雌雄异株精油植物山鸡椒花器官退化分子基础研究

Litsea cubeba, one of Chinese important aromatic plant, has received more attentions owing to multiple applications of essential oil. L. cubeba is a dioecious plant and degeneration of floral organ is a crucial step in sex differentiation. However, the molecular mechanism of floral organ degeneration is unclear. In this study, on the basis of identification of degeneration stage of pistil in staminate flower, the morphological and anatomical characteristics of floral development will be investigated by the stereo microscope observation, scanning electron microscope and paraffin section technology, and the crucial stage of floral organ degeneration in L. cubeba will be determined. The change level of endogenous phytohormone in crucial period of floral organ degeneration will be measured. The effect of exogenous phytohormone on sex differentiation will be studied, and the possibility of using exogenous phytohormone to regulate the sex of L. cubeba will also be discussed. At the same time, the transcriptomic analysis of pistillate flower and staminate flower in degeneration stage will also be undertook using RNA-seq to excavate the differential expression genes which were related with floral organ degeneration. Moreover, the expression patterns of these genes will be detected by in situ hybridization technology. The Hub genes and modules of co-expressed genes that were functionally related will be analyzed by R package for weighted gene co-expression network analysis (WGCNA), and the relationship between phytohormone and differential expression genes will be analyzed. Thus, the molecular mechanism of floral organ degeneration in L.cubeba will be clarified. In conclusions, the result of this research will provide significant theoretical basis for using sexual identification and regulation technologies to optimize the male and female sex ratio, finally improve the seed yields in L.cubeba.

山鸡椒果皮富含精油，是我国重要的香精香料树种之一。山鸡椒是雌雄异株植物，花器官退化是性别分化过程中的关键步骤，然而其退化机制目前尚未明确。本项目拟在掌握山鸡椒花发育外部形态特征、明确雄花中雌蕊退化时期的基础上，观察雌花发育解剖结构特征，阐明山鸡椒花器官退化的细胞学分界点；分析花器官退化关键时期内源激素变化规律，并研究外施激素对性别分化的影响，探讨利用外施激素调控山鸡椒性别的可行性；同时对花器官退化关键时期的雌雄花进行转录组测序，比较分析不同时期差异基因表达情况，挖掘山鸡椒花器官退化相关基因，借助组织原位杂交技术检测这些基因的时空表达特征；利用权重基因共表达网络分析法进行中心基因分析，构建花器官退化相关基因共表达网络图，并综合分析激素响应与退化相关基因之间的关系，最终阐述山鸡椒花器官退化分子机制，这将为通过利用性别鉴定与调控技术优化山鸡椒雌雄株性别比例、并最终提高果实产量奠定理论基础。

山鸡椒（Litsea cubeba）是我国重要的天然香料树种，精油中主要成分柠檬醛是制造香料、日化用品、药品、食品增香剂与防腐剂的原材料。山鸡椒是雌雄异株植物，花器官退化是性别分化过程中的关键步骤，然而其退化机制目前尚未明确。本项目分析了山鸡椒雌雄花发育过程形态特征，挖掘雌花中雄蕊退化的候选调控基因并分析其功能。主要研究结果如下：（1）通过形态发育学观察和解剖结构特征观察，明确了山鸡椒花发育形态学特征及雌花性别分化关键时期——雌花中雄蕊的退化早于小孢子母细胞形成。（2）分析了雌花中退化雄蕊发育中内源激素水平，结果显示相对于其他内源激素，水杨酸在雄蕊退化时期含量达到峰值且在性别间及不同发育时期中差异显著。（3）通过对山鸡椒雌雄花花器官退化关键时期转录组测序，获得了山鸡椒雌花中雄蕊退化过程中差异基因类型及表达变化规律，并筛选到山鸡椒花器官退化候选调控基因--LcTGA10，实时定量PCR和原位杂交结果显示LcTGA10在退化雄蕊组织中特异高表达。（4）进一步利用转基因技术鉴定了LcTGA10调控雄蕊退化的功能，LcTGA10过表达拟南芥（T3代）各株系高度略低，花序和萼片较小；雄蕊数量减少，长度短，花药中有花粉产生，但花粉活性极低；柱头具有可授性；人工杂交过表达拟南芥株系雄蕊花粉育性降低，雌蕊正常可授。（5）山鸡椒转录组共表达网络分析表明，PALs、TGAs和NPRs等SA生物合成和信号转导调控关键基因与雄蕊发育相关的基因如SPL8等密切相关；CMTAs和WRKYs基因在SA的生物合成和信号传导途径中具有的连接性。对野生型拟南芥株系外施SA后，SA下游响应因子AtNPR1、AtTGA9和AtTGA10相对于WT对照组表达量均上调，说明SA可以诱导促进下游信号基因AtNPR1、AtTGA9和AtTGA10的表达。综上所述，LcTGA10可能通过SA途径调控山鸡椒退化雄蕊形成。本项目为深入揭示山鸡椒性别分化机理提供基础，并有助于利用性别鉴定与调控技术优化山鸡椒雌雄株性别比例、并最终提高果实产量。因此，项目的研究成果不仅具有重要理论意义，也具有十分重要的应用价值。