一个新的调控生长、开花和低温响应的基因功能研究

Low temperature is one of the major environmental stresses limiting forage growth. It is important to understand the molecular mechanism of cold tolerance in forage plants. In our previous investigations a novel gene encoding an unknown protein was identified in Medicago falcata, a cold tolerant forage legume germplasm. Transcript of the new gene is highly induced by low temperature, while transgenic tobacco plants overexpressing the gene showed an enhanced biomass and cold tolerance. In addition, Medicago trancatula mutants by Tnt1 insertions showed a phenotype of smaller plant size and leaves with early flowering. Thus we named it GFC (growth, flowering time and cold responsive). The objective of this project is to study the function of MfGFC. MfGFC gene is to be overexpressed in M. trancatula to analyse phenotype in comparison to the gfc mutants as well as the wild type plants. MfGFC interaction proteins will be screened and identified using yeast two hybrid analysis, followed by confirmation with bimolecular fluorescence complementation (BiFC) assay and immunoprecipitation experiments. On the basis of the above studies, transcript profiling and biochemical and physiological changes will be analyzed in transgenic M. trancatula, mutants of gfc and GFC interaction proteins as compared with wild type plants for understanding of the key role of MfGFC and its interaction proteins in regulation of growth, flowering time and cold tolerance. In addition, transgenic Stylosanthes guianensis overexpressing MfGPC will also be generated for improving biomass and chilling tolerance.

低温是限制牧草生长的主要逆境。我们曾从黄花苜蓿发现了一个未知功能新基因，它受低温诱导，过量表达后提高了转基因烟草生物量和抗寒性。其同源基因的蒺藜苜蓿突变体具有植株矮小、叶片小、开花时间提前等表型，因而将该基因暂命名为GFC。本项目拟获得表达MfGFC的转基因蒺藜苜蓿，鉴定转基因蒺藜苜蓿及gfc突变体的表型，筛选、鉴定MfGFC的互作蛋白，进一步筛选鉴定互作蛋白的突变体，比较该突变体与gfc突变体表型的异同。在此基础上，分析比较转基因蒺藜苜蓿、gfc突变体及互作蛋白突变体在基因表达 谱和生理生化等方面的差异，从而揭示MfGFC及其互作蛋白调控植物生长、开花时间和抗冷的机理。将MfGFC过量表达到柱花草中，以改良柱花草生物量和抗冷性。生物量、开花和抗寒性是3个重要农艺性状，本项目研究不仅有望揭示一个重要新基因的功能，具有重要的科学意义，所获得的提高生物量和抗冷性的转基因柱花草也有潜在应用价值。

本项目主要研究了一个黄花苜蓿中调控植物生长和低温响应基因MfLEA-like（过去曾命名为MfGFC）的功能，还研究调控MfLEA-like的转录因子MfERF1、与MfLEA-like相似的MfLEA3的功能。主要结果如下：（1）MfLEA-like表达受低温和一氧化氮（NO）诱导，过量表达MfLEA-like的转基因植物苗期生长加快，耐寒性提高。（2）虽然采用酵母双杂交筛选到MfLEA-like的互作蛋白，但体内验证呈阴性，表明它在细胞内可能没有特异的互作蛋白。（3）转基因植株具有更高的净光合速率（Pn）和GAPDH活性，低温下的Pn也高于野生型，可能与其生长和耐寒性提高有关。（4）获得了过量表达MfLEA-like的转基因柱花草，其生长和耐冷性提高，再次证明了MfLEA-like的功能，表明MfLEA-like具有应用价值。（5）MfERF1能结合到MfLEA-like启动子，其表达受低温诱导；过量表达MfERF1的转基因烟草提高耐寒性，MfERF1的同源基因MtERF1的RNAi植株耐寒性降低，表明MfERF1的具有调控植物耐寒性的功能；经过系列实验证明MfERF1调控耐寒性与其调控低温响应基因表达，多胺合成与代谢的运转及抗氧化酶基因表达有关。（6）MfLEA3受低温、脱水和ABA的诱导，过量表达MfLEA3的转基因烟草提高耐寒和耐旱性；转基因植物比野生型具有更高的过氧化氢酶（CAT）活性，在低温、干旱和氧化胁迫下具有更强的清除活性氧的能力，从而提高耐逆性。（7）MfPIP2-7的表达受低温和ABA诱导，MfPIP2-7具有运输H2O2的功能；过量表达MfPIP2-7转基因烟草中多个低温响应基因表达上调，NIA基因表达也上调，提高了NR活性，影响硝酸盐还原和氮代谢过程，使得大多数游离氨基酸的含量增加，从而提高了植株的耐低氮和耐寒能力。