钙调磷酸酶通过Crz1转录因子调控细胞壁合成在土生隐球酵母耐铝中的作用机制

Aluminum (Al) toxicity is a major factor limiting crop productivity on acidic soil. Researches about mechanism of Al-tolerance are premise and basis for solving Al toxicity problem on acidic soil. Our previous studies showed that gene encoding calcineurin increased aluminum tolerance of Saccharomyces cerevisiae through adsorbing inorganic monomeric Al in the media. Furthermore, Al stress up-regulated gene expression encoding cell wall components. The purpose of this project is to reveal whether calcineurin regulates biosynthesis of cell wall through Crz1 to adsorb Al and to increase resistance of Al. In this project, yeast two-hybrid system is used to verify the interaction between calcineurin and Crz1 transcription factor. The C-terminus of Crz1 is fused with the green fluorescent protein (GFP) gene to analyze whether Crz1 localization in cell is regulated by calcineurin. Fusion of promoter region of fks2 encoding encoding a b-1,3 glucan synthase with lacZ reporter gene is used to assay transcriptional regulation of calcineurin to cell wall synthesis genes. Binding of Crz1 with fks2 promoter DNA was tested using EMSA. Real-time PCR is used to analyze regulation of Crz1 on other genes encoding cell wall components. These researches can further enrich the understanding of the calcineurin signal pathway involving in Al stress and fill in the blanks in signal transduction pathway under aluminum stress.

铝毒害是酸性土壤上作物产量的主要限制因子，研究耐铝机理是解决酸性土壤上铝毒害问题的前提和基础。我们前期研究发现，土生隐球酵母钙调磷酸酶基因通过吸附培养液中的活性铝而增加酿酒酵母的耐铝能力。而且我们还发现，铝胁迫能够诱导细胞壁组分基因表达上调。为了探明钙调磷酸酶是否通过Crz1调控细胞壁的生物合成进而吸附铝来增加耐铝的目的。本项目利用酵母双杂交技术验证钙调磷酸酶与Crz1转录因子之间的互作；将Crz1与GFP融合分析钙调磷酸酶调控Crz1在细胞内的定位；将β-1,3葡聚糖合成酶基因fks2启动子区与lacZ基因融合，分析钙调磷酸酶对细胞壁合成基因的转录调控；利用凝胶阻滞实验验证Crz1与fks2启动子DNA的结合；利用Real-time PCR分析Crz1对其他细胞壁组分基因的调控。这些研究内容可以进一步丰富钙调磷酸酶信号途径参与铝胁迫的认识，填补铝胁迫下信号传递途径的空白。

铝毒害是酸性土壤上限制农作物产量的主要因素，研究耐铝机理是解决酸性土壤上铝毒害问题的基础。我们前期研究发现，土生隐球酵母钙调磷酸酶基因通过吸附活性铝使酿酒酵母耐铝能力提高，铝胁迫能够上调细胞壁组分基因的表达。本项目通过酵母双杂交证明钙调磷酸酶可以与转录因子Crz1互作，铝胁迫处理可以使细胞壁组分葡聚糖和甘露聚糖含量增加，诱导细胞壁合成基因的表达上调。钙调磷酸酶途径阻断使葡聚糖和甘露聚糖含量下降，细胞壁生物合成基因表达下调，说明细胞壁合成是受钙调磷酸酶信号途径调控的。通过转基因酵母研究表明，Crz1转录因子可以增加酵母对铝毒、钙离子、镉和过氧化氢胁迫的抗性。红外光谱分析发现，细胞壁上的氨基、羟基和羰基对铝吸附具有重要作用。这些研究内容证明了钙调磷酸酶信号途径通过转录因子Crz1调控细胞壁合成参与铝胁迫，进一步丰富了钙调磷酸酶信号途径参与铝胁迫的知识。此外，我们还发现铝胁迫下GTPase，蛋白激酶C及MAPK途径基因上调表达，细胞壁组分葡聚糖含量增加。这些结果为我们深入研究铝胁迫下调控细胞壁葡聚糖合成的信号途径打开了一条思路，为后续基金申请奠定了基础。