去除紫花苜蓿叶片高丰度蛋白的方法及其应用

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陈晶, 韩贵清, 尚晨, 张海玲, 李佶恺, 刘慧莹, 张月学. 去除紫花苜蓿叶片高丰度蛋白的方法及其应用.草业学报, 2015,24(7): 131-138
CHEN Jing, HAN Gui-Qing, SHAN Chen, ZHAN Hai-Ling, LI Ji-Kai, LIU Hui-Ying, ZHANG Yue-Xue. Proteomic methods for removing high-abundance proteins in alfalfa leaf.Acta Prataculturae Sinica, 2015,24(7): 131-138 复制到剪切板

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去除紫花苜蓿叶片高丰度蛋白的方法及其应用

陈晶¹, 韩贵清^1,^2*,^*, 尚晨², 张海玲², 李佶恺², 刘慧莹², 张月学²

1.哈尔滨师范大学生命科学与技术学院,黑龙江哈尔滨150025

2.黑龙江省农业科学院草业研究所,黑龙江哈尔滨150080

*通讯作者Corresponding author . E-mail:ccyj200@163.com

作者简介：陈晶（1984-）,女,黑龙江哈尔滨人,在读博士。E-mail:ccyj15@163.com

收稿日期: 2015-01-21

基金: 黑龙江省博士后特殊基金项目（LBH-TZ1209）和“十二五”支撑项目（2011BAD17B04-2）资助

摘要

紫花苜蓿叶片中大量的高丰度蛋白(核酮糖-l,5-二磷酸羧化/加氧酶,Rubisco)干扰了蛋白质的动态分辨率,严重影响蛋白质组学研究中功能蛋白的检测与鉴定。为了探究去除高丰度蛋白的适宜方法,本研究利用Mg/NP-40与聚乙二醇(PEG)预分离紫花苜蓿叶片蛋白,通过双向凝胶电泳法比较了不同浓度PEG对叶片高丰度蛋白的分离情况。电泳图谱显示0,15%,17.5%,20%PEG处理的蛋白质中分别可以检测到(335±17),(417±3),(445±7),(459±11)个蛋白质点,0,15%,17.5%处理组间差异显著( P<0.05),17.5%和20%PEG 处理组间没有差异( P<0.05)。然而,17.5%PEG能够检测到更多的差异蛋白质点,证明其更能有效沉淀高丰度蛋白,便于检测被Rubisco遮盖的蛋白质点。将该方法应用于紫花苜蓿叶片响应低温胁迫的蛋白质组学研究中检验其应用效果,与三氯乙酸/丙酮法提取的全蛋白相比,去除高丰度蛋白后鉴定出8个新的蛋白质差异点,证明该方法适用于实际的蛋白质组学研究。可见,Mg/NP-40与17.5%PEG法是最适宜去除紫花苜蓿叶片高丰度蛋白的方法。

关键词: 高丰度蛋白; 核酮糖-l; 5-二磷酸羧化/加氧酶; 聚乙二醇; 紫花苜蓿; 蛋白质组学

doi: 10.11686/cyxb2015044

Proteomic methods for removing high-abundance proteins in alfalfa leaf

CHEN Jing¹, HAN Gui-Qing^1,^2,^*, SHAN Chen², ZHAN Hai-Ling², LI Ji-Kai², LIU Hui-Ying², ZHANG Yue-Xue²

1.College of Life Sciences and Technology, Harbin Normal University, Harbin 150025, China

2.Institute of Grass Research, Heilongjiang Academy of Agriculture Sciences, Harbin 150080, China

Abstract

The higher content of high-abundance proteins (Ribulose-l, 5-bisphosphate carboxylase/oxygenase; RuBisCo) interferes with the dynamic resolution of proteins in two-dimensional electrophoresis (2-DE), affecting the detection and identification of functional proteins in proteomics. To explore suitable methods for removing high-abundance protein, Mg/NP-40 and different concentrations of polyethylene glycol (PEG) were used for protein pre-fractionation and the treatments’ influence on the separation of proteins in alfalfa leaf was compared. The results indicated that (335±17), (417±3), (445±7) and (459±11) spots were detected in treatments of 0, 15%, 17.5% and 20% PEG respectively. There were significant differences between the 0-17.5% treatments but no significant differences were found between the 17.5% and 20% treatments. More differential protein spots were detected in the 17.5% treatment, which thus proved the most effective way of removing RuBisCo proteins. In order to inspect the applicability of this result, a proteomic study of alfalfa in response to low temperatures was under taken. Compared with total proteins extracted by TCA/acetone, eight new protein spots were identified after PEG treatment to remove high-abundance proteins. The research thus indicates that pre-fractionation with Mg/NP-40 and17.5% PEG is suitable for proteomic studies of alfalfa.

Keyword: high-abundance protein; Rubisco; polyethylene glycol; alfalfa; proteomics

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蛋白质组学是21世纪的前沿热点领域之一。近十年来, 其从高度专业化转变为常规的技术手段应用于植物学研究^[1]。大量蛋白质数据的挖掘在植物基因功能分析和特殊蛋白质鉴定上发挥了重要作用, 也在一定程度上为基因组和代谢组间构建了“ 沟通” 桥梁^[2]。紫花苜蓿(Medicago sativa)是多年生豆科牧草, 其丰富的蛋白质含量及优良的适应性为草业和畜牧业的可持续发展提供了有利条件^[3]。对其非生物性(低温、盐碱、干旱、药害等)逆境因子应答反应和抗、耐性机理的蛋白质组学研究具有重要意义。但紫花苜蓿叶片中大量高丰度蛋白[如, 核酮糖-l, 5-二磷酸羧化/加氧酶(Rubisco)]的存在降低了蛋白质的动态分辨率, 极大地影响了逆境应答和代谢过程中功能蛋白的分析与鉴定。

Rubisco作为一种“ 超大量” 蛋白, 普遍存在于绿色植物中。在对拟南芥(Arabidopsis thaliana)和水稻(Oryza sativa)叶片的蛋白质组学研究中发现, 其高丰度蛋白Rubisco及其变体分别占蛋白总量的12%和35.3%, 极易遮盖邻近蛋白, 使一些低丰度蛋白无法被识别^{[4, 5]}。近年来, 国内外科学家一直致力于寻求提高低丰度蛋白分辨率的方法。Cellar 等^[6]利用Rubisco免疫耗竭柱去除了叶片组织中90%~98%的Rubisco, 增强了低丰度蛋白的检测和鉴定; 但该方法复杂、耗时且费用较高。Cho 等^[7]发现高浓度二硫苏糖醇(dithiothretiol, DTT)可以有效去除水稻叶片中的Rubisco酶, 提高双向凝胶电泳的分辨率; 但DTT 沉淀法原理及其在其他作物中的有效性还需要进一步研究和检验^[8]。另外, 也有研究发现肌醇六磷酸钠结合钙离子法可以去除植物叶片中的大部分Rubisco酶, 其在不同植物中有效性及适宜的反应温度也需要检验和筛选^{[8, 9]}。PEG是一种直链大分子聚合物, 能够破坏蛋白质分子表面的水化层而使蛋白发生沉淀作用, 最早应用于一些细菌和病毒蛋白的提纯^{[10, 11]}。Kim等^[12]在2001年采用聚乙二醇(PEG)沉淀法预分离了水稻叶片蛋白, 去除了大部分Rubisco。此后该方法又被成功应用于拟南芥^[13]、水稻^{[14, 15]}和甜瓜(Cucumis)^[16]的蛋白质组学研究中, 有效提高了低丰度蛋白的分辨率, 也是目前应用最多的去除植物高丰度蛋白的方法。与其他方法相比, PEG沉淀法更加快捷、简单、费用低廉且具有较广的适用性。到目前为止, 尚没有探究紫花苜蓿高丰度蛋白去除方法的相关报道。因此, 本试验在前人的基础上, 利用不同浓度的PEG预分离紫花苜蓿叶片蛋白, 旨在筛选去除高丰度蛋白的适宜方法; 并将该方法应用于紫花苜蓿响应低温的蛋白质组学研究中, 检测其可应用性。

1 材料与方法

1.1 材料

紫花苜蓿WL525HQ种子由黑龙江省农业科学院草业研究所提供。2014年, 紫花苜蓿种子播种于混有草炭土∶ 蛭石=2∶ 1的塑料花盆中, 置于昼/夜温度 25℃/20℃, 光周期 16 h/8 h, 湿度约为80%, 光照强度 12000 lx的人工气候箱中。培养至60 d苗龄, 移至温度 4℃, 其他条件相同的恒温箱中处理12 h。于低温处理0和12 h时分别剪取紫花苜蓿叶片若干, 液氮速冻, -80℃保存备用。

1.2 高丰度蛋白的去除

1.2.1 蛋白质提取

取冻存叶片(低温4℃处理0 h)2 g, 于液氮中研磨成粉末后加入8 mL 预冷的Mg/NP-40蛋白提取液[0.5 mol/L pH 8.3 Tris-HCl, 2%(v/v) NP-40, 0.02 mol/L 氯化镁, 0.001 mmol/L 乙二胺四乙酸(ethylene diamine tetraacetic acid, EDTA), 0.02 mol/L DTT, 0.001 mol/L苯甲基磺酰氟(phenylmethanesulfonyl fluoride, PMSF)]^[5], 在冰上研磨10 min。4℃, 12000 r/min离心30 min后取上清液, 弃沉淀。上清液分为4组, 分别加入适量50%(w/v)PEG4000至终浓度为 0, 15%, 17.5%, 20%, 充分混匀后冰浴30 min。以PEG终浓度 0为对照(Control, CK), 其余3组4℃, 12000 r/min离心1 h, 分别取上清记为S₁、S₂、S₃, 相对应沉淀记为P₁、P₂、P₃, 加入3倍体积量-20℃预冷的10% (w/v)三氯乙酸/丙酮, 混匀, -20℃过夜。4℃, 12000 r/min离心1 h, 弃上清, 沉淀中加入3倍体积量-20℃预冷丙酮, 重悬后置于-20℃ 1 h。 4℃, 12000 r/min离心30 min, 弃上清, 沉淀冻干成粉末。

1.2.2 聚丙烯酰胺凝胶(SDS-PAGE)电泳检测

蛋白质干粉中加入裂解液[7 mol/L尿素, 2 mol/L硫脲, 4%(w/v) 3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐(3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate, CHAPS), 0.04 mol/L DTT], 振荡均匀后置于摇床上持续振荡1 h, 使蛋白完全溶解。2D-Quant 试剂盒进行蛋白质定量, 留取相应体积蛋白溶液, 其余-80℃冰箱冻存。配制12%分离胶和5%浓缩胶用于SDS-PAGE电泳检测。电泳结束后, 考马斯亮蓝R-250染色, 乙醇冰乙酸脱色至条带清晰。白光扫描仪扫描凝胶, 分析蛋白质分离情况。

1.2.3 双向电泳检测

选用13 cm IPG(pH 4~7)胶条, 蛋白质上样量为650 μ g, 加入水化液[8 mol/L尿素, 2%(w/v)CHAPS, 0.3%(w/v)DTT, 1%(v/v)IPG buffer]稀释至250 μ L。第一向等电聚焦程序为:30 V, 12 h; 200 V, 1 h; 500 V, 1 h; 1000 V, 2 h; 8000 V, 3 h; 8000 V, 45000 Vh; 1000 V, 任意。第二向12.5%聚丙烯酰胺凝胶电泳程序为:10 mA/胶条, 0.5 h; 40 mA/胶条, 2.5~4 h。双向电泳后, 考马斯亮蓝R-250染色, 10%(v/v)冰醋酸脱色后, 白光扫描仪扫描凝胶, 使用ImageMaster 2D Platinum凝胶分析软件进行比对分析。

1.3 PEG沉淀法的应用性验证

1.3.1 蛋白质提取

叶片全蛋白提取用传统方法三氯乙酸/丙酮提取法。取冻存叶片(低温4℃处理0, 12 h)各0.5 g置于预冷的研钵中, 加入液氮充分研磨后转移至50 mL离心管中并加入三倍体积-20℃预冷的蛋白提取液[含10%(w/v)三氯乙酸、0.07%(v/v)β -巯基乙醇、1%(v/v)蛋白酶抑制剂的丙酮溶液], -20℃沉淀过夜。4℃, 12000 r/min离心1 h, 弃去上清, 保留沉淀。用三倍于沉淀体积的含有0.07%(v/v)的β -巯基乙醇的冷丙酮将沉淀重悬, -20℃沉淀1 h以上。4℃, 12000 r/min离心1 h, 弃去上清, 保留沉淀。将沉淀冻干成粉末, -80℃保存备用。

低丰度蛋白提取用Mg/NP-40+PEG法, PEG浓度为17.5% (详见1.2.1)。

1.3.2 双向电泳

选用24 cm IPG(pH 4~7)胶条, 蛋白质上样量为1000 μ g, 上样体积为450 μ L。第一向等电聚焦程序为:30 V, 12 h; 200 V, 2 h; 500 V, 2 h; 1000 V, 2 h; 8000 V, 3 h; 8000 V, 65000 Vh; 1000 V, 任意。第二向12.5%聚丙烯酰胺凝胶电泳程序为:1 W/胶条, 1 h; 13 W/胶条, 3.5~4 h。双向电泳后, 考马斯亮蓝R-250染色, 10%(v/v)冰醋酸脱色后, 白光扫描仪扫描凝胶, 使用ImageMaster 2D Platinum凝胶分析软件进行比对分析。

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图1 不同浓度PEG处理下蛋白质样品SDS-PAGE电泳图
P₁~P₃为PEG浓度15%, 17.5%, 20% 蛋白样品的沉淀部分; S₁~S₃为PEG浓度15%, 17.5%, 20% 蛋白样品的上清部分。Fig.1 SDS-PAGE analysis of proteins from the PEG fractionation
P₁-P₃ stand for protein precipitation of treatment with 15%, 17.5% and 20% PEG, respectively. S₁-S₃ stand for protein super nate of treatment with 15%, 17.5% and 20% PEG, respectively.

1.3.3 差异蛋白质点的鉴定

筛选出的差异点通过MADLI_TOF/TOF进行鉴定, 所得结果利用MASCOT软件搜索NCBI Medicago、NCBI Viridiplantae 和Uniprot数据库。

1.4 统计分析

本试验中, 所有处理3次重复, 相关数据通过软件DPS进行差异显著度分析。

2 结果与分析

2.1 不同浓度PEG处理下SDS-PAGE电泳分析结果

为探究不同浓度PEG对紫花苜蓿叶片高丰度蛋白的分离情况, 对各处理下蛋白质的沉淀和上清部分进行了SDS-PAGE检测。图1是以PEG浓度0%为对照组(control), 不同浓度PEG处理蛋白样品后沉淀和上清部分的SDS-PAGE电泳分析对比图。其中P₁~P₃与S₁~S₃分别对应PEG浓度15%, 17.5%, 20% 蛋白样品的沉淀和上清部分。图中对照组高丰度蛋白条带清晰, 聚集在55 kD左右处。而PEG处理后, 沉淀样P₁~P₃可见清晰的高丰度蛋白条带, 并且Rubisco分散为55 kD左右大亚基(large subunit of Rubisco, LSR)和14.4 kD左右小亚基(small subunit of Rubisco, SSR); 上清样S₁~S₃高丰度蛋白条带减弱, 其中S₂和S₃中高丰度蛋白下降更明显, 低丰度蛋白条带清晰稳定。可见, PEG可以沉淀富集紫花苜蓿叶片中的Rubisco, 17.5%, 20%的PEG均可用于去除高丰度。

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	图2 不同浓度PEG处理下2-DE对比图像右侧图像为双向电泳图中红框内蛋白质点的3D图像Fig.2 Comparation of 2-DE gels from the PEG fractionation 3D images on the right showing protein spots in the red box of 2-DE gels.

2.2 不同浓度PEG处理下双向电泳图像分析结果

图2显示不同浓度PEG处理下蛋白质样品上清部分的双向电泳图像。图中红框内为高丰度蛋白点, 用3D图像显示丰度。对照和S₁可见大量高丰度蛋白点, S₂和S₃中高丰度蛋白明显减少。而随着PEG浓度的升高可检测出的蛋白质点数目增多(表1), 与对照组重叠的点数减少, 17.5%和20%的PEG处理均可获得较多蛋白质点, 且二者没有显著差异(P< 0.05); 与对照组相比差异点数变化较大, 差异率分别为(33.33± 0.72)%, (56.62± 0.22)%, (52.28± 0.72)%, 差异显著(P< 0.05)。可见, 17.5%和20%的PEG均可用于提取低丰度蛋白, 与SDS-PAGE分

析结果一致, 但17.5%PEG处理下可检测出更多差异点(表1)。在图2的3D图像中, S₂的蛋白质点形成相对分离的单峰图像, 可观察到清晰的蛋白点和丰度; S₃则蛋白损失过多, 呈现细小尖峰图像。可见, 17.5% PEG更适宜于紫花苜蓿的高丰度蛋白的去除。

2.3 PEG沉淀法在响应低温蛋白质组学研究中的应用

为了验证PEG沉淀法在紫花苜蓿响应低温蛋白质组学研究中的可应用性, 紫花苜蓿叶片的蛋白分别用三氯乙酸/丙酮法和17.5% PEG沉淀法进行提取, 双向电泳后经ImageMaster 2D Platinum软件分析确定响应低温的差异蛋白质点。图3为4℃处理12 h下紫花苜蓿叶片的双向电泳图谱(A为三氯乙酸/丙酮法, B为17.5% PEG沉淀法), 数字分别标注了全蛋白和低丰度蛋白响应低温的差异蛋白质点。与未处理的对照组相比, 经低温后紫花苜蓿叶片蛋白(A)中有

表1 不同处理下2-DE图像蛋白点统计分析 Table 1 Statistical analysis of protein spots from the PEG fractionation

14个点蛋白质丰度变化在1.5倍以上(图3), 而PEG去除高丰度蛋白Rubisco后, 低丰度蛋白(B)中发现8个新的差异点(图3)。经MADLI_TOF/TOF鉴定, 所得结果利用MASCOT软件搜索相关数据库确定其蛋白种类(表2)。由鉴定结果可知, 紫花苜蓿叶片蛋白和低丰度蛋白中Rubisco及其变体分别占差异蛋白的50%和12.5%。去除高丰度蛋白后, 可检测出原本被遮盖或因共迁移影响的蛋白质, 其蛋白种类可见表2。可见, 17.5%的PEG可以清除大部分Rubisco, 并可应用于紫花苜蓿响应低温的蛋白质组学研究中。

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	图3 紫花苜蓿叶片响应低温的全蛋白(A)和低丰度蛋白(B)双向电泳图数字表示差异点编号Fig.3 2-DE gels of holoprotein (A) and low-abundance protein (B) in response to low temperature in alfalfa Digits represent spots numbers.

表2 响应低温的差异蛋白鉴定结果 Table 2 Result of identified proteins in response to low temperature

蛋白质点序号 Spots No.	蛋白质名称 Protein name	NCBI/Uniprot中序列号 Accession No. in NCBI/Uniprot	植物种类 Plant species	蛋白质分子量/等电点 Protein molecular weight (Da)/Protein isoelectric point	蛋白质得分 Protein score
1	Rubisco 活化酶Rubisco activase	gi\|23320705	紫花苜蓿Medicago sativa	30170.2/5.63	100.000
2	Rubisco 活化酶Rubisco activase	gi\|23320705	紫花苜蓿Medicago sativa	30170.2/5.63	100.000
3	Rubisco 活化酶Rubisco activase	RCAB_HORVU	大麦Hordeum vulgare	47425.8/7.59	100.000
4	Rubisco 活化酶Rubisco activase	gi\|23320705	紫花苜蓿Medicago sativa	30170.2/5.63	98.308
5	Rubisco 活化酶Rubisco activase	gi\|223536483	荨麻Ricinus communis	52191.7/5.35	100.000
6	Rubisco 大亚基结合蛋白β 亚基Rubisco large subunit-binding protein subunit beta	RUBB_PEA	豌豆Pisum sativum	63287.4/5.85	99.555
7	Rubisco 大亚基Rubisco large subunit	gi\|1223773	紫花苜蓿Medicago sativa	50684.6/6.22	94.268
8	转酮醇酶 Transketolase	gi\|502121526	鹰嘴豆Cicer arietinum	80412.5/6	100.000
9	转酮醇酶 Transketolase	gi\|502121526	鹰嘴豆Cicer arietinum	79905.7/6.51	100.000
10	转酮醇酶 Transketolase	gi\|502121526	鹰嘴豆Cicer arietinum	80412.5/6	100.000
11	S-腺苷-L甲硫氨酸合成酶 S-adenosyl-L-methionine synthetase	gi\|139478060	黄花苜蓿Medicago falcata	43588/5.77	100.000
12	谷氨酸 1-半醛转氨酶 Glutamate 1-semialdehyde aminotransferase	gi\|345451030	紫花苜蓿Medicago sativa	50260.5/6.36	100.000
13	非典型蛋白 PREDICTED: Uncharacterized protein LOC101499502	gi\|502140419	鹰嘴豆Cicer arietinum	36689.6/6.3	100.000
14	未命名蛋白产物 Unnamed protein product	gi\|297746499	葡萄Vitis vinifera	24323.4/7.63	95.924
L₁	Rubisco大亚基 Rubisco large subunit	gi\|404243904	紫花苜蓿Medicago sativa	21709.9/5.78	100.000
L₂	肽基脯氨酰顺反异构酶 Peptidyl-prolyl cis-trans isomerase CYP20-3	gi\|334186198	拟南芥Arabidopsis thaliana	28403.2/8.97	99.831
L₃	真核翻译起始因子 Eukaryotic translation initiation factor 5A-2	gi\|20138664	紫花苜蓿Medicago sativa	17501.7/5.41	100.000
L₄	2-半胱氨酸过氧化物还原酶 2-Cys peroxiredoxin BAS1-like	gi\|502112098	鹰嘴豆Cicer arietinum	30698.1/6.12	100.000
L₅	2-半胱氨酸过氧化物还原酶 2-Cys peroxiredoxin BAS1-like	gi\|502112102	鹰嘴豆Cicer arietinum	29144.1/6.12	100.000
L₆	5-甲基四氢蝶酰三谷氨酸高半胱氨酸甲基转移酶5-methyltetrahydropteroyltriglutamate-homocysteinemethyltransferase	METE_ARATH	拟南芥Arabidopsis thaliana	84645.6/6.09	100.000
L₇	5-甲基四氢蝶酰三谷氨酸高半胱氨酸甲基转移酶5-methyltetrahydropteroyltriglutamate-homocysteinemethyltransferase-like	gi\|525345100	鹰嘴豆Cicer arietinum	84599.7/6.01	100.000
L₈	放氧增强蛋白 Oxygen-evolving enhancer protein	gi\|502153108	鹰嘴豆Cicer arietinum	35106.8/6.24	100.000

“ L” 表示低丰度蛋白。“ L” represents low-abundance protein.

表2 响应低温的差异蛋白鉴定结果 Table 2 Result of identified proteins in response to low temperature

3 讨论

植物组织中常含有拷贝数众多的高丰度蛋白, 例如, Rubisco作为光合作用中固定CO₂关键酶, 一般占蛋白总量的30%~60%, 严重影响邻近蛋白点的识别和其他蛋白点在凝胶电泳中的共同迁移, 从而降低了低丰度蛋白的分辨率^{[17, 18, 19]}。同样的, 由于高丰度蛋白占据了样品中的一大部分, 使得在双向电泳IPG胶条上样容量一定的情况下, 低丰度蛋白的含量受到限制而不易被检测到^[20]。而低丰度蛋白作为细胞或组织中的受体分子、信号分子等参与基因表达调控, 往往具有十分重要的功能^[19]。因此, 如何去除高丰度蛋白以便分离鉴定低丰度蛋白一直是蛋白质组学研究中亟待解决的问题之一。在以往的文献中发现, 15%~20%PEG可以有效去除植物蛋白中大部分高丰度蛋白, 尤其是Rubisco的大亚基, 但在不同物种或组织中用于预分离的PEG浓度是不同的。在一定范围内, 高浓度PEG对蛋白质的沉淀效率高。Xi等^[13]利用16%的PEG去除了拟南芥中大部分高丰度蛋白; 王莹等^[21]利用20%PEG提取了水稻叶鞘中的低丰度蛋白; 钟俐等^[16]研究发现15%PEG更适宜于甜瓜叶片的低丰度蛋白提取。本研究表明, 适宜的PEG浓度可以去除紫花苜蓿叶片的高丰度蛋白, 且这种筛选是十分必要的。PEG浓度过低无法有效沉淀高丰度蛋白(图2中S₁); 浓度过高则易造成蛋白质过度沉淀, 蛋白质点损失(图2中S₃)。本研究中, 17.5%PEG能够清除大部分高丰度蛋白, 使原本被遮盖的蛋白质点更易于被检测到; 同时避免了蛋白的过度损失。可见, 该浓度最适宜紫花苜蓿叶片高丰度蛋白的去除。

为了验证PEG沉淀法在紫花苜蓿蛋白质组学研究中的可行性, 比较了三氯乙酸/丙酮法与PEG去除叶片高丰度蛋白后响应低温的差异蛋白变化。三氯乙酸/丙酮法是提取植物蛋白质的常用方法之一^{[22, 23, 24, 25]}, 具有降低次生代谢物质的干扰、减少蛋白降解等优点^{[26, 27]}。与其提取的蛋白相比, 17.5% PEG去除了大部分Rubisco, 并检测到8个响应低温的新蛋白质。其中Rubisco大亚基(点L₁)主要参与光合作用过程中CO₂的固定; 肽酰脯氨酰-顺反式异构酶(点L₂)和真核翻译起始因子(点L₃)参与蛋白质的翻译和折叠加工^{[25, 28, 29, 30]}; 2-半胱氨酸过氧化物还原酶(点L₄和L₅)响应低温胁迫下机体内部的氧化还原平衡^[31]; 5-甲基四氢蝶酰三谷氨酸高半胱氨酸甲基转移酶(点L₆和L₇)是一类甲基转移酶, 可为S-甲硫氨酸腺苷途径提供底物; 而放氧增强蛋白(点L₈)作为获光复合体的组成元件, 能通过激发叶绿素而获得更多的光能, 并把能量转移到光化反应中心, 有助于增强低温逆境下的光合作用效率^[32]。这些新发现的蛋白点在紫花苜蓿叶片中由于高丰度蛋白遮盖而无法检测到。可见, 利用17.5%PEG去除高丰度蛋白在紫花苜蓿蛋白质组学研究中有实际的应用意义, 有助于低丰度蛋白的检测。该方法也可用于紫花苜蓿生长发育或逆境应答的蛋白质组学研究, 有助于功能蛋白的分析与鉴定。

4 结论

高丰度蛋白影响了紫花苜蓿叶片中低丰度蛋白的鉴定。本研究采用PEG沉淀法, 比较了0, 15%, 17.5%和20%的PEG对高丰度蛋白的分离情况, 结果显示, 17.5%的PEG最适宜用于紫花苜蓿叶片高丰度蛋白的去除。此法应用于紫花苜蓿低温应答的差异蛋白鉴定中, 获得8个新差异蛋白, 证明其适用于紫花苜蓿的蛋白质组学研究。

The authors have declared that no competing interests exist.

参考文献

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2013

3.637

0.0

Andrew W

, Hiren J

, Joshua L

Managing the green proteomes for the next decade of plant research.

Frontiers in Plant Science, 2013, (4): 501.

Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen Copenhagen, Denmark.

PMID: 24379820

... 近十年来,其从高度专业化转变为常规的技术手段应用于植物学研究^[1] ...

2006

3.262

0.0

... 桥梁^[2] ...

2014

0.0

Kang J

, Zhang T

, Wang M

, et al.

Research process in the quantitative trait loci (QTL) and genomic selection of alfalfa.

Acta Prataculturae Sinica, 2014, 23(5): 304-312.

Although tetraploid alfalfa is one of the most practically important forage legumes, its genetic mapping and the QTL identification of its significant traits are lagging behind other diploid crops due to its complex genetic background. High-density genetic and physical mapping of diploid alfalfa was initiated several years ago and these studies have laid the theoretical foundation for the genetic mapping and QTL identification of tetraploid alfalfa. This new work on tetraploid alfalfa will be greatly accelerated by the rapid development of third generation molecular markers and sequencing technology. On this basis it will be possible to increase the efficiency of alfalfa breeding programs, especially by using marker-assisted selection. This paper reviews the genetic map construction and QTL analysis of alfalfa. It outlines research progress and the prospects of association mapping and genomic selection in the future.

四倍体紫花苜蓿是重要的豆科牧草之一,由于其复杂的遗传背景与二倍体作物相比遗传作图与重要性状数量性状位点(quantitative trait locus,QTL)定位研究相对滞后。然而,二倍体苜蓿的相关研究起步较早,已经建立了高密度遗传图谱和物理图谱,这些研究为四倍体苜蓿遗传作图与QTL定位奠定了基础。随着第三代分子标记与测序技术的快速发展,极大地促进了四倍体苜蓿的高密度遗传图谱构建与QTL定位研究,并借助分子标记辅助育种技术对提高苜蓿选育效率,加速育种进程具有重要意义。本文对苜蓿遗传图谱构建与QTL定位研究及发展趋势进行了总结,并对苜蓿关联作图与全基因组选择的研究进展及应用前景加以概述,旨在为读者就相关研究领域有较全面的了解。

... 紫花苜蓿(Medicago sativa)是多年生豆科牧草,其丰富的蛋白质含量及优良的适应性为草业和畜牧业的可持续发展提供了有利条件^[3] ...

2005

3.973

0.0

... 3%,极易遮盖邻近蛋白,使一些低丰度蛋白无法被识别^[4,5] ...

2006

0.0

... 3%,极易遮盖邻近蛋白,使一些低丰度蛋白无法被识别^[4,5] ...

... 001 mol/L苯甲基磺酰氟(phenylmethanesulfonyl fluoride,PMSF)]^[5],在冰上研磨10 min ...

2008

0.0

Cellar N

, Kuppannan

, Langhorst M

, et al.

Cross species applicability of abundant protein depletion columns for ribulose-1, 5-bisphosphate carboxylase/oxygenase.

Journal of Chromatography, 2008, 861: 29-39.

The Dow Chemical Company, Analytical Sciences, Building 1897, Midland, MI 48667, United States. ncellar@dow.com

In plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an important enzyme in the Calvin cycle, catalyzing the first step of carbon fixation. Because of its critical role in photosynthesis, RuBisCO comprises 30-60% of the total protein content in green leaf tissue and represents a major protein which can interfere with determination of lower abundance proteins in plant proteomics. A potential solution to aid in the determination of low level proteins in plant proteomics are RuBisCO immunodepletion columns. Two formats, spin and LC, of Seppro IgY RuBisCO depletion columns were evaluated for cross species applicability. The spin and LC columns were found to deplete arabidopsis RuBisCO by greater than 90 and 98%, respectively, and automation could be achieved with the LC format. Canola RuBisCO was depleted to a similar extent, and there was evidence suggesting that corn and tobacco RuBisCO were also highly depleted in flow through fractions. Model proteins were spiked into samples to provide insight into the degree of non-specific binding. Finally, improved detection and identification of lower abundance proteins was demonstrated after depletion.

PMID: 18063427

... Cellar 等^[6]利用Rubisco免疫耗竭柱去除了叶片组织中90%~98%的Rubisco,增强了低丰度蛋白的检测和鉴定 ...

2008

1.284

0.0

... Cho 等^[7]发现高浓度二硫苏糖醇(dithiothretiol, DTT)可以有效去除水稻叶片中的Rubisco酶,提高双向凝胶电泳的分辨率 ...

2011

0.0

Li H

, Kang Z

Study of a rapid method for depletion of highly abundant protein Rubisco enzyme from wheat (T. aestivum) leaf for proteome analysis. Journal of Northwest A&

F University (Natural Science Edition), 2011, 39(6): 223-228.

【目的】建立一种小麦叶片蛋白质组分析时去除全蛋白提取物中高丰度蛋白Rubisco酶的方法,为富集低丰度蛋白,提高双向电泳灵敏度提供技术支持。【方法】采用分离技术,以铭贤169小麦苗期叶片为材料,使用不同浓度（1,2,5,10 mmol/L）的肌醇六磷酸钠,结合二水氯化钙在不同温度（4,20,37℃）去除小麦叶片蛋白中的Rubi-sco酶,筛选去除小麦叶片蛋白 Rubisco酶的最佳条件,并采用一维凝胶电泳（1-DE）、Western blot方法检测去除Rubisco酶的有效性,用二维凝胶电泳（2-DE）评估该方法对样品在二维凝胶上分辨率的影响。【结果】在37℃条件下,采用 10 mmol/L肌醇六磷酸钠结合10 mmol/L的二水氯化钙去除小麦叶片蛋白中的Rubisco酶,可以获得较好的去除效果。在上述条件下作用10 min,小麦叶片水溶性蛋白提取物中超过80%的Rubisco酶得以沉淀去除,二维电泳图谱上有更多的低丰度蛋白点显现,且水平条纹明显减少,分辨率有所提高。【结论】得到了一种用于小麦叶片蛋白质组分析时去除高丰度蛋白Rubisco酶的方法,该方法具有快速、高效、成本低的特点。

... 但DTT 沉淀法原理及其在其他作物中的有效性还需要进一步研究和检验^[8] ...

... 另外,也有研究发现肌醇六磷酸钠结合钙离子法可以去除植物叶片中的大部分Rubisco酶,其在不同植物中有效性及适宜的反应温度也需要检验和筛选^[8,9] ...

2009

2.45

0.0

1981

0.0

... PEG是一种直链大分子聚合物,能够破坏蛋白质分子表面的水化层而使蛋白发生沉淀作用,最早应用于一些细菌和病毒蛋白的提纯^[10,11] ...

1971

0.0

Juckes I

Fractionation of proteins and viruses with polyethylene glycol.

Biochimica et Biophysica Acta, 1971, 229: 535-546.

PMID: 5103022

... PEG是一种直链大分子聚合物,能够破坏蛋白质分子表面的水化层而使蛋白发生沉淀作用,最早应用于一些细菌和病毒蛋白的提纯^[10,11] ...

2001

3.161

0.0

Kim S

, Cho K

, Jang Y

, et al.

Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays.

Electrophoresis, 2001, 22: 2103-2109.

Department of Agricultural Chemistry, Gyeongsang National University, Chinju, Korea.

Two-dimensional electrophoresis (2-DE) is known as the most effective as well as one of the simplest methods for separating proteins. However, a few hundred plant leaf proteins out of thousands visualized on a 2-DE gel can be identified by chemical analysis due to the presence of ribulose bisphosphate carboxylase/oxygenase (Rubisco) that limits protein loading. We describe the extraction and fractionation technique with polyethylene glycol (PEG) to analyze rice leaf proteins. Rice proteins were extracted with Mg/NP-40 extraction buffer. The Mg/Nonidet P-40 (NP-40) buffer extract was further fractionated with PEG into three fractions: 10% PEG and 10-20% PEG precipitants and the final supernatant fraction that was precipitated with acetone. Rubisco, the most abundant rice leaf protein, was enriched in the 20% PEG precipitant. This fractionation technique analyzed at least 2,600 well-separated protein spots and exhibited less than 1.2% of noticeable overlapping spots. An immunological approach was used to verify the efficiency whether PEG fractionation technique can detect or enrich signal transduction components such as Galpha, ADP ribosylation factor, small GTP binding protein and 14-3-3. The ADP ribosylation factor (ARF) and Galpha were only detected in the PEG supernatant fraction not in the total protein fraction. The small GTP binding protein (Rab 7) was identified in the 10% PEG fraction and only faintly in the total protein fraction. The 14-3-3 protein was detected in all fractions but was especially prevalent in the 20% PEG fraction.

PMID: 11465512

... Kim等^[12]在2001年采用聚乙二醇(PEG)沉淀法预分离了水稻叶片蛋白,去除了大部分Rubisco ...

2006

3.35

0.0

Xi J

, Wang

, Li S

, et al.

Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome.

Phytochemistry, 2006, 67: 2341-2348.

Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education (Jilin University), Changchun 130021, PR China.

Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which ca. 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants.

PMID: 16973185

... 此后该方法又被成功应用于拟南芥^[13]、水稻^[14,15]和甜瓜(Cucumis)^[16]的蛋白质组学研究中,有效提高了低丰度蛋白的分辨率,也是目前应用最多的去除植物高丰度蛋白的方法 ...

... Xi等^[13]利用16%的PEG去除了拟南芥中大部分高丰度蛋白 ...

2001

3.161

0.0

Sun T

, Kyu S

, Yu S

, et al.

Two-dimensional electrophoresis analysis of rice proteins by polyethylene glycol fractionation for protein arrays.

Electrophoresis, 2001, 22: 2103-2109.

Department of Agricultural Chemistry, Gyeongsang National University, Chinju, Korea.

PMID: 11465512

2007

1.681

0.0

Lee D

, Ahsan

, Lee S

, et al.

An approach to identify cold-induced low-abundant proteins in rice leaf.

Comptes Rendus Biologies, 2007, 330: 215-225.

Division of Applied Life Science (BK21 program), PMBBRC, Gyeongsang National University, Jinju 660-701, Republic of Korea.

A proteomic approach has been adopted to investigate the low-abundant proteins in rice leaf in response to cold stress. Rice seedlings were exposed to different temperatures, such as 5 or 10 degrees C, and samples were collected after different time course. To eliminate the high-abundant proteins in leaf tissues such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), proteins were fractionated by polyethylene glycol (PEG). The elimination of Rubisco from the protein samples was confirmed by Western blot analysis. The PEG fractionated protein samples were separated by 2-DE and visualized by silver or CBB staining. A total 12 up-regulated protein spots were identified using the analysis of MALDI-TOF mass spectrometry or ESI MS/MS. We identified some novel proteins such as cysteine proteinase, thioredoxin peroxidase, a RING zinc finger protein-like, drought-inducible late embryogenesis abundant, and a fibrillin-like protein that had not yet been reported in the earlier reports on cold proteomic analysis. The identification of some novel low-abundant proteins in response to cold stress may provide a new homeostasis to develop enhanced cold tolerance transgenic plants. Thus, we propose that a PEG fractionation system can be used as an influential protein extraction method from the leaf samples, which can lead to knowledge of the expression pattern of low-abundant proteins in response to various biotic or abiotic stresses.

PMID: 17434115

2012

0.0

Zhong

, Ma C

, Liang Y

, et al.

Depletion of Rubisco from cucumis melon leaf and optimization of two-dimensional electrophoresis for proteome.

Plant Physiology Journal, 2012, 48(3): 303-309.

为降低Rubisco的干扰, 建立适合于甜瓜叶片蛋白质的双向电泳技术,本文比较了不同全蛋白提取方法和上样量对双向电泳的影响。结果表明,Mg/NP-40/PEG3350/TCA /丙酮提取法可去除样品中绝大多数的Rubisco,使低丰度蛋白得以检测,适合后续分析。以该法提取的蛋白,采用600、800、1000和 l200μg四种上样量进行双向电泳,上样量为l000μg的样品用pH3～10的IPG胶条,在2-DE胶上分辨出质量好、数量多的蛋白质点(562 个)。因此,Mg/NP-40/PEG3350/TCA/丙酮法是适合甜瓜叶片蛋白质提取的方法,l000μg是最适上样量。

... 钟俐等^[16]研究发现15%PEG更适宜于甜瓜叶片的低丰度蛋白提取 ...

2004

3.973

0.0

Gärg

, Weiss

, Dunn M

Current two-dimensional electrophoresis technology for proteomics.

Proteomics, 2004, 4: 3665-3685.

Department of Proteomics, Technische Universität München, Freising-Weihenstephan, Germany. angelika.gorg@wzw.tum.de

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2-DE enables the separation of complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post-translational modifications. This is in contrast to liquid chromatography-tandem mass spectrometry based methods, which perform analysis on peptides, where Mr and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2-DE technology with IPGs (Görg et al., Electrophoresis 2000, 21, 1037-1053), has overcome the former limitations of carrier ampholyte based 2-DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5-12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow-overlapping IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2-DE/MS workflow including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 2-DE with IPGs; protein detection and quantitation; computer assisted analysis of 2-DE patterns; protein identification and characterization by MS; two-dimensional protein databases.

PMID: 15543535

... 3 讨论植物组织中常含有拷贝数众多的高丰度蛋白,例如,Rubisco作为光合作用中固定CO₂关键酶,一般占蛋白总量的30%~60%,严重影响邻近蛋白点的识别和其他蛋白点在凝胶电泳中的共同迁移,从而降低了低丰度蛋白的分辨率^[17,18,19] ...

2002

7.652

0.0

Lilley K

, Razzaq

, Dupree

Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation.

Current Opinion in Chemical Biology, 2002, 6: 46-50.

Department of Biochemistry, University of Cambridge, Building O, Downing Site, Cambridge CB2 1QW, UK.

A strength of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. This enables identification of the major proteins in a tissue or subcellular fraction by mass spectrometric methods. In addition, 2D PAGE can be used to compare quantities of proteins in related samples, such as those from altered environments or from mutant and wild type, thus allowing the response of classes of proteins to be determined. Those proteins showing a correlated difference in expression may participate in related processes, and this subsequently helps to define protein function. Although there are many limitations of the 2D gel technology that mean it will never be comprehensive in protein coverage, its use for the identification of relatively abundant proteins is now widespread. However, there are still surprisingly few examples of quantitative analysis of changes in protein abundance. In this review we highlight recent advances towards true quantitative analysis of 2D gels that will lead to better prediction of protein function. Despite the development of promising alternatives, 2D PAGE is likely to remain in extensive use for the foreseeable future, because the technology is now simple and readily available to many laboratories.

PMID: 11827822

2003

3.973

0.0

... 而低丰度蛋白作为细胞或组织中的受体分子、信号分子等参与基因表达调控,往往具有十分重要的功能^[19] ...

2007

0.0

Li H

, Shao

, Tan H

, et al.

Establishment and evaluation of the method for removing the high-abundance proteins in cerebrospinal fluid proteomics.

Journal of Modern Clinical Medical Bioengineering, 2007, 13(2): 94-96.

目的建立和评价脑脊液蛋白质组学中高丰度蛋白的去除方法.方法使用Oasis HLB(hydrophilic-lipophilic balance)过滤柱,运用反相固相法,根据蛋白质不同的疏水性分馏脑脊液,去除高丰度蛋白和盐.将含有低丰度蛋白的过滤液冻干,利用双向凝胶电泳(2-DE)分析评价脑脊液蛋白层析去除高丰度蛋白的效果.结果通过Oasis HLB柱处理可将大部分高丰度蛋白去除.2-DE图谱中低丰度蛋白质分辨率明显增加,点数增多59.86%.结论本研究建立的脑脊液高丰度蛋白去除方法可有效地应用于疾病的脑脊液蛋白质组学研究.

... 同样的,由于高丰度蛋白占据了样品中的一大部分,使得在双向电泳IPG胶条上样容量一定的情况下,低丰度蛋白的含量受到限制而不易被检测到^[20] ...

2011

0.0

Wang

, Cui W

, Yang M

, et al.

An approach to detect the low-abundant proteins in rice leaf sheath.

Acta Prataculturae Sinica, 2011, 20(3): 192-197.

蛋白质组学研究中,低丰度蛋白的富集、检测和鉴定是主要的技术难题.植物组织中核酮糖-1,5-二磷酸羧化/加氧酶(rubisco)等高丰度蛋白占细胞全蛋白很大比例,严重影响双向凝胶电泳(2-DE)对低丰度蛋白的检测.为探索一种适用于叶鞘低丰度蛋白的有效检测方法,本研究采用聚乙二醇(PEG)沉淀法以减少rubisco等高丰度蛋白,富集低丰度蛋白.以Mg/NP-40法为对照,分别使用15%和20%PEG分离水稻叶鞘可溶性全蛋白,并将对照和各PEG分离组分进行2-DE.2-DE图谱经软件分析结果表明,20%PEG沉淀法适用于灌浆期叶鞘低丰度蛋白检测.

... 王莹等^[21]利用20%PEG提取了水稻叶鞘中的低丰度蛋白 ...

2008

0.0

... 三氯乙酸/丙酮法是提取植物蛋白质的常用方法之一^{[22,23,24,25]},具有降低次生代谢物质的干扰、减少蛋白降解等优点^[26,27] ...

2010

0.0

Ning S

, Zhao

, Xiang X

, et al.

Proteomics of rice and grain at late growth stage under different nitrogen fertilization levels.

Chinese Journal of Applied Ecology, 2010, 21(10): 2573-2579.

Taking super-rice Liangyoupeijiu as test material, and by the method of two-dimensional gel electrophoresis (2-DE), this paper studied the changes in the leaf and grain proteomics of the variety at its late growth stage under different levels of nitrogen fertilization (1/2 times of normal nitrogen level, 20 mg·L -1 ; normal nitrogen level, 40 mg·L -1 ; 2 times of normal nitrogen level, 80 mg·L -1 ), with the biological functions of 16 leaf proteins, 9 inferior grain proteins, and 4 superior grain proteins identified and analyzed. Nitrogen fertilization could affect and regulate the plant photosynthesis via affecting the activation of photosynthesis-related enzymes and of CO 2 , the light system unit, and the constitution of electron transfer chain at the late growth stage of the variety. It could also promote the expression of the enzymes related to the energy synthesis and growth in inferior grains. High nitrogen fertilization level was not beneficial to the synthesis of starch in superior grain, but sufficient nitrogen supply was still important for the substance accumulation and metabolism. Therefore, rational nitrogen fertilization could increase the photosynthesis rate of flag leaves, enhance the source function, delay the functional early ageing, and promote the grain-filling at late growth stage.

1 School of Crop Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China| 2 School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China

以超级杂交水稻“两优培九”为试验材料，运用蛋白质双向电泳技术研究了水稻生育后期不同氮素水平下（正常供氮水平的1/2，20 mg·L -1 ；正常供氮水平，40 mg·L -1 ；正常供氮水平的2倍，80 mg·L -1 ）叶片和籽粒蛋白质组水平的变化，并鉴定分析了其差异蛋白质点（共鉴定出16个叶片蛋白质、9个弱势粒蛋白质、4个强势粒蛋白质）的生物功能.结果表明：生育后期氮素是通过影响与光合有关的酶的活化、CO 2 的活化及光系统单位和电子传递链构成来影响和调节植物的光合作用；氮素可促进弱势粒中与能量合成和生长相关酶的表达；高氮水平不利于强势粒淀粉的合成，但充足的氮素对水稻物质累积及代谢具有重要作用.因此，在生育后期合理运用氮肥对提高水稻剑叶光合性能、增强源的功能、延缓功能性早衰及强化籽粒灌浆具有积极作用.

... 三氯乙酸/丙酮法是提取植物蛋白质的常用方法之一^{[22,23,24,25]},具有降低次生代谢物质的干扰、减少蛋白降解等优点^[26,27] ...

2010

5.794

0.0

Iker

, Gemma

, Gorka

, et al.

Plant physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L. ).

Journal of Experimental Botany, 2010, 62(1): 111-123.

Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, E-08028 Barcelona, Spain. iker.aranjuelo@gmail.com

Despite its relevance, protein regulation, metabolic adjustment, and the physiological status of plants under drought is not well understood in relation to the role of nitrogen fixation in nodules. In this study, nodulated alfalfa plants were exposed to drought conditions. The study determined the physiological, metabolic, and proteomic processes involved in photosynthetic inhibition in relation to the decrease in nitrogenase (N(ase)) activity. The deleterious effect of drought on alfalfa performance was targeted towards photosynthesis and N(ase) activity. At the leaf level, photosynthetic inhibition was mainly caused by the inhibition of Rubisco. The proteomic profile and physiological measurements revealed that the reduced carboxylation capacity of droughted plants was related to limitations in Rubisco protein content, activation state, and RuBP regeneration. Drought also decreased amino acid content such as asparagine, and glutamic acid, and Rubisco protein content indicating that N availability limitations were caused by N(ase) activity inhibition. In this context, drought induced the decrease in Rubisco binding protein content at the leaf level and proteases were up-regulated so as to degrade Rubisco protein. This degradation enabled the reallocation of the Rubisco-derived N to the synthesis of amino acids with osmoregulant capacity. Rubisco degradation under drought conditions was induced so as to remobilize Rubisco-derived N to compensate for the decrease in N associated with N(ase) inhibition. Metabolic analyses showed that droughted plants increased amino acid (proline, a major compound involved in osmotic regulation) and soluble sugar (D-pinitol) levels to contribute towards the decrease in osmotic potential (Ψ(s)). At the nodule level, drought had an inhibitory effect on N(ase) activity. This decrease in N(ase) activity was not induced by substrate shortage, as reflected by an increase in total soluble sugars (TSS) in the nodules. Proline accumulation in the nodule could also be associated with an osmoregulatory response to drought and might function as a protective agent against ROS. In droughted nodules, the decrease in N(2) fixation was caused by an increase in oxygen resistance that was induced in the nodule. This was a mechanism to avoid oxidative damage associated with reduced respiration activity and the consequent increase in oxygen content. This study highlighted that even though drought had a direct effect on leaves, the deleterious effects of drought on nodules also conditioned leaf responsiveness.

PMID: 20797998

... 三氯乙酸/丙酮法是提取植物蛋白质的常用方法之一^{[22,23,24,25]},具有降低次生代谢物质的干扰、减少蛋白降解等优点^[26,27] ...

2012

5.001

0.0

Zhang

, Feng L

, Jiang

, et al.

Biochemical and proteomic analyses reveal that populus cathayana males and females have different metabolic activities under chilling stress.

Journal of Proteome Research, 2012, 11: 5815-5826.

Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu 610041, China.

Male and female poplars (Populus cathayana Rehd.) respond differently to environmental stresses. However, little is known about sex-dependent responses to chilling at the proteome level. To better understand these differences, a comparative proteomics investigation combined with a biochemical approach was used in the current study. Three-month-old poplar cuttings were treated at 25 or 4 °C for 14 days. Results revealed significant sexual differences in nitrogen metabolic enzymes and free amino acid components in response to chilling. The chilling-treated males showed higher activities of nitrate reductase and glutamine synthetase and higher contents of reduced glutathione, serine, arginine, leucine, glycine, proline and methionine than chilling-treated females. A total of 65 chilling-responsive spots were found, of which 48 showed significant sexual differences. These proteins are involved in photosynthesis, carbon and energy metabolism, metabolic processes of proteins, lipid metabolism, vitamin metabolism, stress defense, and gene expression regulation. The study shows that males have more effective metabolic processes and protective systems to chilling than females.

PMID: 23072643

... 三氯乙酸/丙酮法是提取植物蛋白质的常用方法之一^{[22,23,24,25]},具有降低次生代谢物质的干扰、减少蛋白降解等优点^[26,27] ...

... 肽酰脯氨酰-顺反式异构酶(点L₂)和真核翻译起始因子(点L₃)参与蛋白质的翻译和折叠加工^{[25,28,29,30]} ...

1988

3.161

0.0

... 三氯乙酸/丙酮法是提取植物蛋白质的常用方法之一^{[22,23,24,25]},具有降低次生代谢物质的干扰、减少蛋白降解等优点^[26,27] ...

1999

0.0

Tsugita

, Kamo

2-D electrophoresis of plant proteins.

Methods in Molecular Biology, 1999, 112: 95-97.

Research Institute for Biosciences, Science University of Tokyo, Chiba, Japan.

PMID: 10027232

... 三氯乙酸/丙酮法是提取植物蛋白质的常用方法之一^{[22,23,24,25]},具有降低次生代谢物质的干扰、减少蛋白降解等优点^[26,27] ...

1998

0.0

Schatz

, Oft

, Dascher

, et al.

Interaction of the HIV-1 Rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5.

Proceedings of National Academy of Sciences, 1998, 95: 1607-1612.

Medical Policlinic, Pettenkoferstr. 8a, D-80336 Munich, Germany.

It has previously been shown that interaction of eukaryotic initiation factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope. Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems. Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A. Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity. Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5. Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts.

PMID: 9465063

... 肽酰脯氨酰-顺反式异构酶(点L₂)和真核翻译起始因子(点L₃)参与蛋白质的翻译和折叠加工^{[25,28,29,30]} ...

2006

0.0

... 肽酰脯氨酰-顺反式异构酶(点L₂)和真核翻译起始因子(点L₃)参与蛋白质的翻译和折叠加工^{[25,28,29,30]} ...

2004

13.479

0.0

Thompson J

, Hopkins M

, Taylor

, et al.

Regulation of senescence by eukaryotic translation initiation factor 5A: implications for plant growth and development.

Trends Plant Science, 2004, 9: 174-179.

Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1. jet@sciborg.uwaterloo.ca

Regulation of protein synthesis is increasingly being recognized as an important determinant of cell proliferation and senescence. In particular, recent evidence indicates that eukaryotic translation initiation factor 5A (eIF-A) plays a pivotal role in this determination. Separate isoforms of eIF-5A appear to facilitate the translation of mRNAs required for cell division and cell death. This raises the possibility that eIF-5A isoforms are elements of a biological switch that is in one position in dividing cells and in another position in dying cells. Changes in the position of this putative switch in response to physiological and environmental cues are likely to have a significant impact on plant growth and development.

PMID: 15063867

... 肽酰脯氨酰-顺反式异构酶(点L₂)和真核翻译起始因子(点L₃)参与蛋白质的翻译和折叠加工^{[25,28,29,30]} ...

2011

2.352

0.0

Kim M

, Kim Y

, Kwon S

, et al.

Overexpression of 2-cysteine peroxiredoxin enhances tolerance to methyl viologen-mediated oxidative stress and high temperature in potato plants.

Plant Physiology and Biochemistry, 2011, 49: 891-897.

Environmental Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yusong-gu, Daejeon 305-806, Republic of Korea.

Oxidative stress is one of the major causative factors for injury to plants exposed to environmental stresses. Plants have developed diverse defense mechanisms for scavenging oxidative stress-inducing molecules. The antioxidative enzyme 2-cysteine peroxiredoxin (2-Cys Prx) removes peroxides and protects the photosynthetic membrane from oxidative damage. In this study, transgenic potato (Solanum tuberosum L. cv. Atlantic) expressing At2-Cys Prx under control of the oxidative stress-inducible SWPA2 promoter or enhanced CaMV 35S promoter (referred to as SP and EP plants, respectively) was generated using Agrobacterium-mediated transformation. The transgenic plants were tested for tolerance to stress. Following treatment with 3 μM methyl viologen (MV), leaf discs from SP and EP plants showed approximately 33 and 15% less damage than non-transformed (NT) plants. When 300 μM MV was sprayed onto whole plants, the photosynthetic activity of SP plants decreased by 25%, whereas that of NT plants decreased by 60%. In addition, SP plants showed enhanced tolerance to high temperature at 42 °C. After treatment at high temperature, the photosynthetic activity of SP plants decreased by about 7% compared to plants grown at 25 °C, whereas it declined by 31% in NT plants. These results indicate that transgenic potato can efficiently regulate oxidative stress from various environmental stresses via overexpression of At2-Cys Prx under control of the stress-inducible SWPA2 promoter.

PMID: 21620719

... 2-半胱氨酸过氧化物还原酶(点L₄和L₅)响应低温胁迫下机体内部的氧化还原平衡^[31] ...

1996

0.0

Green B

, Durnford D

The chlorophyll-carotenoid proteins of oxygenic photosynthesis.

Annual Review of Plant Physiology and Plant Molecular Biology, 1996, 47: 685-714.

Department of Botany, University of British Columbia, Vancouver, British Columbia, V6T 1Z4 Canada, Department of Applied Science, Brookhaven National Laboratory, Upton, Long Island, 11973 New York.

The chlorophyll-carotenoid binding proteins responsible for absorption and conversion of light energy in oxygen-evolving photosynthetic organisms belong to two extended families: the Chl a binding core complexes common to cyanobacteria and all chloroplasts, and the nuclear-encoded light-harvesting antenna complexes of eukaryotic photosynthesizers (Chl a/b, Chl a/c, and Chl a proteins). There is a general consensus on polypeptide and pigment composition for higher plant pigment proteins. These are reviewed and compared with pigment proteins of chlorophyte, rhodophyte, and chromophyte algae. Major advances have been the determination of the structures of LHCII (major Chl a/b complex of higher plants), cyanobacterial Photosystem I, and the peridinen-Chl a protein of dinoflagellates to atomic resolution. Better isolation methods, improved transformation procedures, and the availability of molecular structure models are starting to provide insights into the pathways of energy transfer and the macromolecular organization of thylakoid membranes.

PMID: 15012305

... 而放氧增强蛋白(点L₈)作为获光复合体的组成元件,能通过激发叶绿素而获得更多的光能,并把能量转移到光化反应中心,有助于增强低温逆境下的光合作用效率^[32] ...

2014

0.0

康俊梅, 张铁军, 王梦颖, 等. 紫花苜蓿QTL与全基因组选择研究进展及其应用. 草业学报, 2014, 23(5): 304-312.

... 紫花苜蓿(Medicago sativa)是多年生豆科牧草,其丰富的蛋白质含量及优良的适应性为草业和畜牧业的可持续发展提供了有利条件^[3] ...

2011

0.0

李红兵, 康振生. 小麦叶片蛋白质组分析中高丰度蛋白Rubisco酶的快速去除方法研究. 西北农林科技大学学报(自然科学版), 2011, 39(6): 223-228.

... 但DTT 沉淀法原理及其在其他作物中的有效性还需要进一步研究和检验^[8] ...

2012

0.0

钟俐, 马成梅, 梁永增, 等. 甜瓜叶片中Rubisco的去除及双向电泳体系的优化. 植物生理学报, 2012, 48(3): 303-309.

... 钟俐等^[16]研究发现15%PEG更适宜于甜瓜叶片的低丰度蛋白提取 ...

2007

0.0

李焕敏, 邵明, 谭红愉, 等. 脑脊液蛋白质组学高丰度蛋白去除方法的建立与评价. 现代临床医学生物工程学杂志, 2007, 13(2): 94-96.

... 同样的,由于高丰度蛋白占据了样品中的一大部分,使得在双向电泳IPG胶条上样容量一定的情况下,低丰度蛋白的含量受到限制而不易被检测到^[20] ...

2011

0.0

王莹, 崔为同, 杨明峰, 等. 一种有效检测水稻叶鞘低丰度蛋白的方法. 草业学报, 2011, 20(3): 192-197.

... 王莹等^[21]利用20%PEG提取了水稻叶鞘中的低丰度蛋白 ...

2008

0.0

... 三氯乙酸/丙酮法是提取植物蛋白质的常用方法之一^{[22,23,24,25]},具有降低次生代谢物质的干扰、减少蛋白降解等优点^[26,27] ...

2010

0.0

宁书菊, 赵敏, 向小亮, 等. 不同氮素水平下水稻生育后期叶片和籽粒的蛋白质组学. 应用生态学报, 2010, 21(10): 2573-2579.

1 School of Crop Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China| 2 School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China

... 三氯乙酸/丙酮法是提取植物蛋白质的常用方法之一^{[22,23,24,25]},具有降低次生代谢物质的干扰、减少蛋白降解等优点^[26,27] ...