CAREER: Synthetic Genes to Elucidate Glycopeptide Module Function in Extensins and Arabinogalactan-Proteins

富含羟基脯氨酸的糖蛋白（HRGPS）是生长细胞壁中普遍存在的结构成分（高达10-20% dw），涉及植物生长发育的各个方面，包括对胁迫的反应。hrgp的三个主要组代表了糖基化和多肽周期性的连续体，从轻度糖基化的重复富含脯氨酸蛋白（RPRPs），到高度阿拉伯糖基化的交联延伸蛋白，再到高度糖基化和周期性最低的阿拉伯半乳糖蛋白（AGPs）。重复单元通常是小的4-6个残基糖基化基序，假设被视为功能模块。然而，HRGP的功能在分子水平上仍未得到充分的研究。本研究将通过在烟草和番茄培养中表达为重复糖共肽的单糖肽模块的功能分析来确定它们的作用。这些包括（Ser-Hyp4）n, (Ser-Hyp3)n, (Ser-Hyp2)n, (Ser-Hyp)n, (Ser-Hyp)n，以及延伸蛋白和AGPs的相关基序，以及另一种糖蛋白（GAGP）的19个残基一致序列。这涉及到基于连接重叠的寡核苷酸互补的内部重复对和互补的外部3'-和5'-连接对的组合来构建合成基因盒，以形成编码给定数量的模块重复的长双工DNA。将这些合成基因插入到含有延伸蛋白信号序列和增强型绿色荧光蛋白（EGFP）报告基因的修饰的pBIl2l植物转化载体中，将用于转化含有模块翻译后修饰所需的植物脯氨酸羟化酶和糖基转移酶的烟草和番茄细胞培养物。这些模块的表达和分离将允许测试以下关于糖基化和糖模块功能的一般和特定假设：高邻接假说预测的糖基化编码精确地指定了特定HRGP Hyp残基上的糖基化，即：四阿拉伯糖糖加到最高度邻接的残基上，阿拉伯糖基化随着低邻接程度的降低而降低；和阿拉伯半乳多糖添加到重复不连续的Hyp残基中，这些残基代表agp，包括GAGP.2。碳水化合物介导的延伸蛋白单体排列对于它们在细胞壁自组装中的有序组装和交联作用是必要的。作为高周期糖蛋白的类似物，“设计延伸蛋白”和“设计胶”将有助于将位点特异性糖基化与交联、自结合和细胞壁自组装等基本分子功能联系起来。这些类似物还为设计具有可控结构和性能的新型基因工程胶和糖蛋白聚合物提供了一种独特的方法，以增强植物的抗病性，并在农业、食品和纳米技术工业中具有潜在的用途。最后，这项研究将使学生接触到分子生物学中广泛的工具和概念，从基因操作，微生物学和植物生物学，到蛋白质，糖蛋白和碳水化合物化学，并提供一个学习如何整合它们以做出重大科学贡献的绝佳机会。

KieliszewskiHydroxyproline-rich glycoproteins (HRGPS), ubiquitous structural components (up to 10-20% dw) of growing cell walls, are implicated in all aspects of plant growth and development including responses to stress. Three major groups of HRGPs represent a continuum of glycosylation and polypeptide periodicity ranging from the lightly glycosylated repetitive proline-rich proteins (RPRPs), through the highly arabinosylated crosslinked extensins, to the arabinogalactan-proteins (AGPs) that are the most highly glycosylated and least periodic. Repetitive units are generally small 4-6 residue glycosylated motifs viewed hypothetically as functional modules. However, HRGP function remains largely unexplored at the molecular level. This research will define their roles through functional analysis of single glycopeptide modules expressed as repetitive glycopolypeptides in tobacco and tomato cultures. These include the (Ser-Hyp4)n, (Ser-Hyp3)n, (Ser-Hyp2)n, (Ser-Hyp)n, (Ala-Hyp)n, and related motifs of extensins and AGPs, as well as the 19-residue consensus sequence of another glycoprotein (GAGP). This involves construction of synthetic gene cassettes based on ligating sets of overlapping oligonucleotide complementary internal repeat pairs and complementary external 3'- and 5'-linker pairs to form long duplex DNA encoding a given number of module repeats. Insertion of these synthetic genes into a modified pBIl2l plant transformation vector containing an extensin signal sequence and the enhanced green fluorescent protein (EGFP) reporter gene will be used to transform cell cultures of tobacco and tomato that contain the plant prolyl hydroxylases and glycosyltransferases required for module posttranslational modifications. Expression and isolation of these modules will permit tests of the following general and specific hypotheses for glycosylation and glycomodule function:1. Glycosylation codes predicted by the Hyp-contiguity Hypothesis specify precise saccharide addition to specific HRGP Hyp residues, namely: tetraarabinose to the most highly contiguous residues, arabinosylation decreasing with decreasing Hyp-contiguity; and arabinogalactan polysaccharide addition to repetitive noncontiguous Hyp residues that typify AGPs including GAGP.2. Carbohydrate-mediated alignment of extensin monomers is necessary for their orderly assembly and crosslinking roles in cell wall self-assembly. As analogs of highly periodic glycoproteins, "designer extensins" and "designer gums" will help relate site-specific glycosylation to essential molecular functions like crosslinking, self-association and cell wall self-assembly. These analogs also provide a unique approach to the design of novel genetically engineered gums and glycoprotein polymers of controlled structure and properties for enhancing plant disease resistance, and potential use in the agricultural, food and nanotechnology industries. Finally, this research will give students access to a wide range of tools and concepts in molecular biology, ranging from gene manipulation, microbiology and plant biology, to protein, glycoprotein and carbohydrate chemistry, and an excellent opportunity to learn how to integrate them to make significant scientific contributions.