顺式调控变异在重金属抗性植物酸性蔗糖酶基因的胁迫响应转录调控中的作用

Acid invertases are key enzymes for controlling organogenesis of sink organs of plants by providing energe, carbon and hexose signals through hydrolysis of sucrose, and play an important role in rooting, fructification and tolerance maintenance in metallophytes found in uncommonly contaminated environments by heavy metals. The transcription of acid invertase genes is significantly up-regulated by heavy metal stress. It has been recently discovered that, mutation arises in cis-regulation region of acid invertase genes in metallophytes, forming sevreal potential metal response elements (MREs). However, it is not clear whether these MREs are associated with the transcriptional up-regulation of the genes. This project is to identify the cis-regulatory changes,mine new type of MREs, and study their function in the trascriptional regulation in response to heavy metal stress in native Chinese metallophytes found in ancient Cu mines that appeared since the Shang and Zhou dynasties, by means of TAIL-PCR, deletion and site-directed mutagenesis, recombinant plasmid construction, fusions with reporter gene, heterologous expression in Arabidopsis thaliana, Yeast One-hybrid and Electrophoretic Mobility Shift Assays. The novel MREs can be used to construct synthetic promoters for breeding new types of transgenic plants which will have important applications in phytoremediation, ecological restoration and biological treatment of heavy metal wastes. Also, identification of MREs and the associated transcription factors will contribute to elucidation of the regulation network of expressions of acid invertase genes and even other heavy metal responsive genes.

酸性蔗糖酶因水解蔗糖提供能量、碳源和己糖信号是植物库器官建成的关键酶，对重金属极端污染环境中抗性植物生根、结实和抗性维持有重要作用。近期发现，重金属胁迫下抗性植物的该酶基因的转录无例外显著上调，其非编码顺式调控区发生多处突变，出现多个潜在的金属响应元件。但是未知这些顺式元件突变是否与其基因转录上调以响应重金属胁迫相关。本项目用TAIL-PCR、PCR区段缺失和定点突变、重组载体构建、报告基因融合、拟南芥异源表达、酵母单杂交及凝胶阻滞实验等方法，鉴定商周古铜矿遗址抗铜植物该基因顺式调控变异，挖掘新型金属响应元件，研究其在响应重金属胁迫的转录调控中的作用。新型金属响应元件可用于创建人工修饰启动子，培育新型工程植物用于重金属污染环境的植物修复、生态恢复和重金属废弃物生物处理。新型金属响应元件及其转录因子的鉴定还有助于深入阐明重金属胁迫下抗性植物酸性蔗糖酶基因乃至其它重金属响应基因的表达调控网络。

金属型植物是生长在重金属污染区、具有抗重金属性能的植物。它们在植物抗重金属机理与进化、重金属污染土壤的植物修复和生态恢复研究中具有突出地位。酸性转化酶因水解蔗糖提供能量、碳源和己糖信号是植物库器官建成的关键酶，对污染环境中金属型植物生根、结实和抗性维持的特殊生活史形成有重要作用。本项目用TAIL-PCR、PCR区段缺失、重组载体构建、报告基因融合、拟南芥异源表达等方法，鉴定商周古铜矿遗址抗铜植物该基因顺式调控变异，挖掘新型胁迫响应元件，研究其在响应重金属胁迫的转录调控中的作用。研究的金属型植物包括海洲香薷和鸡眼草。酸性转化酶包括细胞壁转化酶和液泡转化酶。研究内容涉及两个物种的抗铜种群和非抗铜种群酸性转化酶基因启动子克隆，启动子的组织特异性，发育阶段性，环境胁迫响应特性，启动子的特异性响应区段鉴定，以及启动子序列甲基化初步研究等。成功克隆获得两物种抗铜种群和非抗铜种群的细胞壁转化酶基因和液泡转化酶基因启动子序列。两物种抗铜种群和非抗铜种群的细胞壁转化酶和液泡转化酶基因启动子成功在模式植物拟南芥中异源表达。海洲香薷抗铜种群液泡转化酶基因启动子的活性受葡萄糖诱导。5’缺失突变体分析发现，其启动子序列的一段39 bp长的插入片段可能为葡萄糖响应片段，其上的AATACTAT序列或富含A/T的序列有可能为葡萄糖响应元件。海洲香薷抗铜种群细胞壁转化酶基因启动子的活性受水杨酸诱导。5’缺失突变体分析发现，水杨酸响应元件可能位于其启动子序列的-1533～-1524bp处，该水杨酸响应元件受CAAT-box调控。海州香薷酸性转化酶基因启动子序列的甲基化水平在抗铜种群中保持相对稳定，而在非抗铜种群中受铜诱导大幅上升或下降。本项目研究结果有助于深入阐明重金属胁迫下金属型植物酸性转化酶基因的表达调控网络。