BACKGROUND Ulva prolifera belongs to green macroalgae and is the dominant species of green tide. It is distributed worldwide and is therefore subject to high-temperature stress during the growth process. However, the adaptation mechanisms of the response of U. prolifera to high temperatures have not been clearly investigated yet. METHODS In this study, isobaric tags for relative and absolute quantitation (iTRAQ) labelling was applied in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) to conduct comparative proteomic analysis of the response of U. prolifera to high-temperature stress and to elucidate the involvement of this response in adaptation mechanisms. Differentially expressed proteins (DEPs) of U. prolifera under high temperature (denote UpHT) compared with the control (UpC) were identified. Bioinformatic analyses including GO analysis, pathway analysis, and pathway enrichment analysis was performed to analyse the key metabolic pathways that underlie the thermal tolerance mechanism through protein networks. Quantitative real-time PCR and western blot were performed to validate selected proteins. RESULTS In the present study, 1223 DEPs were identified under high temperature compared with the control, which included 790 up-regulated and 433 down-regulated proteins. The high-temperature stimulus mainly induced the expression of glutathione S-transferase, heat shock protein, ascorbate peroxidase, manganese superoxide dismutase, ubiquitin-related protein, lhcSR, rubisco activase, serine/threonine protein kinase 2, adenylate kinase, Ca2+-dependent protein kinase (CDPK), disease resistance protein EDS1, metacaspase type II, NDPK2a, 26S proteasome regulatory subunit, ubiquinone oxidoreductase, ATP synthase subunit, SnRK2s, and cytochrome P450. The down-regulated proteins were photosynthesis-related proteins, glutathione reductase, catalase-peroxidase, thioredoxin, thioredoxin peroxidase, PP2C, and carbon fixation-related proteins. Furthermore, biological index analysis indicated that protein content and SOD activity decreased; the value of Fv/Fm dropped to the lowest point after culture for 96 h. However, APX activity and MDA content increased under high temperature. CONCLUSION The present study implied an increase in proteins that were associated with the stress response, oxidative phosphorylation, the cytokinin signal transduction pathway, the abscisic acid signal transduction pathway, and the glutathione metabolism pathway. Proteins that were associated with photosynthesis, carbon fixation in photosynthesis organisms, and the photosynthesis antenna protein pathway were decreased. These pathways played a pivotal role in high temperature regulation. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. These findings significantly improve the understanding of the molecular mechanisms involved in the tolerance of algae to high-temperature stress.

中文翻译：

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石莼对高温胁迫反应的比较蛋白质组学分析。

背景技术 石莼属绿藻类，是绿潮的优势种。它分布于世界各地，因此在生长过程中受到高温胁迫。然而，尚未明确研究 U. prolifera 对高温响应的适应机制。方法 在本研究中，将用于相对和绝对定量 (iTRAQ) 标记的等压标签与液相色谱-串联质谱 (LC-MS/MS) 相结合，对 U. prolifera 对高浓度酶的反应进行比较蛋白质组学分析。温度压力并阐明这种反应在适应机制中的作用。鉴定了与对照 (UpC) 相比在高温下的 U. prolifera (表示 UpHT) 的差异表达蛋白 (DEP)。进行了包括GO分析、途径分析和途径富集分析在内的生物信息学分析，以分析通过蛋白质网络构成耐热机制的关键代谢途径。进行定量实时 PCR 和蛋白质印迹以验证选定的蛋白质。结果在本研究中，与对照相比，在高温下鉴定出1223个DEP，其中包括790个上调蛋白和433个下调蛋白。高温刺激主要诱导谷胱甘肽S-转移酶、热休克蛋白、抗坏血酸过氧化物酶、锰超氧化物歧化酶、泛素相关蛋白、lhcSR、rubisco激活酶、丝氨酸/苏氨酸蛋白激酶2、腺苷酸激酶、Ca2+依赖性蛋白的表达。激酶 (CDPK)、抗病蛋白 EDS1、间半胱天冬酶 II 型、NDPK2a、26S 蛋白酶体调节亚基、泛醌氧化还原酶、ATP 合酶亚基、SnRK2s 和细胞色素 P450。下调的蛋白质是光合作用相关蛋白、谷胱甘肽还原酶、过氧化氢酶-过氧化物酶、硫氧还蛋白、硫氧还蛋白过氧化物酶、PP2C和碳固定相关蛋白。此外，生物指标分析表明蛋白质含量和SOD活性降低；培养96 h后Fv/Fm值降至最低点。然而，高温下APX活性和MDA含量增加。结论 本研究表明与应激反应、氧化磷酸化、细胞分裂素信号转导途径、脱落酸信号转导途径和谷胱甘肽代谢途径相关的蛋白质增加。与光合作用、光合作用生物中的碳固定和光合作用天线蛋白途径相关的蛋白质减少了。这些途径在高温调节中发挥了关键作用。这些新型蛋白质为使用遗传或其他方法进一步研究其功能提供了一个良好的起点。这些发现显着提高了对藻类对高温胁迫耐受性所涉及的分子机制的理解。