An improved method for bright-field imaging of arbuscular mycorrhizal fungi in plant roots

Easy observation methods to assess the colonization levels of arbuscular mycorrhizal (AM) fungi in plant roots are crucial for studying the biology of AM symbiosis and for considering agricultural use. Many AM studies employ Trypan Blue (TB) coupled with lactic acid to stain AM fungal structures as bright-field images; however, TB staining can be difficult to use owing to its noxiousness and high viscosity. Here, we report the development of an easy method for visualizing AM fungal structures as bright-field images using 3,3′-diaminobenzidine (DAB). Wheat germ agglutinin (WGA)-conjugated horseradish peroxidase (HRP), which specifically targets N-acetylglucosamine polymers and detects AM fungal cell walls, penetrated the cortical layers of 10% potassium hydroxide (KOH)-treated soybean roots and stained AM fungal mycelia in the presence of DAB and hydrogen peroxide (H₂O₂). Comparison between DAB and TB staining of soybean (Glycine max L.) roots suggested that the intactness of root systems and image contrast using DAB staining were superior. Background signals in stele observed by DAB staining were negligible as compared with those observed by WGA-fluorescein isothiocyanate staining. DAB staining, which combines the advantages of TB (easy bright-field imaging) and WGA-fluorophore (specific and high-quality) staining, provides a robust imaging method for macro- and micro-level analyses of AM roots and is applicable to at least six crops: soybean, onion (Alium cepa L.), potato (Solanum tuberosum L.), maize (Zea mays L.), rice (Oryza sativa L.), and sunflower (Helianthus annuus L.).     

GNA和ACA双价转基因烟草培育及其抗蚜研究

蚜虫是一种常见的农作物害虫,作为多种植物病毒的载体而成为影响农业发展的重要害虫之一,对蚜虫的防治一直是众多科学工作者所面对的难题。构建了雪花莲凝集素(GNA)和苋菜凝集素(ACA)双价植物表达载体pBI121-GA,在农杆菌介导下,叶盘法转化烟草NC89,得到卡那霉素抗性植株65株。通过基因组PCR、ELISA、RT-PCR和Western-blot等分子生物学检测,从中筛选出外源凝集素不同表达水平的转基因烟草5株。对筛选出的5株转基因烟草植株进行初步蚜虫抗性实验,证实转基因烟草对桃蚜表现出一定的抗性,最高抑制率达85.8%,为农作物双价抗蚜基因的应用研究提供一定的理论基础。     

锯缘青蟹血清凝集素的特性和盐析提取研究

对锯缘青蟹(Scylla serrate)血清凝集素的凝集特性进行了研究。结果显示:锯缘青蟹血清凝集素对人、兔、鼠、鸽子、中华鳖、鲫红细胞均有凝集作用,其中对小鼠红细胞凝集活性最强,血凝活性可达29;凝集素活性最适pH 6.0~7.4;盐度对凝集活力有较大影响,NaC l浓度在0.4~0.5 mol/L时基本失活;未纯化血清凝集素对Ca2+、Mg2+未表现出依赖性;凝集素活性能被N-乙酰葡萄糖胺特异性抑制。对青蟹血清凝集素进行硫酸铵分级沉淀分离后,发现凝集素活性主要分布在25%饱和度硫酸铵沉淀区,该组分凝集比活比血清高27.44倍,SDS-PAGE结果表明主要蛋白带型为79 ku、75 ku、72 ku。     

Variations in Seroprevalence and Host Factors for Bovine Anaplasmosis in Illinois

Hungerford, L.L. and Smith, R.D., 1997. Variations in seroprevalence and host factors for bovine anaplasmosis in Illinois. Veterinary Research Communications, 21 (1), 9-18.The modified card agglutination test (MCAT) was used to detect antibodies to Anaplasma marginale in 4994 samples collected from market cattle through the Brucellosis Eradication Program in Illinois. The MCAT reactor rate in the sample was 6.4%. The statewide prevalence estimate increased to between 7.1% and 10.7% when corrected for sampling distribution and test sensitivity and specificity. While the sample reactor rate measures the proportion of infected cattle moving through the market system and into new herds, consideration of the sampling proportions and test characteristics is important when estimating the overall prevalence. Cows were found to be at decreased risk relative to male cattle in lower-prevalence areas, but the risk was similar in higher-prevalence areas. In both higher- and lower-prevalence areas, the risk for seropositivity increased from 1 year of age onwards, but the rate of increase with age was greater in the higher-prevalence areas. Calves, however, had higher seroprevalence levels than 1-2-year-old animals. The risk was highest for Angus and Angus-Hereford cross cattle, while dairy cattle showed a decreased risk in the whole sample, but not when the analyses were stratified by prevalence level. There was a higher concentration of dairy cattle in areas with lower prevalence levels.     

掌叶半夏凝集素基因表达载体的构建与鉴定

从掌叶半夏(Pinellia pedatisecta Schott)块茎中提取总RNA,反转录合成cDNA.根据GenBank中已报道的天南星科凝集素基因序列,设计带有酶切位点的表达引物,用RT-PCR方法扩增出掌叶半夏凝集素(Pinellia pedatisecta agglutinin,PPA)基因.PPA经双酶切后与表达载体pGEX-6P-3连接,构建pGEX-PPA重组表达载体.通过PCR、双酶切及测序鉴定,结果表明,重组载体pGEX-PPA构建成功,为进一步进行PPA的原核表达及其功能研究奠定了基础.     

大豆凝集素的分子结构及其抗营养作用研究进展

概述了大豆凝集素的研究情况,总结归纳了大豆凝集素的分子结构、理化特性及其对动物和人类造成的抗营养作用。     

牛精子膜麦芽凝集素结合糖蛋白的分离与定位

牛精子膜用非离子去污剂 NP-40增溶,经固相凝集素亲和层析,初步分离了精子膜麦芽凝集素结合糖蛋白,经 SDS-聚丙烯酰胺凝胶电泳、银染色显示出了四条蛋白质带,分子量分别为78,60.42和33 kD。用分离到的糖蛋白制备抗血清,对牛精子进行免疫细胞化学标记,结果顶体区显示为强阳性,与麦芽凝集素亲和细胞化学的染色一致。用抗血清处理精子再进行亲和细胞化学染色时,顶体区的麦芽凝集素结合糖残基多数被封闭,揭示麦芽凝集素结合糖残基是构成抗原决定簇的成分。     

白肋烟转基因育种研究

利用农杆菌介导法将具有广谱抗病的葡萄糖氧化酶(Glucose Oxidase,GO)基因及抗虫半夏凝集素(Pinella Ternata Agglutinin,PTA)基因转化到烟草中,并研究了影响转化和抗性植株再生的一些因素。结果表明,基因型是影响转化和抗性植株再生的关键因素,试验所用的3个品种KY14、KY8959、TN90中,KY14转化效率最高。用于烟草转化的侵染时间以15min为宜,共培养时间以2d最佳。PCR分子检测表明,外源GO基因及PTA基因已转入到烟草KY14中。淀粉-碘化钾显色反应检测到了转基因植株产生的过氧化氢,证实GO基因已经在烟草中发挥功能。