红球藻虾青素几何异构与成因分析

It’s well know that a material functions are determined by its basic molecular structure. Astaxanthin, the most important and economic proporty, can be largely accumulated in aquatic unicellular microalga Haematococcus pluvialis. Studies of astaxanthin accumulation in H. pluvialis will be extended in this applied project from the ordinary molecular level to its sub-molecular structures (geometrical isomers in paticular). Both their common and differential characteristic spectrum of cis/trans geomatrical isomers in H. pluvialis will be extensively compared by using the main global wild strains and their UV-chemical mutants. Using the obtained data of cis/trans geomatrical isomers in H. pluvialis and their original geographic information, the strains’ capacities of accumulating different geomatrical isomers, and their possible phylogenetic evolutionary relationship in H. pluvialis will be further analyzed. Then, some possible alternative changes and the annual variation of cis/trans isomers of astaxanthin during the cell culture and astaxanthin accumulation will be studied in detail from different levels and angles. Many internal and external factors possibly involving in the geomatrial isomerization such as algal species, isomerases, transcriptomics, metabolomics, N/P nutrients, photoperiod (continuous light, light / dark rhythm), light intensity (constant and gradually up/down), light quality, temperature (thermostatic, temperature changes) will be screened out individually by number of experiments. The experiments will be carried out by outdoor mass-scale cultivation with testing at least year-round actual samples, and indoor mimic culture certification by using computor controlled photobioreactor. Based on above studies, the crucial reasons and possible mechanism causing astaxanthin isomerization in H. pluvialis are expected to be determined. Meanwhile, some favourable strains of H. pluvialis with high capacity of producing astaxanthin and certain isomers with specific functions as well as the optimized culture modes will be screened. A theoretical guide for commercial utilization of this microalgal astaxanthin resource, and the scientific standards for judging astxanthin quality and source will be concluded too.

基于物质结构决定着其功能，本申请针对红球藻这一水产微藻的重要经济性状虾青素，拟将其研究从分子水平全面拓展到亚分子几何结构层面。首先，借助已获得的国内外该藻种质资源，全面了解国际上该藻虾青素顺/反几何异构的共性特征和差异谱，分析其亲缘演化关系，了解藻株累积虾青素异构体的能力。.其次，从藻种、异构化酶、转录组学、代谢组、氮磷营养、光照[光周期（连续光照和光/暗节律光照）、光强（恒定与逐渐升降模式）、光质]、以及温度（恒温、变温）等多个层面和不同视角，借助户外规模培养平台实际样品检测与人工控制模拟验证，探讨红球藻虾青素累积过程中几何异构体的交替升降与年际变化规律，分析导致红球藻虾青素发生异构化的关键成因，并认知其异构化波动的可能机理。.本基础研究也为有效开发该资源、定向筛选藻株和特定异构体、建立优化培养模式提供基础理论指导，也为该藻虾青素标准制定、甚至商贸质检和甄别产品来源等提供科学的参考依据。

围绕立项任务在红球藻虾青素分子和亚分子层面开展系统性研究，具体回答了导致虾青素几何异构的主要因素和机制的科学问题，阐明全球红球藻虾青素几何异构体共性特征和差异谱，掌握其累积能力和变化规律，并结合酶学和转录组学信息，探讨导致该藻虾青素各几何异构体的累积变化的成因与机理，达到并超过完成立项目标，并为该资源开发提供了理论支撑，圆满完成项目提出的以下考核指标：. 针对不同培养光温条件下、不同来源的红球藻藻株、不同培养装置和不同季节以及各个发育阶段的红球藻取样，通过模拟和规模化实验获得生理、生化、异构化酶、代谢物组学、酶学和转录组学等多层面的全视角证据和交互验证，全面系统地分析了虾青素及其异构体积累的变化规律，获得红球藻种质资源虾青素顺/反几何异构的共性特征和差异谱，阐明了导致产品虾青素几何异构变化的内因（trans-FPPS和cis-FPPS 、GGPS）和外因（高温和大温差）及其贡献力，检测到FPPS蛋白的一个可信肽段EIVDAVR序列的差异存在，并提出雨生红球藻虾青素异构体积累的路径。. 结合转录组学和代谢组学以及生理生化数据，发现并阐明呼吸作用（包括线粒体、叶绿体和光呼吸）对虾青素积累的作用及调节原理机制，挖掘出可有效提高红球藻虾青素的新途径，并获得减少光损伤、提高生物产量和虾青素含量的新方法。. 构建了从虾青素异构体精准分析方法，为本领域相关研究、产品分析和商贸质量检测提供科学支撑依据。. 项目已形成成果20项，包括发表国家基金标注的基础研究论文16篇（其中SCI论文15篇）、国外出版丛书1章、申请国家发明专利2项，起草并审议通过国家标准（征求意见稿）1项；同时，锻炼了虾青素异构体研究队伍，培养博后1名、博士2名和硕士5名研究生，均顺利出站或毕业并获得了学位；另外受本研究结果启发和基础铺垫，新申请并获得3项国家基金（青年、面上、联合各1项）资助。