失血性休克大鼠肝脏蛋白质的差异分析

 应用蛋白质双向凝胶电泳 (two-dimensional polyacrylamide gel electrophoresis, 2-DE) 技术,分析在急性重度失血性休克 (refractory hemorrhagic shock, RHS) 条件下,大鼠肝脏蛋白质组表达的差异.16 只雄性 Wistar 大鼠随机分成正常对照组 (sham hemorrhage shock, SHS) 和 RHS 模型组,每组 8 只.采用股动脉放血的方法制备模型,在规定时间内处死大鼠并分离肝脏,提取肝脏总蛋白质后进行 2-DE.运用 Image Master 2D Platinum v 5.0 凝胶图像分析软件对 2-DE 凝胶图像进行差异表达分析.有意义的差异蛋白质点用基质辅助激光解析电离飞行时间质谱进行肽质量指纹图谱分析,借助 Swiss-prot 数据库进行蛋白质搜索和鉴定.SHS 组和 RHS 组肝脏的 2-DE 图谱,分别平均识别到 698±11 和 700±13 个蛋白质点,SHS 组和 RHS 组肝脏间平均匹配率达88%～92 %.共发现 10 个差异有意义的蛋白质点,鉴定出了肿瘤抑制性抗原gp96、葡萄糖调节蛋白58、过氧还蛋白Ⅰ、细胞色素b5、谷胱甘肽转移酶、ATP合酶β亚单位、二磷酸果糖酶 B、三磷酸甘油醛脱氢酶等8种蛋白质.结果表明,以 2-DE 技术得到重复性和分辨率都较好的 2-DE图谱,并初步鉴定急性重度失血性休克后大鼠肝脏的差异表达蛋白质,为深入研究失血性休克的生理病理机制及寻找失血性休克预防和治疗的生物标志物提供了依据.     

野生大豆与栽培大豆种子差异蛋白质组学研究

运用蛋白质组学方法比较研究3个野生大豆(Glycinesoja)和3个栽培大豆(Glycinemax)的种子贮藏蛋白差异情况.结果发现,在考马斯亮蓝染色的双向电泳pH4～7的胶上,经过PDQuest图像分析软件平均可检测到550个左右的蛋白质点.进一步分析发现,表达量变化2.5倍以上的点有10个,其中大部分蛋白质仅在栽培大豆中检测到.对这10个蛋白质点进行了胶内酶解,用基质辅助激光解析电离飞行时间质谱测定均得到了肽质量指纹图谱.搜索大豆UniGene库和NCBI库共鉴定出5个蛋白质,主要是与大豆抗性、抗营养以及种子萌发相关的蛋白质,包括大豆血凝素,种子成熟蛋白PM24,糖结合蛋白,胰蛋白酶抑制剂p20以及成熟多肽.对这些蛋白质可能的作用进行了讨论.     

大百合种子休眠特性及休眠破除

对哀牢山自然保护区大百合种子休眠原因、休眠破除方法进行了研究,为大百合的种子繁殖提供理论与技术依据.结果表明:大百合种子休眠的主要原因是胚发育不全,种子休眠类型为复杂的形态生理休眠.温度是影响胚生长和分化的主要因素,经高温到低温的变温[25℃/15℃(60 d)→15℃/5℃(60 d)→4℃(50 d)]层积处理,种子可完成后熟而萌发,所需时间约170 d,比自然条件下层积所需时间缩短了11～12个月.     

樟子松突变丛生枝蛋白质的双向电泳分析

目的：研究樟子松突变丛生枝的发生机理。方法：对其正常枝和突变丛生枝进行蛋白质双向电泳,通过ImageMaster2-DE计算机分析系统对结果进行定性、定量分析。结果：共有59个蛋白点在表达的质和量上有变化,其中35个蛋白点在突变丛生枝2-DE图谱中表达量均高于正常枝的蛋白点,20个蛋白点在突变丛生枝2-DE图谱中表达量均低于正常枝的蛋白点,4个蛋白点在突变丛生枝图谱上发生缺失。结论：差异表达的蛋白点可能与突变丛生枝的发生有关。     

水牛精子蛋白质组双向电泳体系的建立和优化

建立和优化一种适合水牛精子蛋白质组学研究的双向电泳技术。以水牛精子为研究对象,比较两种不同配方的裂解液,以及不同上样量对其2-DE图谱质量的影响。结果显示,以7 mol/L尿素、2 mol/L硫脲、4%CHAPS、1%DTT、0.5%Cocktail of protease inhibitors为裂解液,24 cm胶条上样量200μg时,可获得较好的精子总蛋白质2-DE图谱。运用ImageMaster 2-Dplatinum分析软件检测出约500个蛋白质点,蛋白质大部分分布在等电点5-7之间,分子量范围约40-90 kD。     

慢性应激大鼠海马的比较蛋白质组学研究

慢性应激可造成海马神经细胞丢失、树突萎缩等损伤,但有关其损伤机制仍有很多问题不甚明了.为了寻找应激致海马损伤相关的重要蛋白质、从蛋白质水平揭示应激致海马损伤的分子机制,应用双向凝胶电泳(2-DE)技术分离对照组和束缚应激组大鼠海马组织总蛋白质,图像分析检测差异表达的蛋白质点,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MSS)和数据库检索对差异表达的蛋白质点进行鉴定,并采用半定量的RT-PCR在mRNA水平验证2-DE结果.得到了分辨率较高、重复性较好的对照和束缚应激大鼠海马2-DE图谱,质谱分析和数据库检索鉴定了14个差异表达蛋白质点中的11个蛋白质,大多数差异蛋白的功能涉及能量代谢、信号传递等过程.研究结果为揭示应激致海马损伤的机制、提高机体的应激适应能力提供了理论依据.     

原位鼻咽癌裸鼠模型血清蛋白质组学的研究

利用我室建立的原位鼻咽癌裸鼠模型,建立了稳定性较好的鼻咽癌裸鼠血清蛋白质2-DE方法,并比较了原位鼻咽癌裸鼠血清与对照组裸鼠血清蛋白质2-DE图谱之间的差异.与正常裸鼠血清比较,原位鼻咽癌裸鼠模型组血清增加了4个蛋白质点.利用MALDI-TOF质谱技术对血清差异蛋白点进行鉴定,发现SAA-1前体(serumamyloidA-1 protein precursor)在鼻咽癌裸鼠模型组血清中明显高表达,为寻找鼻咽癌血清生物标志物提供了有利的线索.     

芍药种子下胚轴休眠相关基因的cDNA-AFLP分析

旨在探讨芍药种子下胚轴的休眠机理,采用cDNA-AFLP技术对层积0 d和根部露白两个时期的芍药种子进行基因差异表达分析。利用256对引物组合进行扩增,筛选获得3 600个差异表达转录衍生片段(TDFs),成功回收到1 200个TDFs,选取500个TDFs对其克隆测序,共得到42个有效序列。经BLAST比对发现,其中30个TDFs与已知功能基因同源,分别参与芍药种子下胚轴休眠过程中的基因表达调控、信号转导、逆境胁迫、物质与能量代谢等过程,9个TDFs与功能未知基因及假想蛋白同源,另有3个TDFs为无同源序列,可能是新的未知基因,这些基因有助于更好地阐明芍药种子下胚轴休眠机理。     

应用超高分辨双向凝胶电泳技术进行正常人类肝脏蛋白质组研究

肝脏是人体最大的实质器官, 承担着人体许多关键的生理功能, 在生命活动中占有重要地位. 根据人类肝脏蛋白质组计划, 本实验旨在构建人肝脏蛋白质双向凝胶电泳表达谱, 尽可能分离和鉴定更多的蛋白. 在双向凝胶电泳第一向等电聚焦水平上, 从上样方式、水化液配方、聚焦时间等方面优化了碱性蛋白的分离条件, 利用超放大胶分离技术搭建了高分辨的双向凝胶电泳分离平台, 构建了人类肝脏蛋白质2-DE参考谱, 检测到5481个蛋白质点. 这是目前国际上最为全面的人体器官蛋白质组2-DE参考谱, 为其他肝病的研究提供了较好的参照系. 成功鉴定了429个非冗余蛋白, 对pH 4.0~7.0, 5.0~6.0, 5.5~6.7, 6.0~9.0部分蛋白质点在胶上进行了注释, 由此构建了人肝2-DE蛋白质表达谱数据库. 对蛋白质的理化性质、功能及亚细胞定位进行了全面分析. 研究中构建的人类肝脏蛋白质2-DE图谱将为肝脏比较蛋白质组学研究提供参考.     

不同生长期罗汉果蛋白组图谱比较研究

目的:分析、比较30d和60d两个不同时期罗汉果蛋白质组图谱,寻找与罗汉果发育、成熟及罗汉果甜甙生成密切相关基因,为罗汉果的开发和品种改良提供基础。方法:采用双向电泳(2-DE)技术构建30d和60d罗汉果蛋白质组图谱,应用Im-ageMaster 2D图像分析软件对所得蛋白质组图谱进行分析。结果:30d和60d两个不同时期罗汉果蛋白质组图谱有明显差别,与30d罗汉果蛋白组图谱比较,随果实发育、成熟,60d罗汉果有29个新的蛋白产生,30个蛋白消失,16个蛋白表达3倍升高和15个蛋白表达50%下调。结论:罗汉果生长、发育和成熟的不同阶段受特定基因的表达调控。     

Proteomic analysis of near-isogenic sunflower varieties differing in seed oil traits

Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mass were comparatively analyzed in biological triplicate at 18 days after flowering using two-dimensional (both pI 3-10 and 4-7) Difference Gel Electrophoresis. Additionally, two inbred lines varying in oleic acid content, HA89 (18% oleic) and HA341 (89% oleic), were also analyzed in the same manner. Statistical analyses of these sunflower lines was performed beginning with fitting a mixed effects linear model to the log-transformed optical volume of each spot to account for gel variation, followed by testing the significance between varieties for mean transformed optical spot volumes. The p-values from the spot analysis procedures were then used to find the cutoff point for differential expression using a 10% false-discovery rate (FDR). Comparison of the oil content and oleic acid composition lines revealed 77 and 42 protein spots below the 10% FDR cutoff, respectively, and were therefore declared differentially expressed. Liquid chromatography-tandem mass spectrometry analysis of each of these protein spots resulted in assignments for 44 and 17 spots, respectively. Fructokinase, plastid phosphoglycerate kinase, and enolase proteins were determined to be up-regulated in the high oil line, while phosphofructokinase, cytosolic phosphoglucomutase, and cytsolic phosphoglycerate kinase were up-regulated in the low oil variety. Additionally, four activities involved in amino acid synthesis were up-regulated in the low oil variety in addition to 12S storage proteins and a protein similar to legumin storage protein. Interestingly, two 2-DE spots identified as 14-3-3 proteins were found to be up-regulated in high oleic acid variety. Alteration of glycolytic and amino acid biosynthetic enzymes, as well as storage protein levels, suggests seed oil content is tightly linked to carbohydrate metabolism and protein synthesis in a complex manner.     

Protein changes between dormant and dormancy-broken seeds of Prunus campanulata Maxim

Seed dormancy is regulated by complex networks in order to optimize the timing of germination. However, the biochemical basis of the regulation of seed dormancy is still poorly understood. Many temperate timber species, which are of ecological and/or economic interest, are deeply dormant in seeds, such as Prunus campanulata. Freshly harvested seeds require warm plus cold stratification to break dormancy before they can begin to germinate. According to the results of germination, both warm and cold stratifications are the critical influences for breaking seed dormancy. Significant variations in seed proteins were observed by 2-DE before and after the breaking of seed dormancy. Among the 320, 455, and 491 reproducibly detected spots on the cotyledons, embryos, and testae, respectively, 71 dramatic changes in abundances were observed following warm and/or cold stratification. Among these protein spots, dehydrin, prunin 1 precursor, prunin 2 precursor, and prunin 2 were identified by MS and sequence comparison. The implications of protein changes in relation to the breaking of seed dormancy and germination are discussed. This is the first report of a proteomic analysis of dormancy breaking in woody plant seeds.     

水稻红莲型细胞质雄性不育系小孢子不同发育时期花药总蛋白质比较分析

采用双向凝胶电泳对水稻红莲型细胞质雄性不育的不育系小孢子发育单核期和二核期花药总蛋白进行了分离,通过银染显色,获得了分辨率和重复性较好的双向电泳图谱,且单核期和二核期花药总蛋白质在双向电泳胶上分布的图谱十分相似。PDQuest 2DE图像分析软件在等电点(pI)3.0~10.0、分子量(M.W.)9.0~98.0 kD之间可识别约1 800个蛋白质点。比较分析发现单核期和二核期花药中共有241个差异表达的蛋白质点,其中仅在单核期中表达的点数为125,仅在二核期中表达的为13点;表现为表达量差异的105点,其中在二核期表达下调的点数为70点,表达上调的为33点。还对蛋白质点集中的区域(pI 4.5~8.0,M.W.25.0~70.0 kD)中的41个差异蛋白质点进行了分子量和等电点分析。     

Redox-sensitive proteome and antioxidant strategies in wheat seed dormancy control

Oxidative signalling by ROS has been demonstrated to play a role in seed dormancy alleviation, but the detailed molecular mechanisms underlying this process remain largely unknown. Here, we show dynamic differences in redox-sensitive proteome upon wheat seed dormancy release. Using thiol-specific fluorescent labelling, solubility-based protein fractionation, 2-D IEF PAGE, and MS analysis in conjunction with wheat EST sequence libraries, proteins with reversible oxidoreductive changes were characterized. Altogether, 193 reactive Cys were found in 79 unique proteins responding differentially in dormant, non-dormant, abscisic, or gibberellic acid-treated seed protein extracts from RL4137, a wheat cultivar with extreme dormancy. The identified proteins included groups that are redox-, stress-, and pathogen-responsive, involved in protein synthesis and storage, are enzymes of carbohydrate metabolism, proteases, and those involved in transport and signal transduction. Two types of redox response could be detected: (i) a dramatic increase in protein thiol redox state in seeds during imbibition and hormonal treatment; (ii) higher antioxidant capacity related to sensing of a threshold redox potential and balancing the existing redox pools, in dry dormant versus non-dormant seeds. These results highlight occurrence of the antioxidant defence mechanisms required for the protection of seed during a dormancy stage.     

The accumulation of abundant soluble proteins changes early in the development of the primary roots of maize (Zea mays L.)

A reference database of the major soluble proteins of the primary root of the maize inbred line B73 was generated 5 days after germination (DAG) using a combination of 2-DE and MALDI-TOF MS. A total of 302 protein spots were detected with CBB in a pH 4-7 range and 81 proteins representing 74 distinct Genbank accessions were identified. Only 28% of the major proteins identified in 5 DAG primary roots were identified in similarly analyzed 9 DAG primary roots documenting remarkable changes in the accumulation of abundant soluble proteins early in primary root development.     

Proteomics analysis of mature seed of four peanut cultivars using two-dimensional gel electrophoresis reveals distinct differential expression of storage, anti-nutritional, and allergenic proteins

Profiles of total seed proteins isolated from mature seeds of four peanut cultivars, New Mexico Valencia C (NM Valencia C), Tamspan 90, Georgia Green, and NC-7, were studied using two-dimensional gel electrophoresis coupled with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC–MS/MS). Two-dimensional gels stained with silver nitrate revealed a total of 457, 516, 556, and 530 protein spots in NM Valencia C, Tamspan 90, Georgia Green, and NC-7, respectively. Twenty abundant protein spots showing differences in relative abundance among these cultivars were analyzed by nESI-LC–MS/MS, resulting in identification of 14 non-redundant proteins. The majority of these proteins belonged to the globulin fraction consisting of arachin (glycinin and Arah3/4) and conarachin seed storage proteins as well as other allergen proteins. The expression of some of these identified protein spots was cultivar-specific. For example, allergen Arah3/Arah4 and conarachin protein spots were only detected in Tamspan 90 and NC-7, whereas the Gly1 protein spot was detected only in NM Valencia C and NC-7. Moreover, a galactose-binding lectin protein spot with anti-nutritive properties was only present in Tamspan 90. Other proteins showing differences in relative abundance among the four cultivars included 13-lipoxygenase, fructose-biphosphate aldolase, and glyceraldehyde 3-phosphate dehydrogenase. Together, these results suggest that identified proteins might serve as potential markers for cultivar differentiation and may be associated with underlying sensory and nutritional traits of peanut cultivars.     

利用RIL和CSSL群体检测水稻种子休眠性QTL

利用由梗稻品种Asominori与籼稻品种IR24的杂交组合衍生的重组自交F10。家系(Recombinant Inbred Lines,RIL)群体及其衍生的染色体片段置换系(Chromosome Segment Substitution Lines,CSSL)群体,进行了种子休眠性QTL的检测和遗传效应分析。其中CSSL群体有2个,即CSSLl(以Asominori为背景,置换片段来自IR24)和CSSL2(以IR24为背景,置换片段来自Asominori)。在RIL群体上共检测到3个种子休眠性QTL,分别位于第3、6和9染色体上;在CSSL1群体中检测到分布在第1、3和7染色体上的3个休眠性QTL;而在CSSl2群体上检测到的3个QTL则分别位于第1、2和7染色体上。同时在两套CSSL群体上,分别检测到位于第1、7染色体上位置相近且效应一致的休眠性QTL,分析表明其所在的Asominori片段含对种子休眠性的增效基因,相应的IB24段含有减效基因。     

玉米空间诱变后代SP4选系配合力效应分析

利用返回式卫星“实践八号”搭载3份玉米自交系08-641、RP125和18-599, 从SP4代中选出多个诱变系按不完全双列杂交设计配制杂交组合, 在四川和云南两个环境条件下进行种植鉴定。配合力分析结果表明, 3份玉米自交系经空间诱变后各性状的配合力发生不同程度的变化, 同组诱变系材料在四川和云南两种环境条件下的配合力表现存在较大差异, 且表现配合力差异的性状不同。诱变系C03的穗长、穗行数、行粒数和单株产量4个性状一般配合力(General combining ability, GCA)在该组试验中的正向效应值均表现为最大, 且显著高于基础材料08-641, 可能具有较大育种潜势; 诱变系C01和C04部分产量构成性状的GCA显著高于对照, 但单株产量GCA表现不明显, 需在育种中加以改良利用; 诱变系C06、R18和S22所配杂交组合在产量及产量构成性状上的特殊配合力(Specific combining ability, SCA)表现较优, 这些结果为玉米杂交种的选育提供了重要参考。