细菌双杂交系统的改进

解析蛋白质之间的相互作用,对理解调控和代谢等生物学过程具有重要意义。其中基于互补腺苷酸环化酶功能的细菌双杂交系统是一种研究蛋白质之间相互作用的有效手段。本文在利用该系统时发现存在假阳性高的缺陷。进一步报告基因活性分析表明产生假阳性的原因是不同克隆的生理状态影响了LacZ的输出,即lacZ启动子除了受cAMP信号分子的控制,还受到其他调控蛋白的直接或间接的调控。为了降低生理因素对报告基因的影响,我们向该系统引入用多拷贝lacZ启动子控制的gfp报告基因。这不仅通过增加lacZ启动子的数量,弱化了生理因素的影响;还实现了第二个报告基因gfp与lacZ同时报告蛋白质之间的相互作用情况,提高了输出的准确性。系统的实验验证表明,我们改进的细菌双杂交系统的灵敏性确实得到大幅提高。     

荧光素酶及其应用

介绍了细菌荧光素酶基因(lux)和萤火虫荧光素酶基因(luc)的结构特点和二者的差异,概述了该酶作为报告基因在基础研究和实践(污染物监测、急性毒性和遗传毒性监测、微生物监测等)中的应用,肯定了该酶作为报告基因的广阔应用前景。     

报告基因技术的理论基础及其应用

报告基因技术广泛地应用于监测细胞的信号转导和基因表达,通过把转录控制元件剪接到报告基因,可以直观地“报道”细胞内与基因表达有关的信号级联。这种检测具有敏感性高,方便,可靠适用于大规模检测等优点。随着报告基因技术及其检测方法的不断改进,荧光素酶,绿色荧光蛋白以及新近克同的红色荧光蛋白作为无害性的检测工具,将在检测活体组织和细胞基因表达方面得到越来越广泛的应用。此外,在研究疾病发生的分子机制,基因治疗和药物开发方面,报告基因技术也将发挥出重要作用。本文对报告基因技术的基础理论,常用报告基因的种类和特点,以及其目前的主要应用作了概括性介绍。     

土壤微生物资源管理、应用技术与学科展望

土壤中蕴藏着高度的微生物多样性,在陆地生态系统中发挥着非常重要的功能,加强对土壤微生物资源的综合管理与开发应用是提升生态系统稳定性与生产力及农产品质量的重要途径。首先,土壤微生物多样性具有全球性的重大意义,有待完善对土壤微生物的检测与监测技术研究,进而实现土壤微生物多样性与土壤功能的耦合以及对土壤质量的评定;其次,土壤微生物作为一种宝贵的生产资料和可持续资源,要加强其在土壤肥力强化与保育、土壤障碍消减与调节、土壤污染控制与修复等3个领域的应用研究。最后,未来土壤微生物学发展将会形成土壤微生物系统学、土壤微生物过程学与土壤微生物功能学3个子学科,要建立土壤微生物种质资源库与遗传信息库,推进土壤微生物生理代谢过程、生物化学过程及生态行为过程的研究,联结土壤微生物与土壤功能的关系,并从土壤中的功能微生物出发对环境变化作出积极响应和主动调控。此外,原创性方法的建立与应用是限制土壤微生物学发展的技术瓶颈,联合生物地理学与生物信息学破译重要基因的特定生态功能,并将其应用到生态模型以及生态系统未知领域的研究中去,是土壤微生物学面临的挑战。     

细菌lux荧光报告系统的研究进展

lux报告系统以其灵敏、快速、便捷等特性,在分子生物学、临床微生物和生化检测等领域中占据了一席之地。从lux报告系统的组成、类型及特性出发,介绍该报告系统在环境监测、食品安全、新药发现、肿瘤定位等方面的应用。     

不同分子量壳聚糖对土壤碳、氮及呼吸的影响

考察了不同分子量壳聚糖对土壤微生物量C、N、土壤呼吸及矿质N的影响.研究发现：不同分子量的壳聚糖施入土壤后,土壤的微生物量C、N、呼吸及矿质N均明显提高.微生物量C、N及土壤呼吸有相似的变化趋势： 随壳聚糖用量的增加而增大.低分子量壳聚糖施入土壤后,微生物量C、N及土壤呼吸均先快速增加,然后下降;中等及高分子量壳聚糖施入土壤后则是开始时变化较小,第14天开始快速增加,34d后下降.研究还发现,NO3^--N与NH4^＋-N变化趋势不完全相同,NO3^--N开始时变化较小,第14天开始快速增加,34d后快速下降;低分子量壳聚糖处理时,NH4^＋-N开始时快速增加,之后缓慢下降;中等分子量壳聚糖处理时,因加入量不同而不同;高分子量壳聚糖处理时则是从第24天开始变化显著.     

玉米生长期间土壤微生物量与土壤酶变化及其相关性研究

研究了玉米生长期间土壤微生物量碳、氮与土壤过氧化氢酶、蔗糖酶、脲酶、蛋白酶活性变化及其相关性．结果表明，玉米生长前期和中期，土壤微生物量碳、氮及酶活性迅速上升，并逐渐达到最大值；玉米生长后期，土壤微生物量碳、氮、酶活性下降至某一值后并逐渐趋于平稳．几种处理相比较，以秸秆＋尿素处理的土壤微生物量碳、氮及酶活性为最大．除玉米生长后期，土壤微生物量碳、氮与碱解氮、活性腐殖质、土壤ｐＨ不相关外，土壤微生物量碳、氮与土壤过氧化氢酶、蔗糖酶、脲酶、蛋白酶活性及速效养分在玉米生长期间均相关或极相关     

黄土丘陵区草地土壤微生物C、N及呼吸熵对植被恢复的响应

以野外样地调查和室内分析法研究了黄土丘陵区不同植被恢复年限下草地土壤微生物C、N及土壤呼吸熵的变化.结果表明,土壤微生物量碳明显地随着植被恢复年限的增加而增加.在恢复前23a, 土壤微生物量碳在0～20 cm土层年增加率为24.1%;20～40 cm为104.4%.植被恢复23a后,0～20 cm土层增长率为0.83%,20～40 cm为0.19%.土壤微生物量N表现为在植被恢复的初期略有下降,3a后,开始出现明显增加.0～20 cm土层年增长率为20.14%,20～40 cm为15.11%.在植被恢复23a后,0～20 cm土层的年增长率为0.14%,20～40 cm变化不大.土壤微生物呼吸强度随着恢复年限的增加逐渐加强;土壤呼吸熵随植被封育时间的增加而呈对数降低趋势.土壤呼吸熵(qCO2)在反映土壤的生物质量变化时,显得更加稳定,受植物生长状况影响较小.相关分析表明,土壤微生物量和土壤微生物活性与土壤有机质、碱解氮和粘粒含量显著正相关;与土壤粉粒含量明显负相关;表层土壤pH值对其也有明显影响.草地植被自然恢复过程可增加土壤微生物活性,有利于土壤质量的提高.     

不同森林恢复类型对南方红壤侵蚀区土壤质量的影响

为了探讨主要森林类型对土壤质量的影响 ,对南方红壤侵蚀区 4种主要森林恢复类型下土壤物理、化学、生物学性状进行了比较研究 ,结果表明 :不同的森林恢复类型导致了土壤物理、化学和生物学性质的显著差异。 4种森林类型的土壤质量均比长期干扰下对照的土壤质量高。人工林土壤质量又相对比天然次生林土壤质量低。其土壤质量综合指数分别为 :天然次生林(0 .95 )、油茶林 (0 .6 8)、杉木林 (0 .5 5 )、湿地松林 (0 .36 )、对照 (0 .0 4 )。自然恢复在恢复初期是提高土壤质量的有效途径。导致人工林和对照土壤质量相对较低的主要因素是 :周期性的森林抚育打破土壤物理结构、凋落物质量较低、凋落物量较低、微生物生物量较低、微生物功能较差和土壤养分流失严重。在土壤质量指标选择方面 ,土壤微生物生物量结合微生物功能多样性是反映土壤生物学活性和土壤质量的较好指标。     

洞庭湖区不同利用方式对土壤微生物生物量碳氮磷的影响

以湖南省沅江市典型湖垸为代表,通过密集取样分析,研究了洞庭湖区不同利用方式条件下农田土壤微生物生物量碳、氮、磷的变化及其和土壤碳、氮、磷的关系,发现水田土壤碳、氮和微生物生物量碳、氮明显高于旱地,水田土壤中双季稻高于一季稻;土壤磷的含量旱地稍高于水田,但土壤微生物生物量磷水田稍高于旱地.尽管在水田土壤中微生物生物量碳、氮有明显的不同,但水田土壤微生物生物量磷维持在相对稳定的水平.典型样区土壤微生物生物量碳占有机碳的比例为0.65%～7.24%,平均3.00%;土壤微生物生物量氮占全氮的比例为0.98%～7.41%,平均3.81%;土壤微生物生物量磷占全磷的比例为0.16%～7.54%,平均2.80%.土壤C/N为3.87～17.31,平均9.15;BC/BN为4.06～9.29,平均7.26.土壤微生物生物量碳、氮与土壤碳、氮之间存在极其显著的线性相关关系,但土壤微生物生物量磷占全磷之间相关关系不显著.土壤微生物生物量碳、氮、磷之间的相关关系达到了极显著水平.不同的利用方式和耕作制度导致了土壤碳、氮和微生物生物量碳、氮的差异,土壤微生物生物量碳、氮能够很好地反映洞庭湖区农田土壤碳、氮水平.     

Green fluorescent protein as a molecular marker in microbiology

Molecular markers such as: lacZ (b-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (beta-glucuronidase), gfp (green fluorescent protein), bla (beta-lactamase) and other antibiotic resistance markers, heavy metals resistance genes are commonly used in environmental microorganisms research (Errampaii et al., 1998; Kohler et al., 1999). Most of these markers require one or more substrates, complex media and/or expensive equipment for detection. The gfp gene is widely used as a marker because of its very useful properties such as high stability, minimal toxicity, non-invasive detection and the ability to generate the green light without addition of external cofactors and without application of expensive equipment. Various applications of that reporter gene were showed starting from monitoring of microorganism's survival in complex biological systems such as activated sludge to biodegradation of chemical compounds in soil. GFP allowed the detection, determination of spatial location and enumeration of bacterial cells from diverse environmental samples such as biofilm and water. The gfp as a biomarker was very useful in monitoring of gene expression and protein localisation in bacterial cells, too. The techniques with using gfp marker promise to supply a better understanding of environmental processes. It can make possible to use that knowledge in designing more effective and more efficient methods of biodegradation of toxic compounds from different environments.     

Whole-cell bacterial biosensors for the detection of aromatic hydrocarbons and their chlorinated derivatives (Review)

The review summarizes the data on new directions in biosensor technologies based on whole bacterial cells. Biosensors for the monitoring of mono(poly)aromatic hydrocarbons and their chlorinated derivatives, which are constructed with genetically modified bacterial cells bearing a reporter gene fusion, are considered. The operating principle of these biosensors is based on the expression of reporter genes (luc, lux, gfp, rfp) under the control of a promoter and a regulator that specifically respond to a detected compound.     

Quantitative analysis of gene expression with an improved green fluorescent protein. p6.

The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities. We have increased the sensitivity of GFP detection 320-fold compared to the wild-type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+. Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli. The agreement of GFP+ fluorescence with beta-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo. However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene. Possibilities of using GFP variants beyond this range are discussed.     

Advances in directed molecular evolution of reporter genes

Many reporter genes, such as gfp, gusA, and lacZ, are widely used for research into plants, animals, and microorganisms. Reporter genes, which offer high levels of sensitivity and convenience of detection, have been utilized in transgenic technology, promoter analysis, drug screening, and other areas. Directed molecular evolution is a powerful molecular tool for the creation of designer proteins for industrial and research applications, including studies of protein structure and function. Directed molecular evolution is based mainly on in vitro recombination methods, such as error-prone PCR and DNA shuffling. The strategies of directed evolution of enzyme biocatalysts have been the subject of several recent reviews. Here, we briefly summarize successes in the field of directed molecular evolution of reporter genes and discuss some of the applications.     

Vectors that facilitate the replacement of transcriptional lacZ fusions in Streptococcus mutans and Bacillus subtilis with fusions to gfp or gusA

Plasmid vectors have been constructed for Streptococcus mutans and Bacillus subtilis that make possible rapid replacement of the widely used reporter gene lacZ (encoding beta-galactosidase) with either gfp (encoding green fluorescent protein) or gusA (encoding beta-glucuronidase). The lacZ-->gfp replacement vectors greatly facilitate the analysis of the spatial location of gene expression in biofilms of S. mutans and in sporulating B. subtilis. The lacZ-->gusA replacement vectors facilitate the comparison of two promoters within the same organism. A vector is also described that enables gusA to be replaced with gfp in B. subtilis.     

Green fluorescent protein as a marker for monitoring a pentachlorophenol degrader Sphingomonas chlorophenolica ATCC39723

Sphingomonas chlorophenolica ATCC39723 was successfully labeled with the gfp (green fluorescent protein) gene inserted into the pcpB gene by homologous recombination. As the gfp recombinant was easily distinguished from other indigenous organisms, the population of gfp recombinant was monitored after being released into the soil microcosms. Their population density dropped from 10(8) to 10(6) (cfu/ml) in the non-sterilized soil microcosms during the first 6 days. Moreover, the gfp recombinant was not detected even at lower dilution rates after a certain time period. The recombinant, however, survived for at least 28 days in the sterilized soil microcosms. Although the gfp recombinant did not degrade pentachlorophenol (PCP), this experiment showed the possibility of using gfp as a monitoring reporter system for S. chlorophenolica ATCC39723 and potentially other species of Sphingomonas.     

Multi-scale molecular photoacoustic tomography of gene expression

Photoacoustic tomography (PAT) is a molecular imaging technology. Unlike conventional reporter gene imaging, which is usually based on fluorescence, photoacoustic reporter gene imaging relies only on optical absorption. This work demonstrates several key merits of PAT using lacZ, one of the most widely used reporter genes in biology. We show that the expression of lacZ can be imaged by PAT as deep as 5.0 cm in biological tissue, with resolutions of ～1.0 mm and ～0.4 mm in the lateral and axial directions, respectively. We further demonstrate non-invasive, simultaneous imaging of a lacZ-expressing tumor and its surrounding microvasculature in vivo by dual-wavelength acoustic-resolution photoacoustic microscopy (AR-PAM), with a lateral resolution of 45 μm and an axial resolution of 15 μm. Finally, using optical-resolution photoacoustic microscopy (OR-PAM), we show intra-cellular localization of lacZ expression, with a lateral resolution of a fraction of a micron. These results suggest that PAT is a complementary tool to conventional optical fluorescence imaging of reporter genes for linking biological studies from the microscopic to the macroscopic scales.     

Influence of an arbuscular mycorrhizal fungus on Pseudomonas fluorescens DF57 in rhizosphere and hyphosphere soil

The influence of Glomus intraradices (BEG87) on Pseudomonas fluorescens DF57 in hyphosphere and rhizosphere soil was examined. Cucumis sativus (Aminex, F₁ hybrid) was grown in symbiosis with the arbuscular mycorrhizal fungus G. intraradices in PVC tubes, consisting of a central root compartment and two lateral root-free compartments. Two Tn 5 - lux AB-marked strains of P. fluorescens DF57 were used. Strain DF57-P2, which has an insertion of Tn 5::lux AB in a phosphate starvation-inducible locus, was used as a phosphate starvation reporter. Another lux -tagged strain DF57-40E7, which carries a constitutively expressed lux AB fusion, was used as control for strain DF57-P2 and for measuring the metabolic activity of P. fluorescens DF57. A strain of P. fluorescens DF57, which carries a constitutively expressed gfp gene, was used in studies of attachment between the bacteria and the hyphae. G. intraradices decreased the culturability of P. fluorescens DF57 significantly, both in rhizosphere and hyphosphere soil, whereas the total number of P. fluorescens DF57 measured by immunofluorescence microscopy was decreased in hyphosphere soil only. G. intraradices did not induce a phosphorus starvation response in P. fluorescens DF57, and the metabolic activity of the bacteria was not affected by the fungus after 48 h. P. fluorescens DF57 did not attach to G. intraradices hyphae and was not able to use the hyphae as carbon substrate. The negative effect of G. intraradices on culturability and on number of P. fluorescens DF57 in hyphosphere soil is discussed.