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

doi:10.3864/j.issn.0578-1752.2022.23.013

Abstract

Abstract:

【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.

Key words: nanocomposite packaging, Agaricus bisporus, preservation, proteomics

WANG Chao,FANG DongLu,ZHANG PanRong,JIANG Wen,PEI Fei,HU QiuHui,MA Ning. Physiological Metabolic Rol e of Nanocomposite Packaged Agaricus bisporus During Postharvest Cold Storage Analyzed by TMT-Based Quantitative Proteomics[J].Scientia Agricultura Sinica, 2022, 55(23): 4728-4742.

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Figures/Tables 10

Table 1

Table 2

Fig. 1

Fig. 2

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Table 3

Main differentially expressed proteins of A. bisporus during storage in different packaging groups"

蛋白Accession Protein accession	蛋白描述 Protein description	基因名称 Gene name	差异倍数Fold change
蛋白Accession Protein accession	蛋白描述 Protein description	基因名称 Gene name	6N/6P	10N/10P
XP_006456433.1	吡喃糖脱氢酶受体蛋白Pyranose dehydrogenase receptor protein	AGABI1DRAFT_116682	0.58	/
XP_006457863.1	鸟嘌呤核苷酸结合蛋白Guanine nucleotide-binding protein	AGABI1DRAFT_82206	1.95	1.82
XP_006458170.1	核糖体生物合成蛋白Ribosome biogenesis protein	AGABI1DRAFT_33715	/	0.55
XP_006459487.1	未表征蛋白质Uncharacterized protein	AGABI1DRAFT_114418	0.58	/
XP_006460807.1	PRA家族蛋白PRA family protein	AGABI1DRAFT_112435	0.62	0.55
XP_006463403.1	未表征蛋白质Uncharacterized protein	AGABI1DRAFT_108190	/	1.68
XP_006454671.1	膜组分蛋白Membrane component protein	AGABI1DRAFT_103710	1.76	2.09
XP_006453769.1	未表征蛋白质Uncharacterized protein	AGABI1DRAFT_124747	0.53	0.64
XP_006453957.1	几丁质合成酶Chitin synthase	AGABI1DRAFT_52110	/	0.59
XP_006460122.1	未表征蛋白质Uncharacterized protein	AGABI1DRAFT_127941	0.55	0.66
XP_006462684.1	磷脂酶D Phospholipase D	AGABI1DRAFT_58967	0.68	0.51
XP_006454339.1	ATP激酶ATP kinase	AGABI1DRAFT_110490	1.65	1.51
XP_006454403.1	膜组分蛋白Membrane component protein	AGABI1DRAFT_66797	1.54	1.65
XP_006456787.1	磷脂酸磷酸酯酶Phosphatidic acid phosphatase	AGABI1DRAFT_127573	1.52	1.67
XP_006455355.1	超氧化物歧化酶Superoxide dismutase	AGABI1DRAFT_114765	1.82	1.56
XP_006454789.1	线粒体膜转运蛋白Mitochondrial membrane transporter	AGABI1DRAFT_65989	1.77	1.59
XP_006455653.1	未表征蛋白质Uncharacterized protein	AGABI1DRAFT_115714	/	1.56
XP_006459063.1	脂肪酸合酶Fatty acid synthase	AGABI1DRAFT_61049	1.61	1.87
XP_006454643.1	膜组分蛋白Membrane component protein	AGABI1DRAFT_50813	1.92	1.73
XP_006453790.1	脂肪酶蛋白Lipase protein	AGABI1DRAFT_67808	0.65	0.57
XP_006455684.1	ATP结合蛋白ATP-binding protein	AGABI1DRAFT_102264	1.64	1.55
XP_006456057.1	乌头酸水合酶Aconitate hydratase	AGABI1DRAFT_101681	/	1.82
XP_006456371.1	膜组分蛋白Membrane component protein	AGABI1DRAFT_46804	/	2.12
XP_006458132.1	DNA解旋酶活性蛋白DNA helicase active protein	AGABI1DRAFT_118129	0.36	0.59
XP_006457745.1	膜水解酶活性蛋白Membrane hydrolase active protein	AGABI1DRAFT_124786	0.41	0.61
XP_006459487.1	膜转运蛋白Membrane transporter	AGABI1DRAFT_114418	0.62	0.48
XP_006453826.1	CTP活性蛋白CTP active protein	AGABI2DRAFT_181860	1.56	1.68
XP_006459152.1	柠檬酸合酶蛋白Citrate synthase protein	AGABI1DRAFT_129944	0.64	0.56
XP_006454232.1	RNA结合蛋白RNA binding protein	AGABI1DRAFT_96937	1.59	1.52
XP_006457601.1	氧化还原酶活性Oxidoreductase active protein	AGABI1DRAFT_116005	0.36	0.52
XP_006453831.1	组蛋白H3 Histone H3	AGABI1DRAFT_52286	/	1.87

Table 3

Fig. 6

Fig. 7

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引物名称Primer name	引物序列Primes sequence (5′-3′)
GAPDH	F: ACGACAACGAGTGGGGTTAC	R: TTTCTTCTCGAGCACGGTCC
CS	F: ATCTGGGATGGCTCCGTCTTGG	R: GACCTTCAGGCAATGGCTCTTGG
PLD	F: CAGTCACAGCGTTAGAGCGGATG	R: CCTCCAGCGGTAGCATCAACTTG
CTP	F: CTCCACCCTTGCCCACCATTATTC	R: TGCTGCTGTTGAAGTCGAAGAGTC
PPase	F: CGATGCCTGAAGAAGATGCCTCTG	R: CGTCGTGTACCTCCTGCTCTAATTC
FAS	F: GGACTTGATGACCGCTCTGAACTC	R: GGTGACGATACGCCTATTAGCCTTC
LPS	F: CATTCTCCTACTGCTTCCATTCACTCC	R: CACCACCATAACCTGCCAAGACC

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

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步骤 Step	循环次数 Cycle	温度 Temperature (℃)	时间 Time (s)
预变性 Initial denaturation	1	95	30
循环反应 Cycling stage	40	95	10
循环反应 Cycling stage	40	60	30
融解曲线 Melt curve	1	95	15
		60	60
		95	15

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