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大肠杆菌热激反应研究及其在重组蛋白表达中的应用 总被引:1,自引:0,他引:1
当大肠杆菌所处的环境温度忽然升高时, 细胞体内会激发热激反应, 体内会迅速合成多种热激蛋白, 由热激转录因子调控的热激蛋白主要包括一些分子伴侣、蛋白降解酶、折叠辅助蛋白等。热激蛋白可以促进蛋白正确折叠, 降解错误折叠的蛋白。主要介绍大肠杆菌热激蛋白的表达调控及其功能, 利用热激转录因子发展的新型温控分泌表达系统及其在蛋白可溶性表达中的应用, 以及热激分子伴侣与重组蛋白共表达的研究进展。 相似文献
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35S-Met标记玉米胚蛋白合成结果表明,热激处理(42℃)与对照(25℃)的蛋白合成趋势相近,热激抑制16 DAP的蛋白合成,增加22和34 DAP蛋白合成.SDS-PAGE自显影图谱表明,热激诱导16DAP的胚合成86.4、80.0、73.2 kD等3种分子量较高的热激蛋白,22DAP后热激诱导合成86.4、80.0、73.2、24.4、18.2、16.8和13.6 kD等7种分子量的热激蛋白.2D-PAGE自显影图谱进一步显示,热激诱导22和28 DAP的胚合成近20种热激蛋白,其中超过10种为小分子热激蛋白.特异热激蛋白BiP(HsP70)、PDI(HsP60)Western blot表明,这2种热激蛋白在玉米胚发育过程均有高水平的表达,热激对其合成影响不明显. 相似文献
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钙-钙调素信号系统参与热激信号转导的研究 总被引:3,自引:0,他引:3
根据作者实验室的研究工作结合国内外的研究动态讨论热激信号转导的Ca2 -CaM途径。作者实验室的工作表明,钙一钙调素(Ca^2 -CaM)信号系统参与植物热激信号转导。激光共聚焦扫描显微镜的观察结果表明,37℃热激可引起小麦胞内自由Ca。’浓度迅速提高。在Ca^2 存在条件下,热激也引起小麦CaM基因CaM1-2表达及CaM蛋白含量增加。Ca^2 可促进小麦热激基因hsp26和mp70表达和热激蛋白合成,而Ca^2 螯合剂EGTA、Ca^2 通道阻断剂异搏定和LaCl3、CaM抑制剂W7、TFP和CPZ明显降低热激基因hsp26和mp70表达和热激蛋白合成。EGTA、异搏定、TFP或CPZ也阻止小麦耐热性的获得。小麦CaM基因与热激基因的表达动力学研究表明CaM位于热激信号转导的上游,而Ca^2 是启动热激反应的胞内关键因子。凝胶阻滞分析的结果表明,Ca^2 -CaM在热激信号转导中的作用是通过激活热激转录因子的DNA结合活性来实现的。根据大量实验证据,作者提出在植物细胞内存在一条新的热激信号转导途径——钙一钙调素途径。 相似文献
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高温下线粒体小分子热激蛋白对柠檬酸合成酶、线粒体和花粉粒的保护作用 总被引:9,自引:0,他引:9
选用pGEX-6P-1表达载体,构建GSP与线粒体小分子热激蛋白的融合蛋白表达载体,使用谷胱苷肽Sepharose-4B亲合柱,纯化大肠杆菌中表达的融合蛋白,利用ProScission蛋白酶,将线粒体小分子热激蛋白从融合蛋白中切出,获得纯化的线粒体小分子热激蛋白,分析线粒体小分子热激蛋白对柠檬酸合成酶体外热稳定性的影响,发现线粒体小分子热激蛋白可以减缓柠檬酸合成酶的热变性,并促进热变性的柠檬酸合成酶复性,说明高温下线粒体小分子热激蛋白对柠檬酸合成酶有保护功效,利用转基因方法,将线粒体小分子热激蛋白基因导入烟覃,地.aMV35S启动子的驱动下,导入烟草的线粒体小分子热激蛋白基因可在烟草细胞中组成性地表达,比较正常烟草线粒体和转基因烟草线粒体的氧化磷酸化效率,发现高温下转基因烟草线粒体的氧化磷酸化效率高于对照,说明线粒体小分子热激蛋白对线粒体具有保护作用,此外,。转基因烟草花粉粒的高温萌发率也高于对照,说明线粒体小分子热激蛋白不但可以增加线粒体的耐热性,而且可以提高细胞的抗热能力。 相似文献
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水稻热休克转录因子OsHSF13的克隆与生物信息学的初步分析(简报) 总被引:1,自引:0,他引:1
环境刺激的信号转导是植物信号转导的一个重要研究方向。热激反应(heat-shockresponse,HSR)是动植物细胞或器官在遇到外界热刺激时所产生的一种保护性反应,是正常的蛋白质合成受阻时产生热激蛋白(heat-shockprotein,HSP)的一种细胞生理活动,其表达通过热休克转录因子(heat-shock factor,HSF)来进行调控[1]。编码热激蛋白基因的启动子区域存在着一段保守的DNA序列,是热休克转录因子的结合位点(heat-shockelement,HSE)。当植物受到外界的热刺激时,HSF可以与HSE特异性结合,激活热激蛋白基因的表达, 相似文献
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作为分子伴侣, 热激蛋白可起修复变性蛋白与阻止其他蛋白质聚集的作用。为进一步理解蝶蛹金小蜂Pteromalus puparum热激蛋白家族的分子伴侣功能, 本研究对来自该寄生蜂的热激蛋白Pphsp90, Pphsp70, Pphsc70, Pphsp60, Pphsp40和Pphsp20的基因在大肠杆菌Escherichia coli BL21菌株中进行了过表达。结果表明: 除Pphsp40外, 其余5个基因均得到高效表达, 且表达的重组热激蛋白在高温下(80℃)具可溶性与热稳定性。其中, Pphsp20与Pphsp90在大肠杆菌中的表达显著提高了高温下(50℃, 1 h)细胞的存活率。离体活性实验证实, 利用纯化的融合蛋白Pphsp20可减少高温下荧光素酶的聚集现象。据此认为, Pphsp20与Pphsp90均具有大肠杆菌细胞的热保护功能, 但Pphsp20可以单独发挥作用, 而Pphsp90可能需其他因子协同作用才有保护活性。 相似文献
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选用pGEX-6P-1表达载体,构建GST与线粒体小分子热激蛋白的融合蛋白表达载体,使用谷胱苷肽Sepharose-4B亲合柱,纯化大肠杆菌中表达的融合蛋白,利用ProScission蛋白酶,将线粒体小分子热激蛋白从融合蛋白中切出,获得纯化的线粒体小分子热激蛋白.分析线粒体小分子热激蛋白对柠檬酸合成酶体外热稳定性的影响,发现线粒体小分子热激蛋白可以减缓柠檬酸合成酶的热变性,并促进热变性的柠檬酸合成酶复性,说明高温下线粒体小分子热激蛋白对柠檬酸合成酶有保护功效.利用转基因方法,将线粒体小分子热激蛋白基因导入烟草,在CaMV35S启动子的驱动下,导入烟草的线粒体小分子热激蛋白基因可在烟草细胞中组成性地表达,比较正常烟草线粒体和转基因烟草线粒体的氧化磷酸化效率,发现高温下转基因烟草线粒体的氧化磷酸化效率高于对照,说明线粒体小分子热激蛋白对线粒体具有保护作用.此外,转基因烟草花粉粒的高温萌发率也高于对照,说明线粒体小分子热激蛋白不但可以增加线粒体的耐热性,而且可以提高细胞的抗热能力. 相似文献
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Localization of the heat-shock protein Hsp70 to the synapse following hyperthermic stress in the brain 总被引:7,自引:0,他引:7
Heat-shock proteins are induced in response to cellular stress. Although heat-shock proteins are known to function in repair and protective mechanisms, their relationship to critical neural processes, such as synaptic function, has received little attention. Here we investigate whether the major heat-shock protein Hsp70 localizes to the synapse following a physiologically relevant increase in temperature in the mammalian nervous system. Our results indicate that hyperthermia-induced Hsp70 is associated with pre- and postsynaptic elements, including the postsynaptic density. The positioning of Hsp70 at the synapse could facilitate the repair of stress-induced damage to synaptic proteins and also contribute to neuroprotective events at the synapse. 相似文献
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S. Merino J. Martínez A. Barbosa A. P. Møller F. de Lope J. Pérez F. Rodríguez-Caabeiro 《Oecologia》1998,116(3):343-347
Heat-shock proteins (HSPs) are synthesized by animals and plants in response to various stressors. The level of the HSP60
stress protein was measured from the cell fraction of peripheral blood obtained from nestling house martins (Delichon urbica) to test whether ectoparasitism increased the concentration of stress protein. We assessed HSP from nestlings raised in nests
previously treated with an insecticide or infested with 50 martin bugs (Oeciacus hirundinis). In addition, haematozoa infections were checked in blood smears. Nestlings from parasite-infested nests, or nestlings infected
with trypanosomes, had increased levels of HSP in their blood cells. Nestling growth as determined from wing length was negatively
related to HSP60 levels and within-brood variation in wing length increased with increasing levels of the stress protein independently
of treatment and infection by trypanosomes. These results suggest HSPs may play a role in host-parasite interactions, and
that they can be used reliably for measuring physiological responses to parasites.
Received: 4 February 1998 / Accepted: 4 May 1998 相似文献
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《FEBS letters》1996,390(1):113-118
Heat-shock protein 70 (HSP70)-related proteins are classified in two main subfamilies: the DnaK subfamily and the HSP110/SSE1 subfamily. We have characterized the first plant member of the HSP110/SSE1 subfamily, HSP91. At least two, tightly linked genes encoding HSP91 are present per haploid Arabidopsis genome. HSP91 is constitutively expressed in non-stressed Arabidopsis plants and is transiently induced by heat shock. 相似文献
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Lolli G Thaler F Valsasina B Roletto F Knapp S Uggeri M Bachi A Matafora V Storici P Stewart A Kalisz HM Isacchi A 《Proteomics》2003,3(7):1287-1298
An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor-protein interactions as a complementary approach to two-dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin-dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme. Molecular interactions with the Cdk2 inhibitor were compared for HCT116 cancer cells versus rat pancreatic acinar cells. Proteins interacting with the ligand on the IAC matrix were identified by mass spectrometry. Isothermal calorimetry was used to confirm and quantitatively evaluate the binding affinity of some of the interacting proteins. Heat-shock protein (Hsp) 70 and Hsp27 were the strongest interactors with the inhibitor, displaying binding affinities comparable to those of Cdk2. These results support the use of IAC as a general method for the rapid identification and qualitative evaluation of the in vivo targets and potential side effects of a given drug. 相似文献
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矮牵牛(Petunia hybrida)开花过程中的可溶性蛋白质分析 总被引:4,自引:0,他引:4
在进行矮牵牛(Petunia hybrida)光周期诱导开花的过程中,对叶子中的可溶性蛋白质进行了分析,其中有4条与开花有关的特异蛋白,分子量分别为49.45 kD(a)、35.45 kD(b)、17.98 kD(c)和11.74 kD(d).在不开放的花苞中不含有蛋白质a和d,只有这4种蛋白质全出现时,才能形成花苞并且开放.花开了以后,蛋白质c和d就消失.即使在开花的植株中,各组织中的蛋白质也是不同的.茎中完全不含有蛋白质c和d,叶子和花中的蛋白质组成也是不同的. 相似文献
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California chaparral and South African fynbos are fire-prone communities dominated by species exhibiting remarkable similarities in germination response. In both regions there are a substantial number of species with germination stimulated chemically by charred wood and smoke. This type of germination behaviour has arisen independently in distantly related families and is interpreted as convergent evolution. Heat-shock is also an important germination trigger that is widespread, although in both regions it is most common in the same families. Phylogeney may play an important role in the presence of this postfire germination cue in both regions, but a much more rigorous analysis is required to show that this trait represents a single unique event in each lineage. In both regions, germination response is not randomly distributed across growth forms and there are marked regional similiarities in the type of germination behaviour associated with certain growth forms. Geophytes largely lack refractory seeds, which require fire-type cues for germination, but the presence of fire-stimulated flowering of bulbs and corms may time recruitment to subsequent postfire years. Annuals that cue germination to postfire conditions are predominantly triggered by chemicals from smoke and/or charred wood. 相似文献
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Among the proteins that accumulate as plant seeds desiccate are several protein families that are composed principally of a tandemly repeated 11-mer amino acid motif. Proteins containing the same motif accumulate in the desiccating leaves of a desiccation-tolerant plant species. This motif is characterized by apolar residues in positions 1, 2, 5 and 9, and charged or amide residues in positions 3, 6, 7, 8 and 11. An α helical arrangement of the 11-mer repeating unit gives an amphiphilic helix whose hydrophobic stripe twists in a right-handed fashion around the helix. Should these proteins dimerize via binding of their hydrophobic faces, a right-handed coiled coil would be formed. Such a structure has not previously been observed. A conceivable function for these proteins in ion sequestration in the desiccated state is proposed. 相似文献