PCR-SSCP技术在微生物群落多态性分析中的应用进展

PCR-SSCP技术是新近发展起来的一种分子生物学分析技术,因具有快速、简便、灵 敏和适于大样本筛查的特点,能有效检出碱基置换、缺失、插入等基因变异,因而有广泛和 潜在的应用价值.该文较系统地介绍了PCR-SSCP方法的发展过程、技术优势及其在环境生态 、食品微生物等领域中微生物群落多态性检测的应用进展,并对其发展前景作了展望.     

蛋白质点阵/芯片技术的新进展

蛋白质点阵/芯片技术是分子生物学技术的重要进展,在功能蛋白质组研究方面具有广阔的潜在应用价值.目前发展起来的印迹蛋白微阵列、分子扫描技术和传感器生物芯片质谱,将应用于药靶检测、疾病诊断、蛋白质结构鉴定和/或蛋白质之间的相互作用分析等方面,具有分析速度快、效率高、样品消耗少等特点,将成为生命科学与医学领域新的研究工具.     

分子标记技术在烟草种质遗传多样性研究中的应用进展

DNA分子标记作为新发展起来的一种遗传标记形式,凭借其可靠有效等优点,在农业科学研究中的应用越来越广泛.综述了几种分子标记技术(RAPD、AFLP、ISSR)在烟草种质资源中的应用进展,分析了分子标记技术在烟草种质资源及遗传多样性研究中存在的问题及今后发展方向.     

化学合成的DNA定位断裂工具

化学合成的 DNA 定位断裂工具是80年代初发展起来的一种新型非酶 DNA 定位断裂工具.它由 DNA 识别结合系统及化学断裂系统组成,能在人们预先设计的任何位点断裂 DNA 分子,具有制备简便、价格便宜、不受酶的天然专一性限制等优点.可应用于基因分离、染色体图谱分析、大片段基因的序列分析以及 DNA 定位诱变、肿瘤基因治疗与新的化学疗法等分子生物学领域.     

荧光定量PCR技术在植物研究中的应用

荧光定量PCR技术是近年发展起来的一种用于基因定量分析的PCR方法,自同世以来在基础科学研究、临床诊断、疾病研究及药物研发等领域应用较为广泛.在植物研究方面应用起步较晚,现已开始用于植物基因表达分析、外源基因基因鉴定等方面的研究.介绍了实时荧光定量PCR技术的原理、优缺点和试验中潜在问题和条件的优化,并对其在植物研究中的应用及前景作了探讨.     

土地系统动态模拟方法研究进展

土地系统动态模拟对土地利用规划、国土资源管理具有重要指导价值.基于经验统计的方法、多智能主体分析的方法、栅格邻域关系分析的方法以及近年来发展起来的土地系统动态模拟系统(DLS)方法是当前开展土地系统动态模拟的主要手段.本文综述了上述4种方法的原理、特点与应用案例,概括了相关方法在理论依据和推广应用等方面的优点与不足,展望了DLS在土地系统动态模拟方面的广阔应用前景.     

酵母人工染色体克隆技术及其进展

酵母人工染色体克隆(YAC)是最近几年发展起来的大分子 DNA 克隆技术.文章综述了 YAC 克隆技术的发展,YAC 的分离、分析与鉴定,以及这一技术在分子生物学中的应用.     

我国农药水分散粒剂产品登记现状与改进建议

水分散粒剂(WG)是20世纪80年代从国外发展起来的一种农药新剂型.本文分析了我国水分散粒剂产品登记现状及其存在的问题,提出应加强水分散粒剂产品的研发和登记,适当提高部分产品有效成分含量,规范剂型名称,强化质量监管等改进建议.     

叶绿素荧光分析技术在林木培育中的应用

叶绿素荧光分析技术是近年来在光合作用机理研究中发展起来的一种新型、快速、简便、精确且整体无损伤检测植物光合作用生理状况的新技术.叶绿素荧光信号包含了十分丰富的光合作用过程变化的信息,因而被视为植物光合作用与环境关系的内在探针.该项技术在农业、园艺等方面已得到广泛应用,而在林木培育中的应用较少.本文介绍了叶绿素荧光分析相关参数及其生物学意义,总结了国内外关于叶绿素荧光分析技术在林木栽培、林木逆境生理等方面的应用,并对该技术在林木培育中的应用提出一些初步建议.     

简单重复序列间扩增分子标记技术及其应用

简单重复序列间扩增(ISSR)技术是在PCR基础上发展起来的一种新型的分子标记技术,因其标记通常为显性标记,呈孟德尔遗传,且在进行PCR反应时,稳定性和多态性均很好,而成为是非常理想的分子标记技术.将从ISSR分子标记技术的原理及其在绘制DNA指纹图谱、遗传多样性分析、种质鉴定等领域的应用进行综述.     

A fast and efficient method for isolation of the BAC end

As a new developmental vector system, the bacterial artificial chromosome (BAC) has been used widely in constructing genomic libraries and in generating transgenic animals. Isolation of the BAC insert end is useful to analyze the BAC clone. Here, we describe a fast and efficient method to obtain the BAC end by ligating the BAC fragments digested with Not I and another selected restriction enzyme into universal cloning vector, followed by determining the correct clones with HindIII digestion. Further DNA sequencing analysis verified the results mentioned above.     

Development of a novel bacterial artificial chromosome cloning system for functional studies

Bacterial artificial chromosome (BAC) cloning systems currently in use generate high quality genomic libraries for gene mapping, identification, and sequencing. However, the most commonly used BAC cloning systems do not facilitate functional studies in eukaryotic cells. To overcome this limitation, we have developed pEBAC190G, a new BAC vector that combines the features of the first generation PAC/BAC vectors with eukaryotic elements that facilitate the transfection, episomal maintenance, and functional analysis of large genomic fragments in eukaryotic cells. A number of different cloning strategies may be used to retrofit genomic fragments from existing libraries into the new vector. The system was tested by the retrofitting of a 170kb NotI genomic fragment from the RPCI-11 BAC library into the NotI site of pEBAC190G. Clones from any eukaryotic genomic library harboured in this vector can be transferred from bacteria directly to eukaryotic cells for functional analysis.     

Pyrosequencing analysis of bacterial communities in drinking water biofilters receiving influents of different types

Biological activated carbon (BAC) filters are commonly used in the world for improvement of drinking water quality. The indigenous microbiota in BAC filters can play a crucial role in reduction or biotransformation of contaminants. Molecular analysis can enhance our understanding of ecological functions of the microbial communities in drinking water BAC filters. In this study, three laboratory-scale drinking water BAC filters receiving influents of different types were constructed. Differences of bacterial communities in the three BAC filters were characterized using 454 pyrosequencing analysis. Pyrosequencing analysis illustrated the usefulness in elucidating the bacterial community structure in drinking water biofilter. High bacterial diversity in granular activated carbon (GAC) samples from each BAC biofilter was observed. Proteobacteria was the largest bacterial phylum in each GAC sample, with a marked shift of the proportions of Alpha-, Beta-, and Gammaproteobacteria. The levels of dissolved organic carbon and ammonia nitrogen in the influents could affect the bacterial diversity and community composition in the BAC biofilters. This work might add some new insights into microbial community and its influential factors in drinking water biofilters.     

Heterogeneity of microbial community structures inside the up-flow biological activated carbon (BAC) filters for the treatment of drinking water

Filtration using biological activated carbon (BAC) performs well in the removal of biodegradable dissolved organic carbon from water sources. The application of ozonation followed by up-flow BAC filtration has gained increasing attention in the world scale. In this study, a pilotscale up-flow BAC filtration system was constructed for the treatment of polluted lake water. The operational results indicated that this BAC filtration system could effectively remove organic matter. Spatial heterogeneity of the microbial community structure inside the BAC filtration system was identified using bacterial 16S rRNA clone library analysis. A marked decrease of microbial diversity in the BAC filtration system was observed along the flow path. Alphaproteobacteria, Gammaproteobacteria and Acidobacteria were found to be the major bacterial groups in the BAC filters. Moreover, Novosphingobium aromaticivorans-like microorganisms were detected. This work might add some new insights towards microbial communities in regards to BAC filtration for the treatment of drinking water.     

A cytogenetically characterized, genome-anchored 10-Mb BAC set and CGH array for the domestic dog

The generation of a 7.5x dog genome assembly provides exciting new opportunities to interpret tumor-associated chromosome aberrations at the biological level. We present a genomic microarray for array comparative genomic hybridization (aCGH) analysis in the dog, comprising 275 bacterial artificial chromosome (BAC) clones spaced at intervals of approximately 10 Mb. Each clone has been positioned accurately within the genome assembly and assigned to a unique chromosome location by fluorescence in situ hybridization (FISH) analysis, both individually and as chromosome-specific BAC pools. The microarray also contains clones representing the dog orthologues of 31 genes implicated in human cancers. FISH analysis of the 10-Mb BAC clone set indicated excellent coverage of each dog chromosome by the genome assembly. The order of clones was consistent with the assembly, but the cytogenetic intervals between clones were variable. We demonstrate the application of the BAC array for aCGH analysis to identify both whole and partial chromosome imbalances using a canine histiocytic sarcoma case. Using BAC clones selected from the array as probes, multicolor FISH analysis was used to further characterize these imbalances, revealing numerous structural chromosome rearrangements. We outline the value of a combined aCGH/FISH approach, together with a well-annotated dog genome assembly, in canine and comparative cancer studies.     

Changes of biomass and bacterial communities in biological activated carbon filters for drinking water treatment

Biological activated carbon (BAC) filtration can usually perform well in removal of biodegradable organic compounds in drinking waters. In this study, a pilot-scale down-flow BAC filtration system was constructed for treatment of ozonated waters. The changes of biomass concentration and bacterial community in the BAC filters with contact time and service time were characterized using phospholipid fatty acid (PLFA) analysis and 16S rRNA gene clone library analysis, respectively. The operational results indicated the BAC filtration system could effectively remove dissolved organic carbon (DOC) and assimilable organic carbon (AOC). Biomass concentration decreased with contact time, but showed only a slight change with service time. Contact time and service time could affect the microbial community structure. Alphaproteobacteria was the largest bacterial group and might have important links with the DOC and AOC removal. This work might provide some new insights into microbial community and biological process in the drinking water biofilters.     

Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor.

The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants.     

A Large-Insert (130 kbp) Bacterial Artificial Chromosome Library of the Rice Blast FungusMagnaporthe grisea:Genome Analysis, Contig Assembly, and Gene Cloning

Magnaporthe grisea(Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant–pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitateM. griseagenome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (>25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that ≈26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis ofM. grisea.Copies of colony hybridization membranes are available upon request.     

以BAC为基础的疱疹病毒感染性克隆技术

疱疹病毒（HPVs）庞大而复杂的基因组一直使得HPVs的遗传分析颇具挑战性。近几年发展起来的以细菌人工染色体（BAC）为基础的HPVs全长感染性克隆是全新的技术,促进了在HPVs整个基因组中对单个基因功能的研究。本文以EB病毒为例,介绍了该技术的原理、建立、突变方法及应用。