【作者】 渠柏艳；

【导师】 李晚忱；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 近年来，随着转基因作物品种的逐步推广应用和转基因食品的商品化，转基因作物对食品、环境和农业生产的安全性，越来越受重视。除目的基因而外，转基因操作中所使用选择标记基因的安全性更是倍受关注。除了对选择标记基因进行安全性评价以外，目前转基因研究的一个热点，即是通过共转化、位点特异性重组、转座子介导法重新定位、MAT载体系统等方法，建立可去除选择标记的基因转化体系，在转化筛选以后，去除选择标记基因。 本研究以携带Bt基因的pUBK质粒和具有两个转移DNA（T-DNA）结构域的pCDMAR-Hyg质粒为基础，通过酶切、连接构建可去除选择标记的双T-DNA单子叶植物表达载体pCDMAR-USBK-Hyg，其中一个T-DNA结构域含有外源抗虫基因Bt，另一个T-DNA结构域含有选择标记基因Hpt。用这一双T-DNA质粒转化农杆菌后，通过转化农杆菌菌株与玉米胚性愈伤组织的共培养，使双T-DNA携带的抗虫基因Bt和选择标记基因Hpt共转化玉米，经潮霉素梯度浓度培养基筛选，从阳性克隆分化再生植株。经对T₀代再生植株的PCR和PCR-Southern检测，初步证明外源Bt基因已整合到玉米基因组，并获得了可育的T₁代种子。更多还原

【Abstract】 With commercialization of transgenic plant increasing, the biosafety is always concerned by scientists and governments. Since selectable maker genes are existed in almost all transgenic plants, the biosafety of marker genes are concerned by the public. So various methods and strategies are currently being developed for the maker gene excision and elimination from transgenic plants.Double T-DNA plant expression vector pCDMAR-USBK-Hyg was derived form plasmid vector pUBK and double T-DNA vector pCDMAR-Hyg. One T-DNA construction contains plant selectable maker hygromacin phosphotransferase (hpt) which driven by CaMV35s promoter, the other T-DNA construction contrains Bt gene which was flanked by two MAR sequence. The Bt gene what was obtained from pUBK was driven by ubiquitin promoter. Then the double T-DNA vector was transferred into Type II callus of maize elite inbred via Agrobacterium-medialed. Some fertile transgenic maize had obtained. Result of PCR, PCR-Southem confirmed that transgenic plants carry the foreign gene.By now, T1 seeds had been obtained.更多还原