高羊茅光周期调控基因FaCONSTANS特异性表达与生物学功能分析

Because tall fescue is a long-day plants,it requied a certain period of long-day for floral bud differentiation, otherwise, it will be in the vegetative growth condition. Photoperiodic induction of flowering transition directly determine the development of tall fescue seed.Currently,the study of tall fescue seed production is focused on measures of field management,little is known about the mechanism of photoperiod induction for floral transition and bud differentiation. Based on previous study of cloning whole sequence of photoperiod regulation gene，We will continue to study characteristics of the temporal and spatial expression and biological function of photoperiod regulation gene in floral transition, revealing molecular basis of photoperiod induced for floraltransition in tall fescue, tall fescue photoperiod sensitivity is expected for improving throught regulation of expression level of this gene in the future breeding.So tall fescue seed production can adapt to a wide range of environment, which will provide a theoretical basis for tall fescue seed production and photoperiod adaptability breeding.

由于高羊茅是长日照植物，只有通过一定时期的长日照才能进行花芽分化，否则就处于营养生长状态。所以光周期诱导的开花转变直接决定了高羊茅种子的发育与形成，目前对高羊茅种子生产的研究仅仅集中在田间管理措施上。对高羊茅种子生产中光周期诱导的成花转变与花芽分化的研究几乎空白。本项目基于在克隆了高羊茅光周期调控基因全长的前期研究基础上，利用荧光定量PCR技术分析光周期调控基因FaCONSTANS的时空表达特征，并通过过量表达与基因敲除策略研究其在高羊茅开花转变中的生物学功能，解明光周期诱导高羊茅开花转变的分子基础。可望在将来的育种中通过对光周期调控基因表达水平的调控改良高羊茅光周期的敏感性，使其种子生产能适应很大范围的环境变化，为高羊茅的种子生产和光周期适应性育种提供理论依据。

CONSTANS（CO）是光周期途径中的特有基因，它应答日照长度的变化启动植物开花。本研究以高羊茅叶片的mRNA为模板，综合运用同源扩增、cDNA末端快速扩增从高羊茅中扩增出CONSTANS基因的全长cDNA序列，命名为FaCONSTANS。该序列cDNA全长1557bp，具有完整的开放阅读框（ORF，166~1296bp），编码蛋白为376个氨基酸，FaCONSTANS基因在高羊茅基因组中由2个外显子和1个内含子组成，氨基端具有B-box型锌指结构域，碳端具有CCT (CO, CO-like, TOC1)结构域，B-box1特别保守，B-box2高度异化，缺乏3个保守的半胱氨酸残基。FaCONSTANS与禾本科植物的CONSTANS蛋白亲缘关系较近，与其他植物的亲缘关系较远。亚细胞定位分析表明FaCONSTANS基因所编码的蛋白定位在细胞核，符合它作为转录因子特性。该基因在长日照、短日照、连续光照、连续黑暗条件下表达分析表明FaCONSTANS基因的表达受光周期调控，与生物钟控制的昼夜节律相关。转基因实验表明FaCONSTANS基因在长日照条件下促进拟南芥提前开花，且恢复拟南芥突变体co开花表型。我们利用RNA干扰技术沉默FaCONSTANS，获得转基因植株晚花或者一直处于营养生长阶段。可望在将来的育种中通过对光周期调控基因表达水平的调控改良高羊茅光周期的敏感性，使其种子生产能适应很大范围的环境变化，为高羊茅的种子生产和光周期适应性育种提供理论依据。