草果种子休眠解除关键蛋白的挖掘及基因功能研究

Seed dormancy is considered to be one of the most complicated phenomena in seed biology. Although extensive research has been carried out and achieved fruitful results，there are still many problems to be solved because of the diversity of species and dormancy characteristics. Amomum tsaoko is a kind of commonly used medicinal plants and spice crop, there was obvious dormancy phenomenon in freshly collected A. tsaoko seeds and it is an important limiting factor for its scale cultivation. In our previous study, we found that the seed of A. tsaoko belong to physiological dormancy, while the seed dormancy could be broken after 90 days of stratification. By transcriptionl and RNA-seq sequencing, we found a large number of differentially expressed genes increased during the period of seed dormancy release, and endogenous hormone related gene played an important role in regulating seed dormancy release, implying that the balance of endogenous hormones is an important switch for the transformation of seed germination. But the role of endogenous hormone balance in seed germination transformation and its regulation network, and also the biological function of key genes and key proteins remains unknown. To illustrate the regulation mechanism of endogenous hormones and key genes/proteins in seed dormancy and germination of A. tsaoko, this project intends to carry out the following research: ①Interpreting the regulation of structure and physiological changes in the process of seed dormancy release, through the microstructure observation and determination of endogenous hormone content; ②Excavating the key proteins involved in the regulation of seed dormancy release by using iTRAQ proteomics and targeted protein quantification technology. Screening key genes involved in hormone regulation, by the conjoint analysis of proteome and transcriptome. ③Cloning key genes and verifying the function of key genes by transforming gene to Arabidopsis thaliana. For the purpose to comprehensive interpretation the regulation mechanism of seed dormancy and germination of A. tsaoko in physiological and molecular level, and provide the theory basis for improving crop quality through the molecular breeding.

草果种子具有显著的休眠现象，是其规模化栽培的重要限制因子。我们前期研究发现，草果种子属于生理休眠类型，层积处理能够有效破除种子休眠。通过转录组测序和RNA-Seq测序，发现种子休眠解除过程中伴随着大量差异基因的表达，其中内源激素相关基因发挥重要的调控作用，推测内源激素平衡是种子萌发转变的重要开关。为全面阐释草果种子休眠与萌发的调控机制，本项目拟通过显微/亚显微结构观察和内源激素含量测定，掌握种子休眠解除过程中的结构及生理变化规律。利用iTRAQ蛋白质组学技术和靶向蛋白质定量技术，挖掘参与种子休眠解除的关键蛋白；并将蛋白质组与已有的草果种子转录组进行联合分析，筛选出参与激素调控的关键基因。用RACE法进行基因克隆，构建植物表达载体，转化到拟南芥，进一步验证关键基因的功能。以期从生理和分子水平上全面阐释草果种子休眠与萌发的调控机制，为后续进行遗传改良，提高作物播种品质提供理论依据。

草果种子具有显著的休眠现象，是其规模化栽培的重要限制因子。我们前期研究发现，草果种子属于生理休眠类型，层积处理能够有效破除种子休眠。通过转录组测序和RNA-Seq测序，发现种子休眠解除过程中伴随着大量差异基因的表达，其中内源激素相关基因发挥重要的调控作用，推测内源激素平衡是种子萌发转变的重要开关。为全面阐释草果种子休眠与萌发的调控机制，本项目通过显微结构观察，明确脂类物质降解是草果种子休眠解除的重要特征。采用HPLC法测定种子内源激素含量，发现GA3、IAA、ABA等激素在草果种子休眠解除过程中发挥重要作用。通过蛋白质组学分析技术，挖掘出参与种子休眠解除调控的关键蛋白，它们主要涉及脂质代谢、淀粉和蔗糖代谢、氨基酸合成、激素合成与信号转导。转录组和蛋白质组联合分析结果显示，多个基因和蛋白的表达存在相关性，激素信号转导、温度响应是草果种子休眠解除的重要分子过程。克隆了种子休眠相关基因DRM1和DRM2，以及代谢相关基因SAM1。通过转基因技术证实DRM1和DRM2正向调控种子休眠，其过表达有利于提高植物对生长环境温度的适应性。发现SAM1具有提高种子萌发整齐度的作用，并且ABA合成限速酶NCED被进一步证实具有正向调控种子休眠的作用。本项目从生理和分子水平上阐释了草果种子休眠与萌发的调控机制。