Role of the CRINKLY4 Receptor Kinase in Plant Epidermal Differentiation

96-04426植物叶片被高度专门化的表皮层覆盖，在发育过程中起着界定器官和防止器官融合的作用。草籽的胚乳还含有一层表皮状的细胞层，称为糊粉，它分泌水解酶，在萌发过程中需要重新动员储存的化合物。玉米的crinkly4(CR4)基因参与了叶表皮和糊粉的分化。CR4编码一种与哺乳动物肿瘤坏死因子受体(TNFR)的配体结合域相似的细胞外受体激酶。肿瘤坏死因子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。CR4和TNFR的相似性表明，CR4的配体也可能是一种肿瘤坏死因子类的蛋白质。基因组Southern杂交表明，CR4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CR4基因的分子遗传分析。这包括产生一系列CR4突变的等位基因，并分析CR4基因的表达模式。将使用免疫组织化学和遗传镶嵌分析来研究表达模式。2.分离CR4胞外区的配体。提出了三种不同的方法：A.编码能够结合CR4胞外区的蛋白质的克隆基因的分子鉴定(即相互作用克隆)，B.通过分析与CR4表型相似的突变或通过鉴定增强或抑制CR4突变表型的第二位点突变来鉴定相互作用的蛋白质，以及C.引入可用于基因抑制筛选的特定工程突变的转基因方法。3.确定拟南芥CR4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动子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.