大豆生物钟基因GmCCA1/LHYs耐高pH盐碱胁迫的功能研究

有效利用盐碱滩涂等荒地增加种植面积是提高大豆产量的重要途径。生物钟是植物生命机体的调控中枢，通过整合环境信号、调整资源分配，以平衡生长和抗逆性。CCA1/LHY基因是植物生物钟核心元件，申请者前期研究发现大豆GmCCA1/LHYs基因特异性响应高pH盐碱胁迫，显示其在大豆耐高pH盐碱胁迫中起到重要作用，然而分子机制尚不清楚。因此，本研究将利用基因编辑技术同时敲除四个GmCCA1/LHY基因以确定其功能；并采用免疫共沉淀与质谱法鉴定与其互作的蛋白；同时利用转录组测序及染色质免疫共沉淀技术，鉴定共同参与其耐盐碱胁迫的信号分子，分析可能参与GmCCA1/LHYs耐高pH盐碱胁迫的信号通路；从而深入解析其协同大豆生长和抵抗高pH盐碱胁迫的分子机制；挖掘优良等位变异种质资源，用于耐盐碱大豆分子育种。本项目研究成果不但可以加深对大豆盐碱胁迫分子机制的理解，而且能够为大豆分子育种提供理论基础和育种资源。

Soybean [Glycine max(L.) Merrill] is the primary source of protein and vegetable oil supply for residents in China, and it is mainly dependent on import. It is an important way to increase the planting area and yield of soybean by effectively utilizing the wasteland of salt and alkali marsh. The circadian clock is a central hub in the sophisticated regulatory network in plants, which coordinates the growth and resistance of plants by integrating different environmental signals and adjusting the distribution of resources. CCA1/LHY gene is the core element of plant circadian clock. Previous studies have found that soybean GmCCA1/LHYs gene specifically responds to high-pH alkali stress rather than other adversity stresses, suggesting that it plays an important role in soybean high-pH alkali tolerance; however the molecular mechanism is not clear. In this study, firstly, using the CRISPR-Cas9 gene editing technology the four GmCCA1/LHY genes will be simultaneously disrupted to determine their function. Moreover, immunoprecipitation and ultrahigh resolution Mass spectrometry will be conducted to identify the interacting proteins and determine the signaling pathways involved in the GmCCA1/LHYs mediated high-pH alkali tolerance. In addition, the transcriptome sequencing and chromatin immunoprecipitation combined with the next generation sequencing will be performed to determine the signaling molecules involved in the GmCCA1/LHYs high-pH alkali tolerance and DNA segment associated with GmCCA1/LHYs. Eventually the outcome will greatly facilitate the elucidation of molecular mechanisms of underlying GmCCA1/LHYs in coordinate growth and high-pH alkali tolerance. This study will provide an important theoretical basis for the cultivation of new salt-alkaline tolerant soybean varieties.