Model experimental systems for plant redox regulation and analysis of the stress-tolerance mechanism

为了建立植物氧化还原调控基因表达的模式实验系统，我们利用外源雌激素诱导抗坏血酸过氧化物酶（APX）基因表达的烟草BY-2细胞和拟南芥植株，构建了抗坏血酸过氧化物酶（APX）基因表达的转基因植物。我们使用pMDC 7并开发了pXR作为目标载体。用胞质APX反义构建体转化的烟草BY-2细胞系（命名为AtAS 3）在加入25 μW雌激素后，与未转化的BY-2细胞相比，APX活性降低了40%。AtAS 3细胞内H_2O_2水平显著升高。有趣的是，AtAS 3在雌激素诱导后显示出对盐和热共享的耐受性增加。这些结果表明，这条线是有用的氧化还原基因调控的模型系统。通过抑制性消减杂交（SSH）筛选，收集了几个可能的氧化还原相关基因，包括已知的热休克蛋白（HSP）基因，并通过pXR介导的RNAi沉默拟南芥叶绿体APX基因表达，结果表明叶绿体APX基因表达量和酶活性均较低。沉默对APX表达的影响是稳定的，并持续至少6天后，雌激素治疗。Microsaray分析显示，大约400和250个基因分别上调和下调。其中，与信号转导和代谢相关的基因均被占据，表明有多种可能性，包括叶绿体到核的信号转导。除上述APX抑制系统外，我们还在烟草BY-2和拟南芥中开发了APX过表达系统，证明了APX在细胞氧化还原调控中的重要作用，并成功建立了阐明植物氧化还原基因调控机制的模型实验系统。

To establish a model experimental system for plant redox regulation of gene expression, we made transgenic tobacco BY-2 cells and Arabidopsis plants harboring overexpession and silencing system for ascorbate peroxidase (APX), a key enzyme of H_2O_2-scavenging in plants, inducing by estrogen supplementation. We hired pMDC7 and developed pXR as the vector for the aim. The resultant tobacco BY-2 line transformed with antisense construct for cytosolic APX, named AtAS3, showed 40 % less APX activity compared with untransformed BY-2 cells after addition of 25 μW estrogen. The cellular H_2O_2 level in AtAS3 was significantly increased by the silencing of cytosolic APX. Interestingly, AtAS3 showed increasing tolerance against salt and heat share after estrogen induction. These results indicated that this line is useful fir the model system of redox gene regulation. By the screening of suppressive subtraction hybridization (SSH) method, I have collected several possibilities for redox-responsible genes, including the known genes like HSPs.Furthermore, transgenic Arabidopsis plants silencing chloroplastic APX expression with RNAi by pXR introduction showed lower chloroplastic APX gene expression and its enzyme activity. The effect of the silencing on the APX expression was stable and lasting at least for six days after estrogen treatment. Microsaray analysis showed that approximately 400 and 250 genes were upregulated and downregulated, respectively. Among them, the genes related to signal transduction and metabolism were occupied, indicating there are many possibilities including chloroplast-to-nucleus signal transduction. Beside the APX-suppression systems describing above, we have also developed APX-overexpressors in tobacco BY-2 and Arabidopsis plants.Overall, we have proved that APX plays a central rule on cellular redox regulation, and have successfully developed the model experimental system fir elucidating redox gene regulation mechanism in plants.