基于LC-QTOF-MS代谢组学解析牛樟芝子实体和菌丝体中氨基酸代谢差异

doi:10.13560/j.cnki.biotech.bull.1985.2022-1080

摘要/Abstract

摘要：

为分析氨基酸在牛樟芝子实体和菌丝体代谢中的差异，采用LC-QTOF-MS系统质谱技术对牛樟芝子实体和菌丝体等3组样品进行代谢组分析，运用SIMCA软件（V16.0.2）对差异氨基酸代谢物进行筛选，分析重要氨基酸及其代谢途径，挖掘潜在标记物用于牛樟芝品种多样性和差异性分析。样品间主成分分析（PCA）和正交偏最小二乘判别分析（OPLS-DA）表明样品间在负离子条件下具有显著差异，在t检验P < 0.05，VIP > 1的条件下，牛樟芝子实体（ACFB）与液体培养14 d菌丝体（ACM-14）中有288个显著差异代谢物，其中主要差异氨基酸有15种；在ACFB与液体培养28 d菌丝体（ACM-28）中有332个显著差异代谢物，其中主要差异氨基酸有11种；ACM-14与ACM-28间存在283个显著差异代谢物，其中主要差异氨基酸有14种。牛樟芝子实体与菌丝体间氨基酸代谢差异大于菌丝体间，其中以L-精氨酸差异尤为显著。本研究为挖掘牛樟芝子实体、菌丝体氨基酸标记性代谢物和开发产品等提供理论参考。

关键词: 牛樟芝, LC-QTOF-MS, 子实体, 菌丝体, 氨基酸代谢

Abstract:

To analyze the amino acid differences in the metabolism of the fruiting body and mycelium of Taiwanofungus camphoratus, the LC-QTOF-MS system spectrometry was used to analyze the metabolome of three groups of samples, including the fruiting body and mycelium of T. camphoratus, and the SIMCA software（v16.0.2）was used to screen and identify the main amino acid metabolites, seek important differential metabolites, analyze important metabolic pathways, and tap potential markers for diversity analysis. Principal component analysis（PCA）and orthogonal projections to latent structures- discriminant analysis（OPLS-DA）among the samples demonstrated that there were differences among samples under negative ion conditions. Under the condition of t-test P < 0.05 and VIP > 1, 288 metabolites were significantly different between the fruiting body（ACFB）and the mycelium cultured in liquid for 14 d（ACM-14）, 15 of them were the main amino acids. There were 332 significantly different metabolites between in ACFB and the mycelium cultured in liquid for 28 d（ACM-28）, 11 of them were the main amino acids. There were 283 significantly different metabolites between ACM-14 and ACM-28, of which 14 were the main amino acids. The difference of amino acid metabolism between the fruiting body and mycelia of Taiwanofungus camphoratus was greater than that between the mycelia, especially the difference of L-arginine. All above results may provide a theoretical basis for studying the differences of amino acid metabolism in fruiting body and mycelium of T. camphoratus, mining potential marker metabolites and developing products.

Key words: Taiwanofungus camphoratus, LC-QTOF-MS, fruiting body, mycelium, amino acid metabolism

郑焕, 林冬梅, 刘峻源, 张引莲, 林标声, 林占熺, 李晶. 基于LC-QTOF-MS代谢组学解析牛樟芝子实体和菌丝体中氨基酸代谢差异[J]. 生物技术通报, 2023, 39(5): 254-266.

ZHENG Huan, LIN Dong-mei, LIU Jun-yuan, ZHANG Yin-lian, LIN Biao-sheng, LIN Zhan-xi, LI Jing. Analysis of Amino Acid Metabolism Difference Between Fruiting Body and Mycelium of Taiwanofungus camphoratus by LC-QTOF-MS Metabonomics[J]. Biotechnology Bulletin, 2023, 39(5): 254-266.

图/表 12

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时间 Time/min	流速 Row/（μL·min^-1）	流动相A A/%	流动相B B/%
0.00	300	15	85
1.00	300	15	85
12.00	300	35	65
12.10	300	60	40
15.00	300	60	40
15.10	300	15	85
20.00	300	15	85

时间 Time/min	流速 Row/（μL·min^-1）	流动相A A/%	流动相B B/%
0.00	300	15	85
1.00	300	15	85
12.00	300	35	65
12.10	300	60	40
15.00	300	60	40
15.10	300	15	85
20.00	300	15	85

序号 No.	物质名称 Name	分子式 Molecular formula	保留时间 Retention time/s	VIP	P-value	Log₂fold change及代谢趋势Log₂fold change and metabolic trend	代谢途径 Metabolic pathway
1	L-精氨酸 L-Arginine	C₇H₁₆N₄O₂	919.066	3.0498	3.97E-02	2.12↑	精氨酸代谢、单菌霉素代谢、克拉维酸生物合成、精氨酸和脯氨酸代谢
2	NG,NG'-二甲基-L-精氨酸NG,NG-dimethyl-L-arginine（ADMA）	C₈H₁₉ClN₄O₂	803.221	2.80174	4.89E-04	-1.49 ↓	精氨酸代谢
3	甘氨酰-L-亮氨酸Glycyl-L-leucine	C₈H₁₆N₂O₃	247.711	1.17277	2.18E-04	-2.09 ↓	-
4	酪胺 Tyramine	C₈H₁₁NO	179.924	1.27499	1.80E-05	-2.41↓	酪氨酸代谢、异喹啉生物碱生物合成、碳水化合物代谢
5	L-谷氨酰胺 L-Glutamate	C₅H₈NO₄	523.018	2.31784	1.39E-10	-2.47 ↓	精氨酸合成，嘌呤代谢，嘧啶代谢，丙氨酸、天冬氨酸和谷氨酸代谢、谷氨酸和谷氨酰胺代谢、氮代谢、氨酰合成
6	D-焦谷氨酸 D-Proline	C₅H₉NO₃	332.833	3.88710	4.12E-10	-3.16 ↓	精氨酸和脯氨酸代谢
7	L-酵母氨酸 L-Saccharopine	C₁₁H₁₉N₂O₆	657.875	1.07415	1.40E-07	-3.49 ↓	赖氨酸合成
8	L-苯丙氨酸 L-Phenylalanine	C₉H₁₁NO₂	214.720	1.62936	1.07E-04	-3.58 ↓	苯丙氨酸代谢、苯丙氨酸、酪氨酸和色氨酸生物合成、托烷、哌啶和吡啶生物碱生物合成、芥子油苷生物合成
9	L-高丝氨酸 L-Homoserine	C₄H₉NO₃	414.936	1.00736	3.31E-10	-4.14 ↓	甘氨酸、丝氨酸和苏氨酸代谢、甘氨酸、丝氨酸和苏氨酸代谢、甘氨酸、丝氨酸和苏氨酸代谢、硫代谢
10	L赖氨酸 L-Lysine	C₆H₁₄N₂O₂	815.743	1.10378	3.15E-06	-4.87 ↓	赖氨酸合成与降解、萜类、哌啶和吡啶生物碱生物合成、氨酰生物合成、生物碱合成
11	L-缬氨酸 L-Valine	C₅H₁₁NO₂	305.713	1.36255	2.77E-06	-4.88 ↓	缬氨酸、亮氨酸、异亮氨酸的生物合成与降解、青霉素和头孢菌素生物合成、泛酸盐和CoA生物合成
12	L-丙氨酸 L-Alanine	C₃H₇NO₂	399.437	1.24924	7.60E-07	-5.03 ↓	丙氨酸、天冬氨酸和谷氨酸代谢、半胱氨酸和蛋氨酸代谢、牛磺酸代谢、丙氨酸代谢
13	L-焦谷氨酸 L-Pyroglutamic acid	C₅H₇NO₃	293.365	3.88261	5.43E-17	-5.11 ↓	谷胱甘肽代谢
14	L-脯氨酸L-Proline	C₅H₉NO₂	334.343	1.61681	5.21E-06	-5.72 ↓	精氨酸和普氨酸代谢、碳青霉烯合成
15	羟脯氨酸Hydroxyproline	C₅H₉NO₃	398.864	1.84634	3.68E-06	-7.25 ↓	精氨酸和脯氨酸代谢

序号 No.	物质名称 Name	分子式 Molecular formula	保留时间 Retention time/s	VIP	P-value	Log₂fold change及代谢趋势Log₂fold change and metabolic trend	代谢途径 Metabolic pathway
1	L-精氨酸 L-Arginine	C₇H₁₆N₄O₂	919.066	3.0498	3.97E-02	2.12↑	精氨酸代谢、单菌霉素代谢、克拉维酸生物合成、精氨酸和脯氨酸代谢
2	NG,NG'-二甲基-L-精氨酸NG,NG-dimethyl-L-arginine（ADMA）	C₈H₁₉ClN₄O₂	803.221	2.80174	4.89E-04	-1.49 ↓	精氨酸代谢
3	甘氨酰-L-亮氨酸Glycyl-L-leucine	C₈H₁₆N₂O₃	247.711	1.17277	2.18E-04	-2.09 ↓	-
4	酪胺 Tyramine	C₈H₁₁NO	179.924	1.27499	1.80E-05	-2.41↓	酪氨酸代谢、异喹啉生物碱生物合成、碳水化合物代谢
5	L-谷氨酰胺 L-Glutamate	C₅H₈NO₄	523.018	2.31784	1.39E-10	-2.47 ↓	精氨酸合成，嘌呤代谢，嘧啶代谢，丙氨酸、天冬氨酸和谷氨酸代谢、谷氨酸和谷氨酰胺代谢、氮代谢、氨酰合成
6	D-焦谷氨酸 D-Proline	C₅H₉NO₃	332.833	3.88710	4.12E-10	-3.16 ↓	精氨酸和脯氨酸代谢
7	L-酵母氨酸 L-Saccharopine	C₁₁H₁₉N₂O₆	657.875	1.07415	1.40E-07	-3.49 ↓	赖氨酸合成
8	L-苯丙氨酸 L-Phenylalanine	C₉H₁₁NO₂	214.720	1.62936	1.07E-04	-3.58 ↓	苯丙氨酸代谢、苯丙氨酸、酪氨酸和色氨酸生物合成、托烷、哌啶和吡啶生物碱生物合成、芥子油苷生物合成
9	L-高丝氨酸 L-Homoserine	C₄H₉NO₃	414.936	1.00736	3.31E-10	-4.14 ↓	甘氨酸、丝氨酸和苏氨酸代谢、甘氨酸、丝氨酸和苏氨酸代谢、甘氨酸、丝氨酸和苏氨酸代谢、硫代谢
10	L赖氨酸 L-Lysine	C₆H₁₄N₂O₂	815.743	1.10378	3.15E-06	-4.87 ↓	赖氨酸合成与降解、萜类、哌啶和吡啶生物碱生物合成、氨酰生物合成、生物碱合成
11	L-缬氨酸 L-Valine	C₅H₁₁NO₂	305.713	1.36255	2.77E-06	-4.88 ↓	缬氨酸、亮氨酸、异亮氨酸的生物合成与降解、青霉素和头孢菌素生物合成、泛酸盐和CoA生物合成
12	L-丙氨酸 L-Alanine	C₃H₇NO₂	399.437	1.24924	7.60E-07	-5.03 ↓	丙氨酸、天冬氨酸和谷氨酸代谢、半胱氨酸和蛋氨酸代谢、牛磺酸代谢、丙氨酸代谢
13	L-焦谷氨酸 L-Pyroglutamic acid	C₅H₇NO₃	293.365	3.88261	5.43E-17	-5.11 ↓	谷胱甘肽代谢
14	L-脯氨酸L-Proline	C₅H₉NO₂	334.343	1.61681	5.21E-06	-5.72 ↓	精氨酸和普氨酸代谢、碳青霉烯合成
15	羟脯氨酸Hydroxyproline	C₅H₉NO₃	398.864	1.84634	3.68E-06	-7.25 ↓	精氨酸和脯氨酸代谢

序号 No.	物质名称 Name	分子式 Molecular formula	保留时间 Retention time/s	VIP	P-value	Log₂ fold change 及代谢趋势Log₂fold change and metabolic trend	代谢途径 Metabolic pathway
1	L-精氨酸 L-Arginine	C₇H₁₆N₄O₂	919.066	3.13864	3.43E-02	2.35 ↑	精氨酸代谢、单菌霉素代谢、克拉维酸生物合成、精氨酸和脯氨酸代谢
2	D-脯氨酸 D-Proline	C₅H₁₀ClNO₃	332.833	1.40639	6.85E-04	-1.11 ↓	精氨酸和脯氨酸代谢
3	NG,NG'-二甲基-L-精氨酸NG,NG-dimethyl-L-arginine（ADMA）	C₈H₁₉ClN₄O₂	803.221	2.50100	1.92E-03	-1.35 ↓
4	L-谷氨酰胺 L-Glutamate	C₅H₁₀N₂O₃	523.018	2.09913	3.66E-10	-2.21 ↓	精氨酸合成，嘌呤代谢，嘧啶代谢，丙氨酸、天冬氨酸和谷氨酸代谢、谷氨酸和谷氨酰胺代谢、氮代谢、氨酰合成
5	L-苯丙氨酸L-Phenylalanine	C₉H₁₁NO₂	214.720	1.11750	1.37E-08	-2.56 ↓	苯丙氨酸代谢、苯丙氨酸、酪氨酸和色氨酸生物合成、托烷、哌啶和吡啶生物碱生物合成、芥子油苷生物合成
6	酪胺 Tyramine	C₈H₁₁NO	179.924	1.54963	3.69E-11	-2.87 ↓	酪氨酸代谢、异喹啉生物碱生物合成
7	鸟氨酸 Ornithine	C₅H₁₂N₂O₂	803.355	1.03339	1.06E-04	-4.15 ↓	精氨酸合成、精氨酸和脯氨酸代谢、D-精氨酸和D-脯氨酸代谢、谷胱甘肽代谢
8	L-酵母氨酸L-Saccharopine	C₁₁H₁₉N₂O₆	657.875	1.49179	3.91E-10	-4.31 ↓	赖氨酸合成
9	L-赖氨酸 L-Lysine	C₆H₁₄N₂O₂	815.7425	1.29183	5.70E-07	-5.26 ↓	赖氨酸合成与降解、萜类、哌啶和吡啶生物碱生物合成、氨酰生物合成、生物碱合成
10	L-焦谷氨酸L-Pyroglutamic acid	C₅H₇NO₃	293.365	4.42017	6.95E-11	-5.44 ↓	谷胱甘肽代谢
11	羟脯氨酸 Hydroxyproline	C₅H₉NO₃	398.864	1.37226	1.53E-07	-6.35 ↓	精氨酸和脯氨酸代谢