光促进小球藻葡萄糖转运的作用途径研究

Transmembrane transport of glucose is the first step for its utilization by cells, which has great influence on cell growth. Numerous researches indicated that the cell growth and glucose uptake rate under light were much higher than that in heterotrophic culture. However, studies about the effect of light on the transport of glucose are rare. In our previous study, it was detected that light supply can enhance glucose utilization in Chlorella. Furthermore, the expression of hexose transport gene was significantly promoted by light supply. It is proposed that light may affect glucose transport via two possible pathways: i) ATP generated from photosynthetic electron transport can supply as energy for glucose transport; ii) light can promote glucose uptake via enhance the gene expression of glucose transport. Firstly, the methods of isotope labelling integrated with block electron flow in photosynthesis, and extra addition of ATP will be conducted to confirm the hypothesis that light can supply energy for glucose transport. Secondly, after cloning hexose uptake gene (HUP) and testing its function of glucose uptake, effect of light on HUP gene transcription and translation are investigated through the methods of qPCR and Western blot. This project illuminates the pathway of light promoting glucose transport in the mixotrophic culture of Chlorella and provides the theoretical foundation for the high-density cultivation of algal biomass using glucose.

葡萄糖的跨膜转运是细胞利用糖的第一步，对细胞的生长具有重要影响，众多研究显示小球藻在光下利用葡萄糖可获得比异养培养更高的生长和糖利用速率，但目前关于光影响小球藻葡萄糖转运过程的研究鲜有报道。我们在前期研究中发现光不仅可以促进小球藻对葡萄糖的摄取速率，还可以提高细胞己糖转运基因的表达水平。基于前期实验，本项目提出光可能同时通过两种途径影响小球藻的葡萄糖转运：①光合电子传递产生的ATP可以为糖转运提供能量；②光通过提高葡萄糖转运蛋白的表达水平促进细胞的糖转运。我们拟通过同位素标记结合光合电子传递阻断、外源ATP添加等实验阐明能量供应假说；通过克隆己糖转运基因（HUP）并对其葡萄糖转运功能进行验证，利用qPCR和Western blot在转录和翻译水平研究光对细胞己糖转运蛋白表达的影响，以阐明兼养培养中光对小球藻葡萄糖转运的影响途径。该研究可为高效利用葡萄糖进行微藻高密度培养提供理论指导。

光对小球藻兼养培养过程的葡萄糖利用具有显著影响，而葡萄糖的跨膜转运作为细胞利用糖的第一步，对细胞的葡萄糖利用速率具有重要影响。本项目针对光对葡萄糖转运过程的影响，首先，系统研究了不同光强和葡萄糖浓度下蛋白核小球藻的生长与代谢，发现低光强可以促进葡萄糖的利用，但是进一步增加光强葡萄糖的利用速率和利用效率显著降低。在高光强和高糖条件下，细胞的生长和糖的利用受到光的显著抑制，但是光合电子传递阻断剂的添加可以解除抑制作用，这表明光是通过光合作用间接影响糖的代谢，进而影响糖的摄取。另外，在光照条件下负责葡萄糖转运的己糖转运蛋白的表达水平与黑暗条件下无显著差异，但是阻断光合作用后其表达水平显著提高。综合上述结果，可以得出以下结论：小球藻兼养培养过程中，光可以通过光合产能和提高己糖转运蛋白的表达水平促进葡萄糖的转运，但是，与光对葡萄糖转运过程的影响相比，光对葡萄糖的胞内代谢的影响更大，光通过光合作用对糖的胞内代谢具有显著的抑制作用，进而抑制了葡萄糖的利用。因此，后续需进一步对光抑制葡萄糖的胞内代谢的机制进行深入研究。相关结果对于提高微藻兼养培养过程的能量和物质利用效率，降低生产成本具有重要意义。

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