基于水母发光蛋白的植物低温胁迫Ca2+信号遗传分析

Low temperature is an important environmental factor influencing crop quality and productivity. The immediate response to low temperature in plant is a transient increase in intracellular Ca2+ concentration, and the inhibition of Ca2+ response would hinder cold acclimation. Therefore, it is important to study the mechanism of Ca2+ mediated low-temperature signal transduction. However, it is not yet clear what genes are involved in low-temperature Ca2+ signal transduction, and the crosstalk between low temperature and Ca2+ signaling has rarely been reported. By analysis of low temperature regulated genes from Arabidopsis microarray data, we would select low temperature induced or inhibited genes and obtain the corresponding T-DNA insertion mutants. To address the relationship between those genes and Ca2+, the aequorin would be transformed into the mutants, and then cold-triggered Ca2+ responses would be measured. The functions of the mutant gene which shows the specific changes of Ca2+ would be analyzed through physiology, molecular biology and genetics methods. The relationships of the genes would be analyzed by constructing double or triple mutants which were obtained by crossing similar phenotype mutants. This project will be helpful to define the intermediates of Ca2+ -mediated signal transduction into nucleus and their spatial and temporal positions in low temperature signaling.

低温胁迫严重影响着作物的产量和品质。低温刺激后Ca2+浓度迅速升高，抑制Ca2+浓度升高则破坏冷驯化，因此研究Ca2+介导的低温信号转导机制具有重要意义。但是目前尚不清楚什么基因参与了低温Ca2+信号转导，也未见低温信号与Ca2+信号交互作用（crosstalk）的报道。本项目以基因组学研究为基础，利用生物信息学分析拟南芥中低温调控的基因，选取低温诱导或抑制的基因并获得T-DNA插入拟南芥。将活体Ca2+探针——水母发光蛋白转入突变体中，检测低温刺激后突变体细胞Ca2+浓度变化。对Ca2+浓度特异变化突变体利用生理学、分子生物学和遗传学方法解析突变基因功能。然后选择表型变化相似的突变体构建双突变体或三突变体，多途径多方法解析基因的空间关系，建立低温Ca2+信号转导的网络框架。本研究将初步建立Ca2+信号在低温信号转导中的时空作用模式，为理解低温信号转导机制奠定坚实的细胞学和遗传学基础。

低温胁迫严重影响着作物的产量和品质。低温刺激后植物体内Ca2+浓度迅速升高，抑制Ca2+浓度升高则破坏冷驯化，研究Ca2+介导的低温信号转导机制具有重要意义。以基因组学研究为基础，利用生物信息学数据库提供的信息分析拟南芥中低温调控基因，选取低温特异诱导或抑制的基因并购买相应的T-DNA插入拟南芥突变体。将活体Ca2+探针水母发光蛋白基因转入突变体中，检测低温刺激时突变体细胞内Ca2+浓度变化。对Ca2+浓度特异变化突变体利用生理学、分子生物学和遗传学方法解析突变基因功能。低温刺激之后athcr突变体胞质中Ca2+浓度升高幅度明显大于野生型，但是两者在液泡膜的胞质面Ca2+浓度变化相似。药理学实验证实低温刺激时athcr胞内较高的Ca2+浓度主要来源于细胞外的钙库。表型实验显示athcr突变体比野生型具有更强的低温抗性。在athcr突变体中RD29A、KIN1和COR15A等基因转录活性增强。综上研究结果，定位于质膜上的AtHCR参与了低温信号转导过程，并且作为COR基因的负调节因子参与了基因的转录调节。HCR蛋白功能解析为理解低温信号转导机制奠定坚实的细胞学和遗传学基础。