Purification and Cloning of Hepta-beta Glucoside Elicitor- binding Protein(s) from Soybean

大豆中七-β葡萄糖苷激发子结合蛋白的纯化和克隆

• 批准号: 9723685
• 负责人: Michael Hahn
• 金额: $ 30万
• 依托单位: University of Georgia Research Foundation Inc
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9723685&HistoricalAwards=false
• 关键词: Purification; Cloning; Hepta; beta; Glucoside

9723685 Hahn The long-term goal of this research is to understand how plant cells perceive and respond to extracellular signals. The model system used in these studies is the induction of plant defense responses, specifically phytoalexin accumulation, in soybean (Glycine max) by oligosaccharides (elicitors) originating from the mycelial wall of a phytopathogenic oomycete, Phytophthora sojae var. glycines. Considerable information has accrued about the structure of an elicitor (a branched hepta-beta-glucoside) derived from mycelial wall glucans and about the identity and regulation of elicitor-induced genes encoding enzymes required for the biosynthesis of the phytoalexins. However, little is known about the mechanisms by which plant cells perceive the elicitor, or how that signal is transmitted to the cell nucleus to initiate changes in gene expression. The research described in this proposal focuses on the first step in the elicitor-stimulated signal transduction pathway, namely the recognition of a hepta-beta-glucoside elicitor by a plasma membrane-localized receptor. Recent studies strongly suggest that the 75 kDa polypeptide previously thought to be a hepta-beta-glucoside elicitor-binding protein (EBP) is, in fact, a non-specific glucan- binding protein. Preliminary studies using modified extraction procedures and photo-affinity labeling have now identified a 90 kDa polypeptide as a candidate EBP. The overall goal of the research proposed in this application is to identify, purify, and clone EBPs from soybean root membranes and to obtain evidence that the EBPs are physiological receptors for the hepta-beta-glucoside elicitor. The first specific goal is to identify and purify EBPs. The number and identity of polypeptides in solubilized membrane protein preparations that have hepta-beta-glucoside elicitor-binding sites will be established by photo- affinity labeling. The specificity of the photo-affinity labeling will be rigorously established in l igand competition assays with oligoglucosides structurally related to the hepta-beta-glucoside elicitor. Polypeptides present in the membranes that bind to elicitor-inactive beta-glucans will be eliminated first and then the EBPs will be purified by ligand-affinity chromatography. Partial amino acid sequences will be determined for the polypeptide(s) found to have a specific hepta-beta-glucoside elicitor-binding site. The second specific goal is to obtain antibodies against the EBPs. These antibodies will be used to localize EBPs in plant tissues and to isolate genes encoding EBPs. The third specific goal is to clone cDNA(s) that encode EBPs. Degenerate oligonucleotides will be synthesized and used to amplify, by PCR, a double-stranded DNA probe from a heterogeneous population of single-stranded cDNAs made from soybean root mRNA. This probe will be used to screen a lambda gt10 library for cDNAs encoding EBPs. Alternatively, antibodies generated against affinity-purified EBPs will be used to screen a cDNA expression library for clones expressing EBP sequences. Any cDNA clones that are obtained will be purified and sequenced. The ability of the protein(s) encoded by the cloned cDNA(s) to bind the hepta- beta-glucoside elicitor specifically and with high affinity will be determined. The cDNA clones and their derived sequences will be used to identify possible structural and functional domains in the EBPs that might relate to their role in signal transduction. The research outlined in this renewal application will lay the groundwork for future work that will test whether the EBPs function as physiological receptors and to identify and characterize components that interact with EBPs and might function downstream of the receptor in the cellular signaling pathway that results in the accumulation of phytoalexins in elicited plant cells. The results of these studies should provide additional insight into the mechanisms used by plant cells to perceive and respond to extracellular signals. These studies should also increase our understanding of the roles of carbohydrate signal molecules (oligosaccharins) in the regulation of biological processes in diverse organisms. Plants perceive and respond to pathogen attack. The perception of the infection of a pathogen is mediated by the detection of specific compounds termed elicitors are associated with the attack. Cell surface proteins detect the presence of these elicitors which signal a variety of processes within the plant cell to marshal defense reactions. Dr. Hahn has identified a soybean cell-surface protein that binds an elicitor originating from the pathogen Phytophthora. The binding appears to be highly specific to a single protein and has characteristics that make it a candidate for the elicitor receptor. With this award Dr. Hahn will purify and isolate the receptor protein. Data obtained from the purified protein will be used to obtain a cDNA clone of the elicitor binding protein. The results of this research will yield information on the mechanisms by which plants perceive infection of pathogens. This information is extremely important in understanding how plants maintain growth in a hostile world and it has potential practical applications I improving and modifying a plants response to pathogen challenge. ***

9723685哈恩这项研究的长期目标是了解植物细胞如何感知和响应细胞外信号。这些研究中使用的模型系统是由来源于植物病原卵菌大豆疫霉(Phytophthora Sojae Var)菌丝壁的寡糖(激发子)诱导大豆(Glyine Max)的植物防御反应，特别是植物防御素的积累。甘氨酸。关于菌丝壁葡聚糖的激发子(一种分枝的七β-葡萄糖苷)的结构，以及激发子诱导的基因编码植物抗毒素生物合成所需的酶的鉴定和调控，已经积累了大量的信息。然而，关于植物细胞感知激发子的机制，或者该信号如何传递到细胞核以启动基因表达的变化，人们知之甚少。这项研究集中在激发子刺激的信号转导途径的第一步，即通过质膜定位的受体识别七β-葡萄糖苷激发子。最近的研究有力地表明，以前被认为是七-β-葡萄糖苷激发子结合蛋白(EBP)的75 kDa多肽实际上是一种非特异性的葡聚糖结合蛋白。使用改进的提取程序和光亲和标记的初步研究现已确定90 kDa多肽为候选EBP。本申请中提出的研究的总体目标是从大豆根膜中鉴定、纯化和克隆EBPs，并获得EBPs是七β-葡萄糖苷激发子的生理受体的证据。第一个具体目标是识别和纯化EBP。光亲和标记法将确定具有七β-葡萄糖苷激发子结合位点的可溶性膜蛋白制剂中多肽的数量和特性。光亲和标记的特异性将在L配基竞争分析中得到严格确立，寡糖与七-β-葡萄糖苷激发子在结构上相关。膜中存在的与激发子失活的β-葡聚糖结合的多肽将首先被消除，然后通过配基亲和层析纯化EBPs。部分氨基酸序列将被确定为多肽(S)，发现具有特定的七-β-葡萄糖苷激发子结合部位。第二个具体目标是获得针对EBPs的抗体。这些抗体将被用来定位植物组织中的EBPs，并分离编码EBPs的基因。第三个具体目标是克隆编码EBPS的基因(S)。简并寡核苷酸将被合成并用于通过聚合酶链式反应(PCR)从由大豆根mRNA制成的单链DNA的异质性群体中扩增出双链DNA探针。该探针将用于筛选lambda gt10文库中编码EBPs的cDNA。或者，针对亲和纯化的EBPs产生的抗体将用于筛选表达EBP序列的克隆的cDNA表达文库。获得的任何cdna克隆都将进行纯化和测序。将测定克隆的S编码的蛋白(S)与七β-葡萄糖苷激发子特异性和高亲和力结合的能力。这些克隆及其衍生的序列将被用来确定EBPs中可能与其在信号转导中的作用有关的结构和功能结构域。本次更新申请中概述的研究将为未来的工作奠定基础，这些工作将测试EBPs是否作为生理受体发挥作用，并鉴定与EBPs相互作用的成分，以及可能在细胞信号通路中位于受体下游导致植物细胞中植物抗毒素积累的成分。这些研究的结果应该为植物细胞感知和响应细胞外信号的机制提供更多的洞察力。这些研究还应该增加我们对碳水化合物信号分子(寡糖素)在调节不同生物体的生物过程中的作用的理解。植物能够感知病原体的侵袭并对其做出反应。对病原体感染的感知是通过检测被称为激发子的特定化合物来调节的，这种化合物与攻击有关。细胞表面蛋白检测到这些激发子的存在，这些激发子发出信号，指示植物细胞内的各种过程来组织防御反应。哈恩博士已经确定了一种大豆细胞表面蛋白，它能结合来自疫霉病原菌的激发子。这种结合似乎对单一蛋白质具有高度特异性，并具有使其成为激发子受体候选者的特征。获得这一奖项后，哈恩博士将提纯和分离受体蛋白。从纯化蛋白中获得的数据将用于获得激发子结合蛋白的cDNA克隆。这项研究的结果将提供有关植物感知病原体感染的机制的信息。这些信息对于了解植物如何在恶劣的环境中保持生长是极其重要的，它在改善和修改植物对病原体挑战的反应方面具有潜在的实际应用。***