Hinniger Cécile, Caillet Victoria, Michoux Franck, Ben Amor Mohamed, Tanksley Steve, Lin Chenwei, McCarthy James
Nestlé Research Center, Tours 101, Avenue Gustave Eiffel, BP 49716, 37097 Tours Cedex 2, France.
Ann Bot. 2006 May;97(5):755-65. doi: 10.1093/aob/mcl032. Epub 2006 Feb 27.
Dehydrins, or group 2 late embryogenic abundant proteins (LEA), are hydrophilic Gly-rich proteins that are induced in vegetative tissues in response to dehydration, elevated salt, and low temperature, in addition to being expressed during the late stages of seed maturation. With the aim of characterizing and studying genes involved in osmotic stress tolerance in coffee, several full-length cDNA-encoding dehydrins (CcDH1, CcDH2 and CcDH3) and an LEA protein (CcLEA1) from Coffea canephora (robusta) were isolated and characterized.
The protein sequences deduced from the full-length cDNA were analysed to classify each dehydrin/LEA gene product and RT-PCR was used to determine the expression pattern of all four genes during pericarp and grain development, and in several other tissues of C. arabica and C. canephora. Primer-assisted genome walking was used to isolate the promoter region of the grain specific dehydrin gene (CcDH2).
The CcDH1 and CcDH2 genes encode Y(3)SK(2) dehydrins and the CcDH3 gene encodes an SK(3) dehydrin. CcDH1 and CcDH2 are expressed during the final stages of arabica and robusta grain development, but only the CcDH1 transcripts are clearly detected in other tissues such as pericarp, leaves and flowers. CcDH3 transcripts are also found in developing arabica and robusta grain, in addition to being detected in pericarp, stem, leaves and flowers. CcLEA1 transcripts were only detected during a brief period of grain development. Finally, over 1 kb of genomic sequence potentially encoding the entire grain-specific promoter region of the CcDH2 gene was isolated and characterized.
cDNA sequences for three dehydrins and one LEA protein have been obtained and the expression of the associated genes has been determined in various tissues of arabica and robusta coffees. Because induction of dehydrin gene expression is associated with osmotic stress in other plants, the dehydrin sequences presented here will facilitate future studies on the induction and control of the osmotic stress response in coffee. The unique expression pattern observed for CcLEA1, and the expression of a related gene in other plants, suggests that this gene may play an important role in the development of grain endosperm tissue. Genomic DNA containing the grain-specific CcDH2 promoter region has been cloned. Sequence analysis indicates that this promoter contains several putative regulatory sites implicated in the control of both seed- and osmotic stress-specific gene expression. Thus, the CcDH2 promoter is likely to be a useful tool for basic studies on the control of gene expression during both grain maturation and osmotic stress in coffee.
脱水素，即第2组晚期胚胎丰富蛋白（LEA），是富含甘氨酸的亲水性蛋白，除了在种子成熟后期表达外，还可在营养组织中因脱水、盐浓度升高和低温诱导而产生。为了鉴定和研究咖啡中参与渗透胁迫耐受性的基因，从卡内弗拉咖啡（罗布斯塔）中分离并鉴定了几个编码脱水素（CcDH1、CcDH2和CcDH3）的全长cDNA以及一个LEA蛋白（CcLEA1）。
对从全长cDNA推导的蛋白质序列进行分析，以对每个脱水素/LEA基因产物进行分类，并使用RT-PCR来确定这四个基因在果皮和种子发育过程中以及在阿拉比卡咖啡和卡内弗拉咖啡的其他几种组织中的表达模式。采用引物辅助基因组步移法分离种子特异性脱水素基因（CcDH2）的启动子区域。
CcDH1和CcDH2基因编码Y(3)SK(2)脱水素，CcDH3基因编码SK(3)脱水素。CcDH1和CcDH2在阿拉比卡和罗布斯塔咖啡豆发育的最后阶段表达，但仅在果皮、叶片和花朵等其他组织中能清楚地检测到CcDH1的转录本。CcDH3的转录本也在发育中的阿拉比卡和罗布斯塔咖啡豆中发现，此外在果皮、茎、叶和花中也能检测到。CcLEA1的转录本仅在种子发育的短暂时期被检测到。最后，分离并鉴定了超过1 kb的可能编码CcDH2基因整个种子特异性启动子区域的基因组序列。
已获得三个脱水素和一个LEA蛋白的cDNA序列，并确定了相关基因在阿拉比卡和罗布斯塔咖啡不同组织中的表达。由于脱水素基因表达的诱导与其他植物中的渗透胁迫相关，这里呈现的脱水素序列将有助于未来对咖啡渗透胁迫反应的诱导和控制的研究。观察到的CcLEA1独特表达模式以及相关基因在其他植物中的表达表明，该基因可能在种子胚乳组织发育中起重要作用。已克隆了包含种子特异性CcDH2启动子区域的基因组DNA。序列分析表明，该启动子包含几个推定的调控位点，与种子特异性和渗透胁迫特异性基因表达的控制有关。因此，CcDH2启动子可能是研究咖啡种子成熟和渗透胁迫期间基因表达控制的基础研究的有用工具。
