家蚕品种金秋及其杂交亲本的蛋白质表达谱分析

 【目的】探讨家蚕杂交育成品种及其亲本蛋白水平的差异,从蛋白水平分析育成品种的基因结构,为阐明杂交育种成败的分子机理创造条件。【方法】采用蛋白质组研究技术,分析杂交亲本及其育成品种的丝腺、血淋巴和中肠的蛋白质表达谱。【结果】3种组织器官中育成品种金秋与两个亲本的匹配蛋白斑点比例只达到70%左右,剩余30%左右是各个品种的特异蛋白斑点;在匹配蛋白斑点中,还有9%～24%的蛋白表现上调和下调。这些特异蛋白可能是两个亲本的基因在杂交育成品种中产生了互作而形成的新蛋白,也可能是对相同的蛋白进行了不同的修饰而获得的修饰蛋白。【结论】新品种的育成,除了汇集亲本的优良基因外,还有赖于基因互作产生的新功能蛋白的作用。     

红豆杉细胞蛋白质双向电泳样品制备方法的比较

[目的]探索适用于红豆杉愈伤蛋白质组学研究的样品制备方法。[方法]以红豆杉愈伤组织为材料,分别采角三氯乙酸（TCA）／丙酮沉淀法、Tris—HCl法和酚-甲醇／醋酸铵沉淀法提取其中的总蛋白,通过双向电泳（2DE）检测3种方法对总蛋白的提取效果。[结果]3种方法提取的蛋白样品2DE图谱显示的蛋白点数量均较多。TCA／丙酮沉淀法对蛋白质的提取率较高,2DE图像清晰,条纹少,拖带现象轻;Tris—HCl法提取的蛋白样品2DE图谱个别区域出现较轻的云片状聚集,并有轻微拖带现象;酚-甲醇／醋酸铵沉淀法提取蛋白样品的2DE图谱有条纹和拖带现象。[结论]TCA／丙酮沉淀法提取的蛋白质质量较高,所得图谱背景清晰,蛋白质信息量大。     

Screening and identification of specific serum biomarkers of Parkinson disease

AIM：To screen the possible serum biomarkers of Parkinson’s disease. METHODS：The surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was used to screen the serum samples from 44 cases of Parkinson’s disease and 60 control subjects. The differentially expressed protein peaks were selected and isolated with high-performance liquid chromatography (HPLC), and processed with enzyme before analysis by liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS). The data mining was performed by Xcalibur program component BioWorks 3.2. RESULTS：Three differentially expressed protein peaks were selected as potential serum biomarkers from the patients of Parkinson’s disease: the protein at 8 937 m/z peak showed significant increase (27.47±16.58 in Parkinson’s disease group, and only 5.01±3.47 in control group), and the proteins at 6 636 and 8 697 m/z peaks showed significant decreases (5.43±2.66 and 20.22±9.57, respectively, in Parkinson’s disease group, and 18.85±7.56 and 51.13±26.22, respectively, in control group). The proteins at 6 636, 8 697 and 8 937 m/z peaks were identified as apolipoprotein C-I, apolipoprotein C-III and complement 3a,respectively. Combined use of these 3 biomarkers effectively distinguished the subjects between Parkinsons disease group and control group. The detection rate of the patients with Parkinsons disease was 90.0% (27/30), and the detection rate of the healthy sibkects was 92.5% (37/40). CONCLUSION：The apolipoprotein C-I, apolipoprotein C-III and complement component 3a identified as potential markers of Parkinson’s disease have diagnostic value in clinical application.     

大麦条纹病侵染对大麦叶片蛋白组的影响

大麦条纹病由麦类核腔菌(Pyrenophora graminea)引起,是一种世界性病害.为进一步探索条纹病与大麦(Hordeum vulgare)的相互作用,以大麦品种甘啤6号和Issto为实验材料,在接种麦类核腔菌(代号DWC)7和21d后提取叶片蛋白,运用2-DE和质谱分析技术研究条纹病菌侵染后叶片蛋白质组学的变化.结果显示,与对照相比,甘啤6号和Isotta差异表达量在1.4倍以上的蛋白点28个,其中在甘啤6号中表达上调的蛋白点4个,下调的6个,诱导表达的2个,抑制表达的2个;在Isotta中,表达上调的蛋白点3个,下调的4个,诱导表达的4个,抑制表达的3个.质谱鉴定分析发现,表达上调的蛋白包括二磷酸核酮糖羧化酶A(2号蛋白点)、肌动蛋白(9号蛋白点)、核糖体再循环因子(ribosome-recycling factor,RRF)(10号蛋白点)、ATP合酶γ链(11和27号蛋白点)、琥珀酸脱氢酶(辅酶q)黄素蛋白亚基(15号蛋白点)和假定蛋白(26号蛋白点):表达下调的包括过氧化物还原蛋白(4号蛋白点)、1,5-二磷酸核酮糖羧化酶大亚基(ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit,RuBisCo)(3、5、12、14、16和18号蛋白点)、ATP合酶CF1α亚基(13号蛋白点)、Ycf3蛋白(17号蛋白点)和α-1,4葡聚糖蛋白合酶(alpha-1,4-glucanprotein synthase,UTPG)(28号蛋白点);诱导表达蛋白包括假定蛋白(6号蛋白点)、脂氧合酶2(lipoxygenase 2,LOX2)(7号蛋白点)、捕光叶绿素a/b结合蛋白质(light harvesting chlorophyll a/b-binding protein,LHCP)(19号蛋白点)、3-磷酸甘油酸激酶(3-phosphoglycerate kinase,PGK)(20号蛋白点)、凝集素(21号蛋白点)和ATP合酶γ链(22号蛋白点);抑制表达的蛋白包括RuBisCo(1、8、24和25号蛋白点)和二磷酸核酮糖羧化酶小链(ribulose bisphosphate carboxylase small chain,RuBPCase)(23号蛋白点)等.按照其功能分类,这些差异表达蛋白点分别参与了光合作用、蛋白质生物合成、植物防卫反应、能量代谢和细胞信号转导、细胞结构和纤维素生物合成等生理功能.这些差异表达蛋白可能与大麦响应条纹菌侵染过程有关,研究结果有助于从蛋白质水平揭示不同抗性大麦品种抗条纹病的抗性机制.     

盐碱胁迫下紫花苜蓿叶片蛋白组的初步分析

以紫花苜蓿（Medicago sativa）品种"WL343HQ"为材料,对其幼苗进行150 mmol·L^-1的Na₂CO₃和NaHCO₃混合盐碱胁迫,采用iTRAQ技术结合反相液相色谱与液相串联色谱,分析盐碱胁迫叶片中蛋白表达的变化,并对获得的差异蛋白进行生物信息学分析。结果表明,在盐碱胁迫处理下共鉴定到318个显著差异蛋白,包括172个上调蛋白和146个下调蛋白,这些差异蛋白的功能涉及多种代谢途径,其中与光合作用相关的蛋白质表达量总体下调,与苯丙素生物合成、苯丙氨酸代谢和类黄酮生物合成相关的蛋白质表达量总体上调。通过蛋白组学分析技术可有效筛选紫花苜蓿叶片中差异表达蛋白,可为深入研究紫花苜蓿应对盐碱胁迫的分子机制提供一定的理论基础。     

作物叶片蛋白质组提取与酶切方法比较研究

蛋白质组样品制备主要涉及蛋白质的提取和酶切处理,是蛋白质组学分析的限制环节之一。为了提高作物叶片蛋白质组样品制备的效率和重复性,系统比较了6种缓冲液（pH8.5 Tris-HCl酚、pH7.0磷酸盐、pH9.0碳酸盐、尿素/硫脲、pH8.0 Tris-HCl、pH4.5醋酸盐）和TCA-丙酮处理对水稻、小麦、大豆和玉米叶片蛋白质提取的影响,同时以小麦叶片总蛋白和牛血清白蛋白为样本,比较了传统方法和微波辅助方法的酶切效率。结果表明,TCA-丙酮处理的小麦和水稻样品采用尿素/硫脲方法能获得较高的蛋白得率,而玉米和大豆样品采用尿素/硫脲直接提取时蛋白质得率更高,同时,微波辅助酶切值得用于蛋白质组样品制备。本研究采用适当的蛋白质提取和酶切方法,有效提高了蛋白质的提取率和鉴定率,可为进一步深入开展作物叶片的蛋白质组学研究提供借鉴。     

Alteration and significance of protein expression profile in gastric cancer with preoperative conformal radiation therapy

AIM: To investigate the alteration and significance of the protein expression profile in gastric cancer with preoperative conformal radiation therapy. METHODS: The pathologically-diagnosed gastric cancer patients were randomly chosen to perform conformal radiation therapy before operation. The patients without conformal radiation treatment served as controls. The tumor tissues were collected after operation from the patients. The alteration of the protein expression profile was detected by 2-dimensional electrophoresis and MALDI-TOF-MS. The expression of vascular endothelial growth factor (VEGF), c-erbB-2 and epidermal growth factor receptor (EGFR) was detected by RT-PCR and Western blotting. RESULTS: Compared with the patients without conformal radiation therapy, the protein expression profile of tumor tissues from gastric cancer patients with conformal radiation therapy was obviously altered. The expression of VEGF, c-erbB-2 and EGFR was differentially reduced (P<0.01). CONCLUSION: The preoperative accurate conformal radiation therapy reduces the expression of gastric cancer-related epithelial proteins, indicating new therapeutic targets for gastric cancer.     

cPKCγ-HSP60 signal pathway participates in hypoxic preconditioning in mice

AIM: To identify the proteins interacted with conventional protein kinase Cγ (cPKCγ) which are involved in hypoxic preconditioning (HPC), and to investigate the role of cPKCγ-interacted heat-shock protein 60 (HSP60) in the development of HPC and ischemia (I).METHODS: Healthy male BALB/c mice were randomly divided into normoxia(Norm) and HPC groups. Based on whether middle cerebral artery occlusion (MCAO) was performed, the mice were divided into Norm+sham group, Norm+I group, HPC+sham group and HPC+I group ( all n=6). Immunoprecipitation, two-dimensional electrophoresis and mass spectrometry were applied to identify the proteins interacted with cPKCγ. The changes of HSP60 expression in the brain of the mice after HPC and MCAO were analyzed by Western blotting.RESULTS: The interaction of cPKCγ and HSP60 was confirmed by co-immunoprecipitation result. Compared with Norm groups, the expression level of cPKCγ-interacted HSP60 obviously increased in particulate fraction of cerebral cortex in HPC mice. In the MCAO ischemic animals, the level of HSP60 expression was significantly higher in ischemic core and penumbra in Norm+I group and HPC+I group than that in Norm+sham group. HSP60 expression in ischemic core was lower in HPC+I group than that in Norm+I group.CONCLUSION: cPKCγ-HSP60 signal pathway might be involved in the development of cerebral hypoxic preconditioning in MCAO mice.     

Proteomic analysis of mammary tissues from healthy cows and clinical mastitic cows for identification of disease-related proteins

To investigate the disease-related proteins and understand molecular mechanism of mastitis at the protein level, this project presents the protein changes in the mammary gland between healthy cows and clinical mastitic cows using two-dimensional gel electrophoresis (2-DE), after stained with colloidal Coomassie Bright Blue, six spots of differentially expressed protein were detected by PDQuest software and subjected to ion trap mass spectrometer equipped with a HPLC system, and five proteins were identified. Hemoglobin beta, kappa-casein and tryptophanyl-tRNA-synthetase (TrpRS) in healthy dairy cows, while hemoglobin beta, cytochrome C oxidase and annexin V in clinical mastitic cows were identified, they were involved in binding, transport and catalytic activity. The results may provide valuable information for the investigating of the host mammary immune system response to defense against pathogens at the protein level and potential protein targets for treatment.     

适用于小麦叶片蛋白质组分析的样品制备方法

 【目的】建立适用于小麦叶片蛋白质组分析的样品制备方法。【方法】以Taichung 29小麦苗期叶片为材料，分别采用改进的酚提取-甲醇/醋酸铵沉淀法及尿素/硫脲提取法提取总蛋白，进行双向电泳分离和胶体考染，并选取代表性的蛋白点进行MALDI-TOF质谱分析及数据库搜索。【结果】上述的两种方法可检测到较多的蛋白点数，分别为1 016±203和1 014±281，并能成功地完成代表性蛋白点的鉴定。【结论】作者建议，进行小麦叶片蛋白质组分析时，可采用本实验提供的两种样品制备方法。