百合珠芽形成过程中的蔗糖卸载机制研究

Bulbil is an important asexual reproductive organ for Lilium species. Bulbil propagation can not only retain the characteristics of parent plant, but also be more efficient, making it a promising production. The partitioning and metabolism of sucrose are closely related to the formation of axillary bulbils in our previous study, but the relevant mechanism is unclear. In this study, we are going to use Lilium lancifolium as material. The cellular pathway of sucrose unloading during the bulbil formation will be elucidated by using a combination of TEM observation and CFDA fluorescent tracer technique. The key enzyme of sucrose unloading is determined by analyzing a combination of the activity and gene expression of sucrose unloading related enzymes (SUSY and INVs) and the changes of content of sucrose, glucose and fructose during the bulbil formation. Furthermore, subcellular localization of the key enzyme is explored with immune colloidal gold technique and western blotting, elucidating the enzymatic mechanism of sucrose unloading. In addition, the expression of genes encoding sugar transport proteins are analyzing through qRT-PCR, exploring the mechanisms of sucrose unloading at the expression level. Through the above research, we try to clarify the mechanism of sucrose unloading during the axillary bulbil formation, to provide the theoretical foundation for making a comprehensive analysis of the bulbil formation in lilies and the further application to reproduction through artificial regulating.

珠芽为百合属植物重要的无性繁殖器官，不仅能较好地保留母体的遗传特征，且繁殖效率高，因而，在生产上有很重要的应用前景。前期研究发现蔗糖的分配和代谢与百合腋生珠芽的形成密切相关，但相关机制不清楚。本申请拟在前期工作基础上，以卷丹（Lilium lancifolium）为试材，利用透射电子显微镜及CFDA荧光示踪观察明确珠芽形成过程蔗糖卸载的细胞学路径；通过蔗糖卸载相关酶SUSY和INVs的活性及其基因表达分析，结合蔗糖、葡萄糖和果糖含量变化，确定珠芽形成中蔗糖卸载的关键酶，进一步通过胶体金免疫定位和Western印迹技术研究该酶在“库”组织的亚细胞定位，阐明蔗糖卸载的酶学机制；通过qRT-PCR分析糖转运蛋白在珠芽形成中的表达情况，从表达水平探讨蔗糖卸载机制。项目的实施，可望阐明百合腋生珠芽形成过程中的蔗糖卸载机制，对全面解析百合珠芽形成机理和进一步人工调控百合珠芽形成应用于繁殖提供理论依据。

珠芽为百合属植物重要的无性繁殖器官，不仅能较好地保留母体的遗传特征，且繁殖效率高，因而，在生产上有很重要的应用前景。前期研究发现蔗糖的分配和代谢与百合腋生珠芽的形成密切相关，但相关机制不清楚。本项目基于形态和解剖学观察，将卷丹珠芽的形成分为三个阶段：珠芽原基建立准备阶段（0-4d）；珠芽原基建立阶段（6-8d）和珠芽形成阶段（10-12d）。利用透射电子显微镜在百合珠芽原基建立过程中观察到胞间连丝。CFDA荧光示踪显示在珠芽原基建立准备阶段由韧皮部扩散至周围薄壁细胞的信号较强，后期减弱。通过蔗糖卸载相关酶SUSY和INVs的活性及其基因表达分析，结合蔗糖、葡萄糖和果糖含量变化，确定了珠芽形成中蔗糖卸载的关键酶为蔗糖合酶SUS2，克隆其全长序列为2544bp。制备SUS2的多克隆抗体，通过Western印迹发现该酶的表达量在珠芽形成时显著升高。qRT-PCR分析糖转运蛋白在珠芽形成中的表达，结果发现SUT和STP在珠芽形成后期上调表达。综合上述研究结果，得出在百合珠芽形成前期，蔗糖卸载以共质体途径为主，而珠芽形成后期则以质外体途径为主。百合腋生珠芽形成过程中发生了共质体向质外体的转变。项目的实施阐明了百合腋生珠芽形成过程中的蔗糖卸载机制，为全面解析百合珠芽形成机理和进一步人工调控百合珠芽形成应用于繁殖提供了理论依据。发表SCI论文4篇，授权国家发明专利3项。