杨树SABATH和MES基因家族的功能分化及其在系统获得性抗性反应中的进化意义

Systemic acquired resistance (SAR) is an inducible plant defense response in which a prior foliar pathogen infection activates resistance in noninfected foliar tissues. SAR requires a signal movement from the infected tissue to the systemic tissue through the phloem. Up to date, methyl salicylate (MeSA) was proved to be a mobile SAR signal. In the infected tissue, MeSA is synthesized from salicylic acid (SA) via a reaction catalyzed by salicylic acid methyltransferase (belong to SABATH gene family). After MeSA is moved to noninfected tissue through the phloem, MeSA is converted into SA via a reaction catalyzed by methyl esterase (belong to MES gene family). Thus, both SABATH and MES gene families play the critical roles in the MeSA-mediated SAR signal transduction. In this study, we will focus on Populus SABATH and MES gene families. Firstly, we will investigate the molecular mechanisms contributing to the expansion of SABATH and MES gene families in Populus genomes using the methods of evolutionary genomics and molecular systematics. Secondly, the gene expression patterns of SABATH and MES gene families under normal growth conditions and stress will be examined using RT-PCR and real-time RT-PCR. Thirdly, we will clone all the SABATH and MES genes from Populus genomes, express proteins in E. coli, and purify the SABATH and MES proteins. The biochemical characterization of the purified SABATH and MES proteins will be investigated in detail. Finally, biologic functions of Populus SABATH and MES genes will be investigated in vivo by transgenic Arabidopsis and tobacco mutants. In a word, by integrating all the results about molecular evolution, gene expressions and protein functions, this study will shed light on the functional divergence and evolutional significance of SABATH and MES gene family in the systemic acquired resistance of the Populus.

植物系统获得性抗性（SAR）是某个局部组织受到侵染后，受侵染组织发出的信号传播到其它未被侵染部分，从而引发整个植株的抗性。在受到侵染的组织中，水杨酸被水杨酸甲基转移酶基因家族（SABATH）催化生成水杨酸甲酯，水杨酸甲酯经维管组织转运到其他组织后，被甲酯酶基因家族（MES）催化生成水杨酸，从而引发SAR。因此，在水杨酸甲酯介导的SAR信号转导中，SABATH和MES两个基因家族发挥着至关重要的作用。本课题充分利用植物基因组学与生物信息学的最新成果，综合基因组学、分子系统学、生物化学、分子生物学等多学科研究手段，从"基因组→基因表达→蛋白质结构与功能→体内生理功能"四个层次研究杨树SABATH和MES基因家族的扩张机制、家族内成员间的功能分化、以及功能分化的模式，从而逐步揭示杨树SABATH和MES基因家族内成员间的功能分化机制、以及在杨树系统获得性抗性反应中的进化意义。

在水杨酸甲酯介导的系统获得性抗性信号转导中，SABATH和MES两个基因家族发挥着至关重要的作用。本项目从杨树基因组中鉴定出28个完整的SABATH基因和25个MES基因。28个SABATH基因在杨树染色体上呈不均匀分布，串联重复是杨树SABATH基因家族扩张的主要机制。杨树MES基因在染色体上同样呈现不均匀分布，串联重复也是杨树MES基因家族扩张的主要原因。系统发育树和基因结构分析发现杨树SABATHs分为三个不同的类型。相比于I和II类基因，杨树III类SABATH基因处于更宽松的选择约束。基因家族的表达模式分析发现在II类和III类SABATH基因的表达模式有着显著变异。酶学性质分析发现杨树I类SABATH蛋白具有将吲哚乙酸（IAA）转化为甲基IAA的特异性功能，II类SABATH可将苯甲酸（BA）和水杨酸（SA）转化为甲基苯甲酸和甲基水杨酸，而III类SABATH可以将法呢酸（FA）转化为甲基法呢酸。对于杨树II类SABATH，通过蛋白结构模拟、正向和反向定点突变研究，发现PtSABATH4和PtSABATH24中的单个氨基酸变换可导致底物谱的转换。本项目揭示了杨树SABATH和MES基因家族的扩张机制，以及功能多样化形成的分子机理。