木本植物蛋白提取和SDS-PAGE分析方法的比较和优化

在几种木本植物上对４种常用蛋白提取方法、４种凝胶染色和脱色方法及影响蛋白定量和ＳＤＳＰＡＧＥ电泳的因素进行了比较研究,发现：利用改良丙酮沉降法提取蛋白,不仅提取效率高,而且杂质干扰少,得到的电泳结果,蛋白条带清晰,数量多;将常规凝胶染色和脱色液中的乙醇改用为甲醇可大大改善染色和脱色效果,获得了没有背景或背景很浅的电泳结果。通过研究确定了一套优化的适用于木本植物的蛋白提取、定量、凝胶制备、电泳、染色、脱色以及干胶制备的方法。利用这一优化方法对胡杨细胞盐胁迫蛋白进行了研究,发现了与高水平盐胁迫有关的６６ｋＤ蛋白和与盐胁迫和干旱胁迫均有关的２８ｋＤ蛋白,获得了满意的蛋白ＳＤＳＰＡＧＥ分析结果。     

长角血蜱卵黄蛋白的纯化及其性质

用凝胶过滤与离子交换层析、蛋白质电泳和糖脂蛋白染色等方法提取纯化长角血蜱Haemaphysalis longicornis卵黄蛋白,并对其性质进行了研究。PAGE和SDS-PAGE分析表明,长角血蜱的卵黄蛋白只有一种,由8个亚基组成,亚基的相对分子质量分别为112 kD, 103 kD, 80 kD, 78 kD, 71 kD, 68 kD, 62 kD和52 kD,卵黄蛋白经苏丹黑B和希夫试剂染色呈阳性,表明是一种含血红素的糖脂蛋白。     

卷柏总蛋白质提取方法及双向电泳条件的建立

目的:建立复苏植物——卷柏总蛋白质的提取方法,以及可以对其蛋白质组进行阵列分离的双向电泳条件。方法:通过多种条件的组合与优化,建立了以物理和化学2种方法充分裂解植物组织细胞,并采用低温操作、加入PVP等去除植物组织中大量的酚类物质,最后离心去除不溶性杂质的卷柏蛋白质组双向电泳(2-DE)样品制备方法。第1向电泳为固相pH梯度等电聚焦,第2向电泳为垂直平板SDS-PAGE。结果:通过对样品制备、蛋白定量、第1向和第2向电泳、染色方法等各实验环节进行控制和优化,凝胶经考马斯亮蓝染色后,可分辨蛋白质斑点数约600个。结论:建立了卷柏总蛋白质的提取方法及蛋白质组双向电泳技术,为后续研究卷柏在干旱胁迫下的差异表达奠定了基础。     

杜氏盐藻完整叶绿体的分离及其蛋白提取

应用高压破碎及蔗糖密度梯度离心的方法,分离出杜氏盐藻的完整叶绿体,随后用冻融法和研磨法分别提取叶绿体蛋白,并通过蛋白定量和SDS-PAGE凝胶电泳对两种蛋白提取方法进行比较,确立了一套适用于杜氏盐藻叶绿体分离和蛋白提取、定量以及电泳的方法.结果表明:用高压破碎结合蔗糖密度梯度离心的方法能够获得完整且较纯的盐藻细胞叶绿体;SDS-PAGE凝胶电泳结果显示,冻融法提取叶绿体蛋白效率高,电泳条带清晰,蛋白数量较多,蛋白齐全.为下一步在亚细胞水平进行杜氏盐藻耐盐机制的蛋白组学研究奠定了基础.     

不经染色直接从SDS-PAGE制备凝胶中准确切下所需蛋白条带的方法

蛋白样品的垂直板SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE)不但是一种最常用的蛋白分析方法,也经常用于蛋白质的制备。从电泳凝胶上纯化蛋白,一般都要先用考马斯亮蓝染色,然后切下所需的蛋白条带。这里介绍一种可以不染色,直接从SDS-PAGE制备凝胶上准确切割所需蛋白条带的方法。与染色后切胶的方法相比,这种方法简单、省时,分离到的蛋白容易从胶中洗脱回收,并可明显提高回收率,而且省去了令人烦怖的从回收的蛋白中脱去染色时结合的染料的问题。作者曾用此方法分离过多种蛋白,屡试不爽。这种方法与一般的SDS-PAGE制备电泳的差别主要在电泳结束后对凝胶的处理上。     

种子中Puroindoline蛋白的SDS-PAGE分析

Puroindolne蛋白是小麦中特殊的Triton X-114可溶性蛋白质,对小麦籽粒硬度有着决定性的影响.从二倍体、六倍体小麦材料及小麦近缘种粗山羊草成熟种子中提取Puroindoline蛋白,就对该蛋白的SDS-PAGE及染色条件进行了优化和讨论,建立了浓缩胶T(凝胶浓度)为4、C(交联度)为2.6、电泳电压为128 V;分离胶T为13.5、C为2.6、电泳电压240 V,分离胶电泳时间1.5 h的结果稳定且重复性好的优化的SDS-PAGE条件.同时为降低电泳染色的实验成本,简化实验步骤,用蓝染法代替常用的银染法,并获得了良好的效果,为小麦中Puroindoline蛋白质的进一步研究奠定了实验基础.     

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。     

苹果树腐烂病菌胞外蛋白提取方法

选择一种理想的蛋白提取方法,获得数量多、质量高的苹果树腐烂病菌Valsa mali胞外蛋白用于蛋白质组学分析,为全面解析该病原菌的致病机制奠定基础。采用冷冻干燥透析结合法、脱氧胆酸钠(DOC)-10%TCA沉淀法、硫酸铵沉淀法3种方法分别提取苹果树腐烂病菌的胞外蛋白,并筛选最适的硫酸铵浓度,利用Bradford法测定蛋白总量、SDS-PAGE检测蛋白条带分辨率及不同致病力菌株蛋白差异。结果表明,70%硫酸铵沉淀提取的胞外蛋白量最高、SDS-PAGE电泳可辨认蛋白条带最多。强弱致病菌株胞外蛋白的SDS-PAGE电泳图谱在37-50 kD有4条蛋白条带差异明显,由此表明,70%硫酸铵盐析法更适合用于苹果树腐烂病菌胞外蛋白质差异分析时蛋白质的提取。     

箭毒木种子蛋白质样品制备及双向电泳改良方法

建立箭毒木（Antiaris toxicaria）种子总蛋白的提取方法,以及可以对其蛋白质组进行分析的双向电泳条件。通过各种条件的优化与组合,建立了以TCA-丙酮为基础的Tris—HCl提取法提取总蛋白,第1向电泳为固相pH梯度等电聚焦,第2向电泳为垂直平板SDS-PAGE的双向电泳体系。通过对样品制备、样品溶解、等电聚焦电泳、SDS-聚丙烯酰胺凝胶电泳以及染色方法等关键步骤进行分析,获得了满意的双向电泳图谱。在探索适合箭毒木种子蛋白质组学研究双向电泳方法中,比较了三氯乙酸-丙酮沉淀法、和Tris—HCl法,以及对双向电泳过程中的关键步骤的改良,认为Tris—HCl法为最适方法,所得图谱背景清晰,蛋白质信息量最大,为箭毒木属植物的差异蛋白质组学的后续研究打下了坚实的基础。     

黑木耳漆酶纯化的研究

目的:研究黑木耳(Auricularia auricula)“黑29”的漆酶纯化,为进一步酶学性质的开展、酶应用和酶基因克隆提供理论基础。方法:采用硫酸铵分级沉淀和柱层析技术分离蛋白,通过PAGE和SDS-PAGE电泳检测蛋白。结果:SDS-PAGE电泳检测发现粗酶液含三种漆酶,分子量大小分别为LacA(60kD)、LacB(34kD)、LacC(19kD);纯化后获得纯化的单一漆酶LacA、LacC组分。结论:得漆酶两个单一组分,为进一步漆酶研究奠定基础。     

Changes in the polypeptide patterns of barley seedlings exposed to jasmonic Acid and salinity

Soluble and thylakoid membrane proteins of jasmonic acid (JA)-treated and salt-stressed barley (Hordeum vulgare L.) seedlings were investigated using 15% sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. High JA concentrations induced marked quantitative and qualitative changes in polypeptide profiles concerning mainly the proteins with approximately equal mobility, as in NaCl-stressed plants. The most obvious increase in thylakoid polypeptide band intensity was at 55 to 57 kilodaltons (kD). The relative share of some polypeptides with apparent molecular masses above 66 kD and of polypeptides with lower molecular masses in the region of 20.5 to 15 kD was enhanced. At the same time, one new band at 31 to 31.5 kD was well expressed at 25 and 250 micromolar JA concentrations and became discernible in the 100 micromolar NaCl-treated plants. The intensity of some polypeptides of soluble proteins (molecular masses of 60, 47, 37, 30, and 23.4 kD) increased with increasing JA concentration, whereas the intensities of other polypeptide bands (55, 21.4, and 15 kD) decreased. Enhanced levels of 60-, 47-, 34-, and 30-kD polypeptides and reduced levels of 55- and 15-kD polypeptides were present in NaCl-treated plants. The appearance of one new polypeptide, of 25.1 kD, was observed only in NaCl-treated plants. At 100 millimolar NaCl, an eightfold increase in proline content was observed while at 250 micromolar JA, the proline content was threefold over the control. It is hypothesized that exogenously applied jasmonates act as stress agents. As such, they provoke alterations in the proline content and they can modulate typical stress responses by induction of stress proteins.     

茶树叶片类脱水素蛋白的Western-blot分析

为了解茶树脱水素种类与功能,采用Western-blot技术,研究了不同季节及越冬过程中茶树叶片脱水素蛋白家族的表达模式。结果显示:(1)茶树叶片总蛋白提取采用酚-甲醇/醋酸铵沉淀法,用时短、蛋白浓度高、SDSPAGE电泳条带清晰,背景干净,满足茶树Western-blot技术要求。(2)在不同季节及越冬期中发现14~95kD共9种不同分子量的茶树类脱水素蛋白,其中95、65、48、37、34和14kD等6种蛋白表达量较为稳定,季节与越冬期变化不明显;58kD脱水素仅在冬季表达,越冬期不断上升,2月份增加到最高,表达丰度高;28kD脱水素蛋白在冬季表达量高,越冬期与茶树抗寒力变化规律一致;21kD脱水素在夏季和越冬期后期有较高的表达。研究表明,这3种脱水素可能在茶树抗逆中起着重要作用。     

In vivo and in vitro binding of 2,4-dichlorophenoxyacetic acid to a rat liver mitochondrial protein

2,4-dichlorophenoxyacetic acid (2,4-D) is a hormonal herbicide widely used in the world because of its efficacy in the control of broadleaf and woody plants. In this study we have demonstrated in vivo covalent binding of the phenoxyherbicide 2,4-D to a single protein of 52 kD (from rat liver mitochondrial preparation) detected through immunoblotting studies with the specific antiserum for 2,4-D. The direct involvement of 2,4-D in the formation of the adduct has also been demonstrated in vitro, using liver mitochondrial preparations exposed to 14C-UL-2,4-D. Radiolabeled protein separated by SDS-PAGE and afterwards electroeluted showed a single labeled protein of 52 kD. When mitochondria exposed to radiolabeled xenobiotic were devoid of their outer membrane, the specific activity observed suggest that protein involved in covalent interaction belongs to the inner mitochondrial membrane. We propose that covalent binding of the phenoxyherbicide 2,4-D to a very specific single protein of 52 kD observed in vitro and in vivo may be related to known alterations of the mitochondrial function.     

Stress-mediated alteration in V-ATPase and V-PPase of Butea monosperma

The activity and subunit amounts of V-ATPase and V-PPase in various plants of Butea monosperma Taub. (Fabaceae) (ver. Dhak; Palas) growing as a natural inhabitant in varying stress conditions in southeast Rajasthan were studied. Western blot analysis followed by immunological quantification of V-ATPase subunits using specific polyclonal antibodies showed that the subunits A, B, D, E, and c are clearly detectable in all plants, with A, B, and c appearing as intense bands. The other subunits of V-ATPase, viz., C, a, and d, were also detected in majority of the plants. Various subunits exhibited variations in their protein amount in different plants. Besides, a few other clear bands were also detected. Of these, the 30- and 29-kD bands may possibly be Di and Ei. Furthermore, a clear band of V-PPase corresponding to 67–70 kD was also detected. A comparison of the V-ATPase and V-PPase activity revealed that Butea plants in the upper region of the study site showed 70% and 39% higher activity, respectively. Furthermore, the immunological quantification showed that the V-ATPase and V-PPase protein amounts are also higher in the upper Butea plants which have drought stress and, moreover, are also exposed to stronger light intensities for relatively longer duration.     

Aluminum-Induced Changes in Cell-Wall Glycoproteins in the Root Tips of Al-Tolerant and Al-Sensitive Wheat Lines

To investigate wheat (Triticum aestivumL.) responses to Al stress, KCl- and SDS-extracted glycoproteins (covalently bound proteins isolated by cell-wall digestion by cellulysine–pectolase mixture) and extensins (hydroxyproline-containing glycoproteins, HRGPs) were isolated from cell-wall preparations purified from the root apices of Al-sensitive and Al-tolerant near-isogenic lines ES8 and ET8. Under Al stress conditions, two lines differed mostly in their extensins. The untreated plants of two lines were low in covalently bound extensins, although the content of this protein fraction in ES8 was higher than in ET8. When the seedlings were treated with Al, the extensin content increased in both wheat lines and especially in the Al-tolerant ET8 plants. Using two-dimensional electrophoresis, the authors demonstrated the accumulation of polypeptides with mol wts of 22.2 kD (pI 5.5–6.5), 24.5 kD (pI 5.8–6.0), and 33.1 kD (pI 5.25) and polypeptides of 22.2 kD (pI 6.8–7.6) and 40.5 kD (pI 7.6) in the extensin fraction from the cell walls of the Al-sensitive plants. The regulation of cell responses to Al stress may involve extensin expression.     

Identification and partial characterization of the allergenic proteins of Ricinus communis L. pollen - a new approach

The pollen of Ricinus communis L., a potentially allergenic plant, was extracted to identify the allergenic determinants responsible for causing respiratory disorders. The soluble proteins were extracted and subjected to ammonium sulphate precipitation at 80% saturation and the total protein separated on 12% SDS-Polyacrylamide gel. In order to avoid the time consuming and expensive biochemical methods of column chromatography, each band was directly recovered from the gel by electroelution and the allergenic proteins identified directly by skin tests, without the necessity of Phadezym RAST or ELISA inhibition by reaction with serum IgE, the general procedure to identify the allergens. The fourth and the fifth band in the protein profile of R. communis pollen, RC4 (77 kD) and RC5 (66 kD) were the two major allergenic components. RC3 (91 kD) also induced a considerable amount of reactivity in sensitive patients. Contrary to the earlier reports of protein bands of R. communis ranging from 14 kD to 70 kD, 4 bands above 70 kD i.e. RC1 (123 kD), RC2 (97 kD), RC3 (91 kD) and RC4 (77 kD) are reported here for the first time. Immunodiffusion analysis with pooled sera of patients sensitive to the total extract also revealed similar results.     

杨树细胞色素P450类固醇单加氧酶(CYP90)基因的克隆与分析

拟南芥的CPD基因编码一种与植物油菜素内酯(brassinosteroids,BRs)生物合成有关的细胞色素P450类固醇单加氧酶(CYP90 )。为探讨油菜素内酯这类新型植物激素在多年生木本植物中生物合成及作用的分子机理,以拟南芥CPD基因的一个cDNA片段为探针,从一种杂交杨 (Populustremula×tremudelois)的cDNA文库中分离出一个长 1 442bp的cDNA片段,然后再以这个cDNA的 5′区为探针,从这种杂交杨的基因组文库中分离出一个长 1 900bp的基因组DNA(gDNA)片段。测序结果表明,这段cDNA的 5′区与这段gDNA的 3′区重合; 由这段cDNA和gDNA组成的读框编码一个由 476个氨基酸组成的分子量为 63kD的蛋白质。该蛋白与拟南芥CYP90的同源性为 78 32%,比后者仅长 4个氨基酸,在所有已知的功能结构域,其中包括与BR生物合成密切相关的类固醇结合位点,也具有较高的同源性,表明CPD基因在一年生的草本和多年生的木本植物之间具有很高的保守性。系统树分析还表明,CYP90蛋白与番茄和玉米的矮化基因产物、鱼的all trans retinoicacid4 hydroxy lase及微澡青菌(Synechocystissp. )的细胞色素P450在进化上有较密切的联系。     

耐盐突变体小麦后代耐盐稳定性分析研究

以卫星搭载小麦种子为原始材料,利用其幼穗、幼胚诱导的愈伤组织进行耐盐突变体的筛选,对耐盐愈伤组织再生植株后代进行耐盐稳定性生理生化特性分析。结果表明：(1)耐盐系后代在土壤高盐浓度条件下,游离脯氨酸含量稳定增加,且高于对照系;(2)耐盐系再生植株后代保持较高的K^ ／Na^ 比;(3)与对照相比,种子醇溶蛋白电泳带谱中的b2,b3,b5,b7带为耐盐系所特有,b8带消失;(4)耐盐系再生植株后代可溶蛋白电泳带为26条,而对照系为23条蛋白带。其中98kD、75kD、52kD、49kD和32kD为耐盐系的特有蛋白带。而38kD和35kD蛋白带为对照系所特有。