DNA-binding and transcriptional activation properties of tobacco NIC2-locus ERF189 and related transcription factors
DNA-binding and transcriptional activation properties of tobacco NIC2-locus ERF189 and related transcription factors
复制标题
DOI：
10.5511/plantbiotechnology.11.1216a
复制
发表时间：
2012-01-01
影响因子：
1.6
通讯作者：
Hashimoto, Takashi
中科院分区：
文献类型：
--
作者：
Shoji, Tsubasa;Hashimoto, Takashi
在烟草（Nicotiana tabacum）中，属于 APETALA2/乙烯反应因子 (AP2/ERF) IXa 组的几种转录因子聚集在叶尼古丁水平的 NIC2 调节基因座上，并直接激活尼古丁生物合成和运输所需的结构基因。茄科阿托帕和天仙子植物利用与尼古丁途径部分重叠的酶合成药用托烷生物碱。我们通过电泳迁移率变动测定测试了烟草 NIC2 位点 ERF189 是否与参与尼古丁和托烷生物碱生物合成的各种结构基因启动子中的潜在 ERF 结合位点结合。 ERF189在三个尼古丁结构基因中结合了四个新的GCC盒样序列，但不识别其他候选结合位点。该结合偏好用于将 ERF189 的共有结合序列优化为 5'-(A/C)GC(A/C)NNCC(A/T)-3'。检查五组 IXa ERF（烟草 ERF189、烟草 ERF163、长春花 ORCA3、拟南芥 AtERF13 和拟南芥 AtERF1）是否在体外与烟草腐胺 N-甲基转移酶基因启动子中的 ERF189 识别位点结合，并在瞬时表达中反式激活启动子 使用培养的烟草细胞进行测定。蛋白质序列与 ERF189 越相似，这些 ERF 在这些功能测定中就越有效。瞬时表达测定还鉴定了 ERF189 N 端酸性区域和 AtERF13 C 端富含 Ser 区域的反式激活结构域。
In tobacco (Nicotiana tabacum), several transcription factors belonging to the IXa group of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) cluster at the regulatory NIC2 locus for the leaf nicotine level, and directly activate structural genes required for nicotine biosynthesis and transport. Solanaceous Atropa and Hyoscyamus plants synthesize medicinal tropane alkaloids by utilizing enzymes that partially overlap with the nicotine pathway. We tested whether the tobacco NIC2-locus ERF189 binds to potential ERF binding sites in the promoters of various structural genes involved in biosynthesis of nicotine and tropane alkaloids by an electrophoresis mobility shift assay. ERF189 bound four new GCC-box-like sequences in three nicotine structural genes, but did not recognize other candidate binding sites. This binding preference was used to optimize the consensus binding sequence of ERF189 as 5'-(A/C)GC(A/C)NNCC(A/T)-3'. Five group IXa ERFs (tobacco ERF189, tobacco ERF163, Catharanthus ORCA3, Arabidopsis AtERF13, and Arabidopsis AtERF1) were examined whether they bind to the ERF189-recognition site in the promoter of the tobacco putrescine N-methyltransferase gene in vitro, and transactivate the promoter in a transient expression assay using cultured tobacco cells. The more the protein sequences were similar to ERF189, the more effective these ERFs were in these functional assays. The transient expression assay also identified transactivation domains in an N-terminal acidic region of ERF189, and in a C-terminal Ser-rich region of AtERF13.