基于数字基因表达谱分析的寒兰试管成花分子机理研究

Since the days from sowing to flowering of orchid flora was reduced by flowering in vitro, the related theoretical study and resource utilization of this family/specie had been promoted. However, there were some barriers such as the lower induction rate of flower buds, shorter florescence, bad fertility in flowering in vitro of orchids and other plants. In addition, the cost and technical difficulty would be increased by induction of flowering in vitro. Based on our research foundation on flowering in vitro in Cymbidium kanran, RNA-seq will be performed in terminal bud to obtain the reference sequence, and then digital gene expression tag profiling is conducted in terminal buds during different flower bud differentiation periods. Therefore, the dynamic variation of related gene expression in differentiation of floral bud would be revealed. To fish out genes playing a crucial role in flower induction, Real-time PCR and RNA in situ hybridization is applied for the differential gene expression in DGE. By cloning and transient expression analysis of the promoters of the flowering genes as well as their expression patterns, the regulation characteristic of the target genes is elucidated. Finally, overexpression analysis of the target genes of Arabidopsis thaliana are performed, thus, the function of the flowering genes in Cymbidium kanran would be verified well. This project could provide a deep understanding in molecular mechanism of flowering in vitro, and a solid foundation for molecular breeding of early flowering in Cymbidium kanran mak.

试管成花技术可缩短兰科植物从种子到开花所需的时间，促进该科植物的相关理论研究与资源开发。然而，相关的分子机理尚未明了，体外开花中的花芽诱导率低、花期短、可育性差等问题普遍存在，并且试管开花的诱导将增加操作的成本和技术难度。基于课题组在寒兰试管成花方面的研究基础，本研究拟对寒兰花芽进行RNA-seq结合数字基因表达谱分析，可全面揭示成花过程中基因表达的动态变化；并利用Real-time PCR和RNA原位杂交技术对所得的差异基因进行验证，以期得到在寒兰试管成花中起关键作用的开花基因；再克隆开花基因的启动子，结合其在烟草叶片中的瞬时表达，以明确开花调控特性；最后通过对拟南芥的基因超表达，全面解析所得的寒兰开花基因的功能。本项目的实施，对正确理解以寒兰为代表的兰科植物试管成花的分子机理具有重要意义，同时，为推进兰花的早花育种奠定坚实的基础。

包括寒兰在内的兰科植物从种子到开花所需时间非常长，严重地制约了花发育的相关研究以及新品种培育，所以寒兰试管开花分子机制的研究具有重要意义。项目首先利用qRT-PCR技术结合生物信息分析评价了9个常用的内参基因在寒兰不同组织中以及激素处理和非生物胁迫条件下的表达稳定性，然后对获得的68,699条Unigene进行SSR位点分析，为寒兰基因表达分析与SSR引物开发奠定基础。寒兰试管苗的花芽分化进程可分为花芽分化前期（a）、苞片原基分化期（b）、花原基分化期（c）、花被原基分化形成期（d）、合蕊柱分化期（e）、花粉囊和柱头形成期（f）等6个时期。对寒兰前三个时期的花芽进行DGE分析，通过对三个转录组数据库的比较，共获得23,720个差异表达基因，从中筛选出与成花过程有关的9个基因，分别为：CkFPA，CkGA2OX，CkGID1，CkCO，CkPHYB，CkELF3，CkFRI，CkFLC和CkAP1。通过RT-qPCR技术，验证9个差异表达基因在寒兰成花过程中的表达模式，发现其中CkFPA，CkGA2OX，CkCO，CkFLC和CkAP1 5个基因在寒兰开花过程呈现显著差异及规律的表达模式，表明其与寒兰开花密切相关。为研究AP1基因功能，我们采用瞬时表达和稳定表达两种系统。瞬时表达试验发现，CkAP1蛋白定位在细胞核上，而酵母单杂交试验表明该基因编码的蛋白单独作用时，不具有转录激活活性。我们将基因的编码框插入到双元载体1301-220中，再通过农杆菌介导，将构建好的超表达载体转化拟南芥。经过抗性培养基筛选，GUS染色，RT-PCR检测，鉴定出19个拟南芥转基因株系，转基因T3代用于功能分析。我们发现，无论在长日照还是短日照条件下，转基因植株的花期明显提前，对4个拟南芥开花基因表达的定量分析表明，FT, LFY等多个开花促进因子表达量上升。.项目执行期间，课题组共培养研究生3名，发表标注该基金资助论文4篇，其中SCI论文2篇，CSCD核心1篇，另有1篇已投至molecular breeding，申请国家发明专利2项。