盐胁迫下三倍体小黑杨杂种无性系叶片蛋白质差异表达分析

doi:10.3969/j.issn.1000-2006.201904027

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (2): 59-66.doi: 10.3969/j.issn.1000-2006.201904027

盐胁迫下三倍体小黑杨杂种无性系叶片蛋白质差异表达分析

张恒(), 刘晓婷, 陈嵩, 周雪燕, 司冬晶, 李莹, 赵曦阳^*()

林木遗传育种国家重点实验室,东北林业大学,黑龙江哈尔滨 150040

收稿日期:2019-04-13 修回日期:2019-12-04 出版日期:2020-03-30 发布日期:2020-04-01
通讯作者: 赵曦阳
基金资助:
国家重点研发计划(2016YFD0600404)

Analysis of differentially expressed proteins in leaves of triploid Populus simonii × P. nigra hybrid clones under salt stress

ZHANG Heng(), LIU Xiaoting, CHEN Song, ZHOU Xueyan, SI Dongjing, LI Ying, ZHAO Xiyang^*()

State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China

Received:2019-04-13 Revised:2019-12-04 Online:2020-03-30 Published:2020-04-01
Contact: ZHAO Xiyang

摘要/Abstract

摘要：

【目的】探究三倍体小黑杨生长迅速原因。通过蛋白质差异表达分析,深入研究三倍体小黑杨生长发育机理,为其优质高产育种提供参考。【方法】以三倍体小黑杨杂种无性系为材料,分别用75 mmol/L NaCl溶液胁迫处理0、4、8、12和16 d,利用蛋白质双向电泳和MALDI-TOF/TOF质谱技术,探讨其不同处理条件下叶片差异表达蛋白。【结果】共检测到4 000多个蛋白点,包含96个差异蛋白点,其中盐胁迫4、8、12、16 d与对照相比,分别鉴定出5、21、36、34个差异蛋白点,对60个蛋白点进行质谱鉴定,这些蛋白按功能可分为8类:其中光合作用占25%、能量代谢占20%、氧化还原作用占12%、抗性蛋白占8%、生物合成和代谢占7%、运输蛋白占3%、信号转导占2%和未知蛋白占23%。【结论】三倍体小黑杨杂种无性系受到盐胁迫后,多种蛋白质共同参与应答,其中主要是光合作用和能量代谢相关蛋白,本研究可以为盐胁迫下林木蛋白质组学研究提供基础。

关键词: 三倍体小黑杨, 盐胁迫, 差异蛋白, 蛋白质双向电泳, MALDI-TOF/TOF质谱

Abstract:

【Objective】 Polyploid breeding has widely been used in the genetic improvement of different tree species because of the rapid and extensive growth of polyploid plants. There are many studies describing polyploidy in Populus hybrids, but most of them focused on growth traits, wood properties and stress tolerance, and few studied their growth and development mechanisms. In this study, the salt tolerance mechanisms of polyploid were deeply studied through proteomics analysis, and their metabolic regulation mechanisms were revealed, which could provide a theoretical reference for their high yield breeding. We studied salt tolerance mechanisms of triploid Populus hybrids, which was of great significance for improving salt-tolerance of P. simonii × P. nigra. 【Method】 In this study, we used triploidP. simonii × P. nigra hybrid clones which were treated with 75 mmol/L NaCl solution for 0, 4, 8, 12 and 16 days, and we analyzed the differentially expressed proteins from leaves by two-dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry under salt stress in different treatment conditions. 【Result】 More than 4 000 protein spots were detected in this study. There were 96 protein spots showing different expression patterns after the treatment. We identified 5, 21, 36 and 34 differentially expressed protein spots under salt stress on the 4th, 8th, 12th and 16th day, respectively, compared with the control group. The up-regulated proteins were 1, 7, 17 and 9, respectively, while the rest were down-regulated proteins. Sixty proteins were successfully identified by mass spectrometry. These proteins could be divided into eight categories according to their functions, and 15 photosynthesis-related proteins, including phosphoribulokinase and photosystem Ⅱ oxygen-evolving complex protein two precursor, accounted for 25% of the total protein content. There were 12 energy metabolism-related proteins, including triosephosphate isomerase and fructose-bisphosphate aldolase 1, which accounted for 20% of the total protein content. SOD, glyoxalase I homolog family protein, and seven other redox-related proteins accounted for 12% of the total protein content. H⁺-transporting two-sector ATPase family of proteins and five other resistant proteins accounted for 8% of the total protein content. Four biosynthesis and metabolism proteins, including S-adenosylmethionine synthase 3, accounted for 7% of the total protein content. Two transport proteins accounted for 3% of the total protein content, one belonging to the MD-2-related lipid recognition domain-containing family of proteins, and the other being a temperature-induced lipocalin. The signal transduction protein only contained thylakoid lumeinal 29 ku protein and accounted for 2% of the total protein content, and 14 unknown proteins accounted for 23% of the total protein content. 【Conclusion】 Under salt stress, multiple proteins (mainly photosynthesis and energy metabolism related proteins) were co-involved in the response to salt stress. This study can provide a basis for future studies of tree proteomics under salt stress.

Key words: triploid Populus simonii × P. nigra, salt stress, differential protein, two-dimensional electrophoresis, MALDI-TOF/TOF mass spectrometry

中图分类号:

S792.11

张恒,刘晓婷,陈嵩,等. 盐胁迫下三倍体小黑杨杂种无性系叶片蛋白质差异表达分析[J]. 南京林业大学学报（自然科学版）, 2020, 44(2): 59-66.

ZHANG Heng, LIU Xiaoting, CHEN Song, ZHOU Xueyan, SI Dongjing, LI Ying, ZHAO Xiyang. Analysis of differentially expressed proteins in leaves of triploid Populus simonii × P. nigra hybrid clones under salt stress[J].Journal of Nanjing Forestry University (Natural Science Edition), 2020, 44(2): 59-66.DOI: 10.3969/j.issn.1000-2006.201904027.

图/表 4

图1

图2

表1

盐胁迫下差异蛋白表达的分析结果"

代谢途径 metabolisim		蛋白点 spot number		登录号 accession No		蛋白质名称 protein name	物种名称 plant species		理论等电点/ 分子量/ku theoretical pI/Mrc	观测等电点/ 分子量/ku observed pI/ Mrc
能量代谢 energy metabolism		173		TPIC_SPIOL		磷酸丙糖异构酶(TIM)triosephosphate isomerase	菠菜Spinacia oleracea		6.45/34.79	5.16/24.30
		181		ALFC1_PEA		果糖二磷酸醛缩酶(FBP aldolase1)fructose-bisphosphate aldolase 1	豌豆Pisum sativum		5.83/38.75	5.17/35.06
		189		RPE_ORYSJ		核酮糖磷酸-3-差向异构酶ribulose-phosphate-3-epimerase	粳稻Oryza sativa subsp. japonica		8.65/29.24	6.23/36.67
		196		MDHP_ARATH		苹果酸脱氢酶(NADP-MDH)malate dehydrogenase	拟南芥Arabidopsis thaliana		8.66/42.61	6.15/38.50
		208		TPIC_SPIOL		磷酸丙糖异构酶(TIM)triosephosphate isomerase	菠菜Spinacia oleracea		6.45/34.79	5.45/37.95
		279		PGP1B_ARATH		磷酸甘油磷酸酯酶(phosphatase 1B)phosphoglycolate phosphatase 1B	拟南芥Arabidopsis thaliana		6.89/40.19	5.10/35.56
		367		gi\|743808090		预测:木糖异构酶PREDICTED:xylose isomerase	胡杨P. euphratica		5.45/53.63	5.70/54.15
		402		gi\|566149765		3-异丙基苹果酸脱氢酶(NADP-MDH) 3-isopropylmalate dehydrogenase 2 family protein	毛果杨P. trichocarpa		5.6/43.82	5.05/39.59
		419		GAPN_APIGR		3-磷酸甘油醛脱氢酶(GAPDH) NADP-dependent glyceraldehyde-3-phosphate dehydrogenase	白芹Apium graveolens		7.49/53.88	6.56/53.12
		452		ENO_ORYSJ		烯醇酶enolase	粳稻Oryza sativa subsp. japonica		5.41/48.28	5.87/46.85
		363		gi\|743788904		预测:羧酸酯酶8predicted: probable carboxylesterase 8	胡杨P. euphratica		5.77/36.25	5.15/43.58
		555		gi\|1169528		烯醇酶2enolase 2	ENO2		5.7/48.41	6.32/70.28
光合作用 photosynthesis		72		CX5B1_ARATH		线粒体细胞色素C氧化酶亚基5b-1(C5b-1) cytochrome c oxidase subunit 5b-1,mitochondrial	拟南芥Arabidopsis thaliana		4.91/19.57	4.90/14.30
		88		UCRIA_PEA		细胞色素B6/F铁硫亚基(B6/f) cytochrome b6-f complex iron-sulfur subunit	豌豆Pisum sativum		8.63/24.68	5.12/15
		394		KPPR_ARATH		磷酸核酮糖激酶(PRK)phosphoribulokinase	拟南芥Arabidopsis thaliana		5.71/44.72	5.4/52.23
		111		PREDICTED: ATP synthase subunit d,mitochondrial-like		预测:ATP合酶d亚基(ATP synthase) predicted: ATP synthase subunit d, mitochondrial-like	胡杨P. euphratica		5.31/19.71	5.24/14.58
		147		gi\|224062595		光系统Ⅱ放氧复合体2(OEE2) photosystem Ⅱ oxygen-evolving complex protein 2 precursor	杂交杨P. trichocarpa Quinone oxidoreductase-like protein		7.68/28.34	5.78/30.5
		518		gi\|743804898		预测:转酮醇酶(TK)predicted: transketolase	胡杨P. euphratica		6.00/81.23	5.58/69.85
		529		TKTC_SOLTU		转酮醇酶(TK)transketolase	马铃薯Solanum tuberosum		5.94/80.34	5.90/58.40
		552		TKTC_SOLTU		转酮醇酶(TK)transketolase	马铃薯Solanum tuberosum		5.94/80.34	6.05/66.22
		332		gi\|119905		铁氧还蛋白-NADP还原酶(Fe-S) ferredoxin-NADP reductase,leaf isozyme	豌豆Pisum sativum		8.56/40.453	6.55/38.2
		387		KPPR_MESCR		磷酸核酮糖激酶(PRK)phosphoribulokinase	冰叶日中花 Mesembryanthemum crystallinum		6.03/44.49	5.36/37.2
		75		gi\|118430834		线粒体甘氨酸脱羧酶复合物H蛋白 mitochondrial glycine decarboxylase complex H-protein	美洲山杨P. tremuloides		4.98/17.89	4.96/14.4
		96		gi\|134142794		线粒体甘氨酸脱羧酶复合物H蛋白 mitochondrial glycine decarboxylase complex H-protein	美洲山杨P. tremuloides		4.78/17.72	4.37/15.64
		413		GCSP1_ARATH		甘氨酸脱氢酶(脱羧)1Glycine dehydrogenase (decarboxylating) 1	拟南芥Arabidopsis thaliana		6.51/113.82	5.86/40.34
		532		gi\|705245152		ATP酶亚基1ATPase subunit 1 (mitochondrion)	毛果杨P. trichocarpa		6.11/55.47	6.60/68.34
		566		RUBA_RICCO		磷酸核酮糖缩化酶大亚基蛋白亚基(RuBisCO)RuBisCO large subunit-binding protein subunit alpha (Fragment)	蓖麻Ricinus communi		4.77/52.46	4.90/62.13
氧化还原作用 REDOX reaction		60		gi\|586004		超氧化物歧化酶(SOD)superoxide dismutase [Cu-Zn]	番薯Ipomoea batatas		5.64/15.19	5.88/19.5
		78		SODCP_PINSY		超氧化物歧化酶(SOD)superoxide dismutase [Cu-Zn]	欧洲赤松Pinus sylvestris		5.16/14.54	5.43/14.5
		85		SODCP_PINSY		超氧化物歧化酶(SOD)superoxide dismutase [Cu-Zn]	欧洲赤松Pinus sylvestris		5.16/14.54	5.80/13.23
		233		gi\|283135904		DHAR类谷胱甘肽转移酶(GST) DHAR class glutathione transferase DHAR1	毛果杨P. trichocarpa		4.93/24.52	5.00/28.45
		329		gi\|224078584		乙二醛酶(CAT) glyoxalase I homolog family protein	毛果杨P. trichocarpa		5.21/33.19	5.28/35
		481		gi\|743909002		预测:乳酰谷胱甘肽裂解酶 PREDICTED: putative lactoylglutathione lyase	胡杨P. euphratica		4.87/32.93	6.24/61.23
		354		GSA_SOYBN		谷氨酸-1-半醛2,1-氨基变位酶 glutamate-1-semialdehyde 2,1-aminomutase	大豆Glycine max		5.67/49.84	5.78/37.5
信号转导 signal transduction		136		TL29_SOLLC		类囊体蛋白29 ku thylakoid lumenal 29 ku protein	大番茄Solanum lycopersicum		8.27/37.90	5.25/32.42
运输蛋白 transport protein		58		gi\|224088120		MD-2相关脂类识别蛋白(ML蛋白质) MD-2-related lipid recognition domain-containing family protein	毛果杨P. trichocarpa		5.87/16.94	6.14/11.64
运输蛋白 transport protein		119		gi\|209967467		低温载脂蛋白temperature-induced lipocalin	胡杨P. euphratica		5.73/21.59	6.08/19.88
代谢途径 metabolisim	蛋白点 spot number		登录号 accession No		蛋白质名称 protein name			物种名称 plant species	理论等电点/ 分子量/ku theoretical pI/Mrc	观测等电点/ 分子量/ku observed pI/ Mrc
生物合成和代谢 biosynthesis and metabolism	471		gi\|223635300		S-腺苷甲硫氨酸合成酶3(AdoMet) S-adenosylmethionine synthase 3			毛果杨P. trichocarpa	5.5/43.60	5.82/60.51
	449		gi\|743938030		预测:线粒体加工肽酶亚基β predicted: probable mitochondrial-processing peptidase subunit beta			胡杨P. euphratica	6.14/58.67	5.70/42.89
	67		gi\|743895771		预测:蛋白质3级结构域的部分蛋白 predicted: psbP domain-containing protein 3			胡杨P. euphratica	5.24/19.98	4.90/14.65
	54		MSRB5_ORYSJ		肽-甲硫氨酸亚砜还原酶B5 peptide methionine sulfoxide reductase B5			粳稻Oryza sativa subsp. japonica	6.81/51.72	7.21/50.89
抗性蛋白 resistant protein	458		gi\|743887120		预测:肌动蛋白Predicted: actin-7-like			胡杨P. euphratica	5.24/41.89	5.37/55.41
	443		ACT7_ARATH		肌动蛋白-7 Actin-7			拟南芥Arabidopsis thaliana	5.31/41.94	5.40/43.58
	521		gi\|566190494		H⁺-双阀门三磷腺苷转运酶 H⁺-transporting two-sector ATPase family protein			毛果杨P. trichocarpa	5.96/59.89	5.65/69.85
	524		gi\|566191212		紫色酸性磷酸酶purple acid phosphatase family protein			毛果杨P. trichocarpa	5.78/72.80	5.75/69.85
	527		gi\|743896116		预测:果桃α-L-阿拉伯呋喃糖苷酶抗体 PREDICTED: alpha-L-arabinofuranosidase 1-like			胡杨P. euphratica	5.12/74.37	5.82.67.85
未知蛋白 unknown protein	13		gi\|224108822		假设蛋白hypothetical protein POPTR_0010s16080g			毛果杨P. trichocarpa	5.86/12.02	6.30/10.0
	57		UT107_PEA		unknown protein from spot 107 of 2D-PAGE of thylakoid (Fragment)			豌豆Pisum sativum	9.63/1.58	5.46/11.33
	62		gi\|566191227		假设蛋白hypothetical protein POPTR_0010s16860g			毛果杨P. trichocarpa	5.91/14.69	5.27/31.65
	74		gi\|566258110		假设蛋白hypothetical protein POPTR_0128s00210g			毛果杨P. trichocarpa	8.76/21.99	6.07/26.94
	125		gi\|118489173		unknown			杂交杨P. trichocarpa × P. deltoides	5.72/21.54	6.02/30.23
	199		gi\|224125628		假设蛋白hypothetical protein POPTR_0013s03700g			毛果杨P. trichocarpa	5.28/28.34	4.87/26.60
	236		gi\|224081262		假设蛋白hypothetical protein POPTR_0005s08750g			毛果杨P. trichocarpa	7.71/28.02	5.68/26.4
	240		gi\|743815063		PREDICTED: uncharacterized protein At5g02240			胡杨P. euphratica	8.41/33.98	5.38/26.36
	244		gi\|224093896		假设蛋白hypothetical protein POPTR_0007s06770g			毛果杨P. trichocarpa	8.5/27.83	6.33/27.00
	337		gi\|118487575		unknown			毛果杨P. trichocarpa	7.55/43.08	6.08/39.05
	342		gi\|566210244		假设蛋白hypothetical protein POPTR_0016s14310g			毛果杨P. trichocarpa	8.68/40.80	6.25/35.80
	345		gi\|118489355		unknown			杂交杨P. trichocarpa × P. deltoides	8.17/43.09	6.50/40.12
	372		gi\|566184261		假设蛋白hypothetical protein POPTR_0008s16150g			毛果杨P. trichocarpa	6.67/34.42	5.89/38.90
	374		gi\|118481558		unknown			毛果杨P. trichocarpa	5.78/34.27	6.05/37.50

表1

图3

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盐胁迫下三倍体小黑杨杂种无性系叶片蛋白质差异表达分析

Analysis of differentially expressed proteins in leaves of triploid Populus simonii × P. nigra hybrid clones under salt stress

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