Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Genetics, Faculty of Agriculture, University of Tanta, Tanta, El-Gharbia, Egypt.
Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada.
PLoS One. 2014 Jun 3;9(6):e98524. doi: 10.1371/journal.pone.0098524. eCollection 2014.
In cereals, ADP-glucose transporter protein plays an important role in starch biosynthesis. It acts as a main gate for the transport of ADP-glucose, the main precursor for starch biosynthesis during grain filling, from the cytosol into the amyloplasts of endospermic cells. In this study, we have shed some light on the molecular and biochemical characteristics of barley plastidial ADP-glucose transporter, HvBT1. Phylogenetic analysis of several BT1 homologues revealed that BT1 homologues are divided into two distinct groups. The HvBT1 is assigned to the group that represents BT homologues from monocotyledonous species. Some members of this group mainly work as nucleotide sugar transporters. Southern blot analysis showed the presence of a single copy of HvBT1 in barley genome. Gene expression analysis indicated that HvBT1 is mainly expressed in endospermic cells during grain filling; however, low level of its expression was detected in the autotrophic tissues, suggesting the possible role of HvBT1 in autotrophic tissues. The cellular and subcellular localization of HvBT1 provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM. The study also showed that AMP is another possible exchange substrate. The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.
在谷物中，ADP-葡萄糖转运蛋白在淀粉生物合成中起着重要作用。它作为一个主要的门控，负责将 ADP-葡萄糖，即谷物灌浆期间淀粉生物合成的主要前体，从细胞质转运到胚乳细胞的淀粉体中。在这项研究中，我们揭示了大麦质体 ADP-葡萄糖转运蛋白 HvBT1 的分子和生化特性。几种 BT1 同源物的系统发育分析表明，BT1 同源物分为两个不同的组。HvBT1 被分配到代表单子叶植物 BT 同源物的组中。该组的一些成员主要作为核苷酸糖转运蛋白发挥作用。Southern blot 分析显示，大麦基因组中存在 HvBT1 的单个拷贝。基因表达分析表明，HvBT1 在灌浆期间主要在胚乳细胞中表达；然而，在自养组织中检测到其表达水平较低，这表明 HvBT1 可能在自养组织中发挥作用。HvBT1 的细胞和亚细胞定位提供了额外的证据，表明 HvBT1 靶向胚乳细胞的淀粉体膜。使用大肠杆菌系统对 HvBT1 的生化特性进行了研究，结果表明 HvBT1 能够以 614.5 µM 的亲和力将 ADP-葡萄糖转运到大肠杆菌细胞中，并以 334.7 µM 的亲和力进行 ADP 的反向交换。研究还表明，AMP 是另一种可能的交换底物。非标记的 ADP-葡萄糖和 ADP 对 [α-32P] ADP-葡萄糖摄取率的影响表明了 HvBT1 对 ADP-葡萄糖和 ADP 的底物特异性。
