Analysis of the Plant CR4 Receptor Kinase and Gene Family

项目摘要-项目摘要应包括目标、要采用的方法以及拟议活动对促进知识或教育的意义的陈述。避免使用第一人称来完成此摘要。不要超过一页。(一些项目可能会施加更严格的限制。)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等人，正在准备中)。新的胞外区含有一个39个氨基酸重复序列和一个与哺乳动物肿瘤坏死因子受体(TNFR)胞外配体结合区相似的富含半胱氨酸的区域。在玉米和拟南芥中，含有胞外区编码序列的核苷酸探针与多个基因组片段以低严格率杂交，因此CR4代表了一个新的基因家族。目前的建议有两个目标：1.尝试通过直接分子技术分离信号分子；2.鉴定拟南芥中该基因家族的特征。后者还将导致CR4同源基因的分离，该基因将用于开发研究CR4受体的遗传系统，并可能用于识别信号分子。CR4与TNFR的配体结合域的相似性表明，CR4可能还与一个肽信号分子结合。对于第一个目标，在突变表型首次显现的阶段，将使用叶片中的RNA构建噬菌体展示文库。文库将通过生物扫描来筛选编码CR4胞外结构域结合的多肽的克隆。用玉米CR4的胞外探针筛选低严格率的拟南芥基因组文库，将分离出CR4基因家族的成员。这些克隆将被用来通过RNA凝胶印迹分析来检测拟南芥基因的表达。这些探针还将被用来对基因进行遗传定位，并获得将被测序的cdna克隆。在拟南芥基因家族成员之间以及与玉米基因之间的序列比较将为发现具有重要功能的氨基酸基序提供线索。特别是，将关注类似TNFR的领域。CR4同源基因将通过结构和序列的相似性来确定，并将成为比较CR4在拟南芥和玉米中的发育作用和从功能上剖析CR4蛋白的长期研究的重点。如果直接分子方法不成功，这也可以为遗传筛选提供一种工具来识别信号分子。NSF表格1358(1/94)

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)