低温应答MtLRPK及其同源基因MfLRPK1负调控耐寒性的研究

Low temperature is one of the major environmental stresses limiting growth and productivity of alfalfa and other forage legumes. It is important to improve cold tolerance of alfalfa varieties with high fall dormancy ratings for increased alfalfa growth in temperate regions, which depends on understanding of molecular mechnisms of cold tolerance in alfalfa. In our previous investigations, transcirption of a leucine-rich receptor-like kinase (MtLRPK) gene from Medicago truncatula, a legumeous model plant with cold sensitivity, was found to be induced by low temperature, while the homolorg MfLRPK1 from Medicago falcata, a cold-tolerant germplasm of alfalfa, showed a suppressed expression by low temperature. Mutation of MtLRPK with transposon Tnt1 resulted in enhanced cold tolerance in mtlrpk lines. The data indicated that MtLRPK or MfLRPK1 may negatively regulate cold tolerance. Receptor-like kinases (RLK) play an important role in plant growth and development and may function to perceive environmental signals in plant cell. Thus, it is the aim of this project to investigate the important role of MtLRPK in negative regulation on cold tolerance. To further validate the negative regulation on cold tolerance, MtLRPK and MfLRPK1 driven by MtLRPK promoter will be introduced into a mtlrpk mutant line for evaluation of cold tolerance in transgenic plants. To elucidate the molecular mechanisms of the genes, MtLRPK-interaction proteins and their mutant lines with Tnt1 transposon will be screened and identifed. Transcriptome profiling and relavant biochemical and physiological changes will be analysed using both mtlrpk mutant and double mutant of mtlrpk with mutant of MtLRPK-interaction proteins. The approaches will provide us with new knowledge on the role of RLK in regulation of cold tolerance in falfalfa and a novel clue for improvement on cold tolerance.

低温是影响牧草生长和产量的主要环境因素，改良高秋眠级苜蓿品种耐寒性对提高温带地区苜蓿产量有重要现实意义，这有赖于对苜蓿耐寒分子机理的了解。黄花苜蓿是重要耐寒种质，不耐寒的蒺藜苜蓿是豆科模式植物，申请人在黄花苜蓿耐寒机理研究中，发现蒺藜苜蓿中一个MtLRPK基因表达受低温诱导，而黄花苜蓿同源基因MfLRPK1受低温抑制，MtLRPK的Tnt1插入突变体耐寒性提高，表明该基因负调控耐寒性。RLK是植物细胞感知、传递环境信号的受体蛋白，其重要性不言而喻。本项目拟在前期工作基础上，研究MfLRPK1和MtLRPK基因对mtlrpk突变体的功能恢复，验证其负调控耐寒性的功能，为该基因应用于苜蓿及豆科牧草耐寒性改良提供依据。进一步以蒺藜苜蓿为代表材料，筛选与鉴定MtLRPK的互作蛋白，构建双突变体，并研究它们调控的下游基因网络和生理生化过程，阐明MtLRPK负调控耐寒性的机理，无疑具有重要科学意义。

项目研究了蒺藜苜蓿MtLRPK1和及其黄花苜蓿同源基因MfLRPK1调控植物耐寒性的功能。低温诱导MtLRPK1和MfLRPK1的表达，MtLRPK1定位于细胞质和细胞核内，主要表达于茎和叶脉等输导组织。获得了过表达MtLRPK1转基因蒺藜苜蓿，筛选出3个纯合的mtlrpk1突变体株系，并获得MtLRPK1和MfLRPK1回补突变体的植株，测定了这些植物的耐寒性。结果显示，表达MtLRPK1提高植物耐寒性，突变MtLRPK1降低耐寒性，而MtLRPK1和MfLRPK1能恢复mtlrpk1突变体的耐寒性，表明MtLRPK1和MfLRPK1具有调控耐寒性的功能。与野生型相比，低温下MtLRPK1过表达植株具有更高的MtCBF1, 2, 3, 4及低温响应基因MtCAS15和MtCOR413转录本；MtLRPK1过表达植株具有更高的抗氧化酶活性和脯氨酸含量与其具有更高的抗氧化酶和P5CS基因转录本水平一致，而这些基因的表达、抗氧化酶活性和脯氨酸含量在mtlrpk1突变体中更低。结果表明，MtLRPK1调控耐寒性与其调控CBF途径，进而调控低温响应基因表达、抗氧化保护系统和脯氨酸积累和有关。采用酵母双杂交技术从蒺藜苜蓿cDNA质粒文库筛选MtLRPK1互作蛋白，并进一步利用BiFC进行验证。结果显示，MtLRPK1作用的靶蛋白至少包括MtPIP1;4和MtCYSTM。Tnt1插入MtPIP1;4和MtCYSTM的突变体降低耐寒性，表明这两个基因调控耐寒性，与MtLRPK1功能一致。MtLRPK1与其靶蛋白结合调控耐寒性的机理尚需进一步研究。