盐胁迫响应转录因子MsSPL12调控紫花苜蓿耐盐的分子机制研究

研究苜蓿耐盐机制对苜蓿耐盐育种具有重要意义。研究表明：过表达miR156显著降低盐胁迫下Na+和活性氧积累提高紫花苜蓿的耐盐性，而miR156如何通过其靶基因调控苜蓿耐盐的分子机制不明。MsSPL12是miR156的靶基因，显著响应盐胁迫；而且盐胁迫下MsSPL12在耐盐苜蓿中的上调表达量显著强于敏盐苜蓿。本项目拟在苜蓿中过表达和抑制表达MsSPL12基因，比较盐胁迫下植株表型，检测MsSPL12表达定位及耐盐生理指标验证MsSPL12在苜蓿耐盐中的功能。通过转录组测序，酵母双杂交筛选MsSPL12下游耐盐基因，检测耐盐候选基因表达模式，并通过转基因验证候选基因的耐盐功能等手段，构建以MsSPL12为“桥梁”调节苜蓿“离子平衡”和“活性氧平衡”的基因表达调控通路，揭示MsSPL12介导的苜蓿耐盐分子机制。课题的实施将为紫花苜蓿耐盐育种提供理论支持，储备苜蓿耐盐育种材料和基因资源。

Study on the salt tolerance mechanisms of alfalfa (Medicago sativa) is important on salt tolerant alfalfa breeding. Our previous studies proved that overexpression of miR156 in alfalfa significantly improved salt tolerance of transgenic alfalfa plant by reducing Na+ and ROS accumulation. However the molecular mechanism by which miR156 regulates salt tolerance through its target gene is still unclear. MsSPL12 was identified as a target gene of miR156, which significantly responded to salt stress. Under salt stress, expression of MsSPL12 was significantly up-regulated in alfalfa salt-tolerant cultivar compared to salt-sensitive cultivar, indicating MsSPL12 is involved in the regulation of alfalfa salt tolerance. Based on our previous studies, to verify MsSPL12 function this project will analyze plant phenotype, expression and cellular localization of MsSPL12, and physiological indicators of alfalfa transgenic plants with over-expressing and inhibiting the expression of MsSPL12 under salt stresses. And by the methods of transcriptome sequencing and yeast two-hybrid screen the downstream genes of MsSPL12 in salt tolerance. Test expression and verification of downstream salt-tolerance candidate genes by genetic transformation, construct genes expression and regulation pathways under salt stress, in which the MsSPL12 as a “bridge” in regulation alfalfa “ion balance” and “ROS scavenge”. Explanation the molecular mechanisms of salt tolerance in alfalfa mediated byMsSPL12. The implementation of the project will provide theoretical support for breeding of salt-tolerant alfalfa, and reserve new salt-tolerant germplasm and genes.