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声明，要采用的方法以及拟议活动对知识或教育发展的意义。避免使用第一人称完成此摘要。不要超过一页。 （某些程序可能会施加更严格的限制。）玉米crinkly4（CR4）基因编码参与叶片表皮发展和核的核龙的受体激酶（Becraft等人，准备中）。新型的细胞外域含有39个氨基酸重复基序和一个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的富含半胱氨酸的区域。含有细胞外域编码序列的核苷酸探针在玉米和拟南芥中的几个基因组片段时杂交，因此CR4代表了一个新的基因家族。当前建议的目标是双重的：1。试图通过直接分子技术分离信号分子和2。在拟南芥中表征该基因家族。后者还将导致CR4同源基因的分离，该基因将用于开发遗传系统以研究CR4受体并可能鉴定信号分子。 TNFR的CR4和配体结合结构域之间的相似性表明CR4可能也结合了肽信号分子。朝第一个目标迈进，当突变表型首先变得明显时，将使用叶子的RNA构建噬菌体显示库。该库将通过Biopanning进行筛选，以编码由CR4的细胞外域结合的肽编码的克隆。 CR4基因家族的成员将通过在玉米CR4的细胞外探针下筛选一个拟南芥基因组文库来隔离。这些克隆将通过RNA凝胶印迹分析来检查拟南芥基因的表达。这些探针还将用于遗传绘制基因并获得将被测序的cDNA克隆。拟南芥基因家族成员和玉米基因之间的序列比较将为功能上重要的氨基酸基序提供线索。特别是，将注意类似TNFR的领域。 CR4同源基因将由结构和序列相似性确定，并成为比较CR4与玉米中CR4的发育作用的长期研究的重点，并在功能上剖析CR4蛋白。如果直接分子方法不成功，这也可以为遗传筛选提供一种识别信号分子的工具。 NSF表格1358（1/94）