Biosciences Research Laboratory, USDA-Agricultural Research Service, 1605 Albrecht Blvd., Fargo, ND 58105, USA.
Plant Mol Biol. 2010 May;73(1-2):227-39. doi: 10.1007/s11103-009-9568-9. Epub 2009 Nov 19.
Underground adventitious buds of leafy spurge (Euphorbia esula) undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy. In this study, relationships among genes involved in carbohydrate metabolism and bud dormancy were examined after paradormancy release (growth induction) by decapitation and in response to seasonal signals. Real-time PCR was used to determine the expression levels of carbohydrate metabolism genes at different phases of bud dormancy. Among differentially-regulated genes, expression of a specific Euphorbia esula beta-amylase gene (Ee-BAM1) increased 100-fold after growth induction and 16,000-fold from July (paradormancy) to December (ecodormancy). Sequence data analysis indicated that two genes, Ee-BAM1 and Ee-BAM2, could encode this beta-amylase. However, real-time PCR using gene-specific primer pairs only amplified Ee-BAM1, indicating that Ee-BAM2 is either specific to other organs or not abundant. The deduced amino acid sequences of these two genes are very similar at the N-terminal but differ at the C-terminal. Both contain a nearly identical, predicted 48-amino acid plastid transit peptide. Immunoblot analyses identified a 29 kD (mature Ee-BAM1 after cleavage of the transit peptide) and a 35 kD (unprocessed EeBAM1) protein. Both 35 and 29 kD proteins were constitutively expressed in growth-induced and seasonal samples. Immunolocalization indicated that Ee-BAM1 is in the cytosol of cells at the shoot tip of the bud. Ee-BAM1 also surrounds the amyloplasts in mature cells toward the base of the bud. These observations suggests that Ee-BAM1 may have dual functions; serving as reserve protein in the cytosol and as a degrading enzyme at the surface of amyloplasts.
多枝猪殃殃（Euphorbia esula）的地下不定芽经历了三个明确的休眠阶段，即拟休眠、内休眠和生态休眠。在这项研究中，通过断头处理解除拟休眠（生长诱导）后，以及对季节性信号的反应，研究了参与碳水化合物代谢的基因与芽休眠之间的关系。实时 PCR 用于测定不同休眠阶段芽休眠过程中碳水化合物代谢基因的表达水平。在差异调节基因中，一种特定的多枝猪殃殃β-淀粉酶基因（Ee-BAM1）的表达在生长诱导后增加了 100 倍，从 7 月（拟休眠）到 12 月（生态休眠）增加了 16000 倍。序列数据分析表明，两个基因，Ee-BAM1 和 Ee-BAM2，可以编码这种β-淀粉酶。然而，使用基因特异性引物对进行的实时 PCR 仅扩增了 Ee-BAM1，表明 Ee-BAM2 要么是特定于其他器官的，要么是不丰富的。这两个基因的推导氨基酸序列在 N 端非常相似，但在 C 端不同。两者都包含一个几乎相同的、预测的 48 个氨基酸质体转运肽。免疫印迹分析鉴定出一个 29 kD（成熟 Ee-BAM1 在穿过转运肽后）和一个 35 kD（未加工的 Ee-BAM1）蛋白。35 和 29 kD 蛋白在生长诱导和季节性样品中均持续表达。免疫定位表明，Ee-BAM1 存在于芽的顶端细胞的细胞质中。Ee-BAM1 也存在于芽基部成熟细胞的淀粉粒周围。这些观察结果表明，Ee-BAM1 可能具有双重功能；作为细胞质中的储备蛋白和淀粉粒表面的降解酶。
