四合木抗逆相关的转录因子TmAP2-1基因的克隆及功能分析

AP2/EREBP家族的转录因子在调控植物生长发育和应答环境胁迫方面具有重要作用。利用同源克隆结合RACE(rapid-amplification of cDNA ends)技术, 从四合木(Tetraena mongolica)中克隆了AP2/EREBP家族的基因, 将其命名为TmAP2-1(GenBank登录号: JQ676996)。序列分析结果表明, 该基因的开放阅读框长度为1 452 bp, 编码483个氨基酸; 比对结果显示TmAP2-1有2个AP2/ERF结构域, 属于AP2/EREBP转录因子家族的AP2亚家族。亚细胞定位实验结果表明, TmAP2-1定位在细胞核中。该基因编码的蛋白在酵母中没有转录激活活性。利用Real-time PCR检测发现该基因在根、茎、叶等器官中均表达, 且在叶中表达量最高。此外, TmAP2-1还受到NaCl、低温、PEG和ABA的强烈诱导, 推测TmAP2-1可能参与四合木的逆境胁迫响应。在四合木愈伤组织中过表达该基因能够降低四合木愈伤组织中油脂的含量, 同时提高可溶性糖的含量, 暗示该基因可能通过影响糖代谢过程参与逆境胁迫响应。     

TINY, a dehydration-responsive element (DRE)-binding protein-like transcription factor connecting the DRE- and ethylene-responsive element-mediated signaling pathways in Arabidopsis

Dehydration-responsive element-binding proteins (DREBs) and ethylene-responsive element (ERE) binding factors are two major subfamilies of the AP2/ethylene-responsive element-binding protein family and play crucial roles in the regulation of abiotic- and biotic-stress responses, respectively. In the present work, we have reported a previously identified DREB-like factor, TINY, that was involved in both abiotic- and biotic-stress signaling pathways. TINY was capable of binding to both DRE and ERE with similar affinity and could activate the expression of reporter genes driven by either of these two elements in tobacco cells. The 15th amino acid in the APETALA2 (AP2)/ethylene-responsive element-binding factor domain was demonstrated to be essential for its specific binding to ERE, whereas the 14th and 19th amino acids were responsible for the binding to DRE. The expression of TINY was greatly activated by drought, cold, ethylene, and slightly by methyl jasmonate. Additionally, overexpression of TINY in Arabidopsis resulted in elevated expressions of both the DRE- and the ERE-containing genes. Moreover, the expression of DRE-regulated genes, such as COR6.6 and ERD10, was up-regulated upon ethylene treatment, and the expression of ERE-regulated genes, such as HLS1, was also increased by cold stress, when the expression of TINY was being induced. These results strongly suggested that TINY might play a role in the cross-talk between abiotic- and biotic-stress-responsive gene expressions by connecting the DRE- and ERE-mediated signaling pathways. The results herein might promote the understanding of the mechanisms of specific DNA recognition and gene expression regulation by DREBs.     

茶树冷诱导基因RAV的克隆与表达特性分析

对利用cDNA-AFLP技术所获得的茶树低温诱导差异表达片段TDF,通过RACE方法获得含完整编码区序列的茶树RAV基因cDNA克隆,其开放阅读框编码361个氨基酸,包含两个保守的结构域AP2和B3,与多种植物RAV蛋白具有高度同源性。qRT-PCR分析表明,茶树RAV基因受低温、乙烯、NaCl等上调表达,最大表达量分别是诱导前的5.8、10.0和1.9倍。在成熟叶片、芽、嫩茎中RAV基因表达量相近,花蕾和嫩根中表达较低,而在种子中不表达。推测该基因在组织中的表达受到严格控制以及在响应非生物胁迫中发挥重要作用。     

AP2α is essential for MUC8 gene expression in human airway epithelial cells

Mucins are high molecular weight proteins that make up the major components of mucus. Hypersecretion of mucus is a feature of several chronic inflammatory airway diseases. MUC8 is an important component of airway mucus, and its gene expression is upregulated in nasal polyp epithelium. Little is known about the molecular mechanisms of MUC8 gene expression. We first observed overexpression of activator protein‐2alpha (AP2α) in human nasal polyp epithelium. We hypothesized that AP2α overexpression in nasal polyp epithelium correlates closely with MUC8 gene expression. We demonstrated that phorbol 12‐myristate 13‐acetate (PMA) treatment of the airway epithelial cell line NCI‐H292 increases MUC8 gene and AP2α expression. In this study, we sought to determine which signal pathway is involved in PMA‐induced MUC8 gene expression. The results show that the protein kinase C and mitogen‐activating protein/ERK kinase (MAPK) pathways modulate MUC8 gene expression. PD98059 or ERK1/2 siRNA and RO‐31‐8220 or PKC siRNA significantly suppress AP2α as well as MUC8 gene expression in PMA‐treated cells. To verify the role of AP2α, we specifically knocked down AP2α expression with siRNA. A significant AP2α knock‐down inhibited PMA‐induced MUC8 gene expression. While dominant negative AP2α decreased PMA‐induced MUC8 gene expression, overexpressing wildtype AP2α increased MUC8 gene expression. Furthermore, using lentiviral vectors for RNA interference in human nasal polyp epithelial cells, we confirmed an essential role for AP2α in MUC8 gene expression. From these results, we concluded that PMA induces MUC8 gene expression through a mechanism involving PKC, ERK1/2, and AP2α activation in human airway epithelial cells. J. Cell. Biochem. 110: 1386–1398, 2010. © 2010 Wiley‐Liss, Inc.