微绿球藻生长相变分子机制研究

Nannochloropsis is a microalgal genus containing about 6 species. These microalgae are rich in protein and fatty acids including long chain polyunsaturated fatty acids. Diverse tools and abundant data (e.g., EST and genome sequence) are available for their genetic manipulation and biological studies. These algae have been widely used in aquaculture and food production. Recently, they show a promise for biodiesel production. They are evolving as industrial microalgal models. Cloning of their genes involved in for example the biosynthesis of fatty acids and their genetic transformations are intensive; while description of their biological characteristics associated with their breeding, stable culture and basic research is relatively scarce. Until recently, only two biological characteristics, nuclear monoploidy and asexual reproduction, have been understood, which support their breeding through mutation and elite clone screening. Nannochloropsis are able to survive among extremely different environments (phase variation); but the mechanism of phase variation is not clear. Nannochloropsis contain very rich microsatellite DNA; while the contraction and extension of microsatellite DNA are able to change the plasticity of genome, remodeling gene expression accordingly. Combining these two aspects, we hypothesized that microsatellite DNA determined the phase variation of Nannochloropsis by either contracting or extending and accordingly re-patterning gene expression. We propose to identify those microsatellite DNA varying between fresh water and seawater growth phases and between autotrophic and heterotrophic growth phases and associating genes and metabolism pathways, revealing the molecular mechanism of the phase variation of Nannochloropsis finally. Our findings will provide bases for the stable culture and physio-biochemical researches of Nannochloropsis.

微绿球藻富含蛋白质和油脂，遗传操作工具齐备，基础数据丰富，已用于水产养殖，并有望用于生物柴油生产，逐步成为模式工业微藻。微绿球藻脂肪酸合成关键酶克隆和遗传转化研究较多；但与其良种培育和稳定养殖相关的生物学特征研究较少。目前已阐明微绿球藻细胞核单倍性和无性繁殖方式，为其诱变和克隆化育种提供了依据。微绿球藻能快速适应环境剧烈变化、转变生长状态（生长相变），但相变分子机制不清楚。微绿球藻基因组存在极其丰富的微卫星DNA，而微卫星DNA高效延伸/收缩突变可重塑基因组，改变基因表达模式。课题将两者联系起来，假设微卫星DNA延伸/收缩突变可改变微绿球藻基因表达模式，是其生长相变的分子机制。课题拟确定微绿球藻海水向淡水、自养向异养生长相变前后微卫星DNA延伸/收缩情况，甄别出微卫星DNA延伸/收缩突变及其影响的基因和代谢途径，阐明微绿球藻生长相变分子机制，为其稳定养殖提供依据。

高等植物(尤其是农作物)的抗盐和耐盐性是重要性状，广受关注。同时，具有广盐性生长特性的微藻普遍存在，众多微藻的盐度适应性也较易驯化。因此，具有海水和淡水两种生长状态(或生长相)的微藻是解析植物抗盐和耐盐性生理遗传基础研究的简单而适用的模型。无性繁殖且细胞核单倍性的海洋微拟球藻(Nannochloropsis oceanica)富含蛋白质和油脂，遗传转化工具齐备，基础数据丰富，广泛用于水产生物饵料生产和生物能源研究，已逐步演变成一种模式微藻。本基金课题研究通过人工进化海洋微拟球藻，获得了完全适应淡水生长的海洋微拟球藻藻株，建立了用海洋微拟球藻进行植物抗盐和耐盐性研究的模型。海水和淡水两种生长相的微拟球藻的转录组比较分析发现为适应淡水生长，海洋微拟球藻降解氨基酸、脂肪酸等以合成大量ATP用于离子转运，维持细胞内外钾离子和钙离子稳态；同时降低蛋白质转运与合成，特别是减少核糖体蛋白的合成和核糖体的生成，以维持细胞缓慢生长。这些发现揭示了海洋微拟球藻的海水和淡水生长相变的生理机制。本基金课题研究还建立了海洋微拟球藻的博莱霉素(Zeocin)稳定诱变、转基因、重测序SNP基因型分型和indel基因型分型等技术，并通过实时单分子测序和Hi-C测序完善了海洋微拟球藻基因组参考序列的组装，为创制海洋微拟球藻遗传变异，经混合突变子分析(bulked mutants analysis)、全基因组关联分析(GWAS)等解析海洋微拟球藻的重要性状的遗传基础打下了方法学和数据基础。