【作者】 张燕；

【导师】 吴江生； 刘克德；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2010， 硕士

【摘要】 油菜是我国主要的油料作物之一,在大面积生产过程中常遭受害虫和杂草的危害,严重降低油菜的产量和品质,给油菜生产造成巨大的损失。为了改良油菜抗鳞翅目幼虫和抗除草剂的遗传特性,本研究采用农杆菌介导法,以抗除草剂bar基因为筛选标记,将经过密码子优化、人工合成的抗虫基因Cry2A*、Cry1C*导入甘蓝型油菜品种Westar中,获得一批转基因植株。本研究的主要结果如下：1、通过Basta敏感性试验,确定了油菜品种Westar在诱导愈伤和分化出芽阶段对Basta的适宜筛选浓度为5-7 mg/L2、利用农杆菌介导法,分别获得了18株转化UBI:Cry2A*和42株转化UBI：Cry1C*的转基因植株,转化效率分别为2.02%、3.34%；RT-PCR检测分析了外源基因在RNA水平上的表达,证实了Cry2A*基因和Cry1C*基因在转基因植株中稳定表达；对部分转基因植株进行了Southern检测,结果证明外源基因多以1-3个拷贝插入到了转基因植株基因组中；对两种转基因植株T1代随机取样进行PCR检测,大部分植株检测到外源基因的存在,说明外源基因在转基因后代植株中可以稳定遗传。3、对T0和T1代转基因植株离体叶片进行了二龄小菜蛾的接虫实验,结果表明,Cry1C*转基因植株可以明显抑制小菜蛾幼虫的进食,导致其发育迟缓甚至死亡；而Cry2A*转基因植株则没有表现出明显的抗虫性,小菜蛾幼虫发育正常。4、对T1代转基因植株叶片涂抹不同浓度的Basta稀释液,研究结果表明稀释2500倍的Basta是转基因油菜抗除草剂的临界点；全株喷施2500倍的Basta稀释液,未转化的对照植株死亡,两种转基因油菜均表现出抗性。更多还原

【Abstract】 Rapeseed is one of major oil crops in China, but the yield and quality of which are highly susceptible to pests and weeds during the large scaled production, finally bringing us huge loss to rape production. In order to improve the genetic characterstics of resistance to the larva of Lepidoptera and herbicide, in this research, using two novel synthetic Bt genes Cry2A* and Cry1C* with the modification and optimization in the codon regions of the DNA sequences were transferred into one B.napus cultivar Westar via Agrobacterium-mediated transformation, we got a batch of transgenic plants. The main results of this study are as follows:1. The optimal concentration for screening Westar (B. napus) tolerant to Basta at the callus and differentiation stage was identified as 5-7mg/L by Basta sensitivity test.2. Using Agrobacterium-mediated transformation, we had obtained 18 and 42 transgenic plants with the vector of UBI:Cry2A* and 42 UBI:Cry1C*, transformation efficiency was 2.02% and 3.34%, respectively; RT-PCR analysis confirmed that the Cry2A* and Cry1C* genes are expressed stably at the transcriptional level in transgenic plants; Southern blotting analysis proved that the exogenous genes insert into genome of transgenic plants with 1-3 copies; the T1 generation plants were analyzed based on PCR, which proved that the exogenous gene in the transgenic progeny can be stably inherited.3. Insect bioassay test in To and T1 generation transgenic plants showed that Cry1C* transgenic plants can inhibit larval eating apparently, resulting in growth retardation and even death; on the contrary, Cry2A* transgenic plants didn’t exhibit significant resistance to larvae, the larve developed normally.4. The leaves of transgenic plants at T1 generation were spreaded with herbicide Basta on different concentrations, which suggested that diluted 2500 times Basta is the critical value of herbicide resistance in transgenic plants; after spraying whole plant with diluted 2500 times Basta, transgenic plants with Cry2A* and Cry1C* showed an increasing resistance to Basta compared to non-transgenic plants.更多还原