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

在烟草BY-2细胞中合成了一种荧光素基木葡聚糖低聚物，并将其作为探针来观察将木葡聚糖片段整合到纤维素/木葡聚糖框架中的细胞壁活性。在过度表达反义EXGT- n1 mRNA严重抑制endoxylo葡聚糖转移酶（EXGT）表达的转化细胞中，其活性显著降低。这些结果表明EXGT参与了纤维素/木葡聚糖框架的构建。为了研究它们的易位途径，我们通过gfp融合蛋白程序和间接免疫荧光法追踪了它在烟草by -2细胞中的细胞内定位。在间期，该蛋白通过内质网-高尔基体网络广泛分泌到外质体中。前期，外质体直接运输减少，蛋白质在位于细胞核周围的er -高尔基网状结构域内积累。随着细胞质分裂的进行，蛋白质完全位于膜质体中，并最终被运送到细胞板上。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，分泌酶的膜运输方向以细胞周期依赖的方式精确调节。为了进一步了解植物中单个EXGT基因的功能，我们采用PCR和DNA印迹分析的方法从拟南芥的17000个T-DNA标记系中筛选出DNA池。我们发现了7条独立的T-DNA标记线，其中T-DNA被插入到任何EXGT基因内或附近。测序分析表明，这些标记系包括缺乏EXGT-A2、XTR4或XTR15基因的插入突变体。

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.