Analysis of the Plant CR4 Receptor Kinase and Gene Family

项目摘要 _ 项目总结应包括目标、拟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此摘要。不要超过一页。(Some程序可能会施加更严格的限制。） 玉米crinkly 4（cr 4）基因编码参与叶表皮和籽粒糊粉层发育的受体激酶（Becraft等人，正在筹备中）。新的胞外结构域包含一个39个氨基酸的重复基序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低严格性下与玉米和拟南芥中的几个基因组片段杂交，因此cr 4代表一个新的基因家族。本提案的目标有两个方面：1.开始尝试通过直接分子技术分离信号分子，以及2.来描述拟南芥中的这个基因家族。后者还将导致cr 4同源基因的分离，其将用于开发遗传系统以研究CR 4受体并可能鉴定信号分子。 CR 4和TNFR的配体结合结构域之间的相似性表明CR 4可能也结合肽信号分子。对于第一个目标，将使用来自突变体表型首次变得明显的阶段的叶片的RNA构建噬菌体展示文库。将通过生物淘选筛选编码由CR 4的胞外结构域结合的肽的克隆来筛选文库。 cr 4基因家族的成员将通过用来自玉米cr 4的细胞外探针在低严格性下筛选拟南芥基因组文库来分离。克隆将用于通过RNA凝胶印迹分析检查拟南芥基因的表达。这些探针也将用于基因图谱的基因，并获得cDNA克隆，将测序。拟南芥基因家族成员之间以及与玉米基因的序列比较将为功能上重要的氨基酸基序提供线索。特别地，将关注TNFR样结构域。cr 4同源基因将从结构和序列相似性确定，并成为比较cr 4在拟南芥和玉米中的发育作用以及功能解剖CR 4蛋白的长期研究的焦点。如果直接分子方法不成功，这也可以为遗传筛选提供一种识别信号分子的工具。 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)