SGER: Synthetic Genes for the Design of Hydroxproline-Rich Glycomodules

9805960富含羟脯氨酸的凯氏糖蛋白(HRGP)是生长中植物细胞壁普遍存在的结构成分，通常占植物干重的10-20%，其中多糖占大部分。HRGP参与植物生长发育的方方面面，包括对胁迫的反应。HRGP超家族包括三个主要基团，代表糖基化和周期性的连续体。它们的范围从轻微糖化和高度周期性的重复富含Pro的蛋白质，到高度阿拉伯糖化和周期性的交联型延伸蛋白，到高度糖基化和最不周期性的阿拉伯半乳糖-蛋白质。重复单位很小，通常只有4-6个残基糖基化基序被假设为功能模块。这些独特的植物糖蛋白的功能在很大程度上仍未被探索。我们建议通过对单个糖肽模块的功能分析来确定它们在番茄培养中的作用。我们将人工合成的编码HRGP糖肽模块的基因插入到两个来源于pBI121的植物转化载体中；一个带有报告基因，另一个用于模块功能测试，缺少报告基因。我们将使用这些质粒转化番茄的细胞培养物，其中含有模块翻译后修饰所需的植物脯氨酸羟基酶和糖基转移酶。这些模块的表达和分离将使我们能够测试糖基化和分子功能的一般和特定假说：1.高邻接性假说预测的糖基化密码指定特定HRGP Hyp残基的精确糖添加，即：四阿拉伯糖到最高邻接残基，阿拉伯糖基化随着Hyp-邻接程度的降低而减少；以及典型的AGPs重复的非邻接Hyp残基的多半乳糖化。2.碳水化合物介导的伸展蛋白单体的排列是它们有序组装和细胞壁自组装中的交联作用所必需的。

9805960 Kieliszewski Hydroxyproline-rich glycoproteins (HRGPs) are ubiquitous architectural components of growing plant cell walls, often accounting for 10-20% of their dry weight; polysaccharides account for the bulk. HRGPs are implicated in all aspects of plant growth and development including responses to stress. The HRGP superfamily contains three major groups representing a continuum of glycosylation and periodicity. They range from the repetitive proline-rich proteins that are lightly glycosylated and highly periodic, through the crosslinked extensins that are highly arabinosylated and periodic, to the arabinogalactan-proteins that are the most highly glycosylated and least periodic. The repetitive units are small, often only 4-6 residue glycosylated motifs viewed hypothetically as functional modules. The function of these unique plant glycoproteins remains largely unexplored. We propose to define their roles through functional analysis of single glycopeptide modules when expresssed as repetitive glycopolypeptides in tomato cultures. We shall insert synthetic genes encoding HRGP glycopeptide modules into two plant transformation vectors both derived from pBI121; one with a reporter gene, the other, for test for module function, lacking the reporter. We shall use these plasmids to transform cell cultures of tomato, which contain the plant prolylhydroxylases and glycosyltransferases required for module posttranslational modifications. Expression and isolation of these modules will enable us to test general and specific hypotheses for glycosylation and molecular 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 polygalactosylation of repetitive noncontiguous Hyp residues that typify AGPs. 2. Carbohydrate-mediated alignment of extensin monomers is necessary for their orderly assembly and crosslinking roles in cell wall self-assembly.