Keinänen Sirpa I, Hassinen Viivi H, Kärenlampi Sirpa O, Tervahauta Arja I
University of Kuopio, Institute of Applied Biotechnology, P.O. Box 1627, FIN-70211 Kuopio, Finland.
Tree Physiol. 2007 Sep;27(9):1243-52. doi: 10.1093/treephys/27.9.1243.
Suppression subtractive hybridization (SSH) was used to isolate genes differentially expressed following exposure to copper (Cu) in a naturally selected Cu-tolerant birch (Betula pendula Roth.) clone originating from a disused lead/zinc smelter. Of the 352 cDNA fragments initially isolated, 108 were up-regulated by Cu, of which 55 showed over twofold induction by macroarray analysis. Searches against protein databases (Blastx) and sequence analysis provided the tentative identity of 21 genes. Three fragments lacked homology to any sequences in the databases. Most of the identified genes are involved in cellular transport, regulation or cell rescue and defense. Several genes have not previously been reported to be up-regulated by Cu, e.g., plasma intrinsic protein 2, glutamine synthetase and multi-drug resistance-associated protein (MRP4). The expression of MRP4, a vacuolar sorting receptor-like protein and an unidentified gene was studied in more detail by quantitative real-time PCR. These genes showed stronger up-regulation by Cu in the roots and shoots of the Cu-tolerant birch clone compared with a less tolerant clone. Clear clonal differences in gene expression were observed, e.g., for the regulator of chromosome condensation family protein, DnaJ protein homolog, vacuolar sorting receptor-like protein and MRP4. These findings contribute to our understanding of Cu tolerance in birch, a pioneer plant in metal-contaminated soils.
抑制性消减杂交（SSH）被用于分离来自一个废弃铅锌冶炼厂的自然选择的耐铜桦树（垂枝桦，Betula pendula Roth.）克隆体在暴露于铜（Cu）后差异表达的基因。在最初分离的352个cDNA片段中，108个被铜上调，其中55个通过宏阵列分析显示出两倍以上的诱导。通过对蛋白质数据库（Blastx）的搜索和序列分析确定了21个基因的初步身份。三个片段与数据库中的任何序列均无同源性。大多数已鉴定的基因参与细胞转运、调节或细胞拯救与防御。此前有几个基因尚未被报道会被铜上调，例如血浆内在蛋白2、谷氨酰胺合成酶和多药耐药相关蛋白（MRP4）。通过定量实时PCR对MRP4、一种液泡分选受体样蛋白和一个未鉴定基因的表达进行了更详细的研究。与耐受性较差的克隆体相比，这些基因在耐铜桦树克隆体的根和芽中被铜上调的程度更强。观察到了明显的克隆间基因表达差异，例如染色体凝聚调节家族蛋白、DnaJ蛋白同源物、液泡分选受体样蛋白和MRP4。这些发现有助于我们理解桦树在金属污染土壤中的先锋植物对铜的耐受性。
