茶树ERF类转录因子家族的克隆及功能鉴定

Tea is one of the most popular non-alcoholic beverages worldwide. Camellia sinensis is the species of plant whose leaves and leaf buds are used to produce Chinese tea. It is one of the genus Camellia, a genus of flowering plants in the family Theaceae. However, the tea plant, C. sinensis, is difficult to culture in vitro, and transform, and has a large genome, rendering little genomic information available. Tea plant has to respond to biotic and abiotic stresses and environmental stimuli during their growth and developmental periods. The growth of tea plant is affected by environmental stresses. Abiotic and biotic stresses during tea plant growth session could cause decreased yield and quality. Tea plants respond and adapt to these abiotic stresses (such as low temperature) in order to survive. They develop freezing resistance when exposed to low temperatures. Transcription factor is called trans-factors and binds specific DNA sequence to initiate or block gene expression. Typical transcription factor is composited with DNA-binding domain, transcription regulator domain and nuclear localization signal. Transcription factor utilize these components functions on promoters/cis-acting elements to activate or control transcription. ERF is a large family of plant transcription factors containing the highly conserved ethylene-responsive element-binding factor. The ERF family genes are involved in response to various abiotic and biotic stresses. In this project, transcriptome of "Longjing 43" and "Yinghong 9" shall been sequenced with high-throughput sequence system. All of the putative transcription factor genes in the ERF family shall be identified from the transcriptome database of "Longjing 43" and "Yinghong 9". Then, more than 50 ERF family genes shall be cloned, and expression patterns of those ERF family genes and gene regulatory network shall also been assessed in "Longjing 43" and "Yinghong 9". The putative gene duplication and evolution of ERF family transcription factor in C. sinensis shall also be analyzed. The binding activity with cis-acting elements shall be studied by yeast one-hybrid system. The objective of this study was to establish an extensive picture of the Camellia sinensis ERF gene family.

生物和非生物胁迫，尤其近年频发的低温冻害，严重影响茶树的生长发育，从而影响茶叶的产量和品质。很多基因被这些胁迫诱导表达，以增强茶树对逆境的抗性。转录因子是重要的一类调控基因，通过与顺式元件作用调节植物对各种胁迫的适应。ERF类转录因子与植物细胞周期、生长发育及逆境胁迫应答有关，主要调节植物对激素、逆境等的分子应答反应，调控细胞的生长发育。本项目基于对小叶较耐低温型茶树'龙井43'和大叶低温较敏感型茶树'英红9号'转录组高通量测序，开展茶树ERF类转录因子基因表达和调控研究，分析茶树ERF类转录因子的逆境调控网络，进而探讨茶树ERF类转录因子进化导致的功能分化。从'龙井43'和 '英红9号'中分离50个以上ERF类转录因子，通过与相关顺式元件体外结合和拟南芥转基因研究，筛选抗逆性能强的茶树逆境转录因子。本研究旨在解析茶树抗逆转录因子的调控网络和复制进化机理；同时为茶树抗逆育种奠定基础。

植物在受到高温、低温、干旱和盐害等逆境胁迫时，ERF转录因子作为信号转导因子被诱导并调控其它抗逆基因的表达。茶树是一种重要的自然资源，也是我国重要的经济树种之一，茶树种植受到来自于生物和非生物胁迫的挑战。在茶树中，ERF类家族转录因子参与了对茶树生长发育和抗胁迫多方面的转录调控。本研究基于四个不同品种茶树的转录组数据，对茶树ERF类转录因子进行了全面的分析。检索获得89个拥有完整AP2保守域的茶树AP2/ERF家族转录因子。这些转录因子被分入5个亚家族，即AP2、DREB、ERF、RAV和Soloist。茶树CsAP2/ERF基因在四个品种茶树中呈现相对稳定的表达水平。茶树AP2/ERF基因与温度胁迫相关。这些结果表明茶树ERF类转录因子在茶树中对于响应极端温度胁迫可能发挥重要功能。.从种茶树的cDNA中克隆得到CsERF-B3基因。利用实时定量PCR技术检测该基因在茶树根、茎、叶、花各组织和4种非生物逆境胁迫处理中的表达情况。该基因在茶树根中表达量最高，并且均能快速响应非生物逆境胁迫。通过RT-PCR方法从茶树‘迎霜’叶片的cDNA中克隆得到2个ERF类转录因子基因, CsERF-B1和CsERF-B4。CsERF-B1和CsERF-B4基因均受高低温的诱导表达, 不同的茶树材料, CsERF-B1和CsERF-B4基因表达有显著差异。同属ERF类转录因子的两个成员在三种具有抗寒性差异的茶树品种中的不同响应, 说明ERF转录因子在茶树温度胁迫调控中起重要作用。.本研究对茶树中Hsf和WRKY转录因子家族进行了系统的分析。通过对茶树转录组分析，鉴定出16个CsHsf热激转录因子和50个含有完整WRKY保守域的茶树WRKY转录因子。CsHsf和CsWRKY基因在无胁迫条件下在不同茶树品种中的表达显示差异。温度胁迫促进或抑制所选CsHsf和CsWRKY基因的表达，同一亚族的基因也显示出明显差异。另外，本研究还开展了茶树儿茶素代谢的研究，基于茶树转录组数据，通过RPKM值分析得到217个差异表达基因，其中有150个unigenes是属于类黄酮生物合成途径的结构基因。茶树叶片中儿茶素的组分含量与关键的基因表达结果进行了对比。结合转录组和生理指标分析，鉴定了参与类黄酮合成途径的基因，并且对于儿茶素的生物合成提供了新的见解。