雨生红球藻光合碳代谢和虾青素积累相关基因的克隆和表达研究

Haematococcus pluvialis (Chlorophyta) is an excellent material to accumulate natural astaxanthin, which is a kind of natural antioxidant, so the alga has become one of the most and potential microalgae at present. The current problem is to improve the algal growth rate and astaxanthin content. This project will focus on the above problems: (1) Three carbon metabolism key enzymes of Rubisco, Rubisco activase and carbonic anhydrase in H. pluvialis will be cloned, and their transcription variation under different conditions will be elucidated. Thus, the correlation of three enzymes and algal growth will be compared on the protein and gene levels. (2) The transcriptome will be performed in H. pluvialis by high throughput sequencing technology. The expression profiles of related genes between biomass and astaxanthin accumulation periods will be elucidated by combining with transcriptome and RT-PCR analysis. The genetic transformation system in Haematococcus pluvialis will be constructed, and the function investigation of key genes will performed. (3) The difference of transcriptome will be investigated under different environmental stress, and 10-20 astaxanthin synthesis-related genes will be studied in H. astaxanthin. Through combining with the gene annotation information and KEGG metabolism analysis, the astaxanthin synthetic metabolic networks in H. pluvialis will be constructed, and the preliminarily mechanism of the key genes for astaxanthin accumulation response to outside stimuli will be clarified.

雨生红球藻是生产“天然抗氧化剂”—虾青素的优质原料，是目前研究得最多、最有开发潜力的微藻之一。利用雨生红球藻生产虾青素的关键问题是提高藻的生长速度和虾青素含量。针对以上问题，本项目拟研究：（1）雨生红球藻碳代谢关键酶—Rubisco、Rubisco活化酶和碳酸酐酶的基因克隆及其在不同环境条件下的转录表达规律。从蛋白和基因水平上比较3种酶与藻生长的相关性。（2）利用高通量测序技术对雨生红球藻进行生物量积累期和虾青素积累期转录谱的差异比较，结合RT-PCR技术分析雨生红球藻生长和虾青素积累相关基因的表达特征。建立雨生红球藻遗传转化体系,对目标基因进行功能研究。（3）研究不同环境胁迫条件下雨生红球藻的转录谱差异。重点研究藻生长和虾青素合成相关10-20个基因，结合基因注释信息和KEGG代谢分析，构建雨生红球藻中虾青素合成代谢网络，初步阐明虾青素积累关键基因对外界刺激的响应机制。

雨生红球藻是强抗氧化剂虾青素的天然优质来源，对该藻进行生长和虾青素积累的机理和相关技术研究具有重要意义。围绕着雨生红球藻生长和虾青素积累这两个方面，本项目开展了以下工作：（1）雨生红球藻碳代谢酶和虾青素合成相关基因的研究：高CO2（0.16% CO2）可以促进绿色营养阶段雨生红球藻生物量积累、Rubisco活力、PEPC和MDH活力及其编码基因的转录表达，降低碳酸酐酶活性；高CO2或乙酸钠（0.023 mM）可提高红色孢子阶段雨生红球藻虾青素含量及虾青素合成相关基因—pds、chy和bkt的表达；玉米素可以缓解红色孢子阶段高光对rbcL、rca、ca的下调表达；而水杨酸则促进了虾青素积累相关基因ipi的表达。（2）雨生红球藻基因组测序：共得到1.05×108条clean reads，估算其基因组大小约163.68 Mb，测序深度为92.7倍。组装后共得到49,830条scaffolds，在数据库中比对并注释了35,735条基因。（3）雨生红球藻转录组的工作：转录谱分析表明在绿色营养阶段，高CO2可以促进雨生红球藻光合作用、三羧酸循环、糖酵解、磷酸戊糖途径等相关基因的上调表达；乙酸钠可以上调其三羧酸循环、一碳代谢、硫的吸收和转化、核糖体的合成等途径。在红色孢子阶段，高光胁迫刺激了雨生红球藻虾青素合成途径的关键基因表达，同时抑制了其它分支途径的基因表达；高光胁迫添加碘化钾后其差异基因主要富集在胞吞作用、淀粉和蔗糖代谢、MAPK信号通路、脂肪酸生物合成等代谢途径。（4）雨生红球藻代谢组学结果：高CO2培养雨生红球藻中植物次级代谢物合成、碳代谢、氨基酸代谢等通路显著富集；高光胁迫下棉子糖代谢、谷胱甘肽代谢、嘧啶和嘌呤代谢及磷脂等代谢通路显著变化。综上，补充碳源（CO2或乙酸钠）、高光以及碘化钾或植物激素均可以促进雨生红球藻生长和虾青素的积累，该工作为雨生红球藻的生产实践以及虾青素积累机理研究提供了资料。