碳离子束诱导酿酒酵母甘油脂类代谢异常关键基因的挖掘及功能验证

Eukaryotic lipid metabolism is related to biological processes such as cell energy storage and signal transduction. Lipid metabolism research is one of the effective means to find biomarkers and optimize biological processes. Heavy ion beam irradiation will cause mitochondrial damage in Saccharomyces cerevisiae, resulting in loss of yeast respiratory function and altered lipid metabolism pathway. In previous study, S. cerevisiae BY4743 was irradiated by carbon ion beam, and the genetically stable respiratory defect mutant ResD-4 was obtained. The combined analysis results of lipidomics and transcriptome showed that there was an overlap between elevated levels of glycerides and expressed genes. Combined with the previous results, in order to clarify key genes of glycerides metabolic abnormalities ResD-4, key genes of glycerides metabolism will be screened and gene function will be verified by constructing candidate target knockout and overexpression strain. This study will systematically reveal the molecular mechanisms of carbon ion beam irradiation induced glycerides metabolic abnormalities in respiratory-deficient yeast mutant. It will establish an accurate and effective means to mine the genetic resources associated with abnormalities in metabolites induced by heavy ion beam irradiation.

真核生物脂质代谢与细胞能量贮存、信号转导等生物过程密切相关，脂质代谢的研究是寻找生物标志物及优化生物过程的有效手段之一。重离子束辐照造成酿酒酵母线粒体损伤，导致酵母呼吸功能丧失，脂质代谢途径发生改变。本研究前期利用碳离子束对辐照处理酿酒酵母BY4743，获得了遗传稳定的呼吸缺陷型突变体ResD-4。脂质代谢组和转录组联合分析结果表明，甘油脂类物质含量升高与表达基因之间存在重叠。结合前期研究结果，本项目拟以ResD-4为研究对象，开展甘油脂类代谢关键基因的筛选，通过构建候选目的基因敲除及过表达菌株进行基因功能验证，以期明确引起ResD-4甘油脂类代谢异常的关键基因。本研究将系统的揭示碳离子束辐照诱导的呼吸缺陷型酵母突变体甘油脂类代谢异常的分子机理，同时建立一套准确有效的手段，挖掘与重离子束辐照诱导的代谢产物异常相关联的基因资源。

重离子辐射诱导微生物脂质代谢模式变异及其机理鲜有研究。本项目发现重离子辐射选育的线粒体功能缺陷突变体群体脂质表达模式相似，且甘油脂类脂质含量上调。进一步通过基因组-转录组-脂质组联合分析，揭示了脂质代谢是受线粒体功能缺陷影响的富集程度最高的代谢途径。同时，在突变体群体水平，脂质代谢途径的差异表达基因呈现相似的上调/下调表达模式；通过基因功能注释和差异表达分析选择了ALD4、GUT1和CST26 三个甘油脂类代谢关联基因，作为可能在线粒体功能缺陷影响脂质代谢模式的过程中发挥重要作用的候选关键基因。随后，成功构建了ALD4、GUT1和CST26的过表达株和敲除株，在表型、代谢通路基因表达以及代谢组层面进行了考察以验证ALD4和GUT1的效应。结果表明，甘油脂类代谢通路的ADH4、SFA1、ADH5和ADH3在ALD4敲除菌株中表达量的增幅较大，考虑作为ALD4缺失的代偿性响应；ALD4和GUT1的过表达株和敲除株在整体代谢模式上均呈现明显分化，证实其对酵母整体代谢以及脂质代谢有重要影响。因此，后续可进一步发掘线粒体功能缺陷影响脂质代谢模式过程中的基因网络，并揭示ALD4和GUT1的表型和分子效应。项目结果可补充线粒体功能研究，为微生物油脂类代谢产物的调控研究提供理论依据，为挖掘重离子辐射创制的优良基因资源提供方法学参考。