盐胁迫对燕麦叶片生理指标和差异蛋白组学的影响

doi:10.3724/SP.J.1006.2019.81088

摘要/Abstract

摘要：

为探讨盐胁迫对燕麦叶片生理指标及蛋白组的影响, 对燕麦进行6 d盐胁迫(摩尔浓度NaCl∶Na₂SO₄=1∶1)处理, 测定CK与盐胁迫燕麦叶片MDA含量, SOD、POD活性与游离脯氨酸含量, 并运用Label-Free技术分析叶片差异表达蛋白质。结果表明, 盐胁迫下燕麦叶片MDA含量、SOD、POD活性分别较对照降低了16.7%、23.4%和21.2%, 游离脯氨酸较对照升高1.12%; 满足P-value≤0.05, ratio>2的差异蛋白76个(51个蛋白上调表达, 25个蛋白下调表达); 通过GO注释得到27个差异蛋白显著富集16个代谢路径, 其中氧化还原过程为33.9%, level 3统计富集的生物学过程有氧气结合和氧化还原酶活性; 运用KEGG注释得到22个差异蛋白显著富集10个生化代谢途径, 主要表现在内质网中的蛋白质加工、长寿调节途径、抗原处理和呈现、雌激素信号通路4个过程; STRING蛋白质互作网络显示21个差异蛋白中涉及翻译后修饰、蛋白质周转、分子伴侣功能的有10个, 且HSP70 (F2DYT5)和HSP90(F4Y589)可能在盐胁迫燕麦幼苗的调控中发挥重要作用。

关键词: 盐胁迫, 燕麦, Label-Free, 蛋白组

Abstract:

MDA content, SOD and POD activities, free proline content of oat leaves were determined under salt stress (molar NaCl: Na₂SO₄=1:1), and the differentially expressed proteins in leaves were analyzed by using Label-Free technique. The results showed that the activities of MDA, SOD and POD in oat leaves decreased by 16.7%, 23.4%, and 21.2% respectively, and free proline content increased by 1.12% compared with the control. There were 76 differential proteins with P-value ≤ 0.05 and ratio > 2 (51 proteins up-regulated and 25 proteins down-regulated). The GO annotation indicated that 27 differential proteins were significantly enriched in 16 metabolic pathways, of which the oxidation-reduction process was 33.9%. The biological processes of level 3 statistical enrichment were oxygen binding and oxidoreductase activity. Twenty-two differential proteins were obtained using KEGG annotation, which was significantly enriched in 10 biochemical metabolic pathways, mainly including four processes: protein processing in endoplasmic reticulum, longevity regulating pathway-multiple species, antigen processing and presentation, estrogen signaling pathway. The STRING protein interaction network showed that 10 of the 21 differential proteins involved in post-translational modification, protein turnover, and molecular chaperone function, and HSP70 and HSP90 interacted with the most core proteins of the whole network. It is speculated that the up-regulation of core protein is one of the reasons for the salt tolerance of oats.

Key words: salt stress, oat, Label-Free, proteomic

陈晓晶,刘景辉,杨彦明,赵洲,徐忠山,海霞,韩宇婷. 盐胁迫对燕麦叶片生理指标和差异蛋白组学的影响[J]. 作物学报, 2019, 45(9): 1431-1439.

CHEN Xiao-Jing,LIU Jing-Hui,YANG Yan-Ming,ZHAO Zhou,XU Zhong-Shan,HAI Xia,HAN Yu-Ting. Effects of salt stress on physiological indexes and differential proteomics of oat leaf[J]. Acta Agronomica Sinica, 2019, 45(9): 1431-1439.

图/表 7

图1

图2

图3

表1

BYC与BYS差异蛋白"

功能类 Functional class	蛋白名称 Protein ID	功能描述 Functional description	COG 编号 COG gene ID	可信度 Identity	上调/下调 Up(↑)/Down(↓)
能量生产和转换 Energy production and conversion (9)
C	A0A193CI13	FoF1-type ATP synthase, beta subunit	YP_722959	0.91	↑
C	A9LIN4	Malic enzyme	YP_007218386	0.53	↑
C	I1GZ41	Acyl-CoA reductase or other NAD-dependent aldehyde dehydrogenase	YP_722268	0.61	↑
C	A0A1V0EL65	Hemoglobin-like flavoprotein	YP_007103351	0.30	↓
C	A0A1D5S6L5	Malic enzyme	YP_005607828	0.45	↑
C	A0A1D6CUL3	NADH dehydrogenase, FAD-containing subunit	YP_003322333	0.33	↑
C	I1I051	Coenzyme F420-reducing hydrogenase, beta subunit	YP_007131148	0.55	↓
C	A0A1D6AN16	Ferredoxin-NADP reductase	YP_007118319	0.52	↓
C	W4ZQA0	Hemoglobin-like flavoprotein	YP_003692354	0.33	↓
翻译后修饰、蛋白质周转和分子伴侣 Posttranslational modification, protein turnover, and chaperones (15)
O	A0A165FYR3	Glutathione S-transferase	YP_007096787	0.31	↑
O	Q3I0N4	Molecular chaperone IbpA, HSP20 family	YP_005887446	0.38	↑
O	A0A1C6ZYA4	Molecular chaperone, HSP90 family	YP_634186	0.45	↑
O	I1H6R7	Glutathione S-transferase	YP_007063563	0.28	↑
O	F4Y589	Molecular chaperone, HSP90 family	YP_634186	0.47	↑
O	A0A1D5Y3B7	ATP-dependent Clp protease ATP-binding subunit ClpA	YP_007121403	0.69	↑
O	I1IF07	Molecular chaperone IbpA, HSP20 family	YP_001357091	0.31	↑
O	M8BCN0	Molecular chaperone DnaK (HSP70)	YP_005440675	0.49	↑
O	I1GZ93	Molecular chaperone IbpA, HSP20 family	YP_522114	0.38	↑
O	M0Y631	Chaperonin GroEL (HSP60 family)	YP_006371119	0.62	↓
O	F2E3N4	FKBP-type peptidyl-prolyl cis-trans isomerase	YP_007057296	0.66	↑
O	M8AN59	ATP-dependent Zn proteases	YP_723906	0.37	↑
O	F2DYT5	Molecular chaperone DnaK (HSP70)	YP_005440675	0.49	↑
O	A0A1D5SA32	ATP-dependent Clp protease ATP-binding subunit ClpA	YP_007132111	0.52	↑
O	W5EGU4	Molecular chaperone DnaK (HSP70)	YP_423803	0.71	↑
氨基酸转运和代谢; 辅酶转运和代谢; 一般功能预测; 次生代谢产物的生物合成、转运和分解代谢 Amino acid transport and metabolism; Coenzyme transport and metabolism; General function prediction only; Secondary metabolites biosynthesis, transport, and catabolism (15)
E	A0A1D5XXT6	Monoamine oxidase	YP_005086783	0.30	↓
E	A0A1D6CAG8	Monoamine oxidase	YP_005086783	0.29	↓
E	A0A1D5YV26	3-dehydroquinate dehydratase	NP_867287	0.36	↑
E	I1GLP9	5,10-methylenetetrahydrofolate reductase	YP_112676	0.40	↑
E	A0A1D6BBP7	Aminopeptidase N	YP_006773912	0.36	↑
E	I1HGT7	Threonine synthase	YP_002463167	0.62	↑
EH	I1GU32	3'-phosphoadenosine 5'-phosphosulfate sulfotransferase (PAPS reductase)/FAD synthetase or related enzyme	YP_007109372	0.63	↓
H	I1HWV1	Glutamine amidotransferase PdxT (pyridoxal biosynthesis)	YP_005441479	0.52	↑
HR	F2CYS4	Hydroxymethylpyrimidine pyrophosphatase and other HAD family phosphatases	YP_007159391	0.34	↑
R	A0A1D6APK4	Zn-dependent alcohol dehydrogenase	YP_001983794	0.67	↓
功能类 Functional class	蛋白名称 Protein ID	功能描述 Functional description	COG 编号 COG gene ID	可信度 Identity	上调/下调 Up(↑)/Down(↓)
R	T1MRH6	Predicted oxidoreductase (related to aryl-alcohol dehydrogenase)	YP_007098133	0.45	↑
R	W4ZPA5	Uncharacterized metalloenzyme YdcJ, glyoxalase superfamily	YP_628605	0.26	↑
QR	A0A1D5VID8	NADPH-dependent curcumin reductase CurA	YP_003953479	0.52	↑
Q	I1ICZ4	Carotenoid cleavage dioxygenase or a related enzyme	YP_007064275	0.35	↓
Q	A0A1D5UEP5	Cu²⁺-containing amine oxidase	YP_005086886	0.34	↑
碳水化合物的运输和代谢 Carbohydrate transport and metabolism (7)
G	Q38786	Beta-glucosidase/6-phospho-beta-glucosidase/beta-galactosidase	YP_004449345	0.40	↓
G	Q8S311	Sucrose-6-phosphate hydrolase SacC, GH32 family	YP_002315570	0.29	↑
G	M0WF67	6-phosphogluconate dehydrogenase	YP_007142557	0.47	↓
G	I1IJ14	Ribulose bisphosphate carboxylase small subunit	YP_007111326	0.63	↑
G	I1I5R4	Predicted arabinose efflux permease, MFS family	YP_003405887	0.40	↑
G	A0A1D6RIU0	1,4-alpha-glucan branching enzyme	YP_677957	0.47	↑
G	Q7X9A2	Ribulose bisphosphate carboxylase small subunit	YP_007111326	0.63	↑
翻译、核糖体结构和生物发生 Translation, ribosomal structure, and biogenesis (5)
J	M0WGV4	Ribosome-associated translation inhibitor RaiA	YP_007127538	0.38	↓
J	I1GM81	Ribosomal protein L4	NP_275148	0.41	↓
J	F2DBP2	Ribosomal protein L6P/L9E	YP_007126712	0.54	↑
J	I1IU29	Ribosomal protein L3	YP_004004412	0.39	↓
J	M8CXZ0	RNA recognition motif (RRM) domain	YP_844222	0.44	↓
无机离子转运和代谢 Inorganic ion transport and metabolism (2)
P	I1I9J4	Cu/Zn superoxide dismutase	YP_003290884	0.49	↓
P	R7WAY7	Carbonic anhydrase	YP_004682419	0.38	↓
脂质运输和新陈代谢 Lipid transport and metabolism (3)
I	M7ZQT4	Isopentenyldiphosphate isomerase	YP_006428266	0.39	↑
I	I1IT00	NADPH-dependent2,4-dienoyl-CoA reductase, sulfur reductase, or a related oxidoreductase	YP_824941	0.32	↑
I	O65195	Myo-inositol-1-phosphate synthase	YP_005007067	0.35	↓
信号转导机制 Signal transduction mechanisms (2)
T	I1J3C6	Phosphohistidine swiveling domain of PEP-utilizing enzymes	YP_002508078	0.47	↓
T	I1HYN7	Predicted NTPase, NACHT family domain	YP_003890644	0.30	↑
细胞壁/膜/包膜生物发生 Cell wall/membrane/envelope biogenesis (2)
M	W5BNP0	Glycosyltransferase involved in cell wall bisynthesis	YP_004596549	0.26	↑
M	I1I6H2	Nucleoside-diphosphate-sugar epimerase	YP_007093680	0.72	↑
防御机制 Defense mechanisms (1)
V	I1GTZ4	Enamine deaminase RidA, house cleaning of reactive enamine intermediates, YjgF/YER057c/UK114 family	YP_003319835	0.55	↑
功能未知 Function unknown (1)
S	I1IC12	Uncharacterized protein YjbI, contains pentapeptide repeats	YP_007125802	0.50	↑

表1

图4

图5

图6

参考文献 31

[1]	杨真, 王宝山 . 中国盐渍土资源现状及改良利用对策. 山东农业科学, 2015,47:125-130.
	Yang Z, Wang B S . Current status of saline soil resources in china and countermeasures for improvement and utilization. Shandong Agric Sci, 2015,47:125-130 (in Chinese with English abstract).
[2]	Mahajan S, Tuteja N . Cold, salinity and drought stresses: an overview. Arch Biochem Biophys, 2005,444:139-158.
[3]	张恒, 郑宝江, 宋保华, 王思宁, 戴绍军 . 植物盐胁迫应答蛋白质组学分析. 生态学报, 2011,31:6936-6946.
	Zhang H, Zheng B J, Song B H, Wang S N, Dai S J . Proteomics analysis of plant salt stress response. Acta Ecol Sin, 2011,31:6936-6946 (in Chinese with English abstract).
[4]	Kim D W, Rakwal R, Agrawal G K, Jung Y H, Shibato J, Jwa N S, Iwahashi Y, Iwahashi H, Kim D H, Shim I S, Usui K . A hydroponic rice seedling culture model system for investigating proteome of salt stress in rice leaf. Electrophoresis, 2005,26:4521-4539.
[5]	Parker R, Flowers T J, Moore A L, Harpham N V J . An accurate and reproducible method for proteome profiling of the effects of salt stress in the rice leaf lamina. J Exp Bot, 2006,57:1109-1118.
[6]	Sengupta S, Majumder A L . Insight into the salt tolerance factors of a wild halophytic rice, Porteresia coarctata: a physiological and proteomic approach. Planta, 2009,229:911-929.
[7]	Caruso G, Cavaliere C, Guarino C, Gubbiotti R, Foglia P, Laganà A . Identification of changes in Triticum durum L. leaf-proteome in response to salt stress by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Anal Bioanal Chem, 2008,391:381-390.
[8]	Huo C M, Zhao B C, Ge R C, Shen Y Z, Huang Z J . Proteomic analysis of the salt tolerance mutant of wheat under salt stress. Acta Genet Sin, 2004,31:1408-1414.
[9]	Wang B, Song F B . Physiological responses and adaptive capacity of oats to saline-alkali stress. Ecol Environ, 2006,15:625-629.
[10]	Wang B, Song F B . The effects of saline-alkali stress on water potential, percentage of dry matter and selective absorption to K⁺ and Na⁺ in oats. Syst Sci Compreh Stud Agric, 2006,22:105-108.
[11]	Wang B, Zhang J C, Song F B, Zhao M, Han X Y . Physiological responses to saline-alkali in oats. J Soil Water Conserv, 2007,21:86-89.
[12]	白健慧 . 燕麦对盐碱胁迫的生理响应机制研究. 内蒙古农业大学博士学位论文, 内蒙古呼和浩特, 2016.
	Bai J H . Physiological Response Mechanism of Oats to Saline-Alkali Stress. PhD Dissertation of Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China, 2016 (in Chinese with English abstract).
[13]	Murty A S, Misra P N, Haider M M . Effect of different salt concentrations on seed germination and seedling development in a few oat cultivars. Indian Agric Res J, 1984,18:129-132.
[14]	白宝璋, 史安国, 赵景阳 . 植物生理学. 北京: 北京农业出版社, 2001.
	Bai B Z, Shi A G, Zhao J Y . Plant Physiology. Beijing: Beijing Agricultural Press, 2001 (in Chinese).
[15]	邹琦 . 植物生理学实验指导. 北京: 中国农业出版社, 2000.
	Zou Q. Laboratory Physiology Experiment Guide. Beijing: China Agriculture Press, 2000 (in Chinese).
[16]	Bradford M M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biochem, 1976,72:248-254.
[17]	Tatusov R L, Fedorova N D, Jackson J D, Jacobs A R, Kiryutin B, Kiinin E V, Krylov D M, Mazumder R, Mekhedov S L, Nikolskaya A N, Rao B S, Smirnov S, Sverdlov A V, Vasudevan S, Wolf Y, Yin J, Natale D A . The COG data base: an updated version includes eukaryotes. BMC Bioinform, 2003,4:41.
[18]	Flowers T J, Torke P F, Yeo A R . The mechanism of salt tolerance in halophytes. Annu Rev Plant Physiol, 1977,28:89-121.
[19]	高彩婷, 刘景辉, 张玉芹, 徐寿军, 张玉霞 . 短期盐胁迫下燕麦幼苗的生理响应. 草地学报, 2017,25:337-343.
	Gao C T, Liu J H, Zhang Y Q, Xu S J, Zhang Y X . Physiological response of oat seedlings under short-term salt stress. Acta Agrest Sin, 2017,25:337-343 (in Chinese with English abstract).
[20]	杨舒贻, 陈晓阳, 惠文凯, 任颖, 马玲 . 逆境胁迫下植物抗氧化酶系统响应研究进展. 福建农林大学学报(自然科学版), 2016,45:481-489.
	Yang S Y, Chen X Y, Hui W K, Ren Y, Ma L . Research progress of plant antioxidant enzyme system response under stress. J Fujian Agric For Univ(Nat Sci Edn), 2016,45:481-489 (in Chinese with English abstract).
[21]	周莹, 赵永娟, 黄丽瑾, 唐楠煜, 唐晓清, 王康才 . 荆芥幼苗对盐胁迫的生理响应. 核农学报, 2019,33:166-175.
	Zhou Y, Zhao Y J, Huang L J, Tang N Y, Tang X Q, Wang K C . Physiological response of Nepeta seedlings to salt stress. J Nucl Agric Sci, 2019,33:166-175 (in Chinese with English abstract).
[22]	刘景辉, 胡跃高 . 燕麦抗逆性研究. 北京: 中国农业出版社, 2010.
	Liu J H, Hu Y G. Study on the Resistance of Oats. Beijing: China Agriculture Press, 2010 (in Chinese).
[23]	董靖, 李红丽, 董智, 白文华 . H₂S对NaCl胁迫下草木樨幼苗生理指标及抗氧化酶活性的影响. 草业科学, 2018,35:2430-2437.
	Dong J, Li H L, Dong Z, Bai W H . Effects of H₂S on physiological indexes and antioxidant enzyme activities of alfalfa seedlings under NaCl stress. Pratac Sci, 2018,35:2430-2437 (in Chinese with English abstract).
[24]	Zhao Z, Liu J H, Jia R Z, Bao S, Hai X, Chen X J . Physiological and TMT-based proteomic analysis of oat early seedlings in response to alkali stress. J Proteom, 2019,193:10-26.
[25]	Swindell W R, Huebner M, Weber A P . Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genom, 2007,8:125.
[26]	Jarvis P . Targeting of nucleus-encoded proteins to chloroplasts in plants. New Phytol, 2008,179:257-285.
[27]	Schulze-Lefert P . Plant immunity: the origami of receptor activation. Curr Biol, 2004,14:22-24.
[28]	Panaretou B, Zhai C . The heat shock proteins: their roles as multi-component machines for protein folding. Fungal Biol Rev, 2008,22:110-119.
[29]	Hu W H, Hu G C, Han B . Genome-wide survey and expression profiling of heat shock proteins and heat shock factors revealed overlapped and stress specific response under abiotic stresses in rice. Plant Sci, 2009,176:583-590.
[30]	Cui Y C, Xu G Y, Wang M L, Yu Y, Li M J, Pedro S C, da Rocha F, Xia X J . Expression of OsMSR₃ in Arabidopsis enhances tolerance to cadmium stress. Plant Cell Tissue Organ Cult, 2013,113:331-340.
[31]	Hamilton E W, Heekathorn S A . Mitochondriala daptations to NaCl. Complex I is protected by anti-oxidants and small heat shock proteins, whereas complex II is protected by proline and betaine. Plant Physiol, 2001,126:1266-1274.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed