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

摘要/Abstract

摘要： 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。

关键词: 纳米包装, 双孢蘑菇, 贮藏保鲜, 蛋白质组学

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

王朝,方东路,张攀容,姜雯,裴斐,胡秋辉,马宁. 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[J]. 中国农业科学, 2022, 55(23): 4728-4742.

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.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.23.013

https://www.chinaagrisci.com/CN/Y2022/V55/I23/4728

图/表 10

表1

表2

图1

图2

图3

图4

图5

表3

不同包装组双孢蘑菇贮藏期间主要差异表达蛋白"

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

表3

图6

图7

参考文献 44

[1]	BAN Z J, LI L, GUAN J F, FENG J H, WU M Y, XU X M, LI J. Modified atmosphere packaging (MAP) and coating for improving preservation of whole and sliced Agaricus bisporus. Journal of Food Science and Technology, 2014, 51(12): 3894-3901. doi: 10.1007/s13197-013-0935-9. doi: 10.1007/s13197-013-0935-9
[2]	DOKHANIEH A Y, AGHDAM M S. Postharvest browning alleviation of Agaricus bisporus using salicylic acid treatment. Scientia Horticulturae, 2016, 207: 146-151. doi: 10.1016/j.scienta.2016.05.025. doi: 10.1016/j.scienta.2016.05.025
[3]	徐静, 谷艳杰. 不同包装方式对金针菇冷藏保鲜效果的影响. 山东农业工程学院学报, 2016, 33(12): 122-124.
	XU J, GU Y J. The effect of different packaging methods on Flammulina velutipes refrigerated preservation. Journal of Shandong Agriculture and Engineering University, 2016, 33(12): 122-124. (in Chinese)
[4]	FANG D L, YANG W J, KIMATU B M, AN X X, HU Q H, ZHAO L Y. Effect of nanocomposite packaging on postharvest quality and reactive oxygen species metabolism of mushrooms (Flammulina velutipes). Postharvest Biology and Technology, 2016, 119: 49-57. doi: 10.1016/j.postharvbio.2016.04.012. doi: 10.1016/j.postharvbio.2016.04.012
[5]	YANG W J, SHI C, HU Q H, WU Y Y, FANG D L, PEI F, MARIGA A M. Nanocomposite packaging regulate respiration and energy metabolism in Flammulina velutipes. Postharvest Biology and Technology, 2019, 151: 119-126. doi: 10.1016/j.postharvbio.2019.02.003. doi: 10.1016/j.postharvbio.2019.02.003
[6]	李志啸, 杨文建, 方东路, 裴斐, 马宁, 方勇, 赵立艳, 安辛欣, 胡秋辉. 纳米包装材料对双孢菇细胞壁代谢及品质的影响. 食品科学, 2016, 37(6): 248-253.
	LI Z X, YANG W J, FANG D L, PEI F, MA N, FANG Y, ZHAO L Y, AN X X, HU Q H. Effects of nano-packaging on cell wall metabolism and postharvest qualities of Agaricus bisporus. Food Science, 2016, 37(6): 248-253. (in Chinese)
[7]	ZUO C Z, HU Q H, SU A X, XU H, LI X T, MARIGA A M, YANG W J. Nanocomposite packaging delays lignification of Flammulina velutipes by regulating phenylpropanoid pathway and mitochondrial reactive oxygen species metabolisms. Postharvest Biology and Technology, 2021, 171: 111360. doi: 10.1016/j.postharvbio.2020.111360. doi: 10.1016/j.postharvbio.2020.111360
[8]	剧柠, 苟萌, 张彤彤. 蛋白质组学技术及其在乳及乳制品中的应用研究进展. 食品与发酵工业, 2021, 47(3): 245-251.
	JU N, GOU M, ZHANG T T. Advances in proteomics and its application in milk and dairy products. Food and Fermentation Industries, 2021, 47(3): 245-251. (in Chinese)
[9]	WU X Q, JIANG L, YU M L, AN X J, MA R J, YU Z F. Proteomic analysis of changes in mitochondrial protein expression during peach fruit ripening and senescence. Journal of Proteomics, 2016, 147: 197-211. doi: 10.1016/j.jprot.2016.06.005. doi: S1874-3919(16)30244-5 pmid: 27288903
[10]	陈鹏, 周瑞阳, 蒋利和. 线粒体蛋白质组学技术及其在植物细胞质雄性不育机理研究中的应用. 南方农业学报, 2011, 42(4): 353-357. doi: 10.3969/j.issn.2095-1191.2011.04.002. doi: 10.3969/j.issn.2095-1191.2011.04.002
	CEHN P, ZHOU R Y, JIANG L H. Progress in mitochondrial proteomics technology and its applications in studying plant cytoplasmic male sterility. Journal of Southern Agriculture, 2011, 42(4): 353-357. doi: 10.3969/j.issn.2095-1191.2011.04.002. (in Chinese) doi: 10.3969/j.issn.2095-1191.2011.04.002
[11]	赵巧灵. 金枪鱼在冷藏过程中鲜度变化及差异蛋白质组学研究[D]. 杭州: 浙江工商大学, 2015.
	ZHAO Q L. Freshness variation and differential proteome analysis of tuna (Thunnus Obesus) during refrigerated storage[D]. Hangzhou: Zhejiang Gongshang University, 2015. (in Chinese)
[12]	FANG D L, YANG W J, DENG Z L, AN X X, ZHAO L Y, HU Q H. Proteomic investigation of metabolic changes of mushroom (Flammulina velutipes) packaged with nanocomposite material during cold storage. Journal of Agricultural and Food Chemistry, 2017, 65(47): 10368-10381. doi: 10.1021/acs.jafc.7b04393. doi: 10.1021/acs.jafc.7b04393 pmid: 29111700
[13]	杨波若, 李华健, 苏娅宁, 李霞, 瞿静, 陈韬. 基于微观结构和蛋白质组学分析影响猪肉持水性的差异蛋白. 食品工业科技, 2021, 42(7): 136-144.
	YANG B R, LI H J, SU Y N, LI X, QU J, CHEN T. Analysis of different proteins affecting water holding capacity of pork based on microstructure and proteomics. Science and Technology of Food Industry, 2021, 42(7): 136-144. (in Chinese)
[14]	韩冉, 毛凤鑫, 程敦公, 李豪圣, 刘建军, 曹新有, 宋健民, 辛明明, 郭军, 刘成, 刘爱峰. 全糯性小麦济糯116的籽粒蛋白质组学分析. 山东农业科学, 2021, 53(5): 1-7.
	HAN R, MAO F X, CHENG D G, LI H S, LIU J J, CAO X Y, SONG J M, XIN M M, GUO J, LIU C, LIU A F.Proteomic analysis of waxy wheat Jinuo 116. Shandong Agricultural Sciences, 2021, 53(5): 1-7. (in Chinese)
[15]	BARMAN K, ASREY R, PAL R K, JHA S K, BHATIA K. Post-harvest nitric oxide treatment reduces chilling injury and enhances the shelf-life of mango (Mangifera indica L.) fruit during low-temperature storage. The Journal of Horticultural Science and Biotechnology, 2014, 89(3): 253-260. doi: 10.1080/14620316.2014.11513076. doi: 10.1080/14620316.2014.11513076
[16]	WANG C, CHU J J, FU L L, WANG Y B, ZHAO F, ZHOU D Q. iTRAQ-based quantitative proteomics reveals the biochemical mechanism of cold stress adaption of razor clam during controlled freezing-point storage. Food Chemistry, 2018, 247: 73-80. doi: 10.1016/j.foodchem.2017.12.004. doi: S0308-8146(17)31942-8 pmid: 29277230
[17]	FANG D L, ZHENG Z, MA N, YANG W J, DAI C, ZHAO M W, DENG Z L, HU Q H, ZHAO L Y. Label-free proteomic quantification of packaged Flammulina filiformis during commercial storage. Postharvest Biology and Technology, 2020, 169: 111312. doi: 10.1016/j.postharvbio.2020.111312. doi: 10.1016/j.postharvbio.2020.111312
[18]	HU Y H, CHEN C M, XU L, CUI Y, YU X Y, GAO H J, WANG Q, LIU K, SHI Y, CHEN Q X. Postharvest application of 4-methoxy cinnamic acid for extending the shelf life of mushroom (Agaricus bisporus). Postharvest Biology and Technology, 2015, 104: 33-41. doi: 10.1016/j.postharvbio.2015.03.007. doi: 10.1016/j.postharvbio.2015.03.007
[19]	ZHANG K X, PU Y Y, SUN D W. Recent advances in quality preservation of postharvest mushrooms (Agaricus bisporus): A review. Trends in Food Science & Technology, 2018, 78: 72-82. doi: 10.1016/j.tifs.2018.05.012. doi: 10.1016/j.tifs.2018.05.012
[20]	KAMAL A S M, KHAIR A, BEGUM F, CHOWDHURY K, KARIM R. Effect of respiratory gases (O₂; CO₂) on shelf-life of fresh oyster mushrooms packaged with different sealable polymeric materials. Bangladesh Journal of Scientific and Industrial Research, 2015, 50(3): 205-210. doi: 10.3329/bjsir.v50i3.25587. doi: 10.3329/bjsir.v50i3.25587
[21]	LI W T, FREUDENBERG J, SUH Y J, YANG Y N. Using volcano plots and regularized-Chi statistics in genetic association studies. Computational Biology and Chemistry, 2014, 48: 77-83. doi: 10.1016/j.compbiolchem.2013.02.003. doi: 10.1016/j.compbiolchem.2013.02.003 pmid: 23602812
[22]	KANEHISA M, ARAKI M, GOTO S, HATTORI M, HIRAKAWA M, ITOH M, KATAYAMA T, KAWASHIMA S, OKUDA S, TOKIMATSU T, YAMANISHI Y. KEGG for linking genomes to life and the environment. Nucleic Acids Research, 2007, 36(suppl_1): D480-D484. doi: 10.1093/nar/gkm882. doi: 10.1093/nar/gkm882
[23]	ZHU Z, ZHANG Y B, LIU J, CHEN Y L, ZHANG X J. Exploring the effects of selenium treatment on the nutritional quality of tomato fruit. Food Chemistry, 2018, 252: 9-15. doi: 10.1016/j.foodchem.2018.01.064. doi: S0308-8146(18)30071-2 pmid: 29478567
[24]	ZHU N, LIU J W, YANG J S, LIN Y J, YANG Y, JI L, LI M, YUAN H L. Comparative analysis of the secretomes of Schizophyllum commune and other wood-decay basidiomycetes during solid-state fermentation reveals its unique lignocellulose-degrading enzyme system. Biotechnology for Biofuels, 2016, 9: 42. doi: 10.1186/s13068-016-0461-x. doi: 10.1186/s13068-016-0461-x
[25]	MARTEZ J, ROLAND S, ANDREAS S, PETER K, RONALD E. Perfil de eficacia y seguridad de Echinacea purpurea en la prevención de episodios de resfriado común: Estudio clínico aleatorizado, doble ciego y controlado con placebo. Revista de Fitoterapia, 2013, 13(24): 125-135. doi: 10.1002/jsfa. doi: 10.1002/jsfa
[26]	葛亚东, 潘蔚, 汪劼, 朱国萍. 柠檬酸合酶的分子生物学研究进展. 生物学杂志, 2010, 27(3): 59-62.
	GE Y D, PAN W, WANG J, ZHU G P. Advances in molecular biology of citrate synthase. Journal of Biology, 2010, 27(3): 59-62. (in Chinese)
[27]	杨文建, 单楠, 杨芹, 方勇, 赵立艳, 安辛欣, 马宁, 刘音宏, 辛志宏, 胡秋辉. 纳米包装材料延长双孢蘑菇贮藏品质的作用. 中国农业科学, 2012, 45(24): 5065-5072.
	YANG W J, SHAN N, YANG Q, FANG Y, ZHAO L Y, AN X X, MA N, LIU Y H, XIN Z H, HU Q H. Effect of nano-packing material on improvement of preservation quality of Agaricus bisporus. Scientia Agricultura Sinica, 2012, 45(24): 5065-5072. (in Chinese)
[28]	LI P Y, YIN F, SONG L J, ZHENG X L. Alleviation of chilling injury in tomato fruit by exogenous application of oxalic acid. Food Chemistry, 2016, 202: 125-132. doi: 10.1016/j.foodchem.2016.01.142. doi: 10.1016/j.foodchem.2016.01.142 pmid: 26920276
[29]	马宁, 王鹤潼, 方东路, 赵立艳, 杨文建, 裴斐, 胡秋辉. 基于线粒体能量代谢途径的金针菇采后纳米包装保鲜机制. 中国农业科学, 2020, 53(16): 3356-3371.
	MA N, WANG H T, FANG D L, ZHAO L Y, YANG W J, PEI F, HU Q H. Nano-packaging preservative mechanism of Flammulina filiformis after harvest based on mitochondrial energy status pathways. Scientia Agricultura Sinica, 2020, 53(16): 3356-3371. (in Chinese)
[30]	WU Y Y, HU Q H, LI Z X, PEI F, MUGAMBI M A, YANG W J. Effect of nanocomposite-based packaging on microstructure and energy metabolism of Agaricus bisporus. Food Chemistry, 2019, 276: 790-796. doi: 10.1016/j.foodchem.2018.10.088. doi: 10.1016/j.foodchem.2018.10.088
[31]	GAO H, CHAI H K, CHENG N, CAO W. Effects of 24- epibrassinolide on enzymatic browning and antioxidant activity of fresh-cut lotus root slices. Food Chemistry, 2017, 217: 45-51. doi: 10.1016/j.foodchem.2016.08.063. doi: 10.1016/j.foodchem.2016.08.063
[32]	CHEN Y H, SUN J Z, LIN H T, LIN M S, LIN Y F, WANG H, HUNG Y C. Salicylic acid treatment suppresses Phomopsis longanae Chi-induced disease development of postharvest longan fruit by modulating membrane lipid metabolism. Postharvest Biology and Technology, 2020, 164: 111168. doi: 10.1016/j.postharvbio.2020.111168. doi: 10.1016/j.postharvbio.2020.111168
[33]	YAO W S, XU T T, FAROOQ S U, JIN P, ZHENG Y H. Glycine betaine treatment alleviates chilling injury in zucchini fruit (Cucurbita pepo L.) by modulating antioxidant enzymes and membrane fatty acid metabolism. Postharvest Biology and Technology, 2018, 144: 20-28. doi: 10.1016/j.postharvbio.2018.05.007. doi: 10.1016/j.postharvbio.2018.05.007
[34]	LIU H, SONG L L, YOU Y L, LI Y B, DUAN X W, JIANG Y M, JOYCE D C, ASHRAF M, LU W J. Cold storage duration affects litchi fruit quality, membrane permeability, enzyme activities and energy charge during shelf time at ambient temperature. Postharvest Biology and Technology, 2011, 60: 24-30. doi: 10.1016/j.postharvbio.2010.11.008. doi: 10.1016/j.postharvbio.2010.11.008
[35]	陈牧, 刘锐, 翁屹. 三羧酸循环的发现与启示. 医学与哲学, 2012, 33(1): 71-73.
	CHEN M, LIU R, WENG Y. The discovery and revelation of tricarboxylic acid cycle. Medicine and Philosophy, 2012, 33(1): 71-73. (in Chinese)
[36]	WIENKOOP S, SAALBACH G. Proteome analysis. Novel proteins identified at the peribacteroid membrane from Lotus japonicus root nodules. Plant Physiology, 2003, 131(3): 1080-1090. doi: 10.1104/pp.102.015362. doi: 10.1104/pp.102.015362
[37]	FERNANDEZ-MOYA R, LEBER C, CARDENAS J, DA SILVA N A. Functional replacement of the Saccharomyces cerevisiae fatty acid synthase with a bacterial type II system allows flexible product profiles. Biotechnology and Bioengineering, 2015, 112(12): 2618-2623. doi: 10.1002/bit.25679. doi: 10.1002/bit.25679
[38]	LIN Y X, LIN Y F, CHEN Y H, WANG H, SHI J, LIN H T. Hydrogen peroxide induced changes in energy status and respiration metabolism of harvested longan fruit in relation to pericarp browning. Journal of Agricultural and Food Chemistry, 2016, 64(22): 4627-4632. doi: 10.1021/acs.jafc.6b01430. doi: 10.1021/acs.jafc.6b01430
[39]	ZHOU Y C, PAN X P, QU H X, UNDERHILL S J R. Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development. Journal of Bioenergetics and Biomembranes, 2014, 46(1): 59-69. doi: 10.1007/s10863-013-9538-4. doi: 10.1007/s10863-013-9538-4 pmid: 24390546
[40]	BANCHIO C, SCHANG L M, VANCE D E. Activation of CTP: phosphocholine cytidylyltransferase α expression during the S phase of the cell cycle is mediated by the transcription factor Sp1. The Journal of Biological Chemistry, 2003, 278(34): 32457-32464. doi: 10.1074/jbc.m304810200. doi: 10.1074/jbc.m304810200
[41]	HONG Y Y, ZHAO J, GUO L, KIM S C, DENG X J, WANG G L, ZHANG G Y, LI M Y, WANG X M. Plant phospholipases D and C and their diverse functions in stress responses. Progress in Lipid Research, 2016, 62: 55-74. doi: 10.1016/j.plipres.2016.01.002. doi: 10.1016/j.plipres.2016.01.002 pmid: 26783886
[42]	杨燕婷, 杨芹, 杨方美, 辛志宏, 赵立艳, 郁志芳, 胡秋辉. 纳米包装材料对金针菇的保鲜作用. 中国农业科学, 2009, 42(9): 3250-3258.
	YANG Y T, YANG Q, YANG F M, XIN Z H, ZHAO L Y, YU Z F, HU Q H. Effect of nano-packaging material on quality of Flammulina velutipes. Scientia Agricultura Sinica, 2009, 42(9): 3250-3258. (in Chinese)
[43]	KARAKURT Y, HUBER D J. Ethylene-induced gene expression, enzyme activities, and water soaking in immature and ripe watermelon (Citrullus lanatus) fruit. Journal of Plant Physiology, 2004, 161(4): 381-388. doi: 10.1078/0176-1617-01221. doi: 10.1078/0176-1617-01221 pmid: 15128025
[44]	李一路, 张晴晴, 胡卫芹, 屈钢, 洪月云. 磷脂酸磷酸酶在脂质代谢和信号转导中的作用及其调控. 植物生理学报, 2017, 53(6): 897-904.
	LI Y L, ZHANG Q Q, HU W Q, QU G, HONG Y Y. Roles and regulation of phosphatidic acid phosphatase in lipid metabolism and signaling. Plant Physiology Journal, 2017, 53(6): 897-904. (in Chinese)

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