Dhaubhadel Sangeeta, Farhangkhoee Mana, Chapman Ralph
Southern Crop Protection and Food Research Center, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, N5V 4T3, Canada.
J Exp Bot. 2008;59(4):981-94. doi: 10.1093/jxb/ern046. Epub 2008 Feb 28.
Isoflavonoids are a diverse group of biologically active natural products that accumulate in soybean seeds during development. The majority of isoflavonoids are accumulated in the form of their glyco- and malonyl-conjugates in soybean seeds. The conjugation step confers stability and solubility to isoflavone aglycones enabling their compartmentalization to vacuoles or transport to the site of accumulation. A functional genomic approach was used to identify isoflavonoid specific glycosyltransferase (UGT) and malonyltransferase (MT) from soybean (Glycine max) seeds. An expressed sequence tag database for soybean was searched by key words to make a list of candidate genes. The full-length cDNAs for candidate UGTs and MTs were obtained and cloned into an expression vector for the production of recombinant enzymes. The in vitro enzymatic activity assays were conducted for recombinant UGTs and MTs using uridine diphosphate glucose and malonyl CoA, respectively, as donors with isoflavone substrates. Among several recombinant enzymes, UGT73F2 showed glycosylation activity towards all three soybean isoflavone aglycones and GmMT7 exhibited malonylation activity towards isoflavone glycosides. The subcellular localization study revealed both UGT73F2 and GmMT7 to be in the cytoplasm. The transcripts and protein accumulation patterns for UGT73F2 and GmMT7 genes have provided further support for their in planta function.
异黄酮是一类多样的具有生物活性的天然产物，在大豆种子发育过程中积累。大多数异黄酮在大豆种子中以其糖基和丙二酰基共轭物的形式积累。共轭步骤赋予异黄酮苷元稳定性和溶解性，使其能够分隔到液泡中或运输到积累部位。采用功能基因组学方法从大豆（Glycine max）种子中鉴定异黄酮特异性糖基转移酶（UGT）和丙二酰转移酶（MT）。通过关键词搜索大豆的表达序列标签数据库，列出候选基因清单。获得候选UGT和MT的全长cDNA，并将其克隆到表达载体中以生产重组酶。分别以尿苷二磷酸葡萄糖和丙二酰辅酶A为供体，以异黄酮底物对重组UGT和MT进行体外酶活性测定。在几种重组酶中，UGT73F2对所有三种大豆异黄酮苷元均表现出糖基化活性，而GmMT7对异黄酮糖苷表现出丙二酰化活性。亚细胞定位研究表明UGT73F2和GmMT7均位于细胞质中。UGT73F2和GmMT7基因的转录本和蛋白质积累模式为它们在植物中的功能提供了进一步支持。
