响应多种病原菌诱导型启动子核心顺式作用元件及调控转录因子的鉴定

Pathogen-inducible promoter is not only the key functional elements for constructing the transgenic vector which is used in disease resistance engineering, but also the important resources to understand how plants perceive the stress signals from environment, how the plant defense-related genes are regulated, and how the resistance signal transduction is happened? In previous study, we identified a rice defense-related (DR) gene Os2H16. It encoded an unknown secreted small peptide that significantly enhanced resistance to bacterial blight, banded leaf and sheath blight and drought when it was overexpressed in rice. Also, it could be quickly induced by Xanthomonas oryzae pv. oryzicola, Xanthomonas oryzae pv. oryzae, Rhizoctonia solani and PEG8000 treatment, suggesting that the promoter of Os2H16 is an inducible promoter which could response to multiple pathogens and drought stress. Therefore it may contain one or multiple pathogens-inducible cis-elements. In this study, we focused on functional analysis of the Os2H16 promoter. Step by step, we will telescope the cis-elements to a conservative region by using 5´ deletion assay, and identify the pathogen- and drought-inducible cis-elements and its interaction transcription factors by yeast one-hybrid system. These will reveal the transcriptional regulation mechanisms of Os2H16 under pathogens and drought stresses in rice. Also, they will supply the novel resources as synthetic promoters and transcription factors for crop genetic improvement of biotic- and abiotic- stress.

病原诱导启动子不仅是抗病基因工程重要的载体元件，也是了解植物响应环境胁迫信号、抗病基因的转录调控和抗病信号传递的重要遗传资源。申请人前期鉴定了一个水稻抗病相关基因Os2H16，该基因编码一未知功能的分泌短肽，超量表达该基因显著提高了水稻对白叶枯病、纹枯病和干旱的抗性。同时该基因能够被白叶枯病菌、细菌性条斑病菌、纹枯病菌等病原菌及PEG8000快速诱导表达，暗示Os2H16基因启动子是一个响应多种病原菌及干旱胁迫的诱导型启动子，可能携带一个或多个诱导调控元件。本研究聚焦功能分析Os2H16的启动子，利用5´缺失技术初步定位上述多功能诱导元件所在区间，进一步在该区间内鉴定受多种病原菌及干旱诱导的顺式作用元件，并利用酵母单杂交技术筛选与其互作的转录因子，揭示水稻在病原菌和干旱胁迫条件下调控Os2H16基因转录的可能机制，为进一步利用基因工程改良作物抗逆提供合成启动子及调控转录因子的新资源。

病原诱导启动子不仅是抗病基因工程重要的载体元件，也是了解植物响应环境胁迫信号、抗病基因转录调控和抗病信号传递的重要遗传资源。申请人前期在水稻中鉴定了一个抗病相关基因Os2H16，超量表达该基因能够提高水稻对白叶枯病、纹枯病及干旱的抗性，同时该基因能够被白叶枯病菌、纹枯病菌和干旱胁迫快速诱导表达，暗示了Os2H16基因启动子是一个响应多种病原菌及干旱胁迫的诱导型启动子，可能携带有一个或多个诱导调控元件。在此基础上，本研究对Os2H16基因的启动子进行功能分析，通过截短实验初步确定诱导元件所在区间，并进一步在该区间内精细定位受病原及干旱诱导的顺式作用元件；同时利用酵母单杂交技术筛选与作用元件互作的蛋白，明确互作蛋白在抗病及抗旱中的作用。研究结果表明，通过启动子截短实验，在-411至-309区间鉴定到一个GT-1元件，结合缺失及与35S基础启动子融合的结果，证明GT-1在Os2H16启动子响应病原菌侵染及干旱胁迫中发挥重要作用，是该启动子的核心作用元件。通过酵母单杂交技术筛选到一个与GT-1互作的蛋白OsASR2，该蛋白定位于细胞核且具有转录激活活性，证明其为一个转录因子。过量表达OsASR2基因能够提高水稻对白叶枯病、纹枯病和干旱的抗性，而抑制OsASR2基因使得水稻更加感病及不耐旱，说明OsASR2正调控水稻的抗病性及耐旱性。对OsASR2超量转基因水稻进行ChIP-Seq分析，能够富集到GT-1作用元件且Os2H16基因在超量转基因植株中的表达明显升高，说明OsASR2能够通过靶定GT-1激活Os2H16基因的表达来调节水稻的抗病性及耐旱性。本研究鉴定了GT-1能够响应白叶枯病菌、纹枯病菌和干旱胁迫的新功能，鉴定了GT-1新的调控转录因子OsASR2，揭示了水稻在病原和干旱胁迫条件下Os2H16基因的调控机制，为利用基因工程改良作物抗逆提供了合成启动子及调控转录因子新资源。