AtCHYR1在ABA依赖的逆境胁迫途径中的功能研究

In plants, ABA-dependent stress-responsive pathway plays an important role in sensing and responding to the abiotic stress signal, and its molecular mechanism has been extensively explored. To date, however, the regulatory network is still largely incomplete. Here we found a novel gene up-regulated during ABA and drought treatments, which encodes a protein containing both a functional-unknown domain CHY-zinc finger and a RING finger domain, designated as AtCHYR1 (Arabidopsis thaliana CHY-zinc finger and RING finger containing 1). Sequence analysis showed that several potential AtCHYR1 ortholog genes were found in rice and maize, with yet unknown functions. Preliminary results indicated that AtCHYR1 possessed E3 ubiquitin ligase activity and the AtCHYR1 T-DNA insertion mutants were insensitive to ABA during seed germination. Above all, these results suggest that AtCHYR1 may participate in the ABA and stress-responsive pathway as a new important factor. In this study, we will employ molecular genetics, biochemical and cellular biology manners to characterize the function of AtCHYR1 in ABA-dependent stress-responsive pathway. Besides, we are also interested to find the interaction factor of AtCHYR1 via co-immunoprecipitation and proteomics approaches and clarify their relationship through in vitro ubiquitination assay, thus postulate the molecular mechanism of their functions in plant cell. Additionally, the function of a novel protein type, CHYR (CHY-zinc finger and RING finger containing), will be dissected and thus the knowledge of stress sense and response in plants should be deepened.

ABA依赖的逆境胁迫响应途径是植物感受和应答非生物胁迫的重要方式。其分子机制的研究已经取得一定进展，但其精细的调控网络尚不完整。我们在拟南芥中发现一个受ABA和干旱诱导的基因，其编码蛋白具有CHY锌指和RING结构域且功能未知，命名为AtCHYR1。序列分析发现其同源基因也存在于水稻和玉米基因组中且功能未知。初步研究表明，AtCHYR1蛋白具有泛素连接酶活性；其突变体在种子萌发阶段对ABA信号不敏感。因此AtCHYR1可能是植物响应逆境胁迫和ABA信号途径中一个新的重要因子。本项目将通过分子、生化、细胞生物学等手段研究AtCHYR1在ABA依赖的逆境胁迫响应中的生物学功能；通过免疫共沉淀、蛋白质组技术鉴定其互作蛋白；通过体外泛素化反应，阐明AtCHYR1与其互作蛋白的关系，最终确定其在植物细胞内发挥功能的分子机制。本研究将揭示植物中CHYR类蛋白在逆境信号传递中的作用，加深对该过程的认识。

干旱是植物经常遇到的一种非生物胁迫，脱落酸ABA（Abscisic acid）在植物受到干旱胁迫时会迅速积累，能促进气孔关闭，减少叶片水分散失，帮助植物抵御干旱。ABA信号传导的分子机制的研究已经取得了一定进展，但其精细的调控网络尚不完整。.拟南芥胁迫处理后的基因表达谱数据中，我们发现At5g22920的两个突变体与野生型相比显示萌发对ABA不敏感。该基因编码一个同时含有CHY zinc-finger和RING结构域的蛋白，命名为CHYR1（CHY ZINC-FINGER AND RING PROTEIN 1）。CHYR1显著受ABA和干旱胁迫诱导表达上调，主要在维管组织和气孔中表达，并且在干旱耐受性，ABA诱导的ROS产物及气孔运动中都起正调控作用。.CHYR1是一个有功能的泛素E3连接酶，并且介导形成48位赖氨酸连接的多聚泛素化链。我们通过质谱鉴定CHYR1的免疫沉淀亲和产物找CHYR1可能的互作蛋白SnRK2.6。通过酵母双杂、双分子荧光互补（BiFC）以及免疫共沉淀（Co-IP）等实验进一步验证了他们的互作关系。此外，SnRK2.6磷酸化CHYR1的C端RING结构域中第178位苏氨酸（T178），增强CHYR1的泛素E3连接酶活性。非磷酸化状态的CHYR1转基因植株植株显示类似chyr1突变体的表型，降低植物对干旱胁迫的耐受性。磷酸化状态的CHYR1转基因植株显示类似CHYR1过表达的表型。.拟南芥基因组中有470多个基因编码RING类泛素E3连接酶，但真正解析了生物学功能的却很少，且对它们的互作蛋白更是知之甚少。我们鉴定了一个新的有功能的泛素E3连接酶CHYR1，它在植物对ABA和干旱胁迫响应过程中起正调控作用。并且，本研究阐述了其泛素E3连接酶活性如何被SnRK2.6介导的蛋白磷酸化调控的分子机制。