拟南芥脱腺苷酸化酶AtCAF1s调控抗性基因PR1表达的分子机制研究

mRNA turnover in eukaryotic cells generally begins with shortening of the poly(A) tail at the 3' end, a process called deadenylation. Deadenylation is the limiting step in mRNA decay and thus is important for regulation of various biological events. Compared to other eukaryotic organisms, deadenylation is poorly studied in plants. In our previous work, we found that two deadenylases, AtCAF1a and AtCAF1b, are involved in plant defense response to plant pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000) by positively regulating the expression of pathogenesis-related (PR) gene PR1 in Arabidopsis. What was not understood, however, is how this regulation is accomplished. As the deadenylation activity of these enzymes is essential for the elevated expression of PR1, we speculate that these enzymes are required for degradation of a particular mRNA species specifying a repressor of PR1 transcription. From a forward genetic screen with atcaf1a/atcaf1b, we isolated the eopl1 (elevation of pr1 level 1) mutant in which the amount of PR1 is much elevated at both mRNA and protein level. Importantly, this mutant showed increased resistance to Pst DC3000. Genetic analysis revealed that EOPL1 representing a new repressor of PR1. We aim at cloning the EOPL1 gene and uncovering its biological functions as well as exploring how AtCAF1a and AtCAF1b regulate the expression of PR1 through direct degradation of EOPL1. This study will not only greatly enhance our understanding of how plants respond to pathogen infection and the important roles of mRNA decay in plant defense response, but also lay foundation for controlling plant diseases with transgenic approaches.

脱腺苷酸化通常是真核生物mRNA降解的第一步并且是限速步骤，对于许多生物过程的调节非常重要。与其它真核生物相比，植物中有关脱腺苷酸化的研究还很少。我们在前期研究中发现拟南芥中的脱腺苷酸化酶AtCAF1a与AtCAF1b通过正调控抗性基因PR1的表达而参与了植物对丁香假单胞杆菌的抗性反应，而且该调节依赖于其酶活性。但是到目前为止，AtCAF1s调控PR1表达的机制并不清楚。以atcaf1a/atcaf1b为材料进行遗传学筛选，我们获得了突变体eopl1，该突变体中PR1的水平极大提高，对病原菌的抗性增强。遗传学分析表明EOPL1是PR1的一个新的抑制子。本申请项目将克隆EOPL1基因，研究其生物学功能，阐明AtCAF1s通过降解EOPL1间接调控PR1表达的分子机制。本研究的结果将有助于理解植物对病原菌的抗性反应机制与mRNA降解在植物抗病反应中的作用，以及通过转基因手段控制植物病害的发生。

脱腺苷酸化通常是真核生物mRNA降解的第一步并且是限速步骤，对于许多生物过程的调节非常重要。与其它真核生物相比，植物中脱腺苷酸化的分子机制以及它在抗病反应中的作用并不清楚。在本项目研究中，我们以拟南芥脱腺苷酸化酶AtCAF1a与AtCAF1b的突变体atcaf1a/atcaf1b为材料进行遗传学筛选，获得了抗病基因PR1表达水平升高的突变体，该突变体对丁香假单胞杆菌番茄变种（Pseudomonas syringae pv tomato DC3000, Pst DC3000）的抗性明显增强。图位克隆发现这一表型是由于CBNAC1基因的突变造成的，CBNAC1第231位核苷酸由C突变为A，造成了翻译的提前终止。CBNAC1基因在拟南芥的各个组织器官中都有表达，它编码的蛋白定位于细胞质和细胞核中。定量RT-PCR结果表明，CBNAC1基因受到Pst DC3000侵染以及植物激素水杨酸处理的强烈诱导。CBNAC1编码一个含有NAC结构域的转录因子，通过结合到PR1启动子的TGCTT顺式元件抑制PR1的转录。通过免疫共沉淀鉴定到了AtCAF1的互作蛋白AtPUM6，AtPUM6可以特异性结合到CBNAC1的3'非编码区，招募AtCAF1对CBNAC1脱腺苷酸化。因此，AtCAF1通过对CBNAC1 mRNA的降解正调控PR1的表达水平。AtCAF1是CCR4-NOT复合体的一个组分，但是在拟南芥中该复合体的骨架蛋白AtNOT1的缺失对于AtCAF1所调控的抗病基因PR1的表达以及植物对Pst DC3000的抗性却没有影响。这些研究结果有助于理解植物对病原菌的抗性反应机制与mRNA降解在植物抗病反应中的作用，以及通过转基因手段控制植物病害的发生。