小麦小花高纯度线粒体的分离及其蛋白质双向电泳体系建立

应用差速离心和Percoll不连续密度梯度法分离纯化小麦三核期小花线粒体. 在裂解液选择、IPG胶条pH值范围、SDS-PAGE胶浓度及蛋白质上样量等方面对线粒体蛋白质双向电泳体系进行探索和优化,确立了一套适用于小麦小花高纯度完整线粒体的分离方法及其蛋白质双向电泳的技术体系. 结果表明,采用20%、24%和40% Percoll密度梯度和28% Percoll自形成密度高速离心体系,获得了有活性、高纯度且较完整的线粒体;经TCA-丙酮法提取蛋白,以7 mol/L尿素,2 mol/L硫脲,4% CHAPS(W/V),65 mmol/L DTT,0.5% IPG缓冲液(V/V),0.001% 溴酚蓝(W/V)裂解液溶解蛋白,采用17 cm,pH 4～7 IPG胶条和11% SDS-PAGE分离胶,上样量为160 μg,硝酸银染色法,更适合小麦小花线粒体蛋白质组双向电泳分离. 经PDQuest 2DE 8.0.1软件包统计分析,在2-DE图谱上分辨出约150个蛋白点,蛋白点清晰呈圆形,无横条纹干扰,这为利用双向电泳技术在亚细胞水平对线粒体进行蛋白质组学研究与分析奠定了基础,更为进一步分析研究线粒体与雄性不育的关系提供了理论与技术支撑.     

茅苍术质膜蛋白的制备及其双向电泳蛋白质组学平台的构建

探讨濒危药用植物茅苍术的质膜蛋白质组方法,旨在达到了解其次级代谢物的代谢途径,并对相关药用活性成分进行代谢调控的目的。以茅苍术的根、茎、叶为实验材料,确定了超速离心结合双水相法作为提取、纯化其质膜蛋白的方法 ;进而对这些质膜蛋白的双向电泳分离条件进行了系统的优化和后期的部分蛋白质鉴定。结果表明,分别采用质量分数为6.4%、6.3%和6.1%的聚合物葡聚糖T-500和聚乙二醇PEG 3350(W/W)的双水相体系对超速离心后的茅苍术根、茎、叶粗膜组分进行处理可获得对应纯度高达92.1%、91.5%和90.8%的质膜蛋白;以2%CHAPS和2%Triton X-100作为组合变性剂裂解膜蛋白,等电聚焦总量为80 000 Vhs,浓度为12.5%-15%的梯度SDS-PAGE分离胶对100μg质膜蛋白进行双向电泳,通过Image Master 2D Platinum 7.0分析软件分别在根、茎、叶的质膜蛋白凝胶图谱上识别出了267、297和248个蛋白斑点;进一步比较分析了这3种组织质膜蛋白的异同之处,并选取其中的5个蛋白质利用基质辅助激光解吸/电离飞行时间质谱仪成功进行了鉴定。一套完整的同时适用于茅苍术根、茎、叶各个组织的从高纯度质膜蛋白的制备到膜蛋白的质谱鉴定的蛋白质组学技术平台被成功构建。     

多子房小麦蛋白质双向电泳体系的建立及优化

为建立适用于显性多子房小麦细胞质效应的蛋白质双向电泳体系,以显性多子房小麦材料DUOII与特异细胞质材料TeZhiI杂交的F1幼穗为材料,采用TCA-丙酮法提取蛋白质,并在IPG胶条长度和pH范围、SDS-PAGE凝胶浓度及蛋白质上样量等方面,对多子房小麦幼穗蛋白质双向电泳体系进行了探究与优化.结果表明,本文采用的蛋白质定量方法准确度高(R²=0.9999),确立了17 cm, pH4～7的IPG胶条, 12% SDS-PAGE分离胶,上样量为900 μg的双向电泳方法体系,获得了最适合本研究蛋白质组分析的双向电泳图谱. 经PDQuest 2DE 8.0.1软件分析,2-DE图谱上可分辨出1.444±14个清晰蛋白质点,且重复性较高(95%), 相关系数为0.960. 建立了一套适用于显性多子房小麦细胞质效应研究的蛋白质双向电泳体系.     

发菜蛋白质组双向电泳技术的建立及优化

为建立适用于发菜(Nostoc flagelliforme)蛋白质组研究的双向电泳技术,对发菜蛋白质的提取、裂解、上样量、IEF及SDS-PAGE电泳等关键步骤进行了优化,结果显示:发菜蛋白质主要分布在pH 4～7范围内,采用改良TCA法可提高提取液中蛋白质的含量和双向电泳图谱的分辨率,裂解液含60 mmol/L DTT,24 cm IPG胶条上样量1.5 mg时不仅提高了蛋白质的溶解性,而且改善了双向电泳的分离效果,得到近800个蛋白点,且蛋白点清晰,图谱分辨率较好.采用优化后的双向电泳体系提高了发菜蛋白质双向电泳的分辨率和重复性,建立起一套适用于发菜蛋白质组分析的双向电泳方法.     

蒙古沙冬青叶蛋白质双向电泳体系的建立和优化

开展蒙古沙冬青叶组织的蛋白质组学研究,需要建立和优化叶的蛋白质组双向电泳体系。本研究以蒙古沙冬青(Ammopiptanthus mongolicus)叶片为材料,比较了不同总蛋白提取方法(TCA-丙酮法和Tris-饱和酚法)、不同染色方法(考马斯亮蓝染色和硝酸银染色)和不同蛋白质上样量对双向凝胶电泳蛋白质得率和等电聚焦效果的影响。结果显示,采用TCA-丙酮法提取蒙古沙冬青叶片总蛋白,蛋白质上样量500μg,以硝酸银染色SDS-PAGE胶,双向电泳的分辨率最高,图谱清晰。该方法的建立为开展蒙古沙冬青叶片蛋白质组定量和定性分析奠定了基础。     

水稻根质膜蛋白质组双向电泳水化液的优化

以水稻(Oryza sativa L.)苗期幼嫩根尖作为材料,利用葡聚糖-聚乙二醇两相分配法纯化得到纯度达90%的质膜组分,使用4种不同的水化液溶解质膜蛋白,进行IEF/SDS-PAGE双向电泳和MALDI-TOF/TOF质谱分析.结果显示,4种水化液中,以7 mol/L Urea2、mol/L Thiourea、4%CHAPS、20 mmol/L DTE、1%ASB14的条件对膜蛋白的溶解效果和双向电泳分离效果最好;16个被鉴定蛋白中有9个为质膜相关蛋白,5个为未知蛋白,来自其它细胞器的蛋白仅有2个.研究表明,在常用水化液中添加磺基甘氨酸三甲内盐ASB14有利于植物细胞质膜蛋白质组的分析,并且该优化条件下的双向电泳适合分离水稻质膜中亲水性相对较高的膜附着蛋白.     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率(浓度)高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

荔枝果皮总蛋白质提取及双向电泳体系的建立

用TCA-丙酮、丙酮和酚3种方法提取荔枝(Litchi chinensis Sonn.)果皮的总蛋白质,比较了蛋白产量、单向SDS-PAGE和双向电泳等方面的差异,并对双向电泳体系进行探索.结果表明:酚抽提法最佳,提取的总蛋白得率最高,蛋白在单向SDS-PAGE中形成条带数目最多,最清晰;经双向电泳分离用银染显色,可检...     

适合水稻悬浮培养细胞蛋白质组分析的双向电泳技术

水稻是研究植物蛋白质组学的一个重要试材,从蛋白质的提取,裂解缓冲液的成份和浓度,第一向聚焦的条件,蛋白上样量和二向胶的选择方面,对水稻悬浮培养细胞的双向电泳条件进行了比较和优化。结果表明,采用甲醇/氯仿沉淀蛋白,裂解缓冲液中加入0.5%的两性电解质、并加入盐桥,17cm的胶条、850μg的上样量可达到较好的等电聚焦效果;8%-16%梯度胶分离蛋白较为适宜。     

洋葱伯克霍尔德菌外膜蛋白双向电泳的建立与优化

目的:洋葱伯克霍尔德菌外膜蛋门的分离及双向电泳图谱的建立和优化.方法:用月桂酰基氯酸钠法提取外膜蛋白,以同相pH梯度为第一向和SDS-PAGE为第二向进行双向电泳,对裂解液成分,IPG胶条的pH和凝胶染色方法等进行优化.结果:获得外膜蛋白浓度为2.87μg/μl;最佳裂解液成分为:7mol/L尿素,2mol/L硫脲,4%Chaps,2%pharmalyte,65 mmol/L DTT,0.5%Triton X-100,10mmol/L Tris.结论:提取的外膜蛋白满足双向电泳条件;获得理想的外膜蛋白双向电泳图谱用于后续实验中.     

小麦根蛋白提取与双向电泳方法的优化与应用

为了建立一套适合小麦根蛋白质组分析的双向电泳系统（2-DE）,得到更加清晰的电泳图谱,本研究以小麦幼苗根系为材料,对根蛋白的提取方法、上样量等进行了优化。研究发现,上样量为1200μg时,Trizol抽提法提取的蛋白能够获得图像清晰、分辨率高、重复性好的双向电泳图谱,基本满足小麦根系蛋白质组学的分析和研究。     

两相法分离地被菊叶片质膜的研究

以地被菊(Dendranthema×grandiflorum Kitamura)叶片为材料,通过水溶性聚合物Dextran T-500和PEG 3350所构成的两相分配体系制备质膜。在一定盐浓度（5 mmol.L-1 NaCl）下选用5种不同的聚合物浓度（5.8%、6.0%、6.2%、6.4%、6.6%,W/W）,研究了地被菊叶片质膜在两相体系中的分配情况,在此基础上进一步研究了不同盐浓度（2、4、5、10、20 mmol.L-1 NaCl）对地被菊叶片质膜的纯度及蛋白产率的影响。标志酶鉴定及磷钨酸染色电镜检测的结果表明,地被菊叶片选用6.2%（W/W）聚合物浓度和7 mmol.L-1 NaCl组成的两相分配体系可获得较高纯度的密实正向型质膜囊泡。     

Carbonic anhydrase in the plasma membranes from leaves of C3 and C4 plants

Plasma membranes were isolated from green leaves of maize ( Zea mays ), spinach ( Spinacia oleracea ), Setaria viridis and wheat ( Triticum aestivum cv. Omase) by aqueous two-phase partitioning. Carbonic anhydrase activity was detected in these membranes. The activity was inhibited by specific inhibitors for carbonic anhydrase, acetazolamide and ethoxyzolamide. The carbonic anhydrase activity was markedly enhanced by the addition of Triton X-100 to the plasma membranes. The highest activity was obtained in the presence of 0.015% detergent. The activity was scarcely affected when the plasma membrane vesicles were treated with proteinase K, but largely inactivated by the protease after treating the membranes with Triton X-100. These results indicate that carbonic anhydrase faces the cytoplasmic side of the membrane since plasma membranes purified by aqueous two-phase partitioning are tightly sealed vesicles of right side-out orientation (apoplastic side-out). With leaves of C₄ plants, 20 to 60% of the total carbonic anhydrase activity was found in the microsomal fraction. By contrast, only 1 to 3% of the activity was found in the microsomal fraction from leaves of C₃ plants. Western blot analysis showed that a polypeptide in the spinach plasma membrane cross-reacted with an antiserum raised against spinach chloroplast carbonic anhydrase, and that the molecular mass of the plasma membrane enzyme was higher than that of the chloroplast carbonic anhydrase (28 and 26 kDa, respectively). This indicates the presence of different molecular species of carbonic anhydrase in the chloroplast and the plasma membrane.     

Protein spot recognition on the non-denaturing and denaturing two-dimensional electrophoresis patterns using in situ immunosubtraction via Protein A agarose and antibodies.

Specific proteins in small amounts of human plasma were subtracted from patterns of non-denaturing two-dimensional electrophoresis (non-denaturing 2-DE) by layering a 7 microl aliquot of Protein A agarose-antibody complex on the top of an isoelectric focusing (IEF) gel before the loading of a plasma sample. The Protein A agarose suspension was recovered after non-denaturing IEF and was mixed with 8 M urea-5% 2-mercaptoethanol-1% NP-40 to extract the antibody and the specific plasma protein from Protein A agarose. The extract was then subjected to denaturing two-dimensional electrophoresis (denaturing 2-DE) and the location of the specific polypeptide was determined. The technique can be applied to the extraction and analysis of proteins in small amounts of samples.     

两相分配法制备南极红酵母质膜

用葡聚糖 T-500(Dextran T-500)和聚乙二醇(PEG-3350)两相体系制备南极红酵母(Rhodotorula sp.)菌株 NJ298 的质膜.首先在 2 mmol/L KCl 浓度下,选用5种不同的聚合物浓度(5.6%、5.8%、6.0%、6.2%、6.4%,W/W),研究了 NJ298 质膜在两相体系中的分配情况,在此基础上进一步研究了 KCl 浓度(2 mmol/L、4 mmol/L、6 mmol/L、8 mmol/L、10 mmol/L)对 NJ298 质膜的纯度及得率的影响.结果表明,选用6.0%聚合物浓度,4 mmol/LKCl 的两相分配体系,分离3次可得到相对纯度在 78.2%的南极红酵母质膜组分,标志酶鉴定及磷钨酸染色电镜检测均表明获得了高纯度密实的正向型的质膜囊泡.这为进一步研究该菌株的南极极端环境适应机制奠定了基础.     

两相分配法制备南极红酵母质膜

用葡聚糖T-500(Dextran T-500)和聚乙二醇(PEG-3350)两相体系制备南极红酵母(Rhodotorula sp.)菌株 NJ298的质膜。首先在2 mmol/L KCl浓度下, 选用5种不同的聚合物浓度(5.6%、5.8%、6.0%、6.2%、6.4 %, W/W), 研究了NJ298质膜在两相体系中的分配情况, 在此基础上进一步研究了KCl浓度(2 mmol/L、4 mmol/L、6 mmol/L、8 mmol/L、10 mmol/L)对NJ298质膜的纯度及得率的影响。结果表明, 选用6.0%聚合物浓度, 4 mmol/L KCl的两相分配体系, 分离3次可得到相对纯度在78.2%的南极红酵母质膜组分, 标志酶鉴定及磷钨酸染色电镜检测均表明获得了高纯度密实的正向型的质膜囊泡。这为进一步研究该菌株的南极极端环境适应机制奠定了基础。     

Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.     

Identification and characterization of protein composition in Withania somnifera—an Indian ginseng

To have better understanding of the processes that occur in Withania somnifera L. Dunal, proteome analyses were initiated on two tissues (seeds & leaves) of this plant. Protein extracts were separated by two-dimensional gel electrophoresis (2-DE) across a broad 3.0?C10.0 immobilized pH gradient (IPG) strip that yielded 434 protein spots. A total of 167 individual spots (82 from seeds and 85 from leaves) were excised from the gel and were characterized by peptide mass fingerprinting. From these analyses, 70 individual proteins from seeds and 74 from leaves were identified by protein sequence database interrogation and were catalogued accordingly to different protein functions. A comparative analysis of the two tissues indicated that some enzymes/proteins involved in housekeeping pathways were common to both, whereas some were exclusively tissue specific with specialized metabolic complement. The knowledge gained by this study towards the tissue specific protein expression in W. somnifera would form the basis for our future endeavor of characterization of proteins to understand the physiology and the associated complex metabolic network during its ontogenetic development.     

Subcellular Localization and Kinetic Characterization of ABA Binding Proteins in Maize Root

By means of differential centrifugation, cytosol fraction and microsome were prepared from maize roots which have been grown in dark for 4 d. Highly purified plasma membranes were isolated from the microsome in two-phase aqueous system which is composed of 6.9 % (W/W) Dextran T500 and PEG 3350. The tonoplast was collected from the interface between 1% and 8% (W/W) Dextran T70 after gradient centrifugation. Electron microscopic observation and marker enzyme activities analysis proved that these fractions contained very few other membranes. Microvolume radioactivity ligand binding assay indicated that the specific binding sites of ABA in maize root microsome were mainly distributed on tonoplast and plasma membrane fractions. Their specific binding activity was 2485.4 and 1257.3 fmol/mg protein, respectively, the specific binding activity of cytosol fraction being the lowest (one order of magnitude lower). The dissociation constant (KD) of ABA-BP in plasma membrane was 1.57 nmol/L.     

Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.