茶树低温应答相关转录因子CsICE1和CsCBF1的研究
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摘要
植物遭受低温胁迫时，CBF转录因子通过与CRT/DRE顺式作用元件结合来激活下游一系列冷诱导基因的转录表达，从而提高植物的抗寒性。ICE1是CBF上游的主要激活因子。为了阐明茶树应答低温的分子基础和调控机理，从整体水平上提高茶树的抗寒性，并且为鉴定和选育茶树抗寒品种提供分子水平上的理论依据，本论文进行了以下研究：
（1）根据一段与拟南芥ICE1基因同源性较高的茶树EST，采用RACE技术获得了茶树ICE1基因的cDNA全长，命名为CsICE1。该基因（1964bp）与其同系物一样都含有保守的bHLH（碱性螺旋-环-螺旋）结构域和C端区域。
（2）根据GenBank上已有的序列信息，在茶树中克隆出一个CBF基因，命名为CsCBF1。该基因（1210bp）含有CBF转录因子家族典型的结构域：AP2和其两侧的保守特征序列。
（3）通过实时荧光定量PCR和半定量RT-PCR检测CsICE1和CsCBF1在低温诱导条件下的表达模式，发现与拟南芥的ICE1和CBF表达模式相似。即：CsICE1在4℃处理后与20℃相比转录水平几乎没有变化；CsCBF1在常温下（20℃）不表达，4℃处理后表达量快速而明显大幅增加。
（4）通过原核表达并纯化得到带有6个组氨酸标签的CsCBF1融合蛋白。
（5）采用凝胶迁移分析技术（EMSA）证明了CsCBF1蛋白可以与CRT/DRE顺式作用元件特异性地结合。
以上的研究结果表明：ICE1-CBF冷调控途径存在于茶树中，并且在茶树低温应答过程中起着关键作用。CsICE1和CsCBF1两个转录因子对提高茶树抗寒性起着重要的作用。
CBFs (C-repeat/dehydration-responsive element binding factors) can induce theexpression of a suite of cold-responsive genes to increase plant cold tolerance, and ICE1(Inducer of CBF expression) is a major activator for CBF. Our study is to elucidate themolecular basis and regulatory mechanisms of tea plant cold responses and enhance the teaplant cold tolerance. Besides, the studies provide with theory basis in molecular biologyabout identification and breeding for cold resisting varieties of tea.
We isolated the full-length cDNAs of ICE1and CBF from Camellia sinensis,designated as CsICE1and CsCBF1, respectively. The deduced protein CsICE1contains ahighly conserved basic helix-loop-helix (bHLH) domain and C-terminal region ofICE1-like proteins. CsCBF1contains all conserved domains of CBFs in other plant speciesand can specifically bind to the C-repeat/dehydration-responsive element (CRT/DRE) asconfirmed by electrophoretic mobility shift assay. The transcription of CsICE1had noapparent alteration after chilling treatment (4℃). CsCBF1expression was not detected innormal temperature (20℃) but was induced immediately and significantly by lowtemperature (4℃).
Our results suggest that ICE1-CBF cold-response pathway is conserved in tea plants.CsICE1and CsCBF1, two components of this pathway, play important roles in coldresponses in tea plants.
（1）根据一段与拟南芥ICE1基因同源性较高的茶树EST，采用RACE技术获得了茶树ICE1基因的cDNA全长，命名为CsICE1。该基因（1964bp）与其同系物一样都含有保守的bHLH（碱性螺旋-环-螺旋）结构域和C端区域。
（2）根据GenBank上已有的序列信息，在茶树中克隆出一个CBF基因，命名为CsCBF1。该基因（1210bp）含有CBF转录因子家族典型的结构域：AP2和其两侧的保守特征序列。
（3）通过实时荧光定量PCR和半定量RT-PCR检测CsICE1和CsCBF1在低温诱导条件下的表达模式，发现与拟南芥的ICE1和CBF表达模式相似。即：CsICE1在4℃处理后与20℃相比转录水平几乎没有变化；CsCBF1在常温下（20℃）不表达，4℃处理后表达量快速而明显大幅增加。
（4）通过原核表达并纯化得到带有6个组氨酸标签的CsCBF1融合蛋白。
（5）采用凝胶迁移分析技术（EMSA）证明了CsCBF1蛋白可以与CRT/DRE顺式作用元件特异性地结合。
以上的研究结果表明：ICE1-CBF冷调控途径存在于茶树中，并且在茶树低温应答过程中起着关键作用。CsICE1和CsCBF1两个转录因子对提高茶树抗寒性起着重要的作用。
CBFs (C-repeat/dehydration-responsive element binding factors) can induce theexpression of a suite of cold-responsive genes to increase plant cold tolerance, and ICE1(Inducer of CBF expression) is a major activator for CBF. Our study is to elucidate themolecular basis and regulatory mechanisms of tea plant cold responses and enhance the teaplant cold tolerance. Besides, the studies provide with theory basis in molecular biologyabout identification and breeding for cold resisting varieties of tea.
We isolated the full-length cDNAs of ICE1and CBF from Camellia sinensis,designated as CsICE1and CsCBF1, respectively. The deduced protein CsICE1contains ahighly conserved basic helix-loop-helix (bHLH) domain and C-terminal region ofICE1-like proteins. CsCBF1contains all conserved domains of CBFs in other plant speciesand can specifically bind to the C-repeat/dehydration-responsive element (CRT/DRE) asconfirmed by electrophoretic mobility shift assay. The transcription of CsICE1had noapparent alteration after chilling treatment (4℃). CsCBF1expression was not detected innormal temperature (20℃) but was induced immediately and significantly by lowtemperature (4℃).
Our results suggest that ICE1-CBF cold-response pathway is conserved in tea plants.CsICE1and CsCBF1, two components of this pathway, play important roles in coldresponses in tea plants.
引文
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