【作者】 陈颖；

【导师】 王大志；

【作者基本信息】 厦门大学 ， 环境科学， 2017， 硕士

【摘要】 链状亚历山大藻(Alexandrium catenellla)是一种全球分布的有毒甲藻。研究表明,磷(Phosphorus,P)是链状亚历山大藻细胞生长和藻华形成的限制性因子。目前有关链状亚历山大藻对磷缺乏的生理生态响应已有一些研究,但对于其分子机制尤其是蛋白质层面上的研究仍较少。本论文以链状亚历山大藻(A.catenella)为研究对象,运用iTRAQ(Isobaric tags for relative and absolute quantitation)定量蛋白质组学技术,比较研究了A.catenella对磷缺乏和不同磷源添加的蛋白质组表达变化,筛选差异表达蛋白并分析其功能及参与的生物学过程,确认与磷代谢相关蛋白,并结合生理参数分析,探讨了A.catenell 对磷缺乏的适应策略和不同磷源添加的响应机制。主要研究结果如下:(1)磷缺乏时,A.catenell 可调用细胞内的“储备磷”维持细胞生长。补充不同形态磷源后,A.catenell 均可快速吸收。相较于有机磷源—葡萄糖-6-磷酸(Glucose-6-phosphate,G-6-P),A.catenella对无机磷源—Na2HPO4 吸收更快,但两者对A.catenell 生长的影响没有显著差异;(2)磷缺乏时,A.catenell 细胞密度保持相对稳定,碱性磷酸酶活性(Alkaline phosphatase activity,APA)迅速增加。添加不同形态磷源后,A.catenella恢复生长,APA下降。无机磷添加组,APA下降迅速,而有机磷添加组,APA先上升而后下降,表明A.catenell 对Na2HPO4和G-6-P利用存在差异;(3)在A.catenella中鉴定到6,577个高可信度蛋白质,其中参与核糖体、糖酵解/糖异生、嘌呤代谢和双组分系统等生物学过程的蛋白质丰度最高。磷缺乏时,A.catenell 可通过诱导表达或高表达酸性磷酸酶等转而利用有机磷及利用非磷脂类-鞘糖脂代替磷脂减少细胞对磷的需求等策略来应对环境中磷的缺乏。此外,还通过减少蛋白质翻译、光合碳固定、氮和硫营养元素代谢等生物学过程,调节能量分配以适应低磷环境;(4)添加Na2HPO4或G-6-P后,A.catenell 均能快速恢复生长,但两者相比存在较大差异,相比G-6-P,A.catenell 对Na2HPO4吸收更快;但是G-6-P添加组显著变化的生物学过程较Na2HP04添加组多,核酸代谢等生物学过程显著上调,A.catenella可能存在G-6-P特殊利用机制:细胞吸收G-6-P后,可能通过磷酸戊糖途径,直接用于核酸等物质合成;(5)本论文鉴定到一些与磷代谢相关蛋白质,如酸性磷酸酶和5’-核苷酸酶等,这些蛋白质在A.catenell 适应环境中磷缺乏和变动方面具有重要作用。更多还原

【Abstract】 Alexandrium catenella is a worldwide distributed dinoflagellate species.Studies have shown that phosphorus(P)is a limiting nutrient for cell growth and bloom formation of A.catenella.Much effort has been devoted to eco-physiological responses of A.catenella to ambient P-depletion,however,little is known about the molecular mechanism,particularly at the protein level.This study,applied the quantitative proteomic approach:Isobaric tags for relative and absolute quantitation(iTRAQ)to compare protein expression profiles of A.catenella under different P consitions,characterized the differentially expressed proteins,analyzed their functions and biological processes,identified proteins related to P metabolism and discussed the adaptative strategies of A.catenellato P-depletion and the response mechanism to the resupplement of different P sources,combined with physiological parameter analysis.The main results were as follows:(1)A.catenella cells were able to utilize the intracellular "stored P" to maintain cell growth when ambient P became depletion.After resupplement of different P sources,A.catenella cells rapidly absorbed both inorganic and organic P.Compared with organic P,glucose-6-phosphate(G-6-P),the uptake of inorganic P,Na2HPO4 was faster,but no significant difference on cell growth between the two species.(2)Cell density of A.catenella maintained relatively stable under P-depleted condition,and alkaline phosphatase activity(APA)increased rapidly.After resupplement of P,A.catenella cells recovered growth rapidly and APA decreased.Compared with Na2HPO4,the effect of G-6-P on APA took more time.In Na2HPO4 resupplement group,APA decreased immediately,while in G-6-P resupplement group,APA increased firstly,then decreased,suggesting that the different utilization strategies of A.catenella to Na2HPO4 and G-6-P.(3)A total of 6,577 high-confidence proteins were identified from A.catenella,among them,proteins involved in ribosome,glycolysis/gluconeogenesis,purine metabolism and two-component system were more abundant.In P-depleted condition,A.catenella could induce or up-regulate the expressions of acid phosphatase and other phosphoesterase,utilize organic phosphorus and non-phospholipids-sphingolipids instead of phospholipids to reduce cell demand for P.In addition,protein translations,carbon fixation,nitrogen metabolism,sulfur metabolism and other important biological processes were down-regulated to adjust energy allocation to adapt to ambient P-depletion.(4)A.catenella could quickly recover the growth after addition of Na2HPO4 or G-6-P,but a great difference was observed between the two treatments.A.catenella absorbed Na2HPO4 rapidly compare to G-6-P,but more biological processes changed in DOP-resupplement cells than that in DIP-resupplement cells.Nucleic acid metabolism and other biological processes were significantly up-regulated in DOP-resupplement cells.A.catenella might possess special mechanism to utilize G-6-P:it might directly utlize G-6-P for the synthesis of nucleic acid through the pentose phosphate pathway.(5)P metabolism related proteins,such as acid phosphatase,5’-nucleotidase,were identified in A.catenella.These proteins played a vital role in adaption to ambient P-depletion and in response to the resupplement of different P species.更多还原