A quantitative method for detecting ammonia‐oxidizing bacteria in coastal aquaculture systems

There is a need to quantify autotrophic nitrifiers in coastal aquaculture systems for evolving a bioremediation strategy. Autotrophic nitrifiers are extremely slow‐growing organisms, which cannot be detected by traditional methods as they are notoriously difficult to culture. Molecular techniques based on functional genes could be deployed for the detection of nitrifiers. Ammonia monooxygenase (amoA), that catalyses the oxidation of ammonia to hydroxylamine in the rate‐determining step of nitrification is largely unique to ammonia‐oxidizing bacteria (AOB). In the present study, a quantitative real‐time polymerase chain reaction assay targeting amoA was developed to estimate AOB population size in coastal soil, ammonia‐removing bioaugmentors and the solid matrix. To achieve this objective, different set of primers and a dual labelled probe have been designed for SYBR Green and TaqMan real‐time assays. The abundance of AOB ranged from 10⁴ to 10⁶ order of magnitude in the samples. In the present study, biofilm formation of the consortium of nitrifying bacteria onto bagasse has also been quantified. The results demonstrate that the developed method is a rapid and sensitive tool for the quantitative detection of nitrifying bacteria in aquatic and related environment. This helps in making the bioremediation approach for ammonia removal by immobilization of nitrifying bacteria onto the natural substrate.     

Expression and characterization of the periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida

Cobalamin (vitamin B₁₂) is an essential cofactor in a variety of enzymatic reactions and most prokaryotes contain transport systems to import vitamin B₁₂. A gene coding for a periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida was identified by in silico analysis of sequences from a genomic library. The open reading frame was composed of 834 bp encoding a protein of 277 amino acids. The protein showed 61% identity with the vitamin B₁₂-binding protein precursor of P. profundum , 53% identity with the corresponding protein of Vibrio parahaemolyticus and 43% identity with the periplasmic binding protein BtuF of Escherichia coli. The expression of the native protein was investigated in P. damselae subsp. piscicida , but BtuF was weakly expressed under normal conditions. To characterize the BtuF of P. damselae subsp. piscicida , the recombinant protein was expressed with a C-terminal His₆-tag and purified; the molecular weight was estimated to be approximately 30 kDa. The protein does not contain any free thiol group, consistent with the view that the two cysteine residues are involved in a disulphide bond. The purified BtuF binds cyanocobalamin with an affinity constant of 6 ± 2 μ m .     

Nicotinic acetylcholine receptor chimeras of rat α7 and Drosophila SAD reveal species-specific agonist binding regions

Species-specific agonist binding regions of nicotinic acetylcholine receptors (nAChR) were examined. Imidacloprid and physostigmine (Phy) selectively activated insect nAChR composed of Drosophila second alpha-like subunit (SAD) and chick β2, in contrast to rat α7 nAChR. The Phy-activated currents were α-bungarotoxin (α-BGT) sensitive, suggesting activation at the agonist binding loop C. Several SAD-α7 chimeras were constructed, by switching agonist binding regions, and expressed in oocytes. Though none of the chimeras was activated by a range of nicotinic agonists, [¹²⁵I]α-BGT binding revealed homomeric assembly of all chimeric cDNAs. Phy differentially displaced [¹²⁵I]α-BGT from the nAChR chimeras, suggesting that the β subunit is not involved in Phy binding, and that Phy targets the insect agonist binding loop C.     

Prediction of the binding mode of imidacloprid and related compounds to house-fly head acetylcholine receptors using three-dimensional QSAR analysis

The binding activity of imidacloprid and related compounds to nicotinic acetylcholine receptors (nAChR) of house flies was measured by use of radioactive α-bungarotoxin as a ligand. Variations in the activity were examined three-dimensionally using comparative molecular field analysis (CoMFA). The CoMFA results suggest that one conformer among the four stable ones is active and provide support for one of the proposed binding models for this class of compound, in which the nitrogen atom of the pyridine ring and the nitrogen atom at the 1-position of the imidazolidine ring interact with the hydrogen-donating and electron-rich sites of nAChR, respectively. The CoMFA field map showed that the nitroimino moiety and a portion of the imidazolidine ring were mainly surrounded by a sterically and electrostatically sensitive region of nAChR. © 1998 Society of Chemical Industry     

PRRSV 2b蛋白与LGALS1相互作用的验证研究

旨在研究猪繁殖与呼吸综合症病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)2b蛋白与宿主细胞蛋白之间的相互作用。本研究利用酵母回返试验、GST-pull down试验、免疫共沉淀试验3种方法对LGALS1与PRRSV 2b蛋白间是否相互作用进行验证。结果表明:在酵母回返试验中,含有LGALS1与2b基因的杂交酵母菌株在SD/-Trp-Leu-His-Ade/AbA/X-α-gal的平板上生长出蓝色菌落;在GST-pull down试验中,LGALS1与2b蛋白反应的样品经Western blot可检测到融合蛋白带;在免疫共沉淀试验中,LGALS1与2b蛋白反应的样品经HA单抗沉淀后,经Western blot可检测到相应蛋白带。该研究证明了LGALS1与PRRSV 2b蛋白间发生相互作用,为研究PRRSV感染途径和增殖过程提供了理论基础。     

昆虫嗅觉机制的研究进展

嗅觉机制对于昆虫选择栖息地、获得食物、趋利避害、传递讯息、群集以及繁殖等许多行为起到重要作用。因此,通过研究昆虫嗅觉系统,可以阐释嗅觉发生过程中的普遍机理,还有助于理解昆虫嗅觉活动与其整个生命活动之间的联系,进而为高等动物特别是人的嗅觉研究提供科学依据。并且通过对昆虫嗅觉机理的研究,发展出了一系列新的害虫治理方法,为害虫防治提供了新的思路。近年来,随着生物化学、分子生物学、昆虫行为学和昆虫电生理学的深入研究,科技人员发现了许多相关的嗅觉活性分子和嗅觉相关基因,从分子层面对昆虫嗅觉机制进行解释。本文综述了气味信号通过嗅感器转变为电信号,并由昆虫触角叶编码整合,最终传递到前脑整个过程中所涉及的分子组件及昆虫体内生理生化等方面有关进展。     

带约束拉杆钢管/竹胶板组合空芯短柱的偏心抗压性能

采用冷弯薄壁方型钢管和胶合竹板通过横向约束拉杆和结构胶黏剂复合成顺纹抗压的组合空芯短柱(thin-walled steel tube/bamboo-plywood composite hollow short column with binding bars,SBCCB),采用9个试件研究SBCCB试件的偏心抗压破坏模式、抗压承载力和影响规律。结果表明,SBCCB试件受压破坏形态主要为柱端开胶破坏、横向约束拉杆之间基体胶合界面开胶剥离破坏和胶合竹板局部压屈破坏;SBCCB抗压极限荷载随胶合竹净横截面积增大而提高,随着长细比和荷载偏心率的增大而降低,随空心率增大而增大;约束拉杆可有效延迟SBCCB的开胶剥离破坏,改变屈曲破坏模式,有助于试件抗压承载力的提高;相对于不带约束拉杆试件,SBCCB抗压极限应力提高约17.64%;局部翘曲随约束拉杆间距减小而减小,相对拉杆间距比3.0以下能确保较小的局部翘曲变形。薄壁钢管和胶合竹板能形成较好的复合抗压结构单元,该批试件平均值抗压强度达到18.54 MPa,展现了优异的抗压性能。SBCCB可作为绿色建筑材料广泛应用于现代装配式工程结构,同时拓展竹材的应用途径,实现"以竹代木,以竹代钢",具有很好的工程价值和应用前景。     

小菜蛾触角结合蛋白PxylABP结合模式的研究

为了研究触角结合蛋白(Antennal binding proteins,ABPs)的生理功能,利用RT-PCR方法克隆了小菜蛾Pxyl ABP基因,并在大肠杆菌中表达,纯化的重组蛋白通过荧光竞争结合试验测定其与29种化合物的结合特性,并运用Auto Dock 4.2进行Pxyl ABP蛋白和化合物的分子对接。结果表明,Pxyl ABP与异硫氰酸丙烯酯、己醛、己烷、2-己酮、吲哚和β-紫罗兰酮结合,解离常数分别为0.91,1.81,1.17,2.71,2.74,14.95μmol/L;分子对接显示,Tyr100与异硫氰酸丙烯酯形成1个氢键。说明Pxyl ABP参与了小菜蛾识别十字花科蔬菜和虫害诱导的植物挥发物的生理过程。     

云南省玉溪市玉米致死性坏死病毒原的分子鉴定

玉米致死性坏死病是由玉米褪绿斑驳病毒(Maize chlorotic mottle virus,MCMV)和一种或多种马铃薯Y病毒科病毒复合侵染引起的。2014年1月在对云南省玉溪市玉米病毒病害的调查中发现了一些表现严重花叶、矮化叶片甚至整个植株坏死症状的玉米。对采集样品进行RT-PCR检测,所有样品中都同时检测到了MCMV和甘蔗花叶病毒(Sugarcane mosaic virus,SCMV),在一个样品中同时检测到了MCMV、SCMV和南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus)。