Functional Identification of Genes for Pectin Biosynthetic Galacturonosyltransferases

将确定由推定的果胶生物合成基因编码的蛋白质的酶活性。 果胶是一类结构复杂的细胞壁多糖，在植物生长发育和抗病性中具有多种作用。果胶多糖高半乳糖醛酸聚糖（HGA）、鼠李半乳糖醛酸聚糖-I（RG-I）和取代的半乳糖醛酸聚糖鼠李半乳糖醛酸聚糖II（RG-II）和木半乳糖醛酸聚糖（XGA）和芹菜半乳糖醛酸聚糖都含有1，4-连接的α-D-半乳糖基糖醛酸。 因此，果胶合成部分地由半乳糖醛酸转移酶催化，所述半乳糖醛酸转移酶将半乳糖醛酸从UDP-GalA转移到果胶寡糖/多糖受体上。拟南芥假定半乳糖醛酸转移酶（GalAT）基因的确定，通过胰蛋白酶切割的部分纯化的洗涤剂溶解的拟南芥GalAT含有蛋白质组分，然后通过液相色谱-串联质谱，以获得氨基酸序列。用衍生的氨基酸序列筛选拟南芥基因/蛋白质数据库，基于预测的糖基转移酶和跨膜结构域的存在鉴定了两种推定的GalAT蛋白质/基因。 在HGA存在下，这些基因之一（GALAT 1）在哺乳动物表达系统中的异源表达产生低水平的GalAT活性，表明该基因编码拟南芥UDP-半乳糖醛酸：HGA α-1，4-半乳糖醛酸基转移酶（GALAT 1）。拟南芥基因组的Blast分析鉴定了与GALTA 1具有高度序列相似性的其他基因。这些基因中至少有15个被认为是一个假定的GalAT基因家族的成员。 该项目的目标是克隆所提出的GalAT基因家族的选定成员，异源表达克隆的推定GalAT，并测试表达的蛋白质的GalAT活性。 还将确定在所选推定GalAT基因中通过T-DNA插入物突变的植物的发育表型和壁果胶结构。 长期目标是鉴定和表征果胶生物合成中涉及的基因。 这项研究将导致拟南芥基因的功能特性，催化果胶合成。编码果胶生物合成酶的基因的鉴定将允许对植物进行工程改造以产生具有改变的结构和性质的果胶、具有改善的农业价值的果胶以及基于果胶的营养和药理产品。历史上代表性不足的群体参与研究的方式将是通过格鲁吉亚大学夏季本科生研究计划（SURP）（www.gradsch.uga.edu/rr/）和新的SURP-桥梁计划招募本科生和研究生，该计划吸引了来自全国各地的少数族裔学生。格鲁吉亚。后一项计划为来自代表性不足群体的研究生提供研究生院之前的研究经验，以培养学生的研究技能，并增加他们在研究生院成功的可能性。

The enzyme activity of proteins encoded by putative pectin biosynthetic genes will be determined. Pectin is a family of structurally complex cell wall polysaccharides that has multiple roles in plant growth, development and disease resistance. The pectic polysaccharides homogalacturonan (HGA), rhamnogalacturonan-I (RG-I) and the substituted galacturonans rhamnogalacturonan II (RG-II) and xylogalacturonan (XGA) and apiogalacturonan all contain 1,4-linked alpha-D-galactosyluronic acid. Thus, pectin synthesis is catalyzed in part by galacturonosyltransferases that transfer galacturonic acid from UDP-GalA onto pectic oligosaccharide/polysaccharide acceptors. Arabidopsis putative galacturonosyltransferase (GalAT) genes were identified by trypsin cleavage of partially purified detergent-solubilized Arabidopsis GalAT containing protein fractions followed by liquid chromatography-tandem mass spectrometry to obtain amino acid sequences. Screening of the Arabidopsis gene/protein database with the derived amino acid sequences identified two putative GalAT proteins/genes based on the presence of predicted glycosyltransferase and membrane spanning domains. Heterologous expression of one of these genes (GALAT1) in a mammalian expression system in the presence of HGA yielded low levels of GalAT activity, suggesting the gene encodes an Arabidopsis thaliana UDP-galacturonic acid: HGA a-1,4-galacturonosyltransferase (GALAT1). Blast analysis of the Arabidopsis genome identified additional genes with high sequence similarity to GALTA1. At least 15 of these genes are proposed to be members of a putative GalAT gene family. The goals of the project are to clone selected members of the proposed GalAT gene family, heterologously express the cloned putative GalATs, and test the expressed proteins for GalAT activity. The developmental phenotype and wall pectin structure of plants mutated by T-DNA inserts in selected putative GalAT genes will also be determined. The long-term goals are to identify and characterize genes involved in pectin biosynthesis. The research will lead to the functional characterization of Arabidopsis genes that catalyze pectin synthesis. The identification of genes encoding pectin biosynthetic enzymes will allow the engineering of plants to produce pectins with modified structures and properties, pectins with improved agricultural value, and pectin-based neutraceutical and pharmacological products. The participation of historically underrepresented groups in the research will be achieved by recruiting undergraduate and graduate students through the University of Georgia Summer Undergraduate Research Program (SURP) (www.gradsch.uga.edu/rr/) and the new SURP-Bridge program, which draw in minority students from across Georgia. The latter program offers incoming graduate students from underrepresented groups research experience prior to graduate school in an effort to develop the students' research skills and increase their likelihood of success in graduate school.