Investigation of genes involved in construction of the cell wall architecture in plants

合成了荧光素基木葡聚糖低聚物，并将其用作探针以观察细胞壁将木葡聚糖片段掺入烟草BY-2细胞中的纤维素/木葡聚糖框架中的活性。活性显着降低，其中的表达的内木葡聚糖转移酶（EXGT）的反义EXGT-N1 mRNA的过度表达受到严重抑制的大肠杆菌细胞。这些结果表明EXGT参与了纤维素/木葡聚糖框架的构建，为了研究它们的转运途径，我们通过GFP融合蛋白程序以及间接免疫荧光追踪了EXGT在烟草BY-2细胞中的细胞内定位。在分裂间期，该蛋白通过内质网（ER）-高尔基体网络大量分泌到质外体。在前期，质外体直接运输减少，蛋白质聚集在位于细胞核周围的内质网-高尔基体网络结构域。随着胞质分裂的进行，该蛋白质仅位于成膜体中，并最终运输到细胞板。另一方面，EXGT-N1 mRNA的水平在有丝分裂期和间期之间基本保持不变。这些结果表明，分泌酶的膜运输的方向是精确的细胞周期依赖性的方式进行调控。为了深入了解个别EXGT基因在植物中的功能，我们筛选了来自拟南芥的17000 T-DNA标签系的DNA池，通过PCR和DNA印迹分析。我们发现了7个独立的T-DNA标签系，其中T-DNA已插入任何EXGT基因内或附近。测序分析表明，这些标签系包括EXGT-A2、XTR 4或XTR 15基因缺陷的插入突变体。

A fluoresceinyl xyloglucan oligomer was synthesized and used as a probe to visualize the activity of the cell wall to incorporate xyloglucan fragments into the cellulose/xyloglucan framework in tabacco BY-2 cells. The activity was significantly reduced in transformant cells in which the expression of endoxyloglucan transferase (EXGT) was severely suppressed by over-expression of the antisense EXGT-N1 mRNA. These results indicate the involvement of EXGT in the construction of the cellulose/xyloglucan framework.To examine their translocation pathways, we traced its intracellular localization in tabacco BY-2 cells by means of GFP-fusion protein procedures as well as by indirect immunofluorescence. During interphase, the protein was extensively secreted into the apoplast via the endoplasmic reticulum (ER)-Golgi apparatus network. At prophase, the apoplast-direct transportation was diminished and the protein was accumulated in the ER-Golgi network domain located around the nucleus. As cytokinesis proceeded, the protein was exclusively located in the phragmoplast and eventually transported to the cell plate. On the other hand, levels of the EXGT-N1 mRNA remained mostly unchanged between the mitotic phase and interphase. These results indicate that the direction of membrane trafficking of secretory enzymes be precisely regulated in a cell-cycle-dependent manner.To gain insight into functions of individual EXGT genes in plant, we screened DNA pools derived from 17000 T-DNA tag lines of Arabidopsis by means of PCR and DNA blot analyses. We found 7 independent T-DNA tag lines in which T-DNA had been inserted within or in the vicinity of any EXGT gene. Sequencing analyses have shown that these tag lines include insertion mutants deficient in EXGT-A2, XTR4 or XTR15 gene.