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

研究参与植物细胞壁结构构建的基因

基本信息

批准号：
09440269
负责人：
NISHITANI Kazuhiko
金额：
$ 6.78万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09440269/
关键词：
CELL WALL XYLOGLUCAN ENDOXYLOGLUCAN CELL PLATE Plant Cell Wall xyloglucan gene construction EXGT GFP FITC-xyloglucan シロイヌナズナタバコ培養細胞アンチセンス法形能形成

项目摘要

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.

合成了一种荧光素基木葡聚糖低聚物，并以此为探针，在烟草BY-2细胞中显示了将木聚糖片段整合到纤维素/木聚糖骨架中的细胞壁的活性。在内氧葡聚糖转移酶(EXGT)表达被反义EXGT-N1 mRNA严重抑制的转化细胞中，该酶的活性显著降低。这些结果表明EXGT参与了纤维素/木葡聚糖框架的构建。为了研究它们的转运途径，我们利用GFP融合蛋白程序以及间接免疫荧光的方法追踪了EXGT在烟草BY-2细胞中的定位。在间期，蛋白质通过内质网(ER)-高尔基体网络广泛分泌到质外体中。在前期，质外体直接运输减少，蛋白质积累在位于细胞核周围的ER-高尔基网络结构域中。随着胞质分裂的进行，蛋白质只定位于成膜体中，并最终运输到细胞板。另一方面，EXGT-N1的mRNA水平在有丝分裂期和间期基本保持不变。这些结果表明，分泌酶的膜运输方向是以细胞周期依赖的方式精确调控的。为了深入了解植物中单个EXGT基因的功能，我们通过聚合酶链式反应和DNA印迹分析从17000个拟南芥T-DNA标签系中筛选出DNA库。我们发现了7个独立的T-DNA标签系，其中T-DNA被插入到任何EXGT基因内或其附近。测序分析表明，这些标签系含有缺失EXGT-A2、XTR4或XTR15基因的插入突变体。