Generation of selectable marker-free transgenic rice resistant to chewing insects using two co-transformation systems  

作　　者：Hengxiu Yu Quanhong Yao Ling Wang Zhipeng Zhao Zhiyun Gong Shuzhu Tang Qiaoquan Liu Minghong Gu 

机构地区：[1]Key Laboratory of Plant Functional Genomies of the Ministry of Education, Jiangsu Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou 225009, China [2]Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Academy of Agricultural Science, Shanghai 201106, China [3]China National Rice Research Institute, Hangzhou 310006, China

出　　处：《Progress in Natural Science:Materials International》2009年第11期1485-1492,共8页自然科学进展·国际材料（英文版）

基　　金：supported by the National Natural Science Foundation of China(Grant No.30770131);the Ministry of Education(Grant No.NCET-07-0736);the Education Department of Jiangsu Province(Grant Nos.06KJA21018 and K05015)

摘　　要：To produce selectable marker-free （SMF） transgenic rice resistant to chewing insects, the Bacillus thuringiensis crylA （c） gene （Bt） was introduced into two elite japonica rice varieties by using two Agrobacterium-mediated co-transformation systems. One system is with a single mini-twin T-DNA binary vector in one Agrobacterium strain, which consists of two separate T-DNA regions, one carrying the Bt while the other contains the selectable marker gene, hygromycin resistant gene （HPT）. The other system uses two separate binary vectors in two separate Agrobacterium cultures, containing the Bt or HPT gene on individual plasmids. A lot of independent transgenic rice lines harboring both Bt and selectable marker genes were obtained. The results showed that the co-transformation frequency of the Bt gene and HPT gene was much higher by using the mini-twin T-DNA vector system （29.87%） than that by the two separate binary vector systems （4.52%）. However, the frequency of the SMF transgenic rice plants obtained from the offspring of co-transgenic plants （21.74%） was lower for the mini-twin T-DNA vector system than that for the latter （50-60%）. The data of ELISA implied that the expressed Bt proteins were quantitated as 0.025-0. 103% of total leaf soluble proteins in the transgenic plant. Therefore, several elite transgenic rice lines, free of the selectable marker gene, were chosen. The results from both in vitro and in vivo insect bioassays indicated that the SMF transgenic rice was shown to be highly resistant to the striped stem borer and rice leaf folder. Moreover, in a natural field condition without any insecticide applied, all the transgenic rice plants were found to be not injured by the rice leaf folder, whereas the wild types were impaired seriously.生产可选没有标记(SMF ) 对咀嚼昆虫抵抗的转基因的米饭，杆菌 thuringiensis cryIA (c) 基因(Bt ) 被使用二个调停 Agrobacterium 的合作转变系统介绍进二个精英装饰用的梨树米饭变化。一个系统与在一 Agrobacterium 紧张的单个微型双胞胎 T-DNA 二进制向量，它由二个分开的 T-DNA 区域组成，当其它包含可选的标记基因时，带 Bt 的， hygromycin 抵抗基因(HPT ) 。另外的系统在二种分开的 Agrobacterium 文化使用二分开的二进制向量，包含单个原生质标志上的 Bt 或 HPT 基因。怀有 Bt 和可选的标记基因的很多独立转基因的米饭线被获得。结果证明 Bt 基因和 HPT 基因的合作转变频率由由二个分开的二进制向量系统(4.52%) 比那使用微型双胞胎 T-DNA 向量系统(29.87%) 是高得多的。然而，从 co 转基因的植物(21.74%) 的后代获得的 SMF 转基因的米饭植物的频率为后者比那为微型双胞胎 T-DNA 向量系统是更低的(50 &#x2013； 60%) 。ELISA 的数据暗示表示 Bt 蛋白质作为 0.025 &#x2013 被测定； 0.103% 总数长叶在转基因的植物的可溶的蛋白质。因此，没有可选的标记基因，转基因的米饭衬里的几精英被选择。在 vitro 并且在 vivo 昆虫生物鉴定的从两个的结果显示 SMF 转基因的米饭被显示对有斑纹的茎 borer 和米饭叶文件夹高度抵抗。而且处于没有使用的任何杀虫药剂的一个自然的地条件，所有转基因的大米植物被发现到别被大米叶文件夹伤害，而野类型严重被损害。

关 键 词：Oryza sativa L. Bacillus thuringiensis crylA （c） gene Selectable marker-free （SMF） CO-TRANSFORMATION Insect resistance 

分 类 号：Q949.288.4[生物学—植物学] S511[农业科学—作物学]