用焦锑酸钾沉淀法对45℃和28℃下黄瓜(新泰密刺)授粉后柱头及子房中C a2+分布进行了电镜观察,并采用酶联免疫吸附测定法(EL ISA)测定了叶片中的ABA含量及用等电聚焦聚丙烯酰胺双向电泳(IEF-SDS PAGE)方法对其蛋白合成的变化进行了研究....用焦锑酸钾沉淀法对45℃和28℃下黄瓜(新泰密刺)授粉后柱头及子房中C a2+分布进行了电镜观察,并采用酶联免疫吸附测定法(EL ISA)测定了叶片中的ABA含量及用等电聚焦聚丙烯酰胺双向电泳(IEF-SDS PAGE)方法对其蛋白合成的变化进行了研究.结果表明,28℃时柱头中的C a2+主要分布在细胞间隙中,经45℃处理后细胞间隙和胞内C a2+水平均显著升高,胞内外C a2+浓度梯度逐步丧失;45℃处理1 h后柱头和子房中内源ABA含量显著提高;高温处理后柱头和子房中产生了一些新的蛋白质,如柱头中的(MW26.0 kD,P I 5.4)、(MW90.0 kD,P I5.1)、(MW54.0 kD,P I 4.6)、(MW41.0 kD,P I 6.2),子房中的(MW90.0 kD,P I5.2)、(MW61.0 kD,P I 5.4)、(MW48.0 kD,P I 6.4)、(MW40.5 kD,P I 4.9);另有一些蛋白质与常温相比表达量大幅上调,如柱头中的(MW67.0kD,P I 5.8)、(MW56.5 kD,P I 5.8),子房中的(MW70.0 kD,P I 5.7)、(MW57.0 kD,P I 5.7).上述结果表明,C a2+和ABA信号系统均参与了授粉后黄瓜雌性器官对高温胁迫的反应调节并最终导致基因表达发生变化.展开更多
Proteomics is one of the most active research fields in the post-genomic era. Here we briefly introduce the scientific background of the origination of proteomics and its content, research method. The new developments...Proteomics is one of the most active research fields in the post-genomic era. Here we briefly introduce the scientific background of the origination of proteomics and its content, research method. The new developments of proteomics at the levels of individual plants, tissues, organs and organells, as well as its applications in the area of plant genetic diversity, mutant characterization, and plant physiology, etc are reviewed. At last, the challenge and prospect of proteomics are discussed.展开更多
采用双向凝胶电泳技术优化分离蓝斑背肛海兔(Notarcus leachii cirrosusStimpson,NLCS)口腔神经节(Buccal Ganglion,BG)蛋白质组,并获得约300个蛋白质斑点.用组合基质辅助激光解吸电离化飞行时间(MALD I-TOF)质谱技术和胶内酶解技术测...采用双向凝胶电泳技术优化分离蓝斑背肛海兔(Notarcus leachii cirrosusStimpson,NLCS)口腔神经节(Buccal Ganglion,BG)蛋白质组,并获得约300个蛋白质斑点.用组合基质辅助激光解吸电离化飞行时间(MALD I-TOF)质谱技术和胶内酶解技术测定BG蛋白质组中的96个蛋白质斑点的肽指纹(Peptide m ass fin-gerprint,PMF)图谱.经数据库检索与比对后,发现96种蛋白质中仅有4种蛋白质可获得较高的匹配率,它们分别是微管蛋白(Tubu lin)、肌动蛋白(Actin)和两个1,5-二磷酸核酮糖-羧化酶/加氧酶(R ibu lose-1,5-b i-sphosphate carboxylase/oxygenase,R ibu lose),均属于神经细胞骨架蛋白质;同时还发现一种交配信号肽前体(Peptide m ating pheromone precursor).利用LOC trees软件和分类法对56种蛋白质进行亚细胞定位与分类.展开更多
Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum...Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum and adult worms were obtained from rabbits infected with 1 500 cercariaes on 14 and 42 days after challenge, respectively. Adult male and female worms on 42 days were manually detached and stored into liquid nitrogen until use. Eggs were collected by Percoll TM from the liver of rabbits. ProteoPrep Membrane Extraction Kit TM was employed to extracted membrane proteins by reducing and alkylating with TBP and iodoacetamide from 200 mg of eggs, schistosomulums, adult male worm and female worms, respectively. Immobilized pH gradient strips with a linear pH range of 3-10 (130 mm) were rehydrated together with membrane proteins (30 μg) in 250 μl solution containing 7 mol urea, 2 mol thiourea, 2% SB3-10, 4% CHAPS, 40 mmol Tris, 30 mmol DTT, then separated on 12.5% SDS polyacrylamide gel for the second dimensional electrophoresis. Gels were stained with silver, scanned by Labscan, and analyzed using ImageMaster TM Analysis software. The 2D maps of egg, schistosomulum, adult female worm and male worm were showed 78±3、67±3、108±4 and 122±4 spots respectively. There were 35±1 spots which showed specific expression in female worm as compared with male worm, but 45±2 spots were in male worms. Most differently expressed spots between male and female worms were located in the area of 40-70 kD and pI 4-7. The large number of unique spots from schistosomulum was located in the area of alkalescence. The 2D map of for adult male worms uniquely showed 5 spots as compared with that of schistosomulum and female worm. The female worm showed 4 unique spots as compared with that of schistosomulum, egg and male worm. The unique spots between male and female worms were identified by the database of SWISS 2D-PAGE according to the molecular weight and isoelectronic point. Calreticulin 1, methyltransferase, outer membrane protein tolc and oxygen-evolving enhancer protein 1-1 were uniquely showed in adult male worm after pairing. On the other hand, enolase, outer membrane protein X, ferrienterobactin receptor and heat shock cognate 70 kD protein 3 were uniquely showed in adult female worm. In conclusion, there are different expressions of membrane proteins from egg, schistosomulum, adult male worm and female worms of Schistosoma japonicum. These proteins, which were uniquely expressed between adult male and female worms after pairing, were involved in signal transduction and metabolism展开更多
文摘用焦锑酸钾沉淀法对45℃和28℃下黄瓜(新泰密刺)授粉后柱头及子房中C a2+分布进行了电镜观察,并采用酶联免疫吸附测定法(EL ISA)测定了叶片中的ABA含量及用等电聚焦聚丙烯酰胺双向电泳(IEF-SDS PAGE)方法对其蛋白合成的变化进行了研究.结果表明,28℃时柱头中的C a2+主要分布在细胞间隙中,经45℃处理后细胞间隙和胞内C a2+水平均显著升高,胞内外C a2+浓度梯度逐步丧失;45℃处理1 h后柱头和子房中内源ABA含量显著提高;高温处理后柱头和子房中产生了一些新的蛋白质,如柱头中的(MW26.0 kD,P I 5.4)、(MW90.0 kD,P I5.1)、(MW54.0 kD,P I 4.6)、(MW41.0 kD,P I 6.2),子房中的(MW90.0 kD,P I5.2)、(MW61.0 kD,P I 5.4)、(MW48.0 kD,P I 6.4)、(MW40.5 kD,P I 4.9);另有一些蛋白质与常温相比表达量大幅上调,如柱头中的(MW67.0kD,P I 5.8)、(MW56.5 kD,P I 5.8),子房中的(MW70.0 kD,P I 5.7)、(MW57.0 kD,P I 5.7).上述结果表明,C a2+和ABA信号系统均参与了授粉后黄瓜雌性器官对高温胁迫的反应调节并最终导致基因表达发生变化.
文摘Proteomics is one of the most active research fields in the post-genomic era. Here we briefly introduce the scientific background of the origination of proteomics and its content, research method. The new developments of proteomics at the levels of individual plants, tissues, organs and organells, as well as its applications in the area of plant genetic diversity, mutant characterization, and plant physiology, etc are reviewed. At last, the challenge and prospect of proteomics are discussed.
文摘采用双向凝胶电泳技术优化分离蓝斑背肛海兔(Notarcus leachii cirrosusStimpson,NLCS)口腔神经节(Buccal Ganglion,BG)蛋白质组,并获得约300个蛋白质斑点.用组合基质辅助激光解吸电离化飞行时间(MALD I-TOF)质谱技术和胶内酶解技术测定BG蛋白质组中的96个蛋白质斑点的肽指纹(Peptide m ass fin-gerprint,PMF)图谱.经数据库检索与比对后,发现96种蛋白质中仅有4种蛋白质可获得较高的匹配率,它们分别是微管蛋白(Tubu lin)、肌动蛋白(Actin)和两个1,5-二磷酸核酮糖-羧化酶/加氧酶(R ibu lose-1,5-b i-sphosphate carboxylase/oxygenase,R ibu lose),均属于神经细胞骨架蛋白质;同时还发现一种交配信号肽前体(Peptide m ating pheromone precursor).利用LOC trees软件和分类法对56种蛋白质进行亚细胞定位与分类.
基金国家高技术研发 863项目 (No 2 0 0 1AA2 15 15 1)上海市重大科技攻关项目 (No 0 3DZ192 3 1)资助~~
文摘Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum and adult worms were obtained from rabbits infected with 1 500 cercariaes on 14 and 42 days after challenge, respectively. Adult male and female worms on 42 days were manually detached and stored into liquid nitrogen until use. Eggs were collected by Percoll TM from the liver of rabbits. ProteoPrep Membrane Extraction Kit TM was employed to extracted membrane proteins by reducing and alkylating with TBP and iodoacetamide from 200 mg of eggs, schistosomulums, adult male worm and female worms, respectively. Immobilized pH gradient strips with a linear pH range of 3-10 (130 mm) were rehydrated together with membrane proteins (30 μg) in 250 μl solution containing 7 mol urea, 2 mol thiourea, 2% SB3-10, 4% CHAPS, 40 mmol Tris, 30 mmol DTT, then separated on 12.5% SDS polyacrylamide gel for the second dimensional electrophoresis. Gels were stained with silver, scanned by Labscan, and analyzed using ImageMaster TM Analysis software. The 2D maps of egg, schistosomulum, adult female worm and male worm were showed 78±3、67±3、108±4 and 122±4 spots respectively. There were 35±1 spots which showed specific expression in female worm as compared with male worm, but 45±2 spots were in male worms. Most differently expressed spots between male and female worms were located in the area of 40-70 kD and pI 4-7. The large number of unique spots from schistosomulum was located in the area of alkalescence. The 2D map of for adult male worms uniquely showed 5 spots as compared with that of schistosomulum and female worm. The female worm showed 4 unique spots as compared with that of schistosomulum, egg and male worm. The unique spots between male and female worms were identified by the database of SWISS 2D-PAGE according to the molecular weight and isoelectronic point. Calreticulin 1, methyltransferase, outer membrane protein tolc and oxygen-evolving enhancer protein 1-1 were uniquely showed in adult male worm after pairing. On the other hand, enolase, outer membrane protein X, ferrienterobactin receptor and heat shock cognate 70 kD protein 3 were uniquely showed in adult female worm. In conclusion, there are different expressions of membrane proteins from egg, schistosomulum, adult male worm and female worms of Schistosoma japonicum. These proteins, which were uniquely expressed between adult male and female worms after pairing, were involved in signal transduction and metabolism
