miRNA介导的GA调控板栗雌花形成的生理及分子机制

There are enormous male flowers but less female flowers which is one of main influence factor to cause low yields in chestnut. Gibberellin (GA) regulation of sex differentiation is an important way to increase female flowers and the production. This project focuses on the core research question involved in GA-improving female differentiation in chestnut. Firstly, in current project chestnut bud mutant 'Duanhuayunfeng' with the characteristics of more female flowers and less male flowers, is used to study physiological variation characteristics including endogenous hormones during the flower differentiating process after GA spraying. Secondly, by high-throughput sequencing miRNA library and transcriptome, candidate miRNAs and target genes response to GA are screened to study their gene expression patterns in the process of flower differentiation, and to explore marking miRNA tightly relevant to female differentiation in chestnut. Transformations of over-expression and interference of key miRNA based on vivo and transient techniques are applied to study the interactions between miRNA and target genes, and then to disclose the control mechanisms of GA-promoting female flower differentiation mediated by miRNA. This study lays the foundation for further development of sex differentiation on the chestnut and perennial fruit trees. Furthermore it will provide a theoretical basis for artificial regulation of sex differentiation on fruit trees with unisexual flowers.

板栗雄花多、雌花少，是造成板栗低产的主要因子。赤霉素（GA）调控性别分化是板栗增雌、提高产量的重要途径。本项目围绕板栗GA促雌的核心问题，以板栗芽变'短花云丰'（雌花多，雄花少）为研究试材，研究GA喷施后板栗雌雄花分化进程中内源激素等主要生理特征变化规律；利用高通量测序的板栗雌雄花miRNA文库和转录组，筛选响应GA的候选miRNA分子和靶基因，系统研究miRNA及其靶基因在板栗雌雄花分化过程中的表达特征，发掘与雌花分化有关的标记性miRNA；通过板栗活体瞬时转化技术，研究关键miRNA超表达和干扰对其靶基因的作用机理，解析miRNA介导的板栗GA促雌的调控机制。本项目为进一步开展板栗等多年生果树雌雄花性别分化机理研究奠定基础，同时，为生产上人工调控板栗等雌雄异花果树性别分化提供理论依据。

摘要：本课题围绕板栗GA促雌的核心问题，以‘燕山红栗’为研究试材，首先对雌花分化期的板栗的完全混合芽与不完全混合芽进行石蜡切片观察，确定雌花分化期分为六个阶段，即花序原基分化期、花序原基分化中期、花序原基分化后期、花簇苞片原基形成期、花簇原基分化期及花簇原基形期；进而对雌花分化期的板栗完全混合芽与不完全混合芽中的内源激素以及板栗结果枝果痕上下部的可溶性糖（葡萄糖、果糖、蔗糖及甘露糖）进行测定，发现雌花发育过程中花芽中内源IAA的启动时间早于ABA，雌花分化期韧皮部可溶性糖由高到低依次为果糖、葡萄糖、甘露糖、蔗糖，且葡萄糖及果糖含量均明显高于蔗糖和甘露糖，说明板栗可能是通过葡萄糖的形式进行运输。通过对花芽及雌雄花的miRNA测序及分析，共发现1808个miRNA，其中保守的882个，非保守的926个，花芽分化前后）差异比较发现1686个miRNA，其中差异表达的167个，分化成花后差异比较发现122个miRNA，其中差异表达的84个；对同一批样品的进行转录组测序和分析发现，花芽分化前后差异比较发现27238个基因，其中差异表达的基因3446个，分化成花后差异比较发现26091个基因，其中差异表达的2748个，结合生物信息学，重点针对MADs-box家族成员基因进行了表达分析；对小RNA文库和转录组数据进行联合分析，发现总共有34对miRNA-mRNA对，其中花芽分化前后差异表达14对miRNA-mRNA，对应9个miRNA和9个基因，分化成花后差异表达20对miRNA-mRNA，对应7个miRNA和19个基因。利用RT-PCR方法分析在不同花器官下候选miRNA转录水平的表达情况，结果显示miR164、miR396、miR399、miR535等miRNA在雌雄花中表达量存在显著差异，推测这些miRNA可能参与板栗花发育。综合上述结果，本研究为探讨miRNA调节板栗花发育的分子机理奠定了坚实的工作基础，同时对于认识MADs-box家族成员之间的关系提供了重要的启示性线索。项目资助发表核心论文6篇，SCI收录3篇，待发表1篇。培养硕士生6名，其中4名已经取得硕士学位，2名在读。