效率，当失去鲜重50％时，水稻愈伤组织分化效率可提高2.0-4.6倍。通过改善移栽方法，显著提高了农杆菌转化抗性再生苗的移栽成活率。(3)优化遗传转化体系并获得转基因植株采取分化阶段筛选策略：水稻愈伤组织在增殖阶段对PPT不敏感，而在分化阶段对PPT敏感，PPT选择压力为0.7mg／L。在此条件下筛选，通过农杆菌法转化共获得327株抗性植株，34株为PCR阳性，PCR阳性率平均为10.40％。而在愈伤增殖阶段筛选，通过基因枪法共获得561株抗性植株，8株为PCR阳性，平均PCR阳性率仅为1.43％。更多还原

【Abstract】 Rice is one of the most important crops, but fungi diseases affect its yield and attribute seriously. It was testified that culturing the fungi-resistant varieties is the most economical and basic way. However, the conventional breeding can’t meet the need of cultivating the fungi-resistant varieties because of its long period, short fungi-resistant genes and disappearing resistance with the changes of pathogen. Gene engineering is an available method to breed the fungi-resistant varieties. But there are some problems in rice engineering to resist fungi. One is that a single gene only can restrain a few varieties fungi, and the other is that it is difficult to obtain a large quantity of transgenic plants because of lower rate of transformation.In this study, four cultivars of rice were choosed. Bivalent plant expression vector with chitinase(chi) and ribosome inactivating protein (rip) gene was constructed, and then transferred into rice mediated by Agrobacterium Tumefaciens and particle bombardment. Some factors in plant regeneration and gene transformation were optimized, and plant regeneration system and gene transformation system with high efficiency were established. It could be the base of molecular breeding to resist diseases for rice and other grasses.On the other hand, it could provide gene resources for sexual hybridization breeding.The main results were summarized as follows.(1) Univalent plant expression vectors and bivalent plant expression vector were constructed.Five vectors were constructed in the experiment: One was intermediate clone vector (pUchB), one was plant expression vector cassette (p33EG), one was bivalent plant expression vector (p33ECR), and the other two was univalent plant expression vectors (p33EC and p33ECB).(2) Regeneration system of rice was Optimized.Desiccation can increase differentiation of calli of rice obviously. Differentiation efficiency of rice could be increased 4.6 times at most than control if fifty percent fresh weight were lost. Survival rate of regenerated plants were increased obviously.(3) Transformation system was optimized and transgenic plants were obtained.Bivalent plant expression vector was constructed and transformed into calli of rice.Calli of rice were not sensitive to PPT during their propagating period, but they were sensitive at differentiating period. So the concentration of PPT for selection is 0.7mg/L in the period of shoot differentiation. In the condition, 34 of 327 regenerated plants were PCR positiveafter transforming by Agrobacterium Tumefaciens, and positive rate is 10.40%. However, 8 of 561 regenerated plants were PCR positive after transforming by particle bombardment if they were screened during calli were propagated, and positive rate is only 1.43%..更多还原