Fan Meihua, Sun Xue, Liao Zhi, Wang Jianxin, Li Yahe, Xu Nianjun
1Marine Sciences and Technology College, Zhejiang Ocean University, Zhoushan, Zhejiang, 316000 China.
2Key Laboratory of Marine Biotechnology of Zhejiang Province, School of Marine Sciences, Ningbo University, Ningbo, Zhejiang, 315211 China.
Proteome Sci. 2018 Oct 27;16:17. doi: 10.1186/s12953-018-0145-5. eCollection 2018.
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 to high temperatures have not been clearly investigated yet.
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 to high-temperature stress and to elucidate the involvement of this response in adaptation mechanisms. Differentially expressed proteins (DEPs) of 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.
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, Ca-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.
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.
属于绿藻门大型海藻，是绿潮的优势种。它分布于全球，因此在生长过程中会受到高温胁迫。然而，其对高温响应的适应机制尚未得到明确研究。
在本研究中，采用相对和绝对定量的等压标签（iTRAQ）标记结合液相色谱 - 串联质谱（LC - MS/MS）技术，对其在高温胁迫下的响应进行比较蛋白质组学分析，以阐明该响应在适应机制中的作用。鉴定了高温（记为UpHT）与对照（UpC）相比差异表达的蛋白质（DEPs）。进行了包括基因本体（GO）分析、通路分析和通路富集分析在内的生物信息学分析，以通过蛋白质网络分析耐热机制背后的关键代谢途径。进行了定量实时PCR和蛋白质免疫印迹以验证所选蛋白质。
在本研究中，与对照相比，高温下鉴定出1223个DEPs，其中包括790个上调蛋白和433个下调蛋白。高温刺激主要诱导了谷胱甘肽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、捕光叶绿素a/b结合蛋白（lhcSR）、核酮糖 - 1,5 - 二磷酸羧化酶/加氧酶激活酶、丝氨酸/苏氨酸蛋白激酶2、腺苷酸激酶、钙依赖性蛋白激酶（CDPK）、抗病蛋白EDS1、II型metacaspase、核苷二磷酸激酶2a（NDPK2a）、26S蛋白酶体调节亚基、泛醌氧化还原酶、ATP合酶亚基、蔗糖非发酵相关蛋白激酶2（SnRK2s）和细胞色素P450的表达。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、蛋白磷酸酶2C（PP2C）和碳固定相关蛋白。此外，生物学指标分析表明蛋白质含量和超氧化物歧化酶（SOD）活性降低；培养96小时后，Fv/Fm值降至最低点。然而，高温下抗坏血酸过氧化物酶（APX）活性和丙二醛（MDA）含量增加。
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、光合生物中的碳固定以及光合作用天线蛋白途径相关的蛋白质减少。这些途径在高温调节中起关键作用。这些新发现的蛋白质为利用遗传或其他方法进一步研究其功能提供了良好的起点。这些发现显著提高了对藻类耐受高温胁迫所涉及分子机制的理解。
