兼性金属型植物酸性转化酶基因的变异与功能研究

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 special life history formation of rooting, fructification and tolerance maintenance in metallophytes found in uncommonly contaminated environments by heavy metals. It is recently discovered that, acid invertase genes divergence arises in copper tolerant populations in several metallophyte species showing higher enzyme activities, so that amino acid changes occurred in the enzyme protein and enzyme-substrate affinity increased. However, it is known neither these amino acid changes are correlated with the elevated enzyme activities, nor which of these amino acids are key functional residuals. This project is to study the relationship between potential key point mutations in acid invertase genes and their enzyme characterization as well as functional variations in native Chinese metallophytes found in ancient Cu mines that appeared since the Shang and Zhou dynasties, by means of RT-PCR, PCR site-directed mutagenesis, recombinant plasmid construction, heterologous expression in Pichia pastoris and Arabidopsis thaliana, enzyme characterization and 3-dementional modeling of recombinant enzyme proteins, and phenotype analysis of the transgenic plants. The results could, from one side, elucidate the role of acid invertase variation in the special life history formation and evolution of Cu tolerant populations of the metallophytes. The strategy of comparing the enzyme characterization and functional variations in acid invertases between side-directed mutants and their wild types could, to some extent, confirm their possible evolutionary course occurred naturally in the wild by gene manipulation in the laboratory. This idea might be applied in other similar evolutionary studies.

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，对重金属污染极端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。近期发现，多种金属型植物抗铜种群的该酶基因发生趋异，使酶蛋白出现氨基酸替换、酶-底物亲合力提高且酶活性升髙。但未知这些氨基酸替换是否与酶活性增强相关、其中哪些残基是关键功能位点？本项目用RT-PCR、PCR定点突变、重组质粒构建、毕赤酵母和拟南芥异源表达、重组蛋白酶学特征和3D构型及转基因植株表型分析，研究商周古铜矿遗址原产兼性金属型植物酸性转化酶基因若干关键位点突变与其酶学特征及功能变异的关系，借此从一侧面阐述该酶的变异在抗铜种群特殊生活史形成和进化中的作用。本项目经比较酸性转化酶基因定点突变体与其野生型的异源表达蛋白的酶学特征和功能差异，能在实验室内借助基因操作手段验证该酶在野外可能发生的进化过程。此思路可为其它类似的进化生态学研究借鉴。

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。本项目以我国铜矿污染区原产金属型植物为试材，应用RT-PCR、3'和5'RACE、DNA测序、实时定量PCR、SDS-PAGE、Western Blot、酶蛋白三维模拟、巴斯德毕赤酵母及拟南芥异源表达等技术，比较分析铜矿污染区抗铜种群和对照区非抗铜种群酸性转化酶基因的结构与功能变异。研究的金属型植物包括海洲香薷、鸡眼草和齿果酸模。酸性转化酶包括细胞壁转化酶和液泡转化酶。研究内容涉及三个物种的酸性转化酶基因克隆与转录表达，酶蛋白3D结构、模拟定点突变和分子对接，异源表达，重组蛋白的酶学特征，及启动子的变异和活性。三个物种的抗铜种群的酸性转化酶基因表达无例外地受铜胁迫诱导，而非抗铜种群则被抑制。三个物种的抗铜种群和非抗铜种群之间酸性转化酶蛋白有2-3个氨基酸残基趋异，但这些趋异的氨基酸并不位于酶分子的催化活性中心，也没有显著改变酶分子的构型。海洲香薷和鸡眼草的酸性转化酶基因在毕赤酵母中异源表达获得重组蛋白，其酶学特性及对多种重金属的敏感性在抗铜种群和非抗铜种群之间没有明显差异。因此，抗铜种群酸性转化酶基因编码区的趋异没有对其功能产生显著影响。在海洲香薷酸性转化酶基因启动子序列中发现多个与植物激素、生物和非生物胁迫及糖相关的顺式调控元件，这意味着酸性转化酶基因的表达调控可能受到这些因素的广泛影响。海洲香薷酸性转化酶基因启动子在转基因拟南芥中成功驱动GUS报告基因表达。本项目研究结果初步证实抗铜种群酸性转化酶基因编码区的趋异对其酶学特征和基因表达没有产生显著影响，而其由启动子介导的基因表达调控变异值得深入研究。