Cloning, gene mapping, and functional analysis of a fructan 1-exohydrolase (1-FEH) from Lolium perenne implicated in fructan synthesis rather than in fructan mobilization
Cloning, gene mapping, and functional analysis of a fructan 1-exohydrolase (1-FEH) from Lolium perenne implicated in fructan synthesis rather than in fructan mobilization
复制标题
DOI：
10.1093/jxb/erm053
复制
发表时间：
2007-01-01
影响因子：
6.9
通讯作者：
Morvan-Bertrand, Annette
中科院分区：
文献类型：
--
作者：
Lothier, Jeremy;Lasseur, Bertrand;Morvan-Bertrand, Annette
果聚糖是果糖的 β-(2,1) 和/或 β-(2,6) 连接聚合物，是许多植物中重要的储存碳水化合物。它们通过果聚糖外切水解酶 (FEH) 动员。来自黑麦草 (Lolium perenne L. var.) 的第一个 1-FEH (EC 3.2.1.153) 的克隆、定位和功能分析。这里描述了 Bravo。通过筛选多年生黑麦草cDNA文库，克隆了1-FEH cDNA，命名为Lp1-FEHa。 Lp1-FEHa 推导的蛋白质具有低等电点 (5.22)，它与植物 FEH 和细胞壁型转化酶归为一类。推导的氨基酸序列与小麦1-FEH w2 有75% 的同一性。 Lpl-FEHa 基因被定位在连锁群 3 (LG3) 的远端位置。毕赤酵母中重组蛋白的功能表征表明，其对 1-酮三糖、1,1-酮四糖和菊粉具有高 FEH 活性，但对 6-酮三糖和果聚糖具有低活性。与其他果聚糖植物 FEH 一样，未检测到对蔗糖的水解酶活性，这令人信服地证明该酶不是典型的转化酶。 Lpl-FEHa的表达模式分析揭示了积累果聚糖的叶组织中的转录物积累，而光合组织中的转录物水平较低。该 1-FEH 在果聚糖合成活跃的条件下的高表达水平，以及果聚糖含量低时的低表达水平，表明它可能在果聚糖合成过程中与果聚糖合成酶协同作用，发挥 β-(2,1) 修剪酶的作用。
Fructans, which are beta-(2,1) and/or beta-(2,6) linked polymers of fructose, are important storage carbohydrates in many plants. They are mobilized via fructan exohydrolases (FEHs). The cloning, mapping, and functional analysis of the first 1-FEH (EC 3.2.1.153) from Lolium perenne L. var. Bravo is described here. By screening a perennial ryegrass cDNA library, a 1-FEH cDNA named Lp1-FEHa was cloned. The Lp1-FEHa deduced protein has a low iso-electric point (5.22) and it groups together with plant FEHs and cell-wall type invertases. The deduced amino acid sequence shows 75% identity to wheat 1-FEH w2. The Lpl-FEHa gene was mapped at a distal position on the linkage group 3 (LG3). Functional characterization of the recombinant protein in Pichia pastoris demonstrated that it had high FEH activity towards 1-kestotriose, 1,1-kestotetraose, and inulin, but low activity against 6-kestotriose and levan. Like other fructan-plant FEHs, no hydrolase activity could be detected towards sucrose, convincingly demonstrating that the enzyme is not a classic invertase. The expression pattern analysis of Lpl-FEHa revealed transcript accumulation in leaf tissues accumulating fructans while transcript level was low in the photosynthetic tissues. The high expression level of this 1-FEH in conditions of active fructan synthesis, together with its low expression level when fructan contents are low, suggest that it might play a role as a beta-(2,1) trimming enzyme acting during fructan synthesis in concert with fructan synthesis enzymes.