环境因子调控异叶水蓑衣异形叶形态建成的机制研究

Plants show morphological differences in leaf form in response to changes in the surrounding environment, a phenomenon called heterophylly. Heterophylly provides an ideal process to understand mechanisms by which plants adapt their growth to withstand ecological changes. We found that the aquatic plant Hygrophila difformis is an ideal model for heterophylly study due to its obvious leaf features, sensitivity to environment, and convenience in culture, propagation and gene transformation.Further studies show that expression of KNOTTED1-LIKE HOMEOBOX (KNOX1) orthologs, the genes that regulate leaf morphogenesis and development, are correlated with environmental changes as well as with different leaf forms. Gibberellin (GA) and cytokinin (CK) signaling, regulated by KNOX1, is also correlated with leaf forms. We propose a mechnism that environmental factors regulate KNOX1 orthologs expression thereby modulating gibberellin (GA) and cytokinin (CK) content, and determining leaf form. We aim to elucidate this mechanism through this study. We will use Hygrophila difformis to investigate variations in leaf form in response to environmental factors. We will analyse endogenous hormone levels, and assess the regulationary relationships through qRT-PCR, in situ hybridization, reporter gene assays and EMSA to reveal the regulatory mechanisms of environment, KNOX1, GA and CK biosynthesis. We will also silence and over-express KNOX1 genes, apply exogenous hormones and inhibitors in plants to confirm the role of the KNOX1 genes in altering GA and CK biosynthesis and leaf form.

植物叶形受环境调控产生显著差异的现象叫“异形叶”，它是研究植物生态适应性的理想模型。我们前期研究发现异叶水蓑衣叶形对环境敏感，培养、扩繁和转化简易，是异形叶研究的理想材料。同时发现调控叶形的基因-KNOTTED1-LIKE HOMEOBOX （KNOX1）易受环境调控，其表达与叶形显著相关，受KNOX1基因调控的赤霉素（GA）和细胞分裂素（CK）也与叶形显著相关，可能存在一个“环境调控KONX1的表达，进而调控GA和CK含量并最终决定叶形”的机制。为验证这一机制，本项目拟以异叶水蓑衣为材料，在研究环境因素调控叶形规律基础上，通过内源激素检测、qRT-PCR、原位杂交、报告基因检测和凝胶迁移实验，研究环境变化对KNOX1、GA和CK生物合成的调控关系，还将通过过量表达和敲除KNOX1、施加外源激素和抑制剂研究KNOX1基因对GA和CK生物合成以及叶形的调控作用，以期揭示异形叶发生的机制。

“异形叶”是指基因型相同的植物由于环境变化导致叶形显著差异的现象。异形叶植物的水生叶常为深裂、丝状或线形，而陆生叶的叶形则相对简单。由于缺乏合适的模式植物，其分子机制研究进展缓慢，异形叶调控机制目前仍不清楚。本项目以前期筛选的水生植物异叶水蓑衣（水罗兰）为材料，基于已建立的稳定遗传转化体系，系统研究了KNOTTED1-LIKE HOMEOBOX （KNOX1）基因家族在异叶水蓑衣叶形发育中的功能。本项目首先研究了主要环境因子（陆生/沉水、相对湿度、温度和光照强度）对异叶水蓑衣叶形的调控研究，建立起了一套成熟的异叶水蓑衣叶形与环境因子对应关系的研究体系。随后，对异叶水蓑衣的稳定遗传转化体系进行了进一步的优化，并完成了植物激素对异叶水蓑衣叶形的调控研究。进而我们克隆了KNOX1 基因家族的两个成员HdSTM 和 HdBP基因，研究了HdSTM基因对下游赤霉素（GA）和细胞分裂素（CK）代谢相关基因调控在不同物种中的保守性，分析了HdSTM调控异叶水蓑衣异形叶发育的分子机制。本项目通过一系列的生理、生化和遗传学证据证明了KNOX1基因在异叶水蓑衣的异形叶调控中的重要作用，对后续的异形叶研究提供了重要参考。