莱茵衣藻高细胞生长密度HCD基因的功能分析

Green microalgae are thought to be one of the sources for future biofuels, currently due to the low cell-growth density in photoautotrophic cultures, algae-based biofuel production is not competitive. Hence, increase of biomass density in phototrophic culture is essential for competitiveness of the algae-based biofuel production. Scientists have found that reducing the size of light-harvesting complex can increase the usage of light energy when Chlamydomonas reinhardtii grown under very highlight conditions, leading to high cell-growth density. However, large-scalecultivation system with very highlight intensity is not only costly, but also hard to maintain. To obtain strains that grow with high biomass density under the phototrophic growth condition of continuous light with moderate intensity, we performed forward genetic screening based on passages of a pool of thousands of insertional mutant strains and isolated at least 3 high cell-growth density HCD strains. Biomass production or dry cell weight per liter of the mutant strains is 100% higher than that of wild type; lipid accumulation induced by nitrogen starvation is not affected by mutations; and one of the mutant strains exhibits a wee-like small-sized cell phenotype, suggesting that reduction of cell division threshold volume can increase cell biomass productivity. This project aims to analyze energy metabolic activity and cell cycle regulation in mutant strains for identifying factors that coordinate metabolic activities and the cell cycleand for illumination of molecular mechanisms for increase of biomass by HCD genes in C. reinhardtii. Studies of algal-based energy often focus on the metabolic activities, given that metabolism is confined by cell cycle regulation, our study will discovernew approaches for increase of algal-based energy production.

绿色微藻被认为是未来的生物燃料来源之一，但由于目前光合自养细胞密度过低，微藻能源缺乏竞争力。所以，增加微藻自养生物量是提高微藻能源竞争力的必要条件。 科学家发现，莱茵衣藻的光吸收体变小可以在强光下增加光利用率从而提高生物量。但是，大规模强光养殖投入大，且维护成本高。为了在普通持续光照下提高自养生物量，我们通过连续传代培养含上千个插入突变子的混合衣藻溶液，分离出三株高细胞密度HCD突变体，它们的生物量比野生型高约40%，增加光照后的生物量比野生型高达100%，高细胞密度不影响氮饥饿诱导的油脂积累，其中一株呈类似WEE的小细胞表型，说明降低细胞分裂体积阈值有利于增加细胞生物量。本项目拟通过分析突变子的能量代谢活性和细胞周期调控，找出协调两者的调控因子，阐明HCD基因增加衣藻生物量的分子机制。微藻能源研究通常以提高能量代谢为主，但它受细胞周期调控，所以我们的研究将为提高微藻能源生产提供新的途径。

绿色微藻是潜在的生产生物能源的细胞工厂。细胞工厂藻株应该是具有高细胞生长密度、高油脂含量、高抵逆能力的。本项目以连续传代筛选出来的三株模式莱茵衣藻高细胞密度HCD插入突变子为对象，研究HCD的插入位点及其受影响的基因功能。一株HCD2插入编码区，RNA干扰沉默获得表型拷贝。不过，另两株HCD1和HCD3插入位点在非编码区，比邻基因的干扰或过表达都没能拷贝表型，失去了进一步分析的依据。为此，我们新添了研究富油微拟球藻在缺氮磷应激过程中油脂的代谢流向和积累位点及其分析无壁杜氏盐藻在镉胁迫应激过程中活性氧和抗氧化分子合成途径的内容。HCD1和HCD3的表型似乎与线粒体体积增加有关，HCD2的线粒体体积则与野生型相似，但ATP含量明显增加，这些结果说明细胞密度受多渠道调控。微藻中性油脂含量通常在细胞生长受阻时增加，在内质网膜或叶绿体膜积累。由于位点特异同工酶的分析较少，使得转录组分析无法有效分辨油脂积累的位点。我们通过脂质组与转录组协同分析，根据中性油脂甘油骨架sn2位点的脂肪酸碳链长度，明确了缺氮磷诱导的微拟球藻中性油脂主要在叶绿体积累，少量在细胞质。杜氏盐藻没有细胞壁，是研究环境胁迫应激反应的模式物种。通过代谢组结合转录组分析，我们发现杜氏盐藻在镉的长期胁迫下，一些氧化反应的产物大量积累，比如腺苷在氧化后生成过氧化氢和5’-脱氢腺苷含量，后者在所有代谢物中增量最大，为研究镉胁迫应激反应机制提供了新的途径。同时，谷胱甘肽等抗氧化剂含量也明显增加，以抵御和修复活性氧的侵蚀。因为非质粒CAS9核蛋白的基因编辑技术日趋成熟，使得非模式生物基因修饰与模式生物相差无几，我们的结果为提高叶绿体油脂积累和阻断镉诱导的活性氧合成提供了靶标，加快细胞工厂藻株的构建，保证国家能源安全。

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