小麦逆境相关基因TaSAP1 5'非翻译区内含子对基因表达的影响及机制研究

Fine controlling of transgene expression is a key requirement for transgenic breeding. Introns of many eukaryotic genes are considered to be important elements to optimize the expression of transgenes in that some of them can regulate gene expression in multiple levels. We previously identified two introns in the 5'untranslated region (5UIs) of TaSAP1. Promoter analysis indicated TaSAP1 5UIs might be required for the regulation of gene expression and stress responses. Based on the above results, this project plan to investigate effects of 5UIs on gene expression and stress responses by transformed Brachypodium distachyon. The probable mechanism will be discussed. The main content of this project include: Investigate the effects of 5UIs on gene expression and stress responses through 5UIs deletion mutation; Study the mechanism of TaSAP1 5UIs on gene expression by altering the location, splice site mutation and internal sequence deletion mutation of 5UIs; Evaluate the applications of the mainly functional 5UI by fusing it together with common promoters used in transgenic breeding. The related work of this project will help to elucidate the function of SAP genes 5UIs in stress responses and provide theoretical basis for optimizing the transgene expression in wheat transgenic breeding.

转基因育种的一个关键问题是如何精细地调控外源基因的表达。真核生物内含子能够在多个水平调控基因的表达，是转基因育种中优化外源基因表达的重要元件。项目申请者前期发现TaSAP1 5'非翻译区存在2个内含子(5UIs)。启动子分析表明TaSAP1 5UIs可能参与基因表达调控及逆境响应。本项目拟在此基础上，通过转化二穗短柄草(Brachypodium distachyon)明确TaSAP1 5UIs对宿主基因表达和逆境响应的影响，并探讨其形成机制。主要包括：敲除TaSAP1 5UIs，转化二穗短柄草，研究5UIs对基因表达及逆境响应的影响；通过5UIs位置突变、剪接位点突变、内部序列缺失等方式探讨5UIs影响基因表达的机制；主要功能5UI与常用启动子融合评估TaSAP1 5UIs的应用价值。本研究有助于解析5UIs在SAP家族基因逆境响应中的作用，并为小麦转基因育种中基因表达的优化提供理论依据。

转基因育种的一个关键问题是如何精细地调控外源基因的表达。真核生物内含子能够在多个水平调控基因的表达，是转基因育种中优化外源基因表达的重要元件。本项目在前期研究基础上对TaSAP1 启动子及其5'非翻译区(5’UTR)2个内含子(5UIs)的功能进行了研究。结果表明TaSAP1为组成型启动子，同时又可受干旱、低温和外源ABA诱导上调表达，其中干旱诱导下其活性是对照的10倍；TaSAP1 启动子活性依赖于5’UTR两个启动子，Intron1可维持TaSAP1启动子的基本活性，Intron2对于维持启动子的逆境诱导活性具有重要作用；通过剪接位点突变和位置效应研究，发现Intron1的功能不受其剪接影响，且Intron1和Intron2是在转录水平上发挥增强启动子活性的功能；Intron1突变研究表明其5’端62 bp序列对其功能的发挥至关重要；在拟南芥和短柄草中对两个内含子的应用价值进行了评估，发现Intron1可增强CaMV35S启动子活性约5倍。本项目研究结果有助于进一步揭示内含子调控基因表达的机制，Intron1可用于优化外源基因的表达，Intron2也具有潜在的应用价值。