Isolation and characterization of dominant dwarf mutants, <Emphasis Type="Italic">Slr1-d</Emphasis>, in rice

sd1 is known as the ‘green revolution’ gene in rice because its application in rice breeding has dramatically increased rice yield. Since the ‘green revolution,’ sd1 has been extensively used to produce modern semi-dwarf varieties. The extensive use of limited dwarfing sources may, however, cause a bottleneck effect in the genetic background of rice varieties. To circumvent this problem, novel and useful sources of dwarf genes must be identified. In this study, we identified three semi-dominant dwarf mutants. These mutants were categorized as dn-type dwarf mutants according to the elongation pattern of internodes. Gibberellin (GA) response tests showed that the mutants were still responsive to GA, although at a reduced rate. Map-based cloning revealed that the dwarf phenotype in these mutants was caused by gain-of-function mutations in the N-terminal region of SLR1. Degradation of the SLR1 protein in these mutants occurred later than in the wild type. Reduced interaction abilities of the SLR1 protein in these mutants with GID1 were also observed using the yeast two-hybrid system. Crossing experiments indicated that with the use of an appropriate genetic background, the semi-dominant dwarf alleles identified in this study could be used to alleviate the deficiency of dwarfing genes for breeding applications.     

Sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal

A multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM) principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin was used in this study, M _r6000 Da) was correlated with the shift of resonant frequency of QCM system before and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity, and is sensitive to approx. 1 ng insulin ml^–1 with a linear response for insulin from 10 g ml^–1 to 10 ng ml^–1 in standard solutions. The technique may also be applied for the detection of other small biomolecules.     

酵母三杂交系统的应用研究进展

介绍了酵母三杂交系统的原理、应用、前景和存在的不足.在酵母双杂交基础上发展起来的酵母三杂交系统,将应用范围扩大到研究蛋白质-蛋白质、蛋白质-RNA、蛋白质-小分子药物间的相互作用.     

Adsorption Properties and Activities of Lipase on a Gold Substrate Modified by Self-assembled Monolayers

The adsorption properties, amount and specific activity of lipase D from Rhizopus delemar were investigated by employing a gold substrate modified with seven kinds of thiol monolayer. Quartz crystal microbalance measurements revealed that the amount of the enzyme adsorbed to the hydrophobic monolayers (e.g. benzenethiol) was much higher than that to the hydrophilic monolayers (e.g. 3-mercaptopropanoic acid). In contrast, lipase D adsorbed to the hydrophilic, 2-amino-1-ethanethiol monolayer showed the highest specific activity, the value being 300-fold higher than for the same enzyme dissolved in an aqueous medium.     

Plant ubiquitin-proteasome pathway and its role in gibberellin signaling

The ubiquitin-proteasome system (UPS) in plants, like in other eukaryotes, targets numerous intracellular regulators and thus modulates almost every aspect of growth and development. The well-known and best-characterized outcome of ubiquitination is mediating target protein degradation via the 26S proteasome, which represents the major selective protein degradation pathway conserved among eukaryotes. In this review, we will discuss the molecular composition, regulation and function of plant UPS, with a major focus on how DELLA protein degradation acts as a key in gibberellin signal transduction and its implication in the regulation of plant growth.     

IQCE mRNA与FXR1P在酵母三杂交体系中的相互作用

脆性X相关基因1编码蛋白FXR1P是一种RNA结合蛋白,其所结合的靶RNA目前所知甚少。本研究应用酵母三杂交技术对本课题组从pRH3-cDNA人脑海马RNA表达文库中筛选到的一种可能与FXR1P存在相互作用的RNA IQCE进行研究,以验证该RNA与FXR1P的相互作用。方法为:提取利用酵母三杂交技术初步筛选得到的酵母阳性克隆的质粒,转化大肠杆菌Top10,利用其质粒不相容性分离插入了目的片段的pRH3′-cDNA质粒,将该质粒转化入含目的基因FXR1的酵母菌株L40-ura3/pHyb lex/Zeo-MS2/pYESTrp3/FXR1,进行一对一的酵母三杂交验证,最后将该片段进行测序。测序结果为IQCE的一段编码序列,而目前尚无研究报导FXR1与IQCE的相互关系。结论:提示FXR1P与IQCE mRNA存在相互作用,IQCE可能是FXR1P发育调控网络组成成员之一。     

酵母三杂交系统的应用研究进展

1996年,酵母三杂交技术被研究应用于筛选RNA结合蛋白.本介绍了酵母三杂交系统的原理、应用、前景和存在的不足及局限,并分析其原因,总结了在过去8年中利用此方法所取得的成就.在酵母双杂交基础上发展起来的酵母三杂交系统,其应用范围已扩大到蛋白质-蛋白质、蛋白质-RNA、蛋白质-小分子药物间的相互作用等更加广泛的研究领域.     

发展中的酵母杂交体系

酵母杂交体系包括双杂交、反向双杂交和三杂交等体系。双杂交作为一种新兴的体内研究蛋白质之间相互作用的方法,已经得到了广泛的应用。而反向双杂交和三杂交系统是在双杂交基础上发展起来的两种新技术。反向双杂交除了筛选突变株,以获取蛋白质结合的信息外,还能发现可导致已知蛋白质间特异相互作用发生解离的肽类或其他小分子物质,进一步分析蛋白质间作用位点、调控。三杂交系统则在蛋白质与小分子配基之间以及多种蛋白质之间相     

酵母三杂交系统的原理和应用

酵母双杂交系统自出现以来,广泛用于研究蛋白质之间的相互作用,它是一种具有高灵敏度的研究蛋白质之间关系的技术.在酵母双杂交系统基础上发展的酵母三杂交系统将应用范围扩展到蛋白质-蛋白质、蛋白质-RNA、蛋白质-小分子化合物等更广阔的研究领域.本文着重介绍酵母三杂交系统的原理、应用及局限性.     

Multichannel quartz crystal microbalance array: Fabrication,evaluation, application in biomarker detection

A multichannel quartz crystal microbalance array (MQCM) with three pairs of gold electrodes was fabricated for detection of two biomarkers: acetone and nitric oxide (NO). The gold electrodes were deposited symmetrically on an AT-cut 10 MHz circular quartz plate using photolithography, sputtering, and lift-off technologies. The effect of gold layer thickness on MQCM performance was investigated and the optimized thickness was 101 nm. The simulation values of the electric parameters C₀, C_q, L_q, and R_q in the Butterworth–Van Dike equivalent circuit for the MQCM device were 97 pF, 1.3 pF, 1.05 mH, and 9.8 Ω, respectively. Simulation values were in the theoretical range, which indicated that the fabricated MQCM device had good resonance performance. Two types of nanocomposites, titanium dioxide–multiwalled carbon nanotubes and cobalt (II)phthalocyanine–silica, were synthesized as sensing materials. The sensing mechanism is based on coordination adsorption of target molecules onto the sensing material, resulting in a resonant frequency shift of modified QCM sensor. A linear range from 4.33 to 129.75 ppmv for acetone was obtained and one from 5.75 to 103.45 ppbv for NO.     

ITO石英晶体电极上不同内皮细胞浓度的粘弹性研究及药物作用评估

用石英晶体微天平(quartz crystal microbalance,QCM)和活细胞成像技术实时监测人脐静脉内皮细胞(HUVEC)在ITO石英晶体电极上的动态粘附响应过程。在ITO晶体电极上加入不同浓度的HUVEC,测定细胞在QCM上谐振频率以及耗散的实时变化。通过ITO电极与光学显微镜的联用,监测了HUVEC在药物处理前后的动态变化过程。用细胞粘弹性指数(QCM的动态电阻变化与频移变化之比,CVI=ΔR/Δf)表征细胞的粘弹性变化,同时通过活细胞成像技术的联用,实时监测细胞的形态变化。结果表明:细胞浓度为10万个/m L时,细胞在ITO电极上铺展完全且粘弹性最大。抑制剂y-27632和激动剂凝血酶thrombin药物处理细胞前后,在显微镜的实时监测下细胞形态变化不明显,但CVI粘弹性指数变化较大,说明QCM信号比光学信号更为敏感,且在药物筛选方面有有很大的应用前景。     

Covalent DNA immobilization on polymer-shielded silver-coated quartz crystal microbalance using photobiotin-based UV irradiation.

The use of a commercial, silver-coated quartz crystal microbalance (QCM) as a disposable, low-cost, and reliable DNA sensor is presented. This is an incorporation of polymer-based silver electrode shielding and photochemistry-based surface modification for covalent DNA immobilization. To prevent undesired oxidation, the silver electrodes are coated with thin polystyrene films. The polymer surfaces are then modified by a photoreactive biotin derivative (photobiotin) under UV irradiation. The resulting biotin residues on the polymer-shielded surface react with a tetrameric avidin. Consequently a biotin-labeled DNA probe can be immobilized through a biotin-avidin-biotin bridge. A 14-mer single-stranded biotin-DNA probe and a 70-mer single-stranded DNA fragment containing complementary or noncomplementary sequences are used as a model system for DNA hybridization assay on the proposed sensors. The shielding ability of the polystyrene coatings after photo irradiation is investigated. The DNA probe binding capacity, hybridization efficiency, and kinetics are also investigated.