Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter
Functional dissection of a strong and specific microbe-associated molecular pattern-responsive synthetic promoter
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DOI：
10.1111/pbi.12357
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发表时间：
2016-01-01
影响因子：
13.8
通讯作者：
Hehl, Reinhard
中科院分区：
文献类型：
--
作者：
Lehmeyer, Mona;Kanofsky, Konstantin;Hehl, Reinhard
合成启动子对于转基因植物中的时间和空间基因表达很重要。为了识别用于合成启动子设计的新型微生物相关分子模式（MAMP）响应顺式调控序列，采用了生物信息学和实验方法的结合。鉴定出一个顺式序列，该序列赋予强 MAMP 响应报告基因活性和低背景活性。在拟南芥 DJ1E 基因（人类致癌基因 DJ1 的同源物）的启动子中鉴定出 35 bp 长的顺式序列。在本研究中，该顺式序列被证明是三方顺式调节模块（CRM）。与野生型 DJ1E 启动子相比，具有四个 CRM 拷贝和最小启动子连接的合成启动子可增加 MAMP 响应报告基因的表达。 CRM 由两个 WT 盒（GGACTTTT 和 GGACTTTG）和 GCC 盒变体 (GCCACC) 组成，所有这些都是 MAMP 和水杨酸 (SA) 响应性所必需的。使用仅转录因子 (TF) 的猎物库进行酵母单杂交筛选，鉴定出两个与 CRM 拮抗相互作用的 AP2/ERF：ORA59 和 ERF10。 ORA59激活报告基因活性并需要共有核心序列GCCNCC来激活基因表达。 ERF10 下调 MAMP 反应基因表达。在使用仅 TF 猎物文库的酵母单杂交筛选中，没有选择与 WT 盒 GGACTTTT 和 GGACTTTG 相互作用的 TF。在转基因拟南芥中，合成启动子赋予对活体营养生物、死体营养生物以及 SA 反应的强而特异的报告基因活性。
Synthetic promoters are important for temporal and spatial gene expression in transgenic plants. To identify novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences for synthetic promoter design, a combination of bioinformatics and experimental approaches was employed. One cis-sequence was identified which confers strong MAMP-responsive reporter gene activity with low background activity. The 35-bp-long cis-sequence was identified in the promoter of the Arabidopsis thaliana DJ1E gene, a homologue of the human oncogene DJ1. In this study, this cis-sequence is shown to be a tripartite cis-regulatory module (CRM). A synthetic promoter with four copies of the CRM linked to a minimal promoter increases MAMP-responsive reporter gene expression compared to the wild-type DJ1E promoter. The CRM consists of two WT-boxes (GGACTTTT and GGACTTTG) and a variant of the GCC-box (GCCACC), all required for MAMP and salicylic acid (SA) responsivity. Yeast one-hybrid screenings using a transcription factor (TF)-only prey library identified two AP2/ERFs, ORA59 and ERF10, interacting antagonistically with the CRM. ORA59 activates reporter gene activity and requires the consensus core sequence GCCNCC for gene expression activation. ERF10 down-regulates MAMP-responsive gene expression. No TFs interacting with the WT-boxes GGACTTTT and GGACTTTG were selected in yeast onehybrid screenings with the TF-only prey library. In transgenic Arabidopsis, the synthetic promoter confers strong and specific reporter gene activity in response to biotrophs and necrotrophs as well as SA.