拟南芥CKRW1调节内源生长素水平稳态平衡的分子机理研究

Endogenous auxin homeostasis depends on dynamic controling of auxin biosynthesis and metabolism. Due to recent progress in auxin has unequivocally demonstrated that regulation of auxin biosynthesis and metabolism plays an essential role in every aspects of plant growth, development and morphogenesis, it became one of the research hotspots in auxin research. Our preliminary results indicated that CKRW1 (CYTOKININ -INDUCED ROOT WAVING) loss of function contributes to decrease the level of IAA (Indole-3-acetic acid) and its metabolites in Arabidopsis. This gene encodes an ubiquitin ligase E3, which is involved in ubiquitin modification of some proteins and not an enzyme in auxin biosynthesis and metabolism pathway. We hypothesized that CKRW1 maybe contribute in some unknown way to regulate endogenous auxin homeostasis. Although many researches on regulation of auxin biosynthesis and metabolism have been done, it will be the first time to link the ubiquitin modification and endogenous auxin homeostasis. The project intends to carry on further investigation on CKRW1 and its interaction protein to clarify the molecular mechanism of CKRW1 regulating endogenous auxin homeostasis and reveal the function of CKRW1 in root gravitropic response.

植物内源生长素的稳态平衡依赖于动态调控生长素的生物合成与代谢，目前的研究表明该调控过程对植物的生长发育、形态建成等起重要作用，已经成为当今的研究热点之一。我们的前期工作已经证实蛋白CKRW1（CYTOKININ-INDUCED ROOT WAVING）的功能缺失导致拟南芥突变株内源吲哚-3-乙酸（Indole-3-acetic acid, IAA）及其代谢物的含量降低。基因CKRW1编码的泛素连接酶E3，参与某些蛋白的泛素化修饰，而不参与生长素生物合成及代谢通路，我们推测该酶可能以一种未知的方式调节植物内源生长素稳态平衡。虽然以往对生长素合成/代谢的调控已经做了很多研究，但是我们首次将泛素化修饰和调节生长素稳态平衡联系到了一起。本项目拟通过对蛋白CKRW1及其互作蛋白的深入研究，弄清CKRW1调节内源生长素稳态平衡的分子机理，并揭示CKRW1在根向地性反应中的作用。

拟南芥蛋白CKRW1/WAV3（CYTOKININ INDUCED ROOT WAVING 1 / WAVY ROOT 3）是一个具有Ring-finger结构域的泛素连接酶E3，它与其同源蛋白EDA40（EMBRYO SAC DEVELOPMENT ARREST 40）、WAVH1（WAV3 HOMOLOG 1）及WAVH2（WAV3 HOMOLOG 2）构成一个蛋白亚家族。在项目研究中，我们通过构建CKRW1/WAV3亚家族功能缺失的多突变证实了它们具有功能冗余，多突变体除了表现出生长素缺陷表型外，还表现出和生长素输出载体突变pin2（pin-formed 2）类似的表型，通过显微观察，我们发现PIN2蛋白的亚细胞定位在三突变背景下发生变化。体外实验证实CKRW1/WAV3具有泛素连接酶活性，我们通过酵母双杂交系统筛选得到了与CKRW1/WAV3存在互做的几个蛋白，它们分别为：蛋白磷酸酶PP2C31（type 2C protein phosphatase 31）、E3泛素连接酶SINAT（Arabidopsis homologue of the RING-finger Drosophila protein SINA）蛋白家族、植物分生组织CLV/WUS信号途径相关蛋白WOX9（WUSCHEL-related homeobox 9）、组蛋白H2B单泛素化酶HUB1、钙结合蛋白CML18（putative calcium-binding protein CML18）、热休克蛋白HSP23.6（small heat shock protein 23.6）、微管相关蛋白质MAP70-5（microtubule-associated protein 70-5）。虽然本项目研究没有彻底弄清CKRW1/WAV3调控根向重力性响应的分子机理，但是本项目的研究结果为今后的研究提供了基础。