欧美杨水分利用效率相关基因PdEPF1的克隆及表达

Abstract

提高植物水分利用效率(WUE)是未来解决我国甚至世界干旱缺水的最重要手段之一。在对植物WUE的众多研究方法中,大多集中在生理手段,但通过分子生物学手段研究其表达调控机制的较少。欧美杨(<em>Populus deltoides</em>×<em>Populus nigra</em>)是中纬度地区最适合种植的短轮伐期工业用材集约经营树种之一。近年来我国引进了许多优良的欧美杨无性系用于营造大面积的速生丰产林并取得很好的经济和社会效益。但高耗水量的缺点限制了其进一步的推广。通过基因芯片技术从欧美杨中找到一个可能调控WUE的基因-<em>PdEPF1</em>. 荧光定量表达进一步验证了这一结果。荧光定量表达分析表明该基因受ABA、盐、冷、干旱等胁迫诱导表达。组织特异表达分析说明<em>PdEPF1</em>基因在顶端叶和根中有表达,成熟叶衰老叶中则无表达。克隆到启动子分析表明该启动子含有多种干旱响应元件(drought response elememt),赤霉素响应元件(GA response elememt),低温响应元件(cold response elememt),ABA响应元件(ABA response elememt)等逆境相关的作用元件。;Water-use efficiency (WUE) is crucial to plant survival, yield and vegetation dynamics. Many researches trended to reduce water use by more crop per drop. Althoughmuch progresses have been achieved in determining the physiological mechanisms of WUE, there is few report on the genetic controls of WUE. The first gene ERECTA, which t promotes WUE, was shown to decrease stomatal density and be involved in the positive regulation of photosynthetic capacity.<br> The EPIDERMAL PATTERNING FACTOR 1 (EPF1) gene identified in Arabidopsis is expressed specifically in stomatal cells and precursors. EPF1 controls stomatal patterning through the regulation of asymmetric cell division, its activity depends on the TOO MANY MOUTHS receptor-like protein and ERECTA family receptor kinases, suggesting that EPF1 provides a positional cue interpreted by these receptors. A mutation of EPF1 results in increased stomatal density and violation of the one-cell-spacing rule (clustering of stomata). However, there is no report on the relationship between AtEPF1 and WUE.<br> The cottonwood poplar, Populus deltoides×Populus nigra, is one of the most important species in large-scale forestation in China. However, its high water consumption is a key factor limiting its growth in arid and semiarid regions. Identifcation of genes increasing WUE in this species is important for understanding stress responses and improvement of this species.<br> In this study, microarray analysis between poplar genotypes NE-19 (<em>P. nigra</em>×(<em>P. deltoides</em>×<em>P. nigra</em>)) with the higher WUE and DN-2 (<em>P. deltoides×P. nigra</em>) with a lower WUE,was performed. PdEPF1 from NE-19,one of the WUE-related genes, was identified, strongly expressed in immature leaves and roots, but not in senescent leaves, functional leaves and the stem. This gene is strongly induced by drought, abscisic acid and high salt levels, and enhanced by gibberellic acid. To unvarel the mechanism responsible for the inducible expression of PdEPF1 by different stresses, a 1090-bp PdEPF1 promoter fragment was isolated by the adaptor PCR with reference to the transcriptional start site determined from the PdEPF1 cDNA sequence. The PdEPF1 promoter contains CAAT and TATA motifs located at nucleotides -204 and -35 relative to the transcriptional start site, respectively. Each of these motifs has characteristics of eukaryotic gene promoters. Interestingly,, the 1090-bp promoter region contained several motifs based on the computer prediction, such as a W-box (a GA-responsive element), an MYC binding site, an early response to dehydration binding site, and a controlling guard cell-specific gene expression binding site. This indicate, that the stress-inducible expression of PdEPF1 is regulated at the level of transcription.