雨生红球藻向光素介导的虾青素合成信号转导通路的互作蛋白研究

虾青素是一种具有极强抗氧化性和广泛应用价值的次生类胡萝卜素。雨生红球藻是理想的天然虾青素生产工具，但虾青素的合成总是发生在不适合生物量积累的逆境胁迫条件下，例如蓝光。申请者前期研究发现蓝光受体向光素在雨生红球藻调控虾青素合成过程中发挥重要的作用，并克隆获得向光素（PHOT）基因序列，但PHOT如何介导虾青素合成途径关键限速酶的转录？PHOT有哪些互作蛋白？下游通路中有哪些转录因子参与虾靑素合成基因的表达调控？互作蛋白如何与转录因子进行相互作用？转录因子如何与顺式作用元件相关作用？这正是本项目拟解决的关键科学问题。本项目拟通过以PHOT为切入点，利用免疫共沉淀和酵母双杂交技术筛选互作蛋白并进行功能验证；以虾青素合成相关顺式作用元件为突破口，利用酵母单杂交技术和凝胶阻滞试验筛选转录因子，并进行活性检测，从而构建“蓝光信号-PHOT-互作蛋白-转录因子-顺式作用元件-虾青素合成”信号转导通路。

Astaxanthin, a type of high-value ketocarotenoids, is of great commercial interest for its high anti-oxidant activity with a variety of usages. The unicellular green alga Haematococcus pluvialis accumulates astaxanthin, with a level reaching up to 4% dry weight under environmental stress conditions (e.g., high blue light). In our previous study, we found phototropin(PHOT) played an important role in regulating astaxanthin biosynthesis in Haematococcus pluvialis, and we have cloned a novel homologue gene encoding PHOT. But how the blue light signal to be sensed and how this signal initiates key enzyme functions in astaxanthin biosynthesis pathway via PHOT remain still unclear. The interacting proteins with PHOT are also unknown. None of the transcription factors involved in regulating astaxanthin biosynthesis is known. The interactions between interacting proteins and transcript factors become an very important question. For these above questions, this project is going to carry out functional characterizations of PHOT from H. pluvialis. By using yeast two-hybrid system and immune co-precipitation method, the interacting proteins with PHOT will be characterized. Meanwhile gel retardation experiments and bioinformatics were also applied to verify the interactions between interacting proteins and cis-acting elements. Based on the transcription analysis, the signal transduction pathway “Blue light-PHOT- interacting proteins-transcription factor- cis-acting elements- astaxanthin biosynthesis” would be constructed, which may facilitate the sophisticated understanding of the astaxanthin biosynthesis in Haematococcus pluvialis.