结肠癌蛋白质双向电泳技术的建立

目的:建立结肠癌13cm和24cm非线性分离系统的2-D图谱,分析比较两者的分辨率.方法:提取结肠癌总蛋白,用pH3-10非线性干胶条对样品进行等电聚焦分离,并分别使用13cm和24cm电泳系统进行双向电泳,考马斯亮蓝G250染色,图像分析,比较对比两组2-D图谱,量化分析两种系统的分辨率差异.结果:在等点电3-10,分子量20-170 kD范围内分别分离得到蛋白质斑点873个(13 cm电泳系统)和1349个(24cm电泳系统).对于24cm电泳系统,1 mg蛋白质上样量的电泳图谱清晰,分辨率较好.结论:成功建立了高分辨率、简便易控的结肠癌蛋白质组双向电泳技术平台.     

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.     

小菜蛾血淋巴蛋白质双向电泳技术体系的建立及优化

采用不同方法对小菜蛾Plutella xylostella(L.)血淋巴进行双向电泳研究,建立了一套适用于小菜蛾血淋巴蛋白质组分析的双向电泳体系。从样品处理方案、等电聚焦时间、染色方法等因素对小菜蛾双向电泳结果的影响进行了分析和优化。结果表明,TCA/丙酮沉淀法提取蛋白损失少,图谱均匀清晰,分辨率和重复性更高。不同长度胶条的最佳上样量不同,胶条长度为7、17、24cm时,最佳上样量依次为20～50μg、50～300μg、100～500μg,而聚焦时间则分别以24000、50000、65000vhr为宜。第二向聚丙烯酰胺凝胶的浓度以12%为宜,电泳后蛋白点呈均匀分布。银染的灵敏度明显高于考马斯亮蓝染色,可以检测出更多的蛋白点,但考马斯亮蓝染色在后续蛋白点质谱鉴定中具有优势。     

山黧豆叶片蛋白质双向电泳技术的建立

以山黧豆叶片为材料,比较分析了蛋白质的不同提取方法,在此基础上着重于样品制备。对IPG胶条的选择,第一向等电聚焦和第二向SDS-聚丙烯酰胺凝胶电泳的电泳程序及参数、染色方法等相关技术进行了比较和条件优化。结果显示：采用TCA-丙酮沉淀法提取蛋白质,裂解液中加入Tris-base作为蛋白酶抑制剂,等电聚焦电泳时延长低电压的电泳时间（30V、12h,500V、1h,1000V、2h）以促进盐离子泳出的方法对山黧豆叶片蛋白质进行双向电泳,并用考马斯亮蓝和银染复合染色法进行凝胶染色,能够获得蛋白点清晰的双向电泳图谱,说明用优化后的方法建立起的山黧豆叶片蛋白质双向电泳技术,蛋白质样品制备质量好,电泳分辨率高,完全适合于进一步的蛋白质组学研究。     

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。     

蛋白质组学技术筛选和鉴定白化黑线仓鼠皮肤白化相关差异蛋白质

目的比较黑线仓鼠及其白化突变系背部皮肤蛋白表达的差异,寻找差异蛋白质,从蛋白质水平探讨白化病的发生机制。方法应用双向凝胶电泳技术分离出差异蛋白质,用质谱法分析其结构与组成,通过蛋白质数据库确定差异蛋白的功能。结果从64个表达差异蛋白斑点中发现33个显著差异的蛋白点,其中又有14个差异点匹配到了有意义的蛋白质。14个差异点共鉴定出11个差异蛋白质,这些差异蛋白质按功能可分为4类:(1)糖代谢相关蛋白;(2)运输蛋白;(3)细胞骨架蛋白;(4)其他蛋白。结论黑线仓鼠与其白化突变系背部皮肤蛋白表达存在明显差异,其中一些蛋白与白化病发生相关,并可能成为白化病致病机理研究的分子标志物和药物治疗靶向位点。     

白桦花芽蛋白质双向电泳技术的建立

目的:初步建立白桦花芽蛋白质组研究的双向电泳(2-DE)技术,以提高其分辨率及重复性。方法:以白桦花芽为实验材料,对关键环节如样品处理、上样量和染色方法等进行一系列优化。结果:蛋白上样量为50～60μg、银染剂中采用0.16%的AgNO3染色12min,即可获得蛋白点的形态规则、稳定性高、重复性好的2-DE图谱。结论:建立了适合多糖、醌类等次生代谢物质含量较多的白桦花芽蛋白提取方法,为开展白桦发育生物学研究奠定了基础。     

水稻蛋白质组双向电泳优化流程及方法

双向电泳是分析蛋白质混合物的一种有力手段, 已在蛋白质组研究中得到广泛应用。水稻(Oryza sativa)作为重要的粮食作物, 对其蛋白质组学研究开展较早。但由于技术复杂, 对实验操作要求高, 初学的研究者很难在较短的时间内掌握该实验技术。该文介绍了水稻研究中适合多个组织的双向电泳实验方法和优化流程。该优化流程能使新的研究者逐步优化实验条件, 更快更好地完成双向电泳实验。同时详细介绍了实验关键环节的操作方法。     

水稻蛋白质组双向电泳优化流程及方法

双向电泳是分析蛋白质混合物的一种有力手段,已在蛋白质组研究中得到广泛应用。水稻（Oryzasativa）作为重要的粮食作物,对其蛋白质组学研究开展较早。但由于技术复杂,对实验操作要求高,初学的研究者很难在较短的时间内掌握该实验技术。该文介绍了水稻研究中适合多个组织的双向电泳实验方法和优化流程。该优化流程能使新的研究者逐步优化实验条件,更快更好地完成双向电泳实验。同时详细介绍了实验关键环节的操作方法。     

砂藓总蛋白质提取方法的选取及双向电泳体系的建立

为进行砂藓(Racomitrium canescens)蛋白质组学的双向电泳工作,首要前提是获得质量较好的砂藓总蛋白质,并建立一套完整高效的适用于砂藓的双向电泳体系。本研究以砂藓为研究对象,比较了Tris-酚抽提法、TCA-丙酮法和试剂盒法3种蛋白质提取方法获得砂藓总蛋白质的浓度和完整性,并对砂藓中蛋白质双向电泳的运行条件进行了优化和比较。结果表明:在3种砂藓总蛋白质提取方法中,Tris-酚抽提法获得的砂藓总蛋白质浓度最高,SDS-PAGE电泳结果中条带清晰且较完整,双向电泳后获得的蛋白质点最多,是3种方法中提取砂藓蛋白质浓度最高完整性最好的方法;基于Tris-酚抽提法的基础上进一步对砂藓总蛋白质双向电泳体系进行优化,结果显示:利用pH值范围为pH 4～7的IPG胶条,蛋白质上样量为1 000μg的条件下,获得的砂藓蛋白质双向电泳图谱蛋白质点最多,图谱中可分辨的蛋白质点共(872±15)个,且低丰度蛋白质点清晰。本研究筛选出的方法获得了蛋白质点分布均匀、背景清晰、分辨率高、质量较高的双向电泳图像,建立了砂藓蛋白质组学研究体系的较好方法。本研究结果为砂藓蛋白质组学研究提供了良好基础。     

Tissue and Cellular Distribution of Glutamine Synthetase in Roots of Pea (Pisum sativum) Seedlings

The effect of nitrate application on glutamine synthetase activity in roots of pea (Pisum sativum L.) seedlings (2 weeks old) was studied. Separation of organelles from root fragments by sucrose density-gradient centrifugation revealed that both nitrite reductase and glutamine synthetase activities increased in root plastids as a response to nitrate application and that no such response was induced by ammonium application. Glutamine synthetase activity was also found to increase in plastids with distance from apex in nitrate-treated plants, the highest specific activity being located in the fourth 1-centimeter segment. Separation by SDS-PAGE and characterization by Western blotting showed that cytosolic glutamine synthetase contains one subunit polypeptide (28 kilodaltons) and that plastid glutamine synthetase contains both the 38-kilodalton subunit and a heavier subunit. When nitrate was present in the nutrient solution, the heavier subunit increased in abundance in protein fractions obtained from purified root plastids.     

Induction of Flavonoid Synthesizing Enzymes by Light in Etiolated Pea (Pisum sativum cv. Midfreezer) Seedlings

Etiolated pea (Pisum sativum cv. Midfreezer) seedlings respond to illumination with white light by changes in the activity of phenylpropanoid and flavonoid synthesizing enzymes. Unlike in cell cultures, changes in enzyme activity in pea seedlings are not concerted. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity peaked approximately 18 hours after onset of illumination. The phenylacetate path did not interfere with the measurement of phenylalanine ammonia-lyase activity. Activity of cinnamic acid 4-hydroxylase (EC 1.14.13.11) showed an early peak after 8 hours illumination, declined thereafter sharply, then gradually increased during the remainder of the experiment. Activities of chalcone synthase and UDP glucose:flavonol 3-O-glucosyltransferase (EC 2.4.1.91) increased steadily and reached a plateau after approximately 70 hours illumination time. Activity of 4-hydroxycinnamate:coenzyme A ligase (EC 6.2.1.12) remained relatively unchanged, whereas that of chalcone isomerase (EC 5.5.1.6) declined steadily during the course of the experiment. The relative in vitro enzyme activities suggest that the rate-limiting step for the phenylpropanoid path is the cinnamic acid 4-hydroxylase, that of the flavonoid pathway is the chalcone synthase. Integration of enzyme activity curves, however, show that only the curve deriving from phenylanine ammonia-lyase activity matches closely the production of the flavonol glycosides.     

Characterization of alpha-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings

The most abundant α-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This α-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an α-amylase by polarimetry, substrate specificity, and end product analyses. The purified α-amylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This α-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein. Common cell wall constituents (e.g. β-glucan) are not substrates of the enzyme. A very low amount of this α-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or α-amylase which is integrally associated with the chloroplast.     

Changes in Chloroplast DNA Levels during Development of Pea (Pisum sativum)

Determinations were made of the percentage of chloroplast DNA (ct DNA) in total cell DNA isolated from shoots of pea at different stages of development. Labeled pea ct DNA was reassociated with a high concentration of total DNA; the percentage of ct DNA was estimated by comparing the rate of reassociation of this reaction with that of a model reaction containing a known concentration of unlabeled ct DNA. The maximum change in ct DNA content was from 1.3% of total DNA in young shoots to 7.3% in fully greened shoots. Analyses were also performed on DNA from embryos, etiolated tissue, roots, and leaves. The first leaf set to develop in pea was excised over a growth period of 8 days during which leaf length increased from 4 to 12 millimeters. Young leaves contained about 8% ct DNA; in fully greened leaves the level of ct DNA approached 12%, equivalent to as many as 9,575 copies of ct DNA per cell. Root tissue contained only 0.4% ct DNA.     

Partial Characterization and Subcellular Localization of Three alpha-Glucosidase Isoforms in Pea (Pisum sativum L.) Seedlings

Three isoforms of α-glucosidase (EC 3.2.1.20) have been extracted from pea (Pisum sativum L.) seedlings and separated by DEAE-cellulose and CM-Sepharose chromatography. Two α-glucosidase isoforms (αG1 and αG2) were most active under acid conditions, and appeared to be apoplastic. A neutral form (αG3) was most active near pH 7, and was identified as a chloroplastic enzyme. Together, the activity of αG1 and αG2 in apoplastic preparations accounted for 21% of the total acid α-glucosidase activity recovered from pea stems. The vast majority (86%) of the apoplastic acid α-glucosidase activity was due to αG1. The apparent K_m values for maltose of αG1 and αG2 were 0.3 and 1.3 millimolar, respectively. The apparent K_m for maltose of αG3 was 33 millimolar. The respective native molecular weights of αG1, αG2, and αG3 were 125,000, 150,000, and 110,000.     

热胁迫对豌豆下胚轴生理的一些影响

通过测定热驯和热胁迫下3个豌豆品种幼苗下胚轴生长、细胞膜损伤、抗坏血酸（AsA）和丙二醛（佃A）含量的变化及热激蛋白70（HSP70）表达,探讨热胁迫对豌豆生理的影响。结果表明,在48℃高温胁迫下豌豆种子萌发率下降,幼苗下胚轴生长受抑制,细胞膜受损,AsA含量下降,MDA含量升高;经37℃热驯再48℃热激处理的下胚轴长度和ASA明显高于直接热胁迫的,细胞膜受损程度和MDA含量则低于后者。HSP70测定表明,除台湾品种外,37℃热驯1h不足以诱导HSP70表达;而37℃热驯后常温恢复再48℃热激和直接48℃热激均能诱导HSP70表达,其中蒙自品种经热驯后再热激的HSP70表达量高于直接热激的。     

Mobilization of Phosphorus in the Cotyledons of Young Seedlings of the Garden Pea (Pisum sativum L.)

Changes in the levels of various phosphorus fractions and ofphytase activity in the cotyledons of young pea seedlings grownin the light have been studied. It is shown that from the onsetof germination there is a lag of several days in the hydrolysisof phytic acid and that this is associated with a low levelof phytase activity in cotyledon extracts. Rapid developmentof phytase during the next few days is accompanied by a rapidincrease in the rate of phytic acid break-down and both reachmaximum levels after 6–7 days from soaking the seed. Theamount of phytic acid in the cotyledons becomes negligible afterabout 15 days and at the same time phytase activity declinesmarkedly. At this point protease activity is at a maximum andthe water content of the cotyledons begins to fall. Removal of the shoot 4 days after soaking the seed caused animmediate decrease in export of phosphorus from the cotyledonsbut did not affect the level of phytic acid for several days.Subsequently there was a small, but significant reduction inthe rate of phytic acid hydrolysis in de-shooted seedlings ascompared with intact plants in spite of the fact that phytaseactivity was not affected for several days. Similar effectswere observed when excised cotyledons were cultured on moistfilter-paper. Control mechanisms for phytic acid hydrolysis are discussedand it is concluded that regulation by the axis of the inorganicphosphate concentration at the sites of phytase activity maybe a means of controlling phytic acid hydrolysis.     

Purification of Multiple Forms of Glutathione Reductase from Pea (Pisum sativum L.) Seedlings and Enzyme Levels in Ozone-Fumigated Pea Leaves

Glutathione reductase was purified from pea seedlings using a procedure that included 2′,5′-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chromatofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The K_m for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 μm for plastidic and extraplastidic glutathione reductase, respectively, at 25°C. The K_m for NADPH was 4.8 and 4.0 μm for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L⁻¹ ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozone-fumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.