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该研究基于番茄基因组数据库SGN(Sol Genomic Network)信息,利用RT PCR从栽培番茄‘M82’(Solanum lycopersicum)中成功克隆到番茄SlWRKY6基因(登录号:Solyc02g080890),通过qRT PCR方法和原核表达初步验证其生物学功能。结果表明:(1)生物信息学分析显示,番茄SlWRKY6基因ORF全长1 653 bp,编码550个氨基酸,其蛋白结构含有1个WRKYGQK保守结构域和C2H2锌指结构域,属于IIb类;其基因启动子上游1 500 bp含有多个激素响应元件和非生物胁迫响应元件。(2)进化树分析显示,SlWRKY6与潘那利番茄SpWRKY31 X1(NP_001352691.1)的相似性最高,且定位于细胞核内。(3)qRT PCR结果显示,SlWRKY6基因在番茄根、茎、叶中均有表达,在叶中的表达量最高,且受盐和干旱诱导表达。(4)SDS PAGE及Western blot结果显示,pET 30a SlWRKY6重组蛋白的大小约66 kDa,与预期大小一致。(5)原核表达分析显示,重组菌E. coli BL21∷pET 30a SlWRKY6在不同浓度盐(NaCl)和干旱(Mannitol)胁迫下生长速度显著低于对照菌E. coli BL21∷pET 30a,且在400 mmol/L NaCl、800 mmol/L甘露醇胁迫条件下最为显著;滴板实验初步验证SlWRKY6转录因子能提高重组菌E. coli BL21∷pET 30a SlWRKY6在ABA和pH 9(NaOH)胁迫的耐受性;在400 mmol/L NaCl、pH 5(HCl)、800 mmol/L甘露醇胁迫条件下耐受能力降低。研究表明,SlWRKY6转录因子可能通过参与ABA途径来响应非生物胁迫。 相似文献
106.
用改良的MTT法测定rhG-CSF活性 总被引:1,自引:1,他引:1
MTT测定法是根据线粒体脱氢酶催化MTT形成蓝色甲■的多少来检测活细胞数和功能状态的,但原始方法中存在着一些问题,如敏感性偏低、有机溶剂产生蛋白质沉淀以及产物的溶解度偏低等。为了摸索测定 rhG-CSF活性的最适条件,我们以 NFS-60细胞为对象,比较了多种溶解缓冲液,并且对细胞数、MTT浓度及保留时间、溶解液用量等条件进行了选择。结果表明,DMF-20%SDS和 20%SDS的效果最好,测定时细胞数为每孔 1000个细胞,所加 MTT浓度为 1mg/ml,保留时间为 4 h,溶解液的用量为每孔 100μl。 相似文献
107.
Yu Ban Ying-ying Wu Tao Yu Ning Geng Yong-yue Wang Xiao-guang Liu Ping Gong 《Tissue & cell》2011,43(5):311-317
The process of mechanotransduction of bone, the conversion of a mechanical stimulus into a biochemical response, is known to occur in osteoblasts in response to fluid shear stress. In order to understand the reaction of osteoblasts to various times of flow perfusion, osteoblasts were seeded on three-dimensional scaffolds, and cultured in the following conditions: continuous flow perfusion, intermittent flow perfusion, and static condition. We collected samples on day 4, 8 and 12 for analysis. Osteoblast proliferation was demonstrated by cell proliferation and scanning electron microscopy assay. Additionally, the expression of known markers of differentiation, including alkaline phosphatase and osteocalcin, were tested by qRT-PCR and alkaline phosphatase activity assay, and the deposition of calcium was used as an indicator of mineralization demonstrated by calcium content assay. The results supported that low fluid shear stress plays an important role in the activation of osteoblasts: enhance cell proliferation, increase calcium deposition, and promote the expression of osteoblastic markers. Furthermore, the continuous flow perfusion is a more favorable environment for the initiation of osteoblast activity compared with intermittent flow perfusion. Therefore, the force and time of fluid shear stress are important parameters for osteoblast activation. 相似文献
108.
Xiang T Lundell E Sun Z Liu H 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,858(1-2):254-262
IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions. 相似文献
109.
Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels 下载免费PDF全文
110.
Jun Liu Tao Lin Douglas J. Botkin Erin McCrum Hanspeter Winkler Steven J. Norris 《Journal of bacteriology》2009,191(16):5026-5036
The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of ∼1,280 flagellar motors, a ∼3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.Flagellum-based motility plays a critical role in the biology and pathogenesis of many bacteria (3, 6, 17, 31). The well-conserved flagellum is commonly divided into three physical parts: the flagellar motor, the helically shaped flagellar filament, and the hook which provides a universal joint between the motor and the filament. In most bacteria, counterclockwise rotation of the flagella results in bundling of the helical flagella and propulsion of the cell through liquid or viscous environments. Clockwise rotation of the flagellar motor results in random turning of the cell with little translational motion (“tumbling”). Bacterial motility is thus a zigzag pattern of runs and tumbles, in which chemotactic signals favor running toward attractants and away from repellents (3).Borrelia burgdorferi and other closely related spirochetes are the causative agents of Lyme disease, which is transmitted to humans via infected Ixodes ticks (40). Spirochetes have a distinctive morphology in that the flagella are enclosed within the outer membrane sheath and are thus called periplasmic flagella (6). The flagellar motors are located at both ends of the cell and are coordinated to rotate in opposite directions during translational motion and in the same direction (i.e., both clockwise or both counterclockwise) during the spirochete equivalent of tumbling, called “flexing” (6, 15). Spirochetes are also capable of reversing translational motion by coordinated reversal of the direction of motor rotation at both ends of the cell. Rotation of the flagella causes a serpentine movement of the entire cell body, allowing B. burgdorferi to efficiently bore its way through tissue and disseminate throughout the mammalian host, resulting in manifestations in the joints, nervous system, and heart (40).The flagellar motor is an extraordinary nanomachine powered by the electrochemical potential of specific ions across the cytoplasmic membrane (3). Current knowledge of the flagellar motor structure and rotational mechanisms is based primarily on studies of Escherichia coli and Salmonella enterica and is summarized in several recent comprehensive reviews (3, 22, 31, 39, 42). The flagellar motor is constructed from at least 20 different kinds of proteins. The approximate location of these flagellar proteins has been determined by a variety of approaches and appears to be relatively consistent in a wide variety of bacteria. It can be divided into several morphological domains: the MS ring (FliF, the base for the flagellar motor); the C ring (FliG, FliM, and FliN, the switch complex regulating motor rotation); the export apparatus (multiple-protein complex located at the cytoplasmic side of the MS ring); the rod (connecting the MS ring and the hook); the L and P rings on the rod (thought to serve as bushings at the outer membrane and at the peptidoglycan layer, respectively); and the stator, which is the motor force generator embedded in the cytoplasmic membrane. Electron microscopy studies of the purified flagellar motor have provided a detailed view of the rotor/C-ring assembly (11, 44). However, there is no structural information on the stator and the export apparatus in these reconstructions, because these membrane-associated structures are not retained following detergent extraction during the extensive basal body purification process. The stator and the export apparatus were visualized by using freeze fracture preparations of cytoplasmic membranes. It appears that 10 to 16 stator units form circular arrays in the membrane (9, 20). Part of the export apparatus is located in the central space of the C ring (18). Recently a 7-nm-resolution structure of the intact flagellar motor in situ was revealed by averaging 20 structures obtained using cryo-electron tomography (cryo-ET) of Treponema primitia cells (32). Further analysis of the intact flagellar motor structure would lead to a better understanding of the motor protein distribution, the rotor-stator interaction, and the mechanism of bacterial motility.Cryo-ET has emerged as a three-dimensional (3-D) imaging technique to bridge the information gap between X-ray crystallographic and optical microscopic methods (24, 30). This process involves rapidly freezing viable cells, collecting a series of electron micrographs at different angles, and computationally combining the resulting images into a 3-D density map. Cryo-ET allows investigation of the structure-function relationship of molecular complexes and supramolecular assemblies in their cellular environments without fixation, dehydration, embedding, or sectioning artifacts. Spirochetes are well suited for cryo-ET analysis because of their narrow cell diameter (typically 0.2 to 0.3 μm). Recently the cellular architecture of Treponema primitia, Treponema denticola, and B. burgdorferi, as well as the configuration of the B. burgdorferi periplasmic flagella, were revealed by cryo-ET (7, 16, 26, 33). In combination with advanced computational methods, cryo-ET is currently the most promising approach for determining the cellular architecture in situ at molecular resolution (30). We have developed novel strategies for capturing and averaging thousands of 3-D images of large macromolecular assemblies to obtain ∼2.0-nm-resolution structures (28, 29).In this study, we present the molecular structures of infectious wild-type (WT) and mutant B. burgdorferi organisms and their flagellar motors in situ using high-throughput cryo-ET and 3-D image analysis. By averaging subvolumes of 1,280 flagellar motors from 322 cells, we obtained a ∼3.5-nm-resolution model of the intact flagellar motor, providing a detailed view of rotor-stator interactions. In addition, detergent treatment of intact cells provided a preliminary identification of the rotor and stator structures. Through the comparison of WT and mutant cells, we have also determined the location of the flgI gene product in the B. burgdorferi flagellar motor. 相似文献