Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558

Spike number is one of three yield-related factors and is closely related to wheat yield. In the present study, we found that the inhibited and normal tillers of the 3558 line presented phenotypic differences at the elongation stage by morphological and anatomical analysis. We then initiated a proteomic study using two-dimensional electrophoresis (2-DE) and nanoscale liquid chromatography-high-definition tandem mass spectroscopy, to isolate and identify the key proteins and metabolic pathways related to spike-development inhibition. A total of 31 differentially expressed proteins (DEPs), which were mainly involved in cell cycle regulation, photosynthesis, glycolysis, stress response, and oxidation-reduction reactions, were isolated and identified. 14-3-3-like proteins and proliferating cell nuclear antigen (PCNA), involved in cell-cycle regulation, were dramatically down-regulated in inhibited tillers compared to normal tillers. Six spots corresponding to degraded Rubisco large subunits, involved in photosynthesis, were detected in different locations of the 2-DE gels and were up-regulated in inhibited tillers. In addition, the relative levels of DEPs involved in glycolysis and oxidation-reduction reactions changed dramatically. Development was blocked or delayed at the elongation stage in the inhibited tillers of 3558. Weakened energy metabolism might be one reason that the inhibited tillers could not joint and develop into spikes. These DEPs and related metabolic pathways are significant for understanding the mechanism of spike-development inhibition and studying the spike-development process in wheat.     

Unintended Changes in Genetically Modified Rice Expressing the Lysine-Rich Fusion Protein Gene Revealed by a Proteomics Approach

Development of new technologies for evaluating genetically modified(GM) crops has revealed that there are unintended insertions and expression changes in GM crops.Profiling techniques are non-targeted approaches and are capable of detecting more unintended changes in GM crops.Here,we report the application of a comparative proteomic approach to investigate the protein profile differences between a GM rice line,which has a lysine-rich protein gene,and its non-transgenic parental line.Proteome analysis by two-dimensional gel electrophoresis(2-DE)and mass spectrum analysis of the seeds identified 22differentially expressed protein spots.Apart from a number of glutelins that were detected as targeted proteins in the GM line,the majority of the other changed proteins were involved in carbohydrate metabolism,protein synthesis and stress responses.These results indicated that the altered proteins were not associated with plant allergens or toxicity.     

Separation and Identification of Differentially Expressed Proteins in Pistillate Flowers between Different Mulberry Cultivars

[Objective] This study aimed to explore the proteins related to pistillate flower development in different mulberry cultivars. [Method] The total proteins of the pistillate flowers of two mulberry cultivars Dal0 （Morus atropurpurea Roxb.） and SG01 （Morus muIticaulis Perr.） were extracted, separated and detected through two- dimensional electrophoresis （2-DE） and mass spectrometry. [Result] There was sig- nificant difference in the expression of proteins from the pistillate flowers of different mulberry cultivars. From the 2-DE images of Dal0 and SG01, 445_＋17 and 425_＋12 protein spots were respectively detected. The expression levels of 75 protein spots differed significantly. Thirteen spots those were expressed at high levels and well separated were analyzed by mass spectrometry, and nine of them were identified successfully. The nine proteins are involved in the glycometabolism, protein and amino acid metabolism and defense responses during the development of mulberry pistillate flower after they were pollinated. [Conclusion] The findings will provide reference for further study on the molecular mechanism of mulberry pistillate flower de- velopment.     

Combining Phytate/Ca~（2＋） Fractionation with Trichloroacetic Acid/Acetone Precipitation Improved Separation of Low-Abundant Proteins of Wheat （Triticum aestivum L.） Leaf for Proteomic Analysis

Proteomic assessment of low-abundance leaf proteins is hindered by the large quantity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) present within plant leaf tissues. In the present study, total proteins were extracted from wheat (Triticum aestivum L.) leaves by a conventional trichloroacetic acid (TCA)/acetone method and a protocol first developed in this work. Phytate/Ca2+ fractionation and TCA/acetone precipitation were combined to design an improved TCA/acetone method. The extracted proteins were analysed by two-dimensional gel electrophoresis (2-DE). The resulting 2-DE images were compared to reveal major differences. The results showed that large quantities of Rubisco were deleted from wheat leaf proteins prepared by the improved method. As many as (758±4) protein spots were detected from 2-DE images of protein extracts obtained by the improved method, 130 more than those detected by the TCA/acetone method. Further analysis indicated that more protein spots could be detected at regions of pI 4.00-4.99 and 6.50-7.00 in the improved method-based 2-DE images. Our findings indicated that the improved method is an efficient protein preparation protocol for separating low-abundance proteins in wheat leaf tissues by 2-DE analysis. The proposed protocol is simple, fast, inexpensive and also applicable to protein preparations of other plants.     

Proteomics Identification of Differentially Expressed Leaf Proteins in Response to Setosphaeria turcica Infection in Resistant Maize

Northern corn leaf blight （NCLB）, caused by the heterothallic ascomycete fungus Setosphaeria turcica, is a destructive foliar disease of maize and represents a serious threat to maize production worldwide. A comparative proteomic study was conducted to explore the molecular mechanisms underlying the defense responses of the maize resistant line A619 Ht2 to S. turcica race 13. Leaf proteins were extracted from mock and S. turcica-infected leaves after inoculated for 72 h and analyzed for differentially expressed proteins using two-dimensional electrophoresis and mass spectrometry identification. 137 proteins showed reproducible differences in abundance by more than 2-fold at least, including 50 up-regulated proteins and 87 down-regulated proteins. 48 protein spots were successfully identified by MS analysis, which included 10 unique, 6 up-regulated, 20 down-regulated and 12 disappeared protein spots. These identified proteins were classified into 9 functional groups and involved in multiple functions, particularly in energy metabolism （46%）, protein destination and storage （12%）, and disease defense （18%）. Some defense-related proteins were upregulated such as 13-glucosidase, SOD, polyamines oxidase, HSC 70 and PPIases; while the expressions of photosynthesis- and metabolism-related proteins were down-regulated, by inoculation with S. turcica. The results indicated that a complex regulatory network was functioned in interaction between the resistant line A619 Ht2 and S. turcica. The resistance processes of A619 Ht2 mainly resided on directly releasing defense proteins, modulation of primary metabolism, affecting photosyntesis and carbohydrate metabolism.     

Proteomics Identification of Differentially Expressed Proteins Relevant for Nicotine Synthesis in Flue-Cured Tobacco Roots Before and After Decapitation

Nicotine is a secondary substance synthesized in tobacco roots. In flue-cured tobacco planting, tobacco decapitation is an effective practice to promote nicotine biosynthesis by regulation of the redistribution of total nitrogen amounts. However, proteins relevant to nicotine synthesis in tobacco roots has not been identified and characterized yet. It is important to explore the regulation of nicotine biosynthesis in tobacco roots. To identify the proteins relevant to nicotine synthesis, the protein patterns in roots of flue-cured tobacco （cv. K326） before and after decapitation were analyzed. In the present study, the protein patterns in roots of flue-cured tobacco were analyzed by two-dimensional electrophoresis （2-DE）, and the differentially-expressed spots were identified by matrix assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF-MS）. Paired comparison of 2-DE maps revealed 26 spots of differentially-expressed proteins in roots before and after decapitation. Furthermore, nine differentially-expressed spots were identified. There were four proteins which were enzymes possibly involved in nicotine biosynthesis. In addition, the roles of the four enzymes in nicotine biosynthesis were discussed in a putative network. Our results would contribute to the understanding of the regulation pathway of nicotine biosynthesis and further to the molecular manipulation on the nicotine contents in flue-cured tobacco.     

棉花晋A细胞质雄性不育系及其保持系差异蛋白质组分析

【目的】对棉花晋A细胞质雄性不育系及其同核异质保持系MB177（JB）雄性败育关键时期的花药进行差异蛋白质组研究,为进一步揭示棉花细胞质雄性不育机理奠定基础。【方法】运用双向电泳技术对晋A细胞质雄性不育系及其保持系小孢子发育的造孢细胞时期和小孢子母细胞时期表达的蛋白质进行分离（考马斯亮蓝染色）;采用PDQuest8.0.1软件进行斑点检测,并经统计学分析确定获得差异表达蛋白质;对这些差异蛋白质斑点进行LC-Chip-ESI-QTOF-MS质谱分析,用Mascot软件搜索NCBInr绿色植物,鉴定差异表达蛋白质;对差异表达蛋白质进行GO和KEGG功能分析。【结果】PDQuest8.0.1软件分析表明,晋A不育系和保持系在花药发育的造孢细胞时期和小孢子母细胞时期的花蕾总蛋白质斑点数分别为1 525、1 540和1 554、1 540,这些蛋白质斑点的分子量分布在10-100 kD,等电点分布在3-10。经差异定量研究和统计学分析,共鉴定到15个在晋A不育系与保持系间显著差异表达的蛋白斑点,15个差异蛋白质斑点都得到了质谱特异峰图。对应的蛋白质H（+）-转运ATP合成酶、谷胱甘肽还原酶、线粒体上的ATP酶亚基、核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基和UDP-D葡萄糖脱氢酶在不育系与保持系花药发育2个阶段均差异表达;膜联蛋白、S-甲酸谷胱甘肽水解酶、momilactone A 合成酶类似物、一个具有丙二烯氧化环化酶活性的未命名蛋白质和一个位于线粒体上的保守预测蛋白质仅在不育系和保持系花药发育的造孢细胞时期差异表达;β-羟酰-ACP脱水酶、丙酮酸脱氢酶-α亚基以及与花粉发育相关的一个预测蛋白质仅在不育系和保持系花药发育的小孢子母细胞时期差异表达。这些差异表达蛋白质主要参与小孢子与绒毡层发育过程,它们的下调或上调表达可能造成发育与代谢过程的不协调性,产生不正常的小孢子和绒毡层提前解体,从而导致雄性不育。采用荧光定量PCR分析核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基基因和H（+）转运ATP合成酶基因的转录变化,它们在转录水平与蛋白质表达的趋势一致。【结论】晋A不育系的小孢子败育可能与能量代谢紊乱、茉莉酸合成途径损害、细胞内大量ROS积累、查尔酮合酶活性下降有关。雄性不育的产生是一个复杂的生物学过程,涉及到多个基因的相互作用,构成了一个复杂的调控网络。     

甘蔗叶绿体分离及其蛋白质提取方法研究

【目的】探索甘蔗幼苗叶片完整叶绿体的分离纯化及其叶绿体蛋白质提取方法,为深入研究甘蔗叶绿体蛋白质组学奠定基础。【方法】以甘蔗品种ROC22幼苗叶片为材料,通过差速离心法制备粗叶绿体制品,分别利用蔗糖密度梯度离心和Percoll梯度离心进一步分离纯化完整叶绿体;提取叶绿体蛋白质,并进行电泳分析。【结果】两种密度梯度离心法均可获得纯度和完整率较高的叶绿体,但蔗糖密度梯度离心法具有分离时间较短、成本较低,分离的叶绿体完整率、纯度和含量高等特点,并且提取的叶绿体蛋白质电泳条带清晰,蛋白质数量多且较全。【结论】与Pecoll梯度离心法相比,蔗糖密度梯度离心法更适宜于在亚细胞水平上进行甘蔗蛋白质组学研究。     

抗条锈病小麦品系Taichuang29*6/Yr5接种条锈菌CY32后的蛋白质组学分析

 【目的】了解抗条锈小麦受条锈菌侵染后的蛋白表达变化情况,采用比较蛋白质组学技术鉴定接种条锈菌小种CY32后的抗条锈小麦品系 Taichuang29*6/Yr5和同时期未接种对照之间的差异蛋白。【方法】提取接种48 h和同期未接种小麦的叶片蛋白,进行双向电泳分离和分析,选取差异显著的蛋白点进行MALDI-TOF质谱分析及数据库搜索鉴定。【结果】发现13个体积百分比变化大于1.5倍,符合t检验（P＜0.05）的显著差异蛋白点,通过MALDI-TOF MS获得了这些差异蛋白点的肽指纹图谱,经数据库搜索,共鉴定出11个蛋白,包括Pathogenesis-related homeodomain protein,beta-glucosidase,glutathione transferase等。【结论】经蛋白质功能分析,推测小麦叶片中这些差异蛋白可能与条锈菌小种CY32的侵染有关。     

Proteomic analysis of differentially expressed proteins in poplar leaves induced by Marssonina brunnea f. sp.multigermtubi

Proteomic analysis of differentially expressed proteins in poplar leaves induced by Marssonina brunnea f. sp.multigermtubi     

利用双向电泳分析烯丙异噻唑诱导后水稻的蛋白质变化

本研究应用双向电泳(2-DE)技术分析了烯丙异噻唑诱导后水稻蛋白质的差异表达,所得的双向电泳图谱用PDQuest8.0.1软件进行分析,在诱导4d的双向电泳图谱上找到了10个差异表达蛋白质点,选取其中3个进行质谱(MALDI-TOF-MS)分析,将所得肽段序列在数据库搜索比对后,找到了相匹配的蛋白,它们分别具有泛醌-细胞色素C还原酶、苏氨酸肽链内切酶和苹果酸脱氢酶活性。这些蛋白可能通过提高植物的呼吸作用和启动泛肽依赖性的蛋白质降解途径的活化来参与植物的诱导抗病机制。     

小麦雄性不育系绒毡层异常代谢与小孢子败育的关系

【目的】研究小麦遗传型雄性不育系花药绒毡层降解及营养物质代谢,并揭示其与花粉败育的关系。【方法】以小麦S型1376不育系[(S)-1376(A)]及其保持系[(A)-1376(B)]为试材,在三核期分别用DAPI和KI-I2对花粉粒内淀粉积累进行染色观察;采用半薄切片技术对不育系(S)-1376和保持系1376绒毡层发育过程及多糖、脂类和蛋白等物质积累进行观察和比较;采用软件cellSens Entry和IPP 6.0分别计算图片中小孢子和绒毡层细胞的面积及分析各发育时期图像中的平均光密度值。【结果】与保持系1376比较,(S)-1376花粉粒被KI-I2染成浅黄色,表明三核期其花粉中无淀粉积累,花粉败育彻底;不育系(S)-1376绒毡层较保持系1376绒毡层细胞提前至四分体时期启动细胞程序化死亡（PCD）过程;不育系(S)-1376花粉核发育迟缓,大多发育至单核晚期停止分裂,仅少数可发育至二核期;(S)-1376小孢子在四分体时期和单核早期显著增大;绒毡层细胞在四分体时期也显著大于保持系1376绒毡层细胞。在(S)-1376花粉的发育过程中,除四分体时期外,其余各时期多糖含量（呈红色）均低于保持系对应的各发育时期,不育系(S)-1376绒毡层中多糖在四分体时期多于保持系,在单核早期显著降低。在花粉整个发育过程中,不育系(S)-1376花粉中被染成黑色脂类的含量明显低于保持系,不育系绒毡层中的脂类含量在各个时期也低于保持系。不育系(S)-1376小孢子母细胞四分体时期被染成蓝色的蛋白含量很高,但单核晚期显著降低;在绒毡层细胞中单核早期蛋白含量显著低于保持系,这可能暗示该时期保持系绒毡层开始启动降解。在花粉发育整个过程中保持系1376小孢子母细胞中四分体时期多糖和蛋白的含量都低于不育系(S)-1376,这可能与该时期不育系绒毡层提前降解需要大量的多糖和蛋白有关;单核晚期小孢子中的多糖、脂类和蛋白含量高于不育系,该时期是正常小孢子发育的关键时期,不育系中各种物质供应不足可能是导致花粉败育的主要原因。在二核期和三核期,不育系(S)-1376花粉营养细胞中大液泡不消失,细胞质内含物也不持续增加,淀粉粒、脂类等物质积累终止,最终导致小孢子内没有内含物的积累,呈空壳晶体状。【结论】小麦S型1376A不育系花药绒毡层提前降解所诱发的物质异常代谢,使小孢子在由单核晚期向二核期发育的过程中物质供应不足导致细胞核分裂异常,最终引起花粉败育。     

与黄花马蹄莲互作过程中胡萝卜软腐果胶杆菌的差异表达蛋白分析

通过病原菌胡萝卜软腐果胶杆菌胡萝卜亚种与黄花马蹄莲组织的离体互作,运用双向电泳技术、质谱技术及Im-ageMaster 2D platinum 5.0(GE)软件对互作过程中蛋白质差异表达进行分析。结果表明:70个蛋白点在互作中存在表达差异,占蛋白点总数的18.13%。通过质谱分析发现:2个特异表达蛋白分子伴侣DnaK和1-脱氧木酮糖-5-磷酸合酶(DXS)均为代谢类蛋白;3个显著上调表达蛋白中ATP合酶α亚基和半胱氨酸合酶为代谢类蛋白,而β-内酰胺酶为抗生素类蛋白。     

玉米杂交种与亲本苗期叶片差异表达蛋白谱分析

【目的】建立玉米杂交种与亲本苗期叶片差异表达蛋白谱,探讨叶片大小杂种优势形成的分子机理。【方法】以玉米强优势杂交种Mo17/B73及其亲本发芽后第5天的第3片叶为材料,采用双向电泳技术（2-DE）,结合MALDI TOF MS质谱技术,建立叶片细胞分裂和生长关键区域的差异表达蛋白质谱,并对差异表达蛋白进行质谱鉴定。【结果】在检测到的630个蛋白质点中,有52个蛋白质点在杂交种与亲本之间的表达差异达到显著水平,表现为单亲沉默（15个）、偏高亲（13个）、偏低亲（8个）、杂种上调（6个）、杂种下调（7个）和杂种特异表达模式（3个）。另外,还成功鉴定出了其中的28个差异表达蛋白质点,涉及到代谢、胁迫响应、糖酵解、转录调控、蛋白折叠和降解、三羧酸循环、细胞骨架、发育及未知蛋白质等9个功能类别。【结论】玉米杂交种与亲本在蛋白丰度上存在明显的差异,并且差异表达蛋白涉及到多个功能类别,可能与玉米叶片大小杂种优势的形成有关。     

甘蔗叶绿体分离及其蛋白质提取方法研究

【目的】探索甘蔗幼苗叶片完整叶绿体的分离纯化及其叶绿体蛋白质提取方法,为深入研究甘蔗叶绿体蛋白质组学奠定基础。【方法】以甘蔗品种ROC22幼苗叶片为材料,通过差速离心法制备粗叶绿体制品,分别利用蔗糖密度梯度离心和Percoll梯度离心进一步分离纯化完整叶绿体;提取叶绿体蛋白质,并进行电泳分析。【结果】两种密度梯度离心法均可获得纯度和完整率较高的叶绿体,但蔗糖密度梯度离心法具有分离时间较短、成本较低,分离的叶绿体完整率、纯度和含量高等特点,并且提取的叶绿体蛋白质电泳条带清晰,蛋白质数量多且较全。【结论】与Pecoll梯度离心法相比,蔗糖密度梯度离心法更适宜于在亚细胞水平上进行甘蔗蛋白质组学研究。