基于TMT定量蛋白质组学揭示纳米包装双孢蘑菇采后冷藏生理代谢规律

Physiological Metabolic Role of Nanocomposite Packaged Agaricus bisporus During Postharvest Cold Storage Analyzed by TMT-Based Quantitative Proteomics

【背景】双孢蘑菇采后极易发生开伞、失水及褐变等品质劣变现象,极大地影响了其贮藏品质和商业价值。前期研究已证实纳米包装可有效延缓双孢蘑菇采后的品质劣变,但其保鲜机制仍不清晰。【目的】本研究通过串联质谱标记(TMT)定量蛋白质组学技术,对纳米包装和普通聚乙烯包装的双孢蘑菇贮藏期间的差异表达蛋白进行分析,进一步探究纳米包装保鲜双孢蘑菇的作用机制。【方法】以双孢蘑菇为研究对象,用纳米包装对其进行保鲜,并以普通聚乙烯包装作为对照。对贮藏期间双孢蘑菇进行蛋白提取和胰蛋白酶解,并通过TMT标记及液相色谱串联质谱检测,筛选出差异表达蛋白,结合生物信息学分析,研究差异蛋白所参与的主要代谢途径,同时利用实时荧光定量聚合酶链式反应(qPCR)技术,在基因层面验证差异蛋白的表达水平。【结果】纳米包装有效维持了双孢蘑菇的外观品质,并且延缓了细胞膜透性的增加。随着贮藏时间的增加,两组包装的差异蛋白数目增多,在贮藏中期(6 d)和贮藏末期(10 d),差异蛋白分别达到62个和148个,其中纳米包装和普通包装有共同差异蛋白22个。结合生物信息学分析,发现这些差异蛋白主要与能量代谢和脂代谢等功能途径相关。对脂代谢途径进行重点分析,结果显示纳米包装对双孢蘑菇的膜脂代谢具有调控作用,相较于普通包装组,纳米包装组的脂肪酸合成酶、磷酸胆碱孢苷酰转移酶和磷脂酸磷酸酯酶呈上调趋势,同时下调了膜脂降解关键酶如磷脂酶D和脂肪酶的活性;从基因水平上来看,编码这些蛋白的基因表达与组学结果相一致。【结论】利用TMT定量蛋白质组学技术,能对不同包装双孢蘑菇贮藏期间的差异蛋白进行筛选和分析。纳米包装调节了双孢蘑菇的膜脂代谢,抑制了膜脂降解相关酶的表达,有效延缓了细胞膜透性的增加,维持了细胞膜结构和功能,进而延缓双孢蘑菇贮藏期间的品质劣变。

【Background】Agaricus bisporus(A.bisporus)is prone to quality deterioration,such as umbrella opening,water loss,and browning after harvest,which seriously affects the storage quality and commercial value.Our previous research has confirmed that the nanocomposite packaging material(Nano-PM)could effectively delay the postharvest quality deterioration of A.bisporus,but the preservation mechanism is still unclear.【Objective】In this study,the differentially expressed proteins of A.bisporus in Nano-PM and polyethylene packaging material(Normal-PM)during storage were analyzed by Tandem Mass Tag(TMT)quantitative proteomics technology.The preservation mechanism of Nano-PM on A.bisporus was further explored.【Method】A.bisporus was taken as the research object.The Nano-PM was used for the preservation of A.bisporus,and the Normal-PM was used as the control.The protein extraction and trypsin hydrolysis were performed on A.bisporus during storage.The differentially expressed proteins were screened by TMT labeling and liquid chromatography-tandem mass spectrometry detection.Combined with bioinformatics analysis,the main metabolic pathways involved in differential proteins were studied.Quantitative real-time polymerase chain reaction(qPCR)technology was used to determine the gene expression levels of differential proteins.【Result】The Nano-PM effectively maintained the appearance quality of A.bisporus and delayed the increase of cell membrane permeability.The number of differential proteins in two groups increased during storage.In the middle(6 d)and late(10 d)stages of storage,the numbers of differential proteins were 62 and 148,respectively.Among them,22 differential proteins were common.Combined with bioinformatics analysis,these differential proteins were mainly related to pathways,such as energy metabolism and lipid metabolism.The lipid metabolism pathway was mainly analyzed,and the results showed that the Nano-PM had a regulatory effect on the membrane lipid metabolism of A.bisporus.Compared with the Normal-PM,the protein expression of fatty acid synthase,phosphorylcholine cytidylyltransferase,and phosphatidic acid phosphatase under Nano-PM were up-regulated,while the protein expression of key enzymes in membrane lipid degradation,such as phospholipase D and lipase,were down-regulated.At the gene level,the expression of genes encoding these proteins were consistent with the proteomics results.【Conclusion】The differential proteins of different packaged A.bisporus during storage could be screened and analyzed by TMT-based quantitative proteomics technology.Nano-PM regulated the membrane lipid metabolism of A.bisporus,and inhibited the expression of membrane lipid degradation-related enzymes,which effectively delayed the increase in cell membrane permeability of A.bisporus,maintained the structure and function of the cell membrane,and delayed the quality deterioration of A.bisporus during storage.