mutant axel-4 seedlings. Arabidopsis thaliana

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a ?-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)?selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Keywords: replicate_design Overall design: Computed

本研究以生长素（auxin）响应转基因失调表达为基础，在拟南芥（Arabidopsis）中开展突变体筛选实验。我们采用两种受严格调控的生长素响应启动子——即在未施加生长素处理时表达量极低，经外源生长素处理后表达量显著升高——分别驱动β-葡萄糖醛酸苷酶（β-glucuronidase, GUS）报告基因与潮霉素磷酸转移酶（hygromycin phosphotransferase, HPH）筛选标记基因的表达。
本次筛选获得多个突变体，其中5个突变位点（axe1-1至axe1-5）定位于5号染色体的同一基因座。
通过图位克隆（map-based cloning）技术，我们将axe1突变位点定位到拟南芥的RPD3样组蛋白去乙酰化酶（histone deacetylase）基因，该基因被命名为HDA6（简称AtHDA6）。
与野生型对照植株相比，axe1突变体植株在未施加生长素处理时，GUS与HPH转基因的表达量显著升高，且经生长素诱导后的转基因表达量也进一步提升。
但值得注意的是，突变体植株中多种内源性天然生长素响应基因并未像转基因一样出现表达上调。
DNA甲基化抑制剂5-氮杂-2'-脱氧胞苷（5-aza-2'-deoxycytidine）处理实验结果表明，axe1突变会影响转基因沉默；然而，组蛋白去乙酰化酶抑制剂对突变体或野生型对照植株中的转基因沉默均无显著影响。
AtHDA6突变对生长素响应转基因的特异性影响，提示这种RPD3样组蛋白去乙酰化酶参与了转基因沉默过程。
此外，AtHDA6对转基因沉默的调控作用可能不依赖于其组蛋白去乙酰化酶活性。
重复实验设计类型指通过开展一系列重复实验以评估结果可重复性，或作为预实验以确定后续实验所需的合适重复次数。
关键词：重复实验设计（replicate_design） 整体实验设计：已完成计算。