具槽帕拉藻对不同形态磷源的生理响应

	具槽帕拉藻对不同形态磷源的生理响应
	于倩 1,2
学位类型	硕士
导师	赵建民
	2014-12
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	海洋生物学
关键词	具槽帕拉藻磷源磷酸酶代谢组学差异代谢物
摘要	目前，国内外学者已对具槽帕拉藻(Paralia sulata)在不同海域的丰度和生态位展开了相关研究，但其对不同磷源利用能力以及代谢水平的研究尚未报道。本研究以海洋环境指示物种具槽帕拉藻为研究对象，从生理学角度研究了磷酸二氢钾(KH2PO4)、β-甘油磷酸钠(Sodium-β-glycerophosphate，β-G-P)、葡萄糖-6-磷酸(Glucose-6-phosphate，G-6-P)、卵磷脂(Lecithin，LEC)和三磷酸腺苷二钠(Adenosine disodium triphosphate，ATP)分别为单一磷源时对具槽帕拉藻生长的影响；建立了基于核磁共振(Nuclear magnetic resonance，NMR)技术的具槽帕拉藻代谢组学研究方法，并使用该方法筛选出具槽帕拉藻利用不同形态磷源的差异代谢物，探讨了其对不同形态磷源的代谢响应差异。研究结果为揭示具槽帕拉藻对不同形态磷源的利用特征和机制提供了科学依据。论文主要内容如下： 1. 不同形态磷源对具槽帕拉藻生长和磷酸酶活性的影响分别采用KH2PO4、β-G-P、G-6-P、ATP和LEC作为f/2 培养基的磷源，探讨了不同形态磷源对具槽帕拉藻生长及磷酸酶活性的影响。结果表明，具槽帕拉藻能够有效利用上述五种磷源，且溶解有机磷(Dissolved organic phosphorus，DOP)比KH2PO4 更有利于具槽帕拉藻的生长；对碱性磷酸酶(Alkaline phosphatase，AP)活性分析发现，大分子DOP(LEC)可能需要通过磷酸酶的水解作用才能被吸收利用。在胞外磷源充足的情况下，具槽帕拉藻能够在胞内储存磷，而在磷限制时则可利用胞内储存的磷维持藻细胞生长。 2. 具槽帕拉藻代谢组提取方法的比较研究采用两种细胞破碎方式和三种溶剂系统对均一生物样本的代谢物进行提取，结合核磁共振波谱分析和主成分分析(Principal components analysis，PCA)，对不同的代谢物提取方法进行了评估。结果表明：甲醇/水(1:1)与匀浆相结合的方法具有高提取产率、高重现性、易操作和速度快等特性，是比较适合具槽帕拉藻代谢物提取的方法，可为其后续代谢途径的研究提供参考。 3. 不同形态磷源对具槽帕拉藻生理响应的代谢组学研究将匀浆、甲醇/水(1:1)的提取方法应用于具槽帕拉藻代谢物的提取，结合NMR波谱图和正交偏最小二乘判别分析(Orthogonally partial least squares－discriminant analysis，O-PLS-DA)模型，研究了不同形态磷源条件下具槽帕拉藻代谢物产物的变化。结果表明，具槽帕拉藻可以通过改变其体内代谢途径，从而更好的利用不同形态的磷源；相比于以KH2PO4为磷源时藻细胞中的代谢物，LEC为磷源时藻细胞代谢物与其不具有差异性，而相比与其它物质为磷源时藻细胞的代谢物具有显著差异性。分析发现，乳酸盐为β-G-P处理组中具槽帕拉藻的差异代谢物，葡萄糖为G-6-P处理组中具槽帕拉藻的差异代谢物，糖原、酪氨酸、组氨酸和苯丙氨酸为ATP处理组中具槽帕拉藻的差异代谢物。以上结果可为揭示具槽帕拉藻对不同形态磷源的利用特征和机制提供科学依据。
其他摘要	Currently, the abundance and the ecological niche of Paralis sulata have been previously reported. However, the physiological response of P. sulata to different phosphorus substrates has been not reported. In this dissertation, the growth and phosphatase activity of the marine environment indicator species(P. sulata) under different phosphorus substrates including, KH2PO4, sodium-β-glycerophosphate (β-G-P), glucose-6-phosphate(G-6-P), lecithin(LEC) and adenosine disodiumtriphosphate(ATP) were investigated. In addition, extraction methods for metabolome were evaluated based on nuclear magnetic resonance(NMR) in P. sulata. Taking this outcome into the metabolomic studies of P. sulata, we intend to find out stable metabolomic biomarkers in P. sulata under different phosphorus substrates. All the findings provide important insights into physiological effects of P. sulata to different phosphorus substrates. The results of the studies are as follows. 1. Effects of different phosphorus substrates on the rowth and phosphatase activity of P. sulata Five phosphorus substrates, including KH2PO4, β-G-P, G-6-P, LEC and ATP, were used to explore their effects on the growth and phosphatase activity of P. sulata. It was found that P. sulata could utilize all five phosphorus substrates to support the growth, and dissolved organic phosphorus(DOP) was better than rthophosphate for the growth of P. sulata. The changes of alkaline phosphatase(AP) activity suggested that large molecular DOP(LEC) could be utilized by phosphatase hydrolysis. Furthermore, high external phosphorus concentration would eventually cause high intracellular phosphorus in P. sulata. It was suggested that P. sulata could utilize intracellular phosphorus to sustain growth under depletion of dissolved phosphorus. 2. Evaluation of metabolome extraction strategies from P. sulata The metabolites of P. sulata were extracted by using three solvent extraction systems and two ways of cell disruption, and then assessed with one dimensional NMR spectroscopy combined with principal components analysis(PCA). It was found that the extraction strategy of methanol/water(1:1, homogenized) was preferable for the metabolite extraction due to its highest yield, reproducibility, ease and speed. Validation of extraction protocol will provide a reference for further detection of metabolic pathway of P. sulata. 3. A metabolomic study on the effects of different phosphorus substrates in P. sulata The metabolites of P. sulata were extracted by using methanol/water solvent extraction systems and homogenate cell disruption, and then analyzed with one dimensional proton NMR spectroscopy combined with Orthogonally-Partial Least SquaresDiscriminant Analysis(O-PLS-DA). Metabolomics study revealed that P. sulata had significant differences between metabolic pathways on uptake and utilization different phosphorus substrates. The metabolites between LEC and KH2PO4 group were not significantly different; however, the metabolites of other treatment group had obvious difference compared with KH2PO4 group. Metabolic responses demonstrated that lactate was the difference metabolite of β-G-P treatment; glucose could be used as the difference metabolite of G-6-P treatment, while glycogen, tyrosine, histidine, and phenylalanines were the difference metabolite of ATP treatment in P. sulata. These findings provide important insights into physiological effects of P. sulata to different phosphorus substrates at molecular level using metabolomics.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/7945
专题	中国科学院烟台海岸带研究所知识产出_学位论文
作者单位	1.烟台海岸带研究所 2.中国科学院研究生院
推荐引用方式 GB/T 7714	于倩. 具槽帕拉藻对不同形态磷源的生理响应[D]. 北京. 中国科学院研究生院,2014.

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