锥栗花性别分化激素调控机理研究

Castanea henryi is an important woody grain tree species in China. However, the imbalance proportion of male and female flowers restricted the development of the industry. So it’s an urgency to understand the mechanism of sex differentiation of C. henryi, and regulate the ratio of female flower to male flowers to increase the production. The preliminary study was found that flower sex expression in Castanea henryi could be regulated, besides, hormone played an important role in sex expression procedure. However, there are some problems still not clear. What are the major hormones associated with floral sex differentiation? How these key genes of hormones biosynthesis and signal transduction affect the sex differentiation? In this study, the mechanism of sex expression in C. henryi would be investigated from cytology, physiology (hormone) and molecular biology aspects, by using tissue dissection techniques, HPLC-MSn analysis technology, immune electron microscopy, fluorescence quantitative PCR technology, transcriptomics, bioinformatics analysis and so on. Firstly, we plan to explore the dynamic variation rules and the distribution of endogenous hormone in male and female flower during C. henryi differentiation, and figure out the major hormone which effects sex differentiation in C. henryi. Secondly, we plan to identify the key gene which plays a key role in the process of sex differentiation of C. henryi. Lastly, we intend to study how the key hormone to regulate the key gene expression of C. henryi flower organ formation and development. We want to elaborate the regulation mechanism of hormone on sex differentiation of C. henryi. It will be significant to explain the mechanism of sexual differentiation and to facilitate the production application in C. henryi.

我国重要的木本粮食树种锥栗存在雄花量过大、雌花量不足、单产低的现象，如何调节雌雄比例以提高产量是生产中急需解决的重要问题。项目组初步研究发现锥栗花性别分化具有可塑性，激素对栗花性别分化起着重要作用。然而它们调控花性别分化的机制却尚不清楚。本项目以发现的锥栗特异种质为试材，应用组织解剖技术、HPLC-MSn分析、荧光定量PCR技术、转录组学和生物信息学等研究手段，从细胞水平、激素代谢和分子水平上，探究栗花在性别分化前后内源激素的分布及消长动态变化，确定影响雌、雄花性别分化的主要激素；对影响雌雄花分化的激素生物合成的基因进行鉴别和表达分析，确定其关键基因；研究影响雌雄花分化的关键激素对锥栗花器官建成、发育关键基因表达的调控，阐明激素对栗花性别分化的调控机理。研究结果具有重要的理论和实践价值。

项目针对我国锥栗生产中存在的雄花量过大、雌花量不足、单产低等亟需解决的关键问题，为探明锥栗花性别分化激素调控机制，以锥栗特异种质‘华栗2号’为试材，采用组织解剖显微技术、HPLC-MSn分析技术、转录组学、生物信息学、荧光定量PCR技术和转基因技术等研究手段，从细胞水平、激素代谢和分子水平，分析了锥栗雌花形成过程中形态特征、内源激素含量动态变化、激素合成及信号途径相关基因表达，并将这些特征与规律进行关联分析，阐明激素对栗花性别分化的调控机理。主要结果：（1）解析了锥栗花性别分化形态学过程，确定了锥栗花性别分化具有可塑性，明确了雄蕊原基到雌蕊原基分化期为调控锥栗花性别分化的关键时期；（2）建立了锥栗花性别分化过程中激素的变化模式，确定了影响锥栗花性别分化的主要激素为细胞分裂素和赤霉素，其中细胞分裂素可以促进锥栗雌花的形成，赤霉素促进锥栗雄花的形成；（3）制定了锥栗促雌增产的人工调控技术：于芽萌动期、芽萌动后第1，2周均喷施350 mg/L 6-BA可诱导混合花序产生3~6朵雌花，于花芽萌发后的第1~3周各喷施100 mg/L CPPU可诱导雄花序转变为全雌花序；（4）外源细胞分裂素和赤霉素处理通过改变锥栗内源ZT、GA3、ABA含量和（GA3+ZT）/（IAA+ABA）比值来参与调控锥栗性别分化过程；（5）通过转录组学及WGCNA分析鉴定了12个细胞分裂素代谢基因、4个花发育基因和3个转录因子是调控锥栗雌花形成的关键候选基因；（6）阐明了细胞分裂素调控锥栗花性别分化机制：外源细胞分裂素处理后，调控了内源细胞分裂素合成途径相关基因（ChCKX3、ChCKX5和CYP735A）的表达，从而降低了内源激素的含量，而信号传导途径基因（ChHK4、ChHK5、ChRR5、ChRR7、ChRR9、ChRR11和ChRR12）上调表达改变了细胞分裂素靶基因的转录水平，最终促使了花器官发育相关基因（SEP1、AGL11、SPL6和SPL16）的显著性差异表达，从而诱导了锥栗雌蕊原基的分化、发育，使得锥栗雄花转变为了雌花。结论：细胞分裂素促进锥栗雌花分化，采取适宜的人工调控措施可以增加锥栗雌雄花比，大幅度提高产量。