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

富含羟脯氨酸的糖蛋白（HRGPS）是生长细胞壁中普遍存在的结构组分（高达10- 20%dw），涉及植物生长和发育的各个方面，包括对胁迫的响应。HRGP的三个主要组代表了糖基化和多肽周期性的连续体，范围从轻度糖基化的重复富含脯氨酸的蛋白质（RPRP），通过高度阿拉伯糖基化的交联延伸蛋白，阿拉伯半乳聚糖蛋白（AGP）是最高度糖基化和最少的周期性。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然很大程度上未被探索。本研究将通过对烟草和番茄培养物中表达为重复糖肽的单个糖肽模块的功能分析来确定它们的作用。这些包括（Ser-Hyp 4）n、（Ser-Hyp 3）n、（Ser-Hyp 2）n、（Ser-Hyp）n、（Ala-Hyp）n和延伸蛋白和AGP的相关基序，以及另一种糖蛋白（GAGP）的19个残基的共有序列。这涉及基于重叠寡核苷酸互补内部重复序列对和互补外部3 '-和5'-接头对的连接组以形成编码给定数目的模块重复序列的长双链DNA来构建合成基因盒。将这些合成基因插入到含有延伸蛋白信号序列和增强的绿色荧光蛋白（EGFP）报道基因的修饰的pBI 12 1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酰羟化酶和糖基转移酶的烟草和番茄的细胞培养物。这些模块的表达和分离将允许测试糖基化和糖基化功能的以下一般和特定假设：1.由Hyp-contiguity Hypothesis预测的糖基化代码指定了对特定HRGP Hyp残基的精确糖添加，即：对最高度连续的残基添加四阿拉伯糖，阿拉伯糖基化随着Hyp-contiguity的降低而降低;以及对代表包括GAGP在内的AGP的重复非连续Hyp残基添加阿拉伯半乳聚糖多糖。碳水化合物介导的伸展蛋白单体的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。作为高度周期性糖蛋白的类似物，“设计者延伸蛋白”和“设计者树胶”将有助于将位点特异性糖基化与基本分子功能如交联、自缔合和细胞壁自组装相关联。这些类似物还提供了一种独特的方法来设计新型基因工程树胶和糖蛋白聚合物，其具有受控的结构和特性，用于增强植物抗病性，并在农业、食品和纳米技术工业中具有潜在的用途。最后，这项研究将使学生获得广泛的工具和概念，在分子生物学，从基因操作，微生物学和植物生物学，蛋白质，糖蛋白和碳水化合物化学，以及一个很好的机会，学习如何整合它们作出重大的科学贡献。

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.