水稻细胞壁关联蛋白激酶（WAK）应答铜胁迫的功能及作用机理研究

Wall-associated kinases (WAKs) are a type of receptor-like kinases which are known to directly bind to cell walls. In this study, a wall-associated kinase of rice is identi?ed by two-dimensional PAGE as a Cu-induced up-regulated protein. Similar results are observed in expression analysis of WAK under Cu stress as determined by semi-quantitative RT-PCR.The above-mentioned results show that the wall-associated kinase may be involved in responding to Cu stress in rice. On this basis, a wall-associated kinase gene, WAK and its promoter in rice will need to clone, and the characterization of WAK expression remains to be investigated. What are their physiology ligands and down stream substrates, and the relationship between WAK and other signals are also required to investigate. These ?ndings may lead to a better understanding of the physiological functions and mechanisms of wall-associated kinase in responses to copper exposure in plants. The research will fill a scientific gap of WAK in tolerance to heavy metal, and provid the theory basis for the cloning of heavy metal tolerance genes and increasing the tolerance of heavy metals in plants with transgenic engineering technology.

细胞壁关联蛋白激酶是一类特殊的类受体蛋白激酶,本实验室利用2D-PAGE技术，发现Cu胁迫引起了水稻细胞壁关联蛋白激酶（WAK）的表达显著增加；这与半定量RT-PCR分析结果是一致的，这说明WAK可能参与了水稻对铜胁迫的响应。本项目将在此基础上通过克隆水稻WAK基因及其启动子、研究该基因的表达调控特性；同时明确其胞外的结合配基和胞内调控的靶蛋白、及与其他信号分子的关系，旨在阐明WAK在植物耐铜性中的生理功能及作用机理；这将填补植物重金属耐性机理研究领域中的空白，同时研究结果也可为进一步克隆植物重金属耐性相关基因、并通过转基因技术提高敏感植物对重金属的抗性提供理论依据。

细胞壁关联蛋白激酶（Cell all associated kinase, WAK）是类受体蛋白激酶（Receptor-like kinase, RLK）家族成员。本项目克隆了模式植物水稻OsWAK11基因，并对该基因及其上游启动子序列（WAKpro）进行了分析。发现在OsWAK11启动子区可能包含铜响应调控元件（CuRE)，脱落酸（ABA）调控元件（ABRE)，热激蛋白调控元件（HSE），以及TATA-box、CAAT-box核心元件等，将该启动子区序列（WAKpro）融合GUS基因成功转入了烟草中。研究发现，CuSO4，AlCl3和NaCl处理均上调水稻体内OsWAK11基因的表达，也诱导转基因烟草中GUS的活性，机械伤害也强烈诱导烟草中GUS活性。同时，本研究还发现，OsWAK11激酶结构域片段（WAKD）具有体外激酶活性。本项目又借助RNA干扰技术，获得了OsWAK11基因沉默表达的水稻转基因株系。OsWAK11基因沉默引起水稻根系及叶片的细胞伸长受到明显的抑制，从而导致幼苗生长矮小。另外，OsWAK11突变体幼苗表现出对过量铜的敏感，与野生型水稻相比，过量铜显著抑制了突变体水稻幼苗的生物量、株高、根系伸长。突变体水稻幼苗根系及地上部铜浓度显著高于野生型。OsWAK11基因突变导致细胞壁结合Cu的能力下降，从而导致进入细胞的铜含量增加，最终导致oswak11突变体幼苗铜毒害的症状较野生型明显；在此过程中，果胶甲酯酶发挥重要的作用。