Zhang Xinxin, Liu Shenkui, Takano Tetsuo
Asian Natural Environment Science Center, The University of Tokyo, Midori Cho 1-1-1, Nishitokyo City, Tokyo, Japan.
Plant Mol Biol. 2008 Sep;68(1-2):131-43. doi: 10.1007/s11103-008-9357-x. Epub 2008 Jun 4.
Two cysteine proteinase inhibitors (cystatins) from Arabidopsis thaliana, designated AtCYSa and AtCYSb, were characterized. Recombinant GST-AtCYSa and GST-AtCYSb were expressed in Escherichia coli and purified. They inhibit the catalytic activity of papain, which is generally taken as evidence for cysteine proteinase inhibitor function. Northern blot analyses showed that the expressions of AtCYSa and AtCYSb gene in Arabidopsis cells and seedlings were strongly induced by multiple abiotic stresses from high salt, drought, oxidant, and cold. Interestingly, the promoter region of AtCYSa gene contains a dehydration-responsive element (DRE) and an abscisic acid (ABA)-responsive element (ABRE), which identifies it as a DREB1A and AREB target gene. Under normal conditions, AtCYSa was expressed in 35S: DREB1A and 35S: AREB1 plants at a higher level than in WT plants, while AtCYSa gene was expressed in 35S: DREB2A plants at the same level as in WT plants. Under stress conditions (salt, drought and cold), AtCYSa was expressed more in all three transgenic plants than in WT plants. Over-expression of AtCYSa and AtCYSb in transgenic yeast and Arabidopsis plants increased the resistance to high salt, drought, oxidative, and cold stresses. Taken together, these data raise the possibility of using AtCYSa and AtCYSb to genetically improve environmental stresses tolerance in plants.
对来自拟南芥的两种半胱氨酸蛋白酶抑制剂（胱抑素）AtCYSa和AtCYSb进行了表征。重组GST-AtCYSa和GST-AtCYSb在大肠杆菌中表达并纯化。它们抑制木瓜蛋白酶的催化活性，这通常被视为半胱氨酸蛋白酶抑制剂功能的证据。Northern印迹分析表明，AtCYSa和AtCYSb基因在拟南芥细胞和幼苗中的表达受到高盐、干旱、氧化剂和寒冷等多种非生物胁迫的强烈诱导。有趣的是，AtCYSa基因的启动子区域包含一个脱水响应元件（DRE）和一个脱落酸（ABA）响应元件（ABRE），这表明它是DREB1A和AREB的靶基因。在正常条件下，AtCYSa在35S:DREB1A和35S:AREB1植物中的表达水平高于野生型植物，而AtCYSa基因在35S:DREB2A植物中的表达水平与野生型植物相同。在胁迫条件下（盐、干旱和寒冷），AtCYSa在所有三种转基因植物中的表达均高于野生型植物。AtCYSa和AtCYSb在转基因酵母和拟南芥植物中的过表达提高了对高盐、干旱、氧化和寒冷胁迫的抗性。综上所述，这些数据增加了利用AtCYSa和AtCYSb在基因上提高植物对环境胁迫耐受性的可能性。
