FT基因对高羊茅开花转变的生物学功能研究

As one of the key pasture and turfgrass seeds，tall fescue features high value of economic development. However, it requires long-day (LD) photoperiod conditions to differentiate flowers and buds, and then produce seeds. Currently its seeds are mainly imported from Europe and the US with long exposure to sunshine, whereas China cannot produce them efficiently. Therefore, it’s of great significance to conduct in-depth study on the role of photoperiod-dependent floral reversion and flower bud differentiation during its seed production in promoting its localized seed production. Based on the early study on the expressions of the FT upstream regulating gene—FaCONSTANS under varying light conditions，and the functional verification in its flowering transformation, the project was intended to conduct investigations as follows: 1）analyze FT activity difference and tissue specificity in the vegetative and reproductive phases of Brachypodium distachyon through Laser Scanning Confocal Microscopy; 2）construct the FT and GFP fusion expression vectors to transform Brachypodium distachyon and screen the strains in high and stable expression of phloem , and comparatively analyze the long distance mobility and the role of FaFT proteins in flowering transition to expound the molecular basis of the FT gene regulation in tall fescue floral transition. The results are expected to explore the role and mechanism of the FT regulation in its photoperiod-dependent seed production as well as provide a theoretical basis for its adaptive photoperiod breeding.

作为重要的牧草及草坪草种之一，高羊茅具有较高的经济开发价值，但它需要长日照才能进行花芽分化从而产生种子；目前高羊茅草种子主要从长时照的欧美地区进口，在我国不能有效生产种子。因此，深入研究高羊茅种子生产过程中光周期依赖性成花转变与花芽分化机制对于高羊茅草种本地化生产具有十分重要的意义。本项目拟在前期研究对不同光照条件下FT上游调控基因FaCONSTANS表达差异及调节开花转变功能验证的基础上，首先利用激光共聚焦分析FT基因在二穗短柄草营养生长期和生殖期的活性差异及组织特异性；同时构建FT和GFP融合表达载体转化二穗短柄草并筛选出在二穗短柄草韧皮部高效稳定表达的植株，对比分析FaFT蛋白在植物开花转变过程中长距离的移动性及其作用途径，解明FT基因调控高羊茅开花转变的分子基础。研究结果可望深入阐明FT基因在高羊茅光周期依赖性种子生产中作用及机制，同时也为高羊茅的光周期适应性育种提供理论依据。

FT基因是光周期应答途径中的关键基因，调控下游开花基因SOC1和AP1的表达促进开花。贵州属于短日照地区，在研究光周期适应性育种上，长日照高羊茅一方面生产种子周期很长，另一个方面，转化的高羊茅其外源基因的稳定遗传有所障碍。为揭示成花转变过程中高羊茅FaFT基因的功能，本研究以‘黔草1号’高羊茅为实验材料，运用同源克隆获得高羊茅FaFT基因的cDNA全长序列，高羊茅FaFT基因序列全长5045bp，G+C含量为58.33%，含有2个内含子和3个外显子。FaFT基因cDNA序列全长1073 bp，开放阅读框为537 bp，位于154 bp~689 bp，编码178个氨基酸残基。其编码的蛋白为亲水性蛋白，含有α螺旋、无规则卷曲等丰富的二级结构，有一个保守的PEBP结构域。多重比对结果显示：高羊茅FaFT基因编码的氨基酸与黑麦草的同源性最高为98.84%，其次是二穗短柄草97.73%，高度保守。.无论是长日照还是短日照其表达量在叶中最高，其次是茎和根。长日照下的表达量高于短日照，但表达高峰出现的时间都在ZT8。长日照转连续光照和连续黑暗，表达高峰出现在ZT0，而短日照转连续光照和连续黑暗表达高峰出现在ZT4和ZT12。光照/黑暗循环模式下表达高峰出现在ZT4~ ZT16。构建高羊茅FaFT基因与绿色荧光蛋白融合表达载体1300-GFP-FT转化烟草，激光共聚焦显微镜下检测融合载体的瞬时表达，结果显示高羊茅FaFT基因编码的蛋白定位在细胞核。酵母双杂交系统和双分子荧光互补证实FaFT与FaFD存在相互作用。农杆菌介导转化拟南芥发现：LD下，FaFT基因过量表达转基因拟南芥植株表现出早花，比野生型平均开花时间提前约4d，比突变体植株提前约3d，功能补偿后的突变体ft能恢复了野生型的开花表型，但突变体植株ft本身无开花现象，一直停留在营养生长期。过表达FaFT基因拟南芥莲座叶数量比野生型、突变体要多。这表明FaFT基因促进拟南芥提前开花，恢复了ft突变体的晚花表型。通过对高羊茅FT基因的生物学功能进行系统研究，旨在为更进一步解明高羊茅开花转变中光周期诱导应答途径的分子基础，为高羊茅光周期适应性育种提供理论依据。