雨生红球藻转录因子MYB1和HB1在虾青素合成中的调控机制研究

Haematococcus pluvialis is the ideal resource of natural astaxanthin. But its large-scale cultivation still faces low growth rate, low yield of astaxanthin, and other bottlenecks. To obtain the ideal algal strains for astaxanthin production, it is in urgent need of identifying effective regulatory factors of astaxanthin synthesis and clarify its regulation mechanism. In Haematococcus pluvialis, the astaxanthin biosynthesis is regulated at transcriptional level. Through RNA-seq, we have found two transcription factor: HpMYB1 and HpHB1 which strongly induced expressed under high-light and high-salt treatments. What is the biological functions of transcription factors HpMYB1 and HpHB1 in Haematococcus pluvialis? How dose HpMYB1 and HpHB1 regulate astaxanthin synthesis? By which molecular mechanism HpMYB1 and HpHB1 regulate astaxanthin synthesis? All the questions above will be answered after this project performed. The project prepares to use RACE to obtain complete gene of HpMYB1 and HpHB1. EMSA (Electrophoretic Mobility Shift Assay) will be employed to reveal the reaction between HpMYB1 or HpHB1 and the promoters of genes encoding key enzymes in astaxanthin biosynthesis in Haematococcus pluvialis. The yeast one-hybrid will be performed to determine the transcription activation of HpMYB1 and HpHB1. At the same time, we will analyze the biological functions of HpMYB1 and HpHB1 by over-expressing them in Haematococcus pluvialis and functional complementation in mutants. The study will not only set a scientific foundation for transcriptional regulation mechanism of astaxanthin biosynthesis, but also provide the theoretical basis for genetic breeding of fine algal species.

雨生红球藻是天然虾青素理想来源，其规模化养殖仍面临生长慢、虾青素得率低等瓶颈。为获得用于生产的理想藻株，亟需找到虾青素合成的有效调控手段并厘清其调节机制。雨生红球藻虾青素合成的调控能发生在转录水平。通过转录组测序，我们找到了受到强光和高盐强烈诱导表达的转录因子：HpMYB1和HpHB1。它们具有什么样的生物学功能，如何调控虾青素的合成？又通过何种分子机制来进行转录调控？这些都将在本项目中得到回答。本项目拟通过RACE获得基因全长，通过EMSA揭示转录因子与虾青素合成关键酶基因启动子的相互作用，采用酵母单杂交分析转录因子的转录激活活性，通过突变体功能互补或在雨生红球藻中过量表达有效基因，分析其生物学功能。研究结果不仅为阐明虾青素合成的转录调控机制奠定科学基础，还将为优良藻种选育提供理论依据。

雨生红球藻是虾青素天然的“生产工厂”，其规模化养殖仍面临生长慢、虾青素得率低等瓶颈。为获得用于生产的理想藻株，亟需找到虾青素合成的有效调控手段并厘清其调节机制。虾青素合成关键酶基因启动子中存在多个MYB和HB转录因子的结合位点，并且通过高通量测序我们找到了HpMYB1和HpHB1这两个受到强光和高盐强烈诱导表达的转录因子。通过本项目的实施，我们分析了HpMYB1和HpHB1基因在强光、高盐和缺氮下转录和翻译水平的变化，发现HpMYB1和HpHB1受到强光和缺氮胁迫的上调表达。高盐胁迫可以促进HpHB1表达，但不影响HpMYB1基因表达。强光处理8小时内，HpHB1和HpMYB1蛋白质都有一个先上调后下降的趋势。通过RACE法克隆了HpHB1和HpMYB1。亚细胞定位显示它们都位于细胞核中，与基因的转录相关。通过酵母杂交实验，找到了与HpMYB1结合的虾青素合成关键酶PDS，以及与HpHB1结合的胁迫相关蛋白质HpHSP90和HpPKTC。采用凝胶阻滞方法获得了与HpHB1结合的STRE顺式元件。通过本项目的实施，共发表论文6篇，申请专利4项，其中1项授权，培养博士后2名，博士生1名，硕士研究生4名。研究结果不仅为阐明虾青素合成的转录调控机制奠定科学基础，还将为优良藻种选育提供理论依据。