Role of the CRINKLY4 Receptor Kinase in Plant Epidermal Differentiation

Becraft 96-04426 植物叶片被高度特化的表皮层覆盖，其在发育中起着划分器官和防止器官融合的作用。禾本科植物种子的胚乳也含有一个表皮样的细胞层，称为糊粉层，它能分泌水解酶，在萌发过程中使贮藏化合物再活化。玉米crinkly 4（cr 4）基因与叶表皮和糊粉层的分化有关。cr 4编码与哺乳动物肿瘤坏死因子受体（TNFR）的配体结合结构域具有胞外相似性的受体激酶。肿瘤坏死因子（TNF）是一种小的蛋白质信号分子，参与免疫系统和其他组织的发育过程。CR 4和TNFR之间的相似性表明CR 4的配体也可能是TNF类蛋白。基因组southern杂交表明cr 4是一个基因家族的成员。本研究有三个具体目标：1。完成玉米cr 4基因的分子遗传学分析。这包括产生cr 4突变体的等位基因系列和分析cr 4基因的表达模式。将使用免疫组织化学和遗传镶嵌分析研究表达模式。2.分离CR 4胞外结构域的配体。提出了三种不同的方法：A.分子鉴定编码能够结合CR 4胞外结构域的蛋白质的克隆基因（即相互作用克隆），B.通过分析与cr 4具有相似表型的突变体，或通过鉴定增强或抑制cr 4突变表型的第二位点突变，遗传鉴定相互作用蛋白，和C.一种转基因方法，引入可用于遗传抑制筛选的特定工程突变。3.描述拟南芥cr 4基因家族的特征，以确定该家族的范围，是否所有成员都编码受体激酶以及每个成员的表达谱。

Becraft 96-04426 Plant leaves are covered by a highly specialized epidermal layer which functions in development to delimit organs and prevent organ fusions. The endosperm of grass seeds also contains an epidermis-like cell layer called the aleurone which secretes hydrolytic enzymes required for remobilization of storage compounds during germination. The crinkly4 (cr4) gene of maize is involved in the differentiation of leaf epidermis and aleurone. cr4 encodes a receptor kinase with extracellular similarity to the ligand binding domain of tumor necrosis factor receptors (TNFRs) from mammals. Tumor necrosis factors (TNFs) are small protein signal molecules that are involved in developmental processes in the immune system and other tissues. The similarity between CR4 and TNFR suggests that the ligand for CR4 might also be a protein of the TNF class. Genomic southern hybridization suggests that cr4 is a member of a gene family. The proposed research has three specific aims: 1. Complete a molecular genetic analysis of the maize cr4 gene. This includes generating an allelic series of cr4 mutants and analyzing the expression pattern of the cr4 gene. The expression pattern will be studied using immunohistochemistry and genetic mosaic analyses. 2. Isolate a ligand for the extracellular domain of CR4. Three different approaches are proposed: A. molecular identification of a cloned gene that encodes a protein able to bind the extracellular domain of CR4 (i.e. interaction cloning), B. genetic identification of an interacting protein by analysis of mutants with similar phenotypes to cr4, or by identification of second site mutations that enhance or suppress the cr4 mutant phenotype, and C. a transgenic approach to introduce specifically engineered mutations that can be used in genetic suppressor screens. 3. Characterize the cr4 gene family in arabidopsis to determine the extent of the family, whether all members encode receptor kineses and the expression profiles for each member.