大豆GmMCU1基因响应盐胁迫的分子机制研究

Salt stress is a key factor seriously limiting yield and quality of crops. Calcium as a second messenger, plays an important role in the process of responding to salt stress in plant. MCU (mitochondria calcium uniporter) is reported as an improtmant Ca2+ channel protein on mitochondrial inner membrane. I have isolated 10 soybean MCU genes, and found that the expression of GmMCU1 was significantly induced by Ca2+ or salt stress in the soybean root, and subcellular localization analysis showed GmMCU1 was a mitochondrial protein. The yeast and tobacco with heterologous expression of GmMCU1 showed a more salt-tolerant phenotype than the control, but the function of GmMCU1 on Ca2+ transport and the subsequent physiological effects are unclear. In this project, we will employ soybean hairy-root system and transgenic soybean plants, using technology of calcuim ion measurement by mitochondrial fluorescent Ca2+ probe Rhod-2 or calmodulin 4mt-YC3.6, combined with analysis of the cotent of active substance and metabolic products under salt stress in soybean, to detect the GmMCU1 whether involving in mitochondrial Ca2+ dynamics and the regulatory mechanism of GmMCU1 on Ca2+ transport. This project is espected to clarify the function of GmMCU1 responding to salt stress, and provide theoretical basis for futher exploration in soybean salt tolerance molecular mechanism and salt tolerance breeding.

盐胁迫是限制作物产量和品质的重要因素。Ca2+作为第二信使，在植物抵御盐胁迫的过程中发挥重要作用。MCU（mitochondria calcium uniporter）是线粒体内膜上单向转运Ca2+的通道蛋白。申请人从大豆基因组中查找到10个MCU同源基因，研究发现，只有GmMCU1在大豆根系中的表达均受Ca2+或盐胁迫诱导显著升高。GmMCU1亚细胞定位于线粒体，其过量表达显著提高酵母和烟草的耐盐性，但对线粒体内钙信号的形成机制和生理作用尚不清晰。本项目基于大豆发根表达系统和稳定转基因材料，利用线粒体钙敏荧光染料Rhod-2以及钙调荧光蛋白4mt-YC3.6为内标的Ca2+测定技术，配合活性物质生成和代谢产物积累量的分析，研究GmMCU1在大豆受到盐胁迫时对Ca2+转运的调控功能和生理作用，以期探明GmMCU1响应盐胁迫的作用机制，为大豆耐盐分子机理研究和育种提供新的思路。

大豆是我国重要的粮食和油料作物，属于中度耐盐植物，是中轻度盐渍土开发利用的重要作物品种。盐渍化条件下过多的盐分影响大豆光合作用及新陈代谢，抑制其植株的生长发育，最终导致产量减少和品质下降。近年国家农业结构调整明确指出要扩大大豆种植面积，且我国有大量受盐渍影响的中低产田，但是适合种植的耐盐大豆品种极少。本项研究从大豆耐盐基因资源挖掘开始，分析钙离子胁迫诱导基因GmMCU1的特征和耐盐机理。发现其主要在根中表达，并且受盐胁迫诱导上调表达，主要定位于线粒体，为钙离子通道的蛋白，异源过表达此基因能发现，GmMCU1能提高酵母菌和烟草的耐盐胁迫能力。利用发状根系统和拟南芥突变体研究发现，MCU1能影响钙离子在线粒体内外的运输。酵母双杂交实验显示GmMCU1能与钙离子结合蛋白GmMICU6相互作用。所以推断GmMCU1和GmMICU6协同调控盐胁迫条件下大豆线粒体内的钙离子稳态，从而避免钙离子过载造成的活性氧积累，提高大豆的耐盐性。本研究克隆盐胁迫应答的钙离子转运蛋白基因，研究其分子功能和生理作用，将进一步完善大豆耐盐相关的生物学理论，为大豆耐盐新品种的培育提供科学依据。