Functional Identification of Genes for Pectin Biosynthetic Galacturonosyltransferases

由果胶生物合成基因编码的蛋白质的酶活性将被测定。果胶是一类结构复杂的细胞壁多糖家族，在植物生长发育和抗病中具有多种作用。果胶多糖高半乳糖醛酸(HGA)、鼠李半乳糖醛酸-I(RG-I)、取代半乳糖醛酸脂II(RG-II)、木糖半乳糖醛酸(XGA)和半乳糖醛酸脂均含有1，4-连接的α-D-半乳糖糖醛酸。因此，果胶的合成部分是由半乳糖醛酸基转移酶催化的，半乳糖醛酸基转移酶将半乳糖醛酸从UDP-GALA转移到果胶低聚糖/多糖受体上。拟南芥半乳糖醛酸基转移酶(Galat)基因通过胰酶裂解部分纯化的半乳糖醛酸基转移酶(Galat)蛋白组分，再经高效液相色谱-串联质谱(LC-MS)获得氨基酸序列。利用推导的氨基酸序列对拟南芥基因/蛋白数据库进行筛选，根据预测的糖基转移酶和跨膜结构域的存在，鉴定了两个可能的Galat蛋白/基因。其中一个基因(GALAT1)在HGA存在的哺乳动物表达系统中异源表达，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 a-1，4-半乳糖醛酸基转移酶(GALAT1)。对拟南芥基因组的BLAST分析发现了与GALTA1序列高度相似的其他基因。这些基因中至少有15个被认为是Galat基因家族的成员。该项目的目标是克隆所建议的Galat基因家族中选定的成员，异源表达克隆的假定Galat，并测试表达的蛋白的Galat活性。还将确定被选定的可能的Galat基因的T-DNA插入突变的植物的发育表型和壁果胶结构。长期目标是识别和表征与果胶生物合成有关的基因。这项研究将导致拟南芥催化果胶合成的基因的功能特征。识别编码果胶生物合成酶的基因将使植物工程能够生产结构和性质改变的果胶、提高农业价值的果胶，以及以果胶为基础的中性和药理产品。历史上代表性不足的群体将通过佐治亚大学暑期本科生研究计划(www.gradsch.uga.edu/rr/)和新的SURP-Bridge计划招收本科生和研究生来实现这项研究，该计划吸引了佐治亚州各地的少数民族学生。后一项计划为来自代表性不足群体的应届研究生提供研究生入学前的研究经验，以努力发展学生的研究技能，增加他们在研究生院取得成功的可能性。

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.