Rapid isolation of promoter sequences by TAIL-PCR:: the 5′-flanking regions of Pal and Pgi genes from yams (Dioscorea)
Rapid isolation of promoter sequences by TAIL-PCR:: the 5′-flanking regions of Pal and Pgi genes from yams (Dioscorea)
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DOI：
10.1007/s004380051201
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发表时间：
2000-04-01
期刊：
影响因子：
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通讯作者：
Kahl, G
中科院分区：
文献类型：
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作者：
Terauchi, R;Kahl, G
使用改良的 TAIL-PCR 技术，成功分离了薯蓣的苯丙氨酸解氨酶 (Pal) 基因和 D. tokoro 的磷酸葡萄糖异构酶 (Pgi) 基因的 5' 侧翼区域。这里介绍的 TAIL-PCR 程序的两个新修改，即 (1) 使用一组随机 10 聚体（RAPD 引物）作为短任意引物，以及 (2) 使用总共 5 个嵌套的基因特异性引物，可以从具有大基因组的生物体中快速分离任何基因的 5' 侧翼区域。将分离的 5' 侧翼区域与 gus 基因融合，并测试烟草 BY2 细胞中的瞬时表达。所有分离的 5' 侧翼区域均显示驱动报告基因表达。三个 Pal 启动子对水杨酸做出反应，可能是 MYB 转录激活因子与这些区域中存在的多个 MRE（Myb 识别元件）结合的结果。
Using a modified TAIL-PCR technique, the 5'-flanking regions of the phenylalanine ammonia lyase (Pal) genes of a yam species, Dioscorea bulbifera, and the phosphoglucose isomerase (Pgi) gene of D. tokoro were successfully isolated. Two novel modifications of the TAIL-PCR procedure introduced here, namely (1) the use of a battery of random 10-mers (RAPD primers) as short arbitrary primers, and (2) the use of a total of five nested, gene-specific primers, allow the rapid isolation of the 5'-flanking region of any gene from organisms with large genomes. Isolated 5'-flanking regions were fused to the gus gene, and tested for transient expression in tobacco BY2 cells. All the isolated 5'-flanking regions were shown to drive reporter gene expression. Three Pal promoters responded to salicylic acid, presumably as a result of the binding of a MYB transcriptional activator to the multiple MREs (Myb Recognition Elements) present in these regions.