Visual selection allows immediate identification of transgenic rice calli efficiently accumulating transgene products
Visual selection allows immediate identification of transgenic rice calli efficiently accumulating transgene products
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DOI：
10.1007/s00299-009-0671-9
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发表时间：
2009-04-01
影响因子：
6.2
通讯作者：
Toki, Seiichi
中科院分区：
文献类型：
--
作者：
Saika, Hiroaki;Toki, Seiichi
在遗传转化系统中，抗生素抗性基因通常用作从周围的非转化细胞中选择转化细胞的有力标记。然而，同时使用编码绿色荧光蛋白（GFP）的基因和抗生素抗性基因有利于选择过程，因为它允许转化细胞的可见选择。在这里，我们报告了一个可视化的选择系统的发展，转化细胞使用GFP标记，而不选择对抗生素后，农杆菌介导的水稻转化。通过视觉选择分离的愈伤组织中的GFP蛋白水平和GFP荧光均高于在潮霉素（Hyg）上选择的愈伤组织中的GFP蛋白水平和GFP荧光，这表明通过使用GFP荧光本身而不是Hyg抗性进行选择来有效地获得超积累GFP的转基因愈伤组织。此外，GFP的成绩单在愈伤组织中分离的视觉选择比Hyg选择下更丰富，相反，在视觉上选择的愈伤组织中的hpt的转录水平是可比的Hyg选择下获得的。这些结果表明hpt和gfp表达水平之间没有相关性，尽管事实上它们在通过GFP荧光或Hyg抗性选择后在整合的基因座上串联排列。这一事实表明，位置效应可以不同地影响每个转基因的表达，即使当它们串联位于同一基因座时。总之，基于我们的研究结果，我们讨论了一个模型系统的水稻细胞培养转化生产重组蛋白，使用视觉选择。
In genetic transformation systems, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. However, simultaneous use of the gene encoding green fluorescent protein (GFP) and an antibiotic resistance gene facilitates the selection process, since it allows visible selection of transformed cells. Here, we report the development of a visual selection system for transformed cells using a GFP marker without selection against antibiotics after Agrobacterium-mediated transformation in rice. Both GFP protein levels and GFP fluorescence in calli isolated by visual selection were higher than in calli selected on hygromycin (Hyg), suggesting that transgenic calli hyper-accumulating GFP were efficiently obtained by selection using GFP fluorescence itself rather than Hyg resistance. Furthermore, gfp transcripts in calli isolated by visual selection were more abundant than under Hyg selection; in contrast, transcript levels of hpt in calli selected visually were comparable to those obtained under Hyg selection. These results suggest that there was no correlation between hpt and gfp expression levels, despite the fact that they are aligned in tandem on an integrated locus after selection by either GFP fluorescence or Hyg resistance. This fact indicates that positional effects can influence the expression of each transgene differently, even when they are located in tandem at the same locus. In summary, based on our results, we discuss a model system for rice cell culture transformation for the production of recombinant proteins using visual selection.