Analysis of the Plant CR4 Receptor Kinase and Gene Family

植物 CR4 受体激酶和基因家族分析

• 批准号: 9634145
• 负责人: Philip Becraft
• 金额: $ 3.5万
• 依托单位: Iowa State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-15 至 1997-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9634145&HistoricalAwards=false
• 关键词: Analysis; Plant; CR4; Receptor; Kinase

PROJECT SUMMARY _ The Project Summary should include a statement of objectives, methods to be employed, and the significance of the proposed activity to the advancement of knowledge or education. Avoid use of first person to complete this summary. DO NOT EXCEED ONE PAGE. (Some Programs may impose more stringent limits.) The maize crinkly4 (cr4) gene encodes a receptor kinase involved in the development of leaf epidermis and the aleurone of kernels (Becraft et al., in preparation). The novel extracellular domain contains a 39 amino acid repeat motif and a cysteine-rich region with similarity to the extracellular ligand-binding domain of mammalian tumor necrosis factor receptors (TNFRs). A nucleotide probe containing the extracellular domain coding sequence hybridizes at low stringency to several genomic fragments in both maize and Arabidopsis, thus cr4 represents a new gene family. The objectives of the current proposal are twofold: 1. to initiate attempts to isolate the signal molecule by direct molecular techniques and 2. to characterize this gene family in Arabidopsis. The latter will also result in the isolation of the cr4 cognate gene which will be used to develop a genetic system to study the CR4 receptor and possibly to identify the signal molecule. The similarity between CR4 and the ligand-binding domain of TNFR suggests that CR4 probably also binds a peptide signal molecule. Toward the first objective, a phage display library will be constructed using RNA from leaves at the stage when the mutant phenotype first becomes apparent. The library will be screened by biopanning for clones encoding peptides bound by the extracellular domain of CR4. Members of the cr4 gene family will be isolated by screening an Arabidopsis genomic library at low stringency with the extracellular probe from the maize cr4. The clones will be used to examine expression of the Arabidopsis genes by RNA gel blot analysis. These probes will also be used to genetically map the genes and obtain cDNA clones which will be sequenced. Sequence comparisons among Arabidopsis gene family members and with the maize gene will provide clues to functionally significant amino acid motifs. Particular, attention will be paid to the TNFR-like domain. The cr4 cognate gene will be determined from structure and sequence similarity, and become the focus of longer term studies comparing the developmental role of cr4 in Arabidopsis with maize and to functionally dissect the CR4 protein. This could also provide a tool for genetic screens to identify the signal molecule if direct molecular methods are unsuccessful. NSF FORM 1358 (1/94)

项目摘要_项目摘要应包括目标、采用的方法，以及拟议活动对促进知识或教育的重要性的说明。避免使用第一人称来完成这个总结。不要超过一页。（有些项目可能会有更严格的限制。）玉米crinkly4 （cr4）基因编码一种参与叶表皮和籽粒糊粉发育的受体激酶（Becraft et al., in preparation）。新的细胞外结构域包含一个39个氨基酸的重复基元和一个富含半胱氨酸的区域，与哺乳动物肿瘤坏死因子受体（TNFRs）的细胞外配体结合结构域相似。含有胞外结构域编码序列的核苷酸探针在玉米和拟南芥中与几个基因组片段进行低强度杂交，因此cr4代表了一个新的基因家族。当前提案的目标是双重的：1。开始尝试通过直接分子技术分离信号分子；来描述这个基因家族在拟南芥中的特征。后者也将导致cr4同源基因的分离，这将用于建立一个遗传系统来研究cr4受体，并可能识别信号分子。CR4与TNFR配体结合域的相似性表明，CR4可能也结合肽信号分子。为了实现第一个目标，在突变表型首次显现的阶段，将利用叶片中的RNA构建噬菌体展示库。该文库将通过生物筛选筛选编码CR4胞外结构域结合肽的克隆。cr4基因家族成员将通过低严格筛选拟南芥基因组文库，利用玉米cr4的细胞外探针进行分离。这些克隆将用于通过RNA凝胶印迹分析来检测拟南芥基因的表达。这些探针还将用于基因图谱和获得cDNA克隆，这些克隆将被测序。拟南芥基因家族成员之间的序列比较以及与玉米基因之间的序列比较将为研究具有重要功能的氨基酸基序提供线索。特别要注意的是TNFR-like域。从结构和序列相似性方面确定cr4同源基因，并将成为比较cr4在拟南芥和玉米中的发育作用以及功能解剖cr4蛋白的长期研究重点。如果直接分子方法不成功，这也可以为基因筛选识别信号分子提供工具。NSF表格1358 （1/94）