Lu Nan, Carter Jared D, Boluarte Medina Tatiana, Holt Sarah H, Manrique-Carpintero Norma Constanza, Upham Kendall T, Pereira Andy, Shulaev Vladimir, Veilleux Richard E
Department of Horticulture, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA.
Plant Cell Rep. 2014 Jul;33(7):1203-16. doi: 10.1007/s00299-014-1610-y. Epub 2014 Apr 12.
Diploid strawberry and potato transformed with a transposon tagging construct exhibited either global (strawberry) or local transposition (potato). An activation tagged, compact-sized strawberry mutant overexpressed the gene adjacent to Ds. As major fruit and vegetable crops, respectively, strawberry and potato are among the first horticultural crops with draft genome sequences. To study gene function, we examined transposon-tagged mutant strategies in model populations for both species, Fragaria vesca and Solanum tuberosum Group Phureja, using the same Activation/Dissociation (Ac/Ds) construct. Early somatic transposition during tissue culture occurred at a frequency of 18.5% in strawberry but not in potato transformants. Green fluorescent protein under a monocot promoter was a more reliable selectable marker in strawberry compared to potato. BASTA (gluphosinate herbicide) resistance served as an effective selectable marker for both species (80 and 85% reliable in strawberry and potato, respectively), although the effective concentration differed (0.5% for strawberry and 0.03% for potato). Transposons preferentially reinserted within genes (exons and introns) in both species. Real-time quantitative PCR revealed enhanced gene expression (670 and 298-fold expression compared to wild type in petiole and leaf tissue, respectively) for an activation tagged strawberry mutant with Ds inserted about 0.6 kb upstream from a gene coding for an epidermis-specific secreted glycoprotein EP1. Our data also suggested that endopolyploid (diploid) cells occurring in leaf explants of monoploid potato were the favored targets of T-DNA integration during transformation. Mutants obtained in these studies provide a useful resource for future genetic studies.
用转座子标签构建体转化的二倍体草莓和马铃薯表现出全局转座（草莓）或局部转座（马铃薯）。一个激活标签型紧凑型草莓突变体过表达了与Ds相邻的基因。草莓和马铃薯分别作为主要的水果和蔬菜作物，是最早拥有基因组草图序列的园艺作物之一。为了研究基因功能，我们使用相同的激活/解离（Ac/Ds）构建体，在两种植物的模式群体——森林草莓和马铃薯栽培种Phureja中研究了转座子标签突变体策略。组织培养过程中的早期体细胞转座在草莓转化体中发生频率为18.5%，而在马铃薯转化体中未发生。与马铃薯相比，单子叶植物启动子驱动的绿色荧光蛋白在草莓中是更可靠的选择标记。BASTA（草铵膦除草剂）抗性对两种植物都是有效的选择标记（在草莓和马铃薯中分别有80%和85%的可靠性），尽管有效浓度不同（草莓为0.5%，马铃薯为0.03%）。转座子在这两种植物中都优先重新插入基因内部（外显子和内含子）。实时定量PCR显示，对于一个激活标签型草莓突变体，Ds插入到一个编码表皮特异性分泌糖蛋白EP1的基因上游约0.6 kb处，该突变体在叶柄和叶片组织中的基因表达增强（分别比野生型高670倍和298倍）。我们的数据还表明，单倍体马铃薯叶片外植体中出现的内多倍体（二倍体）细胞是转化过程中T-DNA整合的首选靶标。这些研究中获得的突变体为未来的遗传学研究提供了有用的资源。
