N-连糖基化修饰对类受体激酶DIS1和DIS2调控水稻花序发育的功能分析

The rice inflorescence development is directly related with the production, so it’s significant in theory and reality to study the functional genes controlling this developmental progress. The previous works showed that knocking-down of two CrRLK1L receptor-like kinase coding genes DIS1 and DIS2 resulted in short and sparse inflorescence. The function of CrRLK1L receptor-like kinases in rice is poorly understood. Here we will firstly confirm that DIS1 and DIS2 redundantly control the inflorescence development in rice through observing the phenotypes of their single and double mutants. N-linked glycosylation is important for the selection of secretary and membrane proteins, we found nine N-linked glycosylation sites on the extracellular region of DIS1 and DIS2, respectively, and will secondly analyze the importance of these N-linked glycosylations for their functions. Through transforming different form of single N-linked glycosylation site mutant DIS1 and DIS2 into dis1-1 dis2 double mutant, we will find the key sites essential for the function of DIS1 and DIS2 in regulating inflorescence development, and that will provide information for their extracellular signaling molecules.

水稻的花序发育与产量密切相关，研究调控花序发育的基因具有重要的理论和现实意义。前期工作表明，两个编码CrRLK1L类受体激酶的基因DIS1和DIS2同时降低表达后，水稻呈现花序短小稀疏的表型。CrRLK1L类受体激酶在单子叶植物水稻中的功能未见报道。本项目首先通过观察DIS1和DIS2双重突变体dis1-1 dis2，明确它们功能冗余地调控水稻花序的发育。据报道，N-连糖基化修饰对膜蛋白的分选和功能都很重要，但CrRLK1L类受体激酶胞外域N-连糖基化修饰的功能未知，本项目将以DIS1和DIS2的双重突变体dis1-1 dis2的花序发育为背景材料，通过遗传转化将不同N-连糖基化位点突变的DIS1和DIS2回转双突，重点解析胞外N-连糖基化修饰对于CrRLK1L类受体激酶功能的重要性，找到对DIS1和DIS2调控花序发育贡献最大的关键位点，为探索DIS1和DIS2的胞外信号分子奠定基础。

水稻的花序发育与其产量密切相关，研究调控花序发育的基因促进分子育种工作的开展，提高水稻产量。本项目两个编码CrRLK1L类受体激酶基因DRUS1和DRUS2的单突和双突进行观察发现，单突与野生型相比没有明显异常，而双突植株严重矮化，花序极其短小稀疏，完全败育。这比DRUS1和DRUS2基因敲减株系的表型更加严重，然而drus1-1+/- drus2-/-株系呈现比敲减株系更弱的表型，这些突变体材料花序中DRUS1和DRUS2的蛋白表达情况与表型正相关。表达模式分析发现DRUS1和DRUS2是两个遍在表达的蛋白，但在花序中的表达量比较高。生化实验分析发现DRUS1和DRUS2都具有激酶活性，亚细胞定位发现它们都定位在细胞质膜上。我们推断DRUS1和DRUS2在水稻生殖发育过程功能冗余地调控花序发育，同时这种调控作用在蛋白水平存在一个阈值。几乎所有类受体激酶都经由蛋白分泌途径最终定位到细胞质膜，在此过程完成N-连糖基化修饰，DRUS1和DRUS2也不例外。本项目已经创制了DRUS1N1，2Q和DRUS1N9Q的回补株系，初步观察这三个位点的N-连糖基化修饰对于DRUS1的功能贡献比较小，其他N-连糖基化修饰位点单突和九个位点全部突变的回补株系都在生产中，后续将对这些位点的功能进行详细阐述。总之，本项目对DRUS1和DRUS2调控水稻花序功能发育的生物学和分子机制进行了探究，为后续挖掘由它们介导的信号通路其他信号组分奠定了基础。