贫氮-富磷耦合诱导小球藻油脂积累及调控机制

发布时间：2018-03-10 05:14

本文选题：原始小球藻　切入点：生物量　出处：《湘潭大学》2016年硕士论文　论文类型：学位论文

【摘要】：本研究考察了不同氮磷耦合诱导模式(即富氮-富磷、贫氮-富磷、富氮-贫磷、贫氮-贫磷、贫氮-磷限制)对小球藻(Chlorella protothecoides)生物量及油脂积累的影响,并考察了最佳氮磷耦合诱导模式下小球藻细胞油脂高效富集的调控机制。结果表明,富氮-富磷耦合诱导模式下小球藻最大生物量为5.92 g/L,高于贫氮-富磷(2.99 g/L)、富氮-贫磷(2.38 g/L)、贫氮-贫磷(0.76 g/L)及贫氮-磷限制(2.81 g/L)耦合诱导模式的生物量;贫氮-贫磷模式下,小球藻最大油脂含量为55.8%,优于富氮-富磷(22.17%)、贫氮-富磷(52.5%)、富氮-贫磷(32.77%)及贫氮-磷限制(53.7%)耦合诱导模式的油脂含量;贫氮-富磷模式下,最大油脂产率为224.14 mg/L/d,这一油脂产率分别是对照组、富氮-贫磷、贫氮-贫磷、贫氮-磷限制耦合诱导模式的1.19~3.70倍。这证实贫氮-富磷耦合模式是促进小球藻(C.Protothecoides)细胞油脂积累的潜力诱导模式。进一步地,采用二维荧光差异凝胶电泳定量蛋白质组学手段揭示贫氮-富磷耦合诱导模式下小球藻细胞油脂积累相关调控机制。采用贫氮-富磷耦合模式诱导的藻细胞中,鉴定出27个差异表达蛋白质,包括8个下调蛋白和19个上调蛋白。根据代谢通路分析得出,糖酵解途径中尿苷二磷酸-葡萄糖-6-脱氢酶和葡萄糖-1-磷酸腺苷转移酶是贫氮-富磷耦合诱导模式下小球藻细胞油脂富集的关键调控因子,这两种酶可催化细胞中的淀粉转化为葡萄糖,为糖酵解途径的开始提供切入点;磷酸戊糖途径(PPP)中表达上调的转酮醇酶将五碳酮糖和醛糖转化为七碳酮糖和3-磷酸甘油醛(G3P);二磷酸核酮糖盐羧化酶(RuBis CO)是提高卡尔文循环的关键酶,这种酶表达上调可以促进3-磷酸甘油醛(G3P)进入糖酵解途径;苹果酸脱氢酶(ME)表达上调能有效提高小球藻细胞油脂产率,可据此推测关键节点调控的脂质合成积累路径;氮饥饿信号调控因子—硝酸还原酶(NR)是小球藻细胞油脂产率提高的潜在调控因子;谷氨酸盐脱氢酶(GLDH)表达上调可有效抵御培养条件失衡。这些研究发现为人工干预微藻油脂积累、推进微藻生物柴油规模化发展提供基础,同时也为其它藻类油脂代谢研究提供参考。综上所述,贫氮-富磷耦合模式是促进小球藻细胞油脂积累的有效诱导方式,这一模式为实现微藻高油脂产量提供了可能。而这些调控小球藻油脂积累的关键酶的发现为建立高效产油机制和基于微藻制备生物柴油的工业化生产奠定了基础。
[Abstract]:In this study, the effects of different nitrogen and phosphorus coupling induction models (i.e., rich in nitrogen and phosphorus, low in nitrogen and phosphorus, rich in nitrogen and phosphorus, low in nitrogen and phosphorus, and low in nitrogen and phosphorus) on the biomass and oil accumulation of Chlorella protothecoides were investigated. The regulation mechanism of oil enrichment in Chlorella vulgaris cells under the optimal nitrogen and phosphorus coupling induction model was investigated. The maximum biomass of Chlorella vulgaris was 5.92 g / L, which was higher than 2.99 g / L of nitrogen and phosphorus rich, 2.38 g / L, 0.76 g / L and 2.81 g / L of N / P and 2.81 g / L, respectively. The maximum oil content of Chlorella vulgaris was 55.8, which was better than that of nitrogen-rich phosphate-rich chlorella 22.17, nitrogen-rich phosphorus-rich 52.5, nitrogen-poor phosphorus-rich (32.77) and nitrogen-phosphorus limiting (53.775)) -induced oil content, while in the nitrogen-phosphorus-rich model, The maximum oil yield is 224.14 mg / L / d, which is the control group, which is rich in nitrogen and poor in phosphorus, low in nitrogen and poor in phosphorus. The results show that the coupling model is a potential model for promoting the accumulation of lipid in C. Protothecoides cells. The regulation mechanism of lipid accumulation in Chlorella vulgaris cells induced by N-rich coupling was revealed by two-dimensional fluorescence differential gel electrophoresis (FDGE) quantitative proteomics, and the effect of nitrogen-phosphorus-rich coupling model on lipid accumulation in Chlorella vulgaris cells was investigated. Twenty-seven differentially expressed proteins were identified, including 8 down-regulated proteins and 19 up-regulated proteins. In glycolysis pathway, uridine diphosphate glucose-6-dehydrogenase and glucose-1-phosphate transferase are the key regulating factors of lipid enrichment in Chlorella vulgaris cells under the coupling induction of nitrogen and phosphorus deficiency. These two enzymes can catalyze the conversion of starch in cells to glucose, which provides a starting point for the beginning of glycolysis pathway. The up-regulated transketol enzyme expressed in the pentose phosphate pathway (PPP) converts pentacarbonose and aldose to heptose and glyceraldehyde 3-phosphate (G3PN), and ribonose diphosphate carboxylase (RuBis COS) is the key enzyme to improve the Calvin cycle. The up-regulation of the enzyme expression can promote the glycolysis pathway of glyceraldehyde-3-phosphate (G3P3), and the increase of malate dehydrogenase (MEE) expression can effectively increase the oil production rate of Chlorella vulgaris cells, which can be used to speculate the accumulation pathway of lipid synthesis regulated by the key nodes. Nitrogen starvation signal regulator, nitrate reductase (NR), is a potential regulator for the increase of oil production in Chlorella vulgaris cells. The up-regulated expression of glutamate dehydrogenase (GLDH) can effectively resist the imbalance of culture conditions. These findings provide a basis for the artificial intervention of microalgae oil accumulation and the promotion of large-scale development of microalgae biodiesel. In conclusion, the coupling model of nitrogen-rich phosphorus is an effective inductive way to promote the accumulation of lipid in Chlorella vulgaris cells. The discovery of these key enzymes to regulate the accumulation of Chlorella microalgae oil may provide a basis for the establishment of efficient oil production mechanism and the industrialized production of biodiesel based on microalgae.
【学位授予单位】：湘潭大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TE667

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