柿花性别分化的基因表达和激素调控机理研究

Most varieties of persimmon (Diospyros kaki L.) bear female flowers, while a small number of varieties are monoecious. Additionally, a few varieties bear male, female and hermaphrodite flowers on a same tree, and a very few varieties just bear male flowers. The regulatory mechanism of sex differentiation of persimmon flowers has not been investigated. The lack of male resources strongly restricts hybridization of persimmon. In order to cultivate male resources artificially, the regulatory mechanism of persimmon flower sex differentiation should be investigated. Thus, female, male and hermaphrodite flowers will be used to observe the development of pistil and stamen through paraffin method and scanning electron microscope, through which the critical morphological period of sex differentiation can be determined. Subsequently, flowers with three different sex types collected from a same ‘Zenjimaru’ tree will be used for transcriptome sequencing, comparative analysis and phytohormone determination. Thus, the crucial gene and phytohormone as well as their relationship, which regulate sex differentiation will be revealed. Then, the expression pattern of crucial gene in the female and male flowers during development will be investigated. Finally, crucial phytohormone will be sprayed on female and male flowers, and the difference of sex differentiation and gene expression level of these treated flowers will be determined, through which the regulatory mechanism of gene expression and phytohormone on the sex differentiation of persimmon flowers will be clarified. These results will be helpful to improve the hybridization of persimmon through promoting the cultivation of male resources artificially.

柿（Diospyros kaki L.）绝大部分品种仅开雌花，少数雌雄同株或雌花、雄花、完全花混生（如“禅寺丸”），极少数只开雄花，柿开花特性复杂，其原因尚未阐明。雄性资源缺乏严重限制了柿杂交育种工作的开展。要培育雄性资源，首先需要探索柿花性别分化的调控机理。因此本研究采集不同发育阶段的雌花、雄花、完全花，用石蜡切片和扫描电镜观察其性别分化的形态学关键时期；再以该时期为依据，从同一株既开雌花又开雄花和完全花的“禅寺丸”单株上采集三种类型的花，分析其基因表达和激素含量差异，找出调控柿花性别分化的关键基因和关键激素及二者的相关性，同时分析关键基因在雌雄花完整发育过程中的表达模式；最后利用关键激素分别处理柿雌花和雄花，分析处理后的花的性别分化变化情况和关键基因的表达量差异，从而阐明柿花性别分化的基因表达和激素调控机理。本研究可为人工培育柿雄性资源提供理论依据，进而促进柿杂交育种工作的深入开展。

柿（Diospyros kaki Thunb.）栽培品种绝大部分仅开雌花，少数雌雄同株或雌花、雄花、完全花混生，完全雄株零星分布于野生种质中。柿开花特性复杂，其原因尚未阐明。雄性资源缺乏严重限制了柿杂交育种工作的开展。要培育雄性种质，首先需要探索柿花性别分化的调控机理。本研究利用石蜡切片、扫描电镜、液相色谱质谱联用、转录组测序、实时定量PCR及植物生长调节剂注射等技术，从形态、生理和分子生物学层面对柿花性别分化的调控机理进行系统揭示。结果如下：柿雌、雄花发育从6月持续到次年5月，整个过程可从形态上划分为11个阶段，其中阶段2和阶段8为柿花性别分化的形态学关键时期；在阶段8，雌、雄蕊原基的选择性败育是形成单性花的直接原因。以形态学发育阶段为依据，分析各阶段雌、雄花芽中内源激素含量与性别分化的相关性，发现吲哚乙酸（IAA）、玉米素（ZT）和脱落酸（ABA）具有促进雌花发育作用，而赤霉素（GA3）具有促进雄花发育作用。采集性别分化关键时期的雌、雄花芽，利用转录组测序对比分析，筛选出与激素合成、信号转导、花器官发育、转录调控和细胞程序性死亡等过程相关的性别分化关键调控基因25个，并确定其中6个基因的分子调控关键时期。基于以上结果，开展外施植物生长调节剂、土壤水分控制和花芽碳氮比调控对柿花性别分化影响试验。发现外施乙烯利能显著降低雄花的花粉粒直径和花粉体外萌发的花粉管长度，外施GA3能显著提高雌花中退化雄蕊的长度。与土壤体积含水率为20%的对照相比，土壤体积含水率为55%和45%可显著提高柿雌花的雌蕊长度，花芽高碳氮比也有促进雌花中雌、雄蕊长度的作用。此外，还发现多种处理可显著改变性别分化关键调控基因的表达模式，并改变花芽同化物、丙二醛含量及抗氧化酶活性，这可能是引起表型变化的原因。本研究在提升柿乃至植物性别分化理论水平的同时，为人工调控柿性别类型奠定了重要基础。