基因芯片技术及其在微生物检测中的应用

基因芯片技术是20世纪90年代发展起来的一门高新技术。应用该技术可以对大量的遗传信息进行快速、高通量、并行检测,解决了传统检测方法中遇到的难题,已广泛应用于基因表达分析、突变检测、核酸多态性分析、基因测序和药物筛选等几乎所有的生物学研究领域。作为一种高通量的基因检测方法,基因芯片技术在微生物检测和鉴定方面的应用越来越多,具有巨大的应用潜力。文章简要介绍了基因芯片技术的基本概念和技术流程,综述其在微生物检测和鉴定中的应用。     

Ethics and Genetic Selection in Purebred Dogs

There is an ongoing revolution in medicine that is changing the way that veterinarians will be counselling clients regarding inherited disorders. Clinical applications will emerge rapidly in veterinary medicine as we obtain new information from canine and comparative genome projects ( Meyers‐Wallen 2001 : Relevance of the canine genome project to veterinary medical practice. International Veterinary Information Service, New York). The canine genome project is described by three events: mapping markers on canine chromosomes, mapping gene locations on canine chromosomes ( Breen et al. 2001 : Genome Res. 11, 1784–1795), and obtaining the nucleotide sequence of the entire canine genome. Information from such research has provided a few DNA tests for single gene mutations [ Aguirre 2000 : DNA testing for inherited canine diseases. In: Bonagura, J (ed), Current Veterinary Therapy XIII. Philadelphia WB Saunders Co, 909–913]. Eventually it will lead to testing of thousands of genes at a time and production of DNA profiles on individual animals. The DNA profile of each dog could be screened for all known genetic disease and will be useful in counselling breeders. As part of the pre‐breeding examination, DNA profiles of prospective parents could be compared, and the probability of offspring being affected with genetic disorders or inheriting desirable traits could be calculated. Once we can examine thousands of genes of individuals easily, we have powerful tools to reduce the frequency of, or eliminate, deleterious genes from a population. When we understand polygenic inheritance, we can potentially eliminate whole groups of deleterious genes from populations. The effect of such selection on a widespread basis within a breed could rapidly improve health within a few generations. However, until we have enough information on gene interaction, we will not know whether some of these genes have other functions that we wish to retain. And, other population effects should not be ignored. At least initially it may be best to use this new genetic information to avoid mating combinations that we know will produce affected animals, rather than to eliminate whole groups of genes from a population. This is particularly important for breeds with small gene pools, where it is difficult to maintain genetic diversity. Finally, we will eventually have enough information about canine gene function to select for specific genes encoding desirable traits and increase their frequencies in a population. This is similar to breeding practices that have been applied to animals for hundreds of years. The difference is that we will have a large pool of objective data that we can use rapidly on many individuals at a time. This has great potential to improve the health of the dog population as a whole. However, if we or our breeder clients make an error, we can inadvertently cause harm through massive, rapid selection. Therefore, we should probably not be advising clients on polygenic traits or recommend large scale changes in gene frequencies in populations until much more knowledge of gene interaction is obtained. By then it is likely that computer modelling will be available to predict the effect of changing one or several gene frequencies in a dog population over time. And as new mutations are likely to arise in the future, these tools will be needed indefinitely to detect, treat and eliminate genetic disorders from dog populations. Information available from genetic research will only be useful in improving canine health if veterinarians have the knowledge and skills to use it ethically and responsibly. There is not only a great potential to improve overall canine health through genetic selection, but also the potential to do harm if we fail to maintain genetic diversity. Our profession must be in a position to correctly advise clients on the application of this information to individual dogs as well as to populations of dogs, and particularly purebred dogs.     

Non-lactational traits of importance in dairy cows and applications for emerging biotechnologies

Dairy cattle have traditionally been selected for their ability to produce milk and milk components. The traditional single-minded approach to selection of dairy cattle has now changed and secondary traits are being included in selection indices by decreasing the emphasis on production. Greater emphasis on non-production traits reflects the industry's desire for functional dairy cattle. Six broad categories of non-lactational traits are discussed in this review. They are: type; growth, body size and composition; efficiency of feed utilisation; disease resistance, e.g. udder health as measured by somatic cell score; reproduction; and management. Most of these traits can be found within selection indices worldwide, although relative emphasis varies. The non-lactational traits mentioned above are quantitative, meaning that the phenotype in the whole animal represents the sum of lesser traits that cannot be easily measured. The physiological mechanisms that underlie quantitative traits are extremely complex. Genetic selection can be applied to quantitative traits but it is difficult to link successful genetic selection with the underlying physiological mechanisms. The importance that the bovine genome sequence will play in the future of the genetics of dairy cattle cannot be understated. Completing the bovine genome sequence is the first step towards modernising our approach to the genetics of dairy cattle. Finding genes in the genome is difficult and scanning billions of base pairs of DNA is an imperfect task. The function of most genes is either unknown or incompletely understood. Combining all of the information into a useable format is known as bioinformatics. At the present time, our capacity to generate information is great but our capacity to understand the information is small. The important information resides within subtle changes in gene expression and within the cumulative effect that these have. Traditional methods of genetic selection in dairy cattle will be used for the foreseeable future. Most non-lactational traits are heritable and will be included in selection indices if the traits have value. The long-term prognosis for genome science is good but advances will take time. Genetic selection in the genome era will be different because DNA sequence analysis may replace traditional methods of genetic selection.     

Non-lactational traits of importance in dairy cows and applications for emerging biotechnologies

Dairy cattle have traditionally been selected for their ability to produce milk and milk components. The traditional single-minded approach to selection of dairy cattle has now changed and secondary traits are being included in selection indices by decreasing the emphasis on production. Greater emphasis on non-production traits reflects the industry's desire for functional dairy cattle. Six broad categories of non-lactational traits are discussed in this review. They are: type; growth, body size and composition; efficiency of feed utilisation; disease resistance, e.g. udder health as measured by somatic cell score; reproduction; and management. Most of these traits can be found within selection indices worldwide, although relative emphasis varies.

The non-lactational traits mentioned above are quantitative, meaning that the phenotype in the whole animal represents the sum of lesser traits that cannot be easily measured. The physiological mechanisms that underlie quantitative traits are extremely complex. Genetic selection can be applied to quantitative traits but it is difficult to link successful genetic selection with the underlying physiological mechanisms. The importance that the bovine genome sequence will play in the future of the genetics of dairy cattle cannot be understated. Completing the bovine genome sequence is the first step towards modernising our approach to the genetics of dairy cattle.

Finding genes in the genome is difficult and scanning billions of base pairs of DNA is an imperfect task. The function of most genes is either unknown or incompletely understood. Combining all of the information into a useable format is known as bioinformatics. At the present time, our capacity to generate information is great but our capacity to understand the information is small. The important information resides within subtle changes in gene expression and within the cumulative effect that these have.

Traditional methods of genetic selection in dairy cattle will be used for the foreseeable future. Most non-lactational traits are heritable and will be included in selection indices if the traits have value. The long-term prognosis for genome science is good but advances will take time. Genetic selection in the genome era will be different because DNA sequence analysis may replace traditional methods of genetic selection.     

猪伪狂犬病毒TK缺失株PRV/TK~-的构建及其生物学特性

为了构建伪狂犬病毒容A株(PRV-Ra)TK基因缺失重组病毒,本研究利用PCR从PRV-Ra株基因组分别扩增TK基因两侧的序列LTK和RTK,将LTK和RTK克隆到pUC19载体,构建转移质粒pUC19-TK/HEK。进一步将增强型绿色荧光蛋白基因(EGFP)表达盒克隆到pUC19-TK/HEK质粒中,构建转移质粒pUC19-TK/EGFP。用pUC19-TK/EGFP与PRV-Ra株基因组共转染BHK21细胞,通过噬斑纯化获得重组病毒PRV/TK-/EGFP;用pUC19-TK/HEK与PRV/TK-/EGFP病毒基因组共转染BHK21细胞,噬斑纯化获得不含EGFP基因的重组病毒PRV/TK-。该重组病毒在体外传30代后仍其有良好的遗传稳定性;PRV/TK-体外增殖速度、对家兔的毒力以及对仔猪的安全性和抗体反应性研究表明,重组毒株与原始株(PRV-Ra)在BHK21上具有相似的增殖规律;PRV/TK-对家兔的毒力比PRV-Ra下降了10000倍;以105.0TCID50PRV/TK-免疫小猪后4周,体内产生的平均中和抗体水平达101.9,略高于对照疫苗产生的抗体水平(101.8)。本试验为PRV基因功能的研...     

Alpharma Beef Cattle Nutrition Symposium: nutrition and the genome

It has long been appreciated that animals fed the same diet may perform differently. This is due to the ability of nutrients to interact with and affect molecular pathways that result in differences in BW gain, production performance, or disease resistance. To understand these effects, studies are being undertaken to discover how the differential expression and function of genes occur with different diets. These studies are using new technologies, genomic resources, and analysis techniques that have recently become available for domestic animals. Nutrigenomics and nutrigenetics are new research approaches that strive to optimize health by looking beyond the diet to understand the effects of food at the genetic and epigenetic levels. Nutrigenomics is focused on the effects of diet on health through an understanding of how bioactive chemicals in foods and supplements alter gene expression or the structure of the genome of an animal. Nutrigenetics focuses on how the genetic composition (i.e., genetic variation) of an animal influences their response to a given diet. Results from these studies will aid in formulating nutritionally appropriate diets that may be optimized for animals based on their genomic underpinnings. Nutrigenomics and nutrigenetics unite many fields: nutrition, bioinformatics, molecular biology, genomics, functional genomics, epidemiology, and epigenomics. The use of multi-disciplinary tools promises new opportunities to investigate the complex interactions of the genome and the diet of an animal. Through these new approaches, the partnerships of the genome and nutrition will be revealed resulting in improved efficiency of diets, enhanced sustainability of animals as a protein source, and improved methods for preventing illnesses.     

表达红色荧光蛋白的重组鸭肠炎病毒的构建及鉴定

鸭肠炎病毒(Duck enteritis virus, DEV),又称为鸭瘟病毒,是一种只感染雁形目禽类的疱疹病毒。DEV具有基因组大、非必需基因多、能插入外源基因的容量大、遗传稳定等优点,其庞大而复杂的基因组为外源基因提供了诸多可插入位点,以DEV为载体,成功表达了禽流感病毒HA蛋白_^[1]、鸭病毒性肝炎病毒VP0蛋白_^[2]、鸭坦布苏病毒E基因_^[3]以及鹅细小病毒VP2蛋白_^[4]。构建重组DEV的重要一步是将报告基因插入到基因组中,目前常用的报告基因有绿色荧光蛋白(GFP)、增强型绿色荧光蛋白(EGFP)、红色荧光蛋白(RFP)以及LacZ报告基团。本研究用RFP报告基团插入DEV UL2基因中,获得表达红色荧光的重组病毒,一步生长曲线表明,RFP对DEV的生长无影响;连续传代12代,RFP能够稳定表达,为DEV载体研究奠定基础。     

植物抗旱基因工程研究进展

干旱是影响农作物产量的主要胁迫因素之一。高通量生物技术的使用促成了新的干旱胁迫相关基因的发现,一些重要基因已转化到植物中,通过专一性或广谱性地应答路径来调节其干旱耐受性。最近的一些研究进展,进一步加深了对植物通过调控干旱相关基因的表达而抵御干旱胁迫的理解,并对植物干旱胁迫的复杂调控网络有了更深刻的认识,同时逐渐探索出一些具体的植物抗旱基因工程研究的途径。本文主要综述了信号分子、转录因子、小RNA分子、渗透调节分子、多胺类分子,活性氧清除分子方面的基因工程研究进展,并对其研究中存在的问题及应用前景进行了讨论。     

分析基因表达的新方法

公共和私有的EST计划为大量物种的基因表达研究提供了大量利用EST的机会。为了估计EST数据库中基因的表达水平，可以通过计算每一种基因在不同组织、不同发育阶段和不同条件下构建的cDNA文库中的出现次数，来获得基因表达的有关信息。这些信息水平的量与常规的Northern blots不同，可以允许不同基因之间表达水平的比较。而且当EST资料来源于不同发育阶段的cDNA文库时，还可以了解不同发育阶段的基因表达谱。由电子Northern杂交分析所获得的基因表达资料可以通过应用高密度DNA点阵得到证实并扩展于高表达基因领域之外。相关ESTS可以标定于尼龙膜或玻璃上并用相关组织的一链总cDNA作为探针进行鉴定。双色荧光标记可以得到精确的mRNA比率测定。在不远的将来，包含一种机体所有基因互补物的高密度DNA点阵将成为整个基因组范围基因表达类型分析的有效工具。     

转录组测序技术在植物乳杆菌细菌素合成研究中的应用研究进展

细菌素因具备抵御食源性致病菌和腐败菌的特性,可作为天然无毒抗菌剂应用在食品中。生物信息学是以计算机科学和应用数学为基础理论,将生命科学领域测得的生物信息经搜索比对,分析相关的核酸信息和蛋白结构基因,从而定位功能基因的一门科学。其中,转录组学即从整体转录水平分析不同细胞或组织在特定条件下所转录的所有RNA,从而揭示生物学通路和调控分子机制。将转录组测序结果中获得的海量数据与参考基因组比对,即可从基因表达水平上定量分析转录本的遗传信息。通过差异表达倍数筛选出差异基因,对其进行GO（Gene Ontology）功能注释和KEGG通路富集分析,进而推测其功能特性,并揭示细菌素等代谢产物的合成机理。由于转录组测序不存在安全隐患且属于较为经济的调控方式,因此该技术有望为细菌素大规模投入生产提供新思路。     

小RNA病毒基因表达调控研究进展

小RNA病毒科属于正链RNA病毒,该科各属病毒基因组结构和基因表达机制具有保守性。文章对小RNA病毒科病毒基因表达调控,包括非依赖帽状结构的翻译起始、宿主细胞翻译的关闭、病毒多聚蛋白的加工处理和RNA的复制研究进行综述,为阐明该科病毒的致病机理和研究真核生物的基因表达调控提供可借鉴的资料。     

隆林山羊肌细胞生成素基因的序列分析与结构预测

为了丰富优良地方品种隆林山羊的肌细胞生成素(myogenin,MyoG)基因遗传学基础信息,并探究其序列结构及其对山羊肌肉的发育分化调控机理。本研究设计1对特异性引物,采用PCR和克隆技术成功获得隆林山羊MyoG基因2419 bp长的DNA序列。结果表明,该基因包括3个外显子、2个内含子、部分5'UTR和3'UTR区,编码区DNA序列长675 bp,共编码224个氨基酸,该氨基酸序列无信号肽序列和跨膜结构。经同源性分析可知,隆林山羊MyoG编码区核苷酸序列及其编码的氨基酸序列与其他物种比对结果和氨基酸系统进化树的聚类结果相符,均显示隆林山羊与哺乳动物中的绵羊、牛和野猪的亲缘关系最近,氨基酸同源性达到95%以上。因此隆林山羊MyoG基因在哺乳动物中具有较高的保守性,而MyoG基因的克隆、序列分析和结构预测将为今后该基因的表达与调控、肉质改良、山羊开发利用等研究提供了更充分的分子生物学基础信息和理论依据。     

Genes controlling vaccine responses and disease resistance to respiratory viral pathogens in cattle

Farm animals remain at risk of endemic, exotic and newly emerging viruses. Vaccination is often promoted as the best possible solution, and yet for many pathogens, either there are no appropriate vaccines or those that are available are far from ideal. A complementary approach to disease control may be to identify genes and chromosomal regions that underlie genetic variation in disease resistance and response to vaccination. However, identification of the causal polymorphisms is not straightforward as it generally requires large numbers of animals with linked phenotypes and genotypes. Investigation of genes underlying complex traits such as resistance or response to viral pathogens requires several genetic approaches including candidate genes deduced from knowledge about the cellular pathways leading to protection or pathology, or unbiased whole genome scans using markers spread across the genome. Evidence for host genetic variation exists for a number of viral diseases in cattle including bovine respiratory disease and anecdotally, foot and mouth disease virus (FMDV). We immunised and vaccinated a cattle cross herd with a 40-mer peptide derived from FMDV and a vaccine against bovine respiratory syncytial virus (BRSV). Genetic variation has been quantified. A candidate gene approach has grouped high and low antibody and T cell responders by common motifs in the peptide binding pockets of the bovine major histocompatibility complex (BoLA) DRB3 gene. This suggests that vaccines with a minimal number of epitopes that are recognised by most cattle could be designed. Whole genome scans using microsatellite and single nucleotide polymorphism (SNP) markers has revealed many novel quantitative trait loci (QTL) and SNP markers controlling both humoral and cell-mediated immunity, some of which are in genes of known immunological relevance including the toll-like receptors (TLRs). The sequencing, assembly and annotation of livestock genomes and is continuing apace. In addition, provision of high-density SNP chips should make it possible to link phenotypes with genotypes in field populations without the need for structured populations or pedigree information. This will hopefully enable fine mapping of QTL and ultimate identification of the causal gene(s). The research could lead to selection of animals that are more resistant to disease and new ways to improve vaccine efficacy.     

Defining and interpreting intraspecific molecular variation.

Defining the extent and character of intraspecific genetic variation provides important information about gene function and organismal history. Powerful tests may be applied to sequenced alleles in order to critically examine whether natural selection is responsible for limiting or elevating intraspecific polymorphism in particular genes. Unconventional patterns of sequence variation and unusual allelic frequency distributions can be used to test whether genes encoding parasite antigens are being diversified by immune selection. The strikingly limited genetic variation in the falciparum malaria genome, and in human chromosomes encoding resistance to severe malaria, date the emergence of this disease to within the last few thousand years, illustrating the power of population genetic analysis to elucidate the history of host-parasite interactions. Coupling phylogenetic and geographic information and analyzing the rate of diversification in intraspecific gene trees provides new and rich sources of information on microbial evolution and epidemiology.     

利用基因组信息提高畜禽生产性能研究进展

为了满足人们对畜产品需求的快速增长，必须在加快畜禽产业发展的同时把对环境的影响降到最低，提高畜禽遗传特性有望促进这一问题的解决。进入21世纪以来，以基因组选择为核心的分子育种技术迎来了发展机遇，利用该技术可实现早期准确选择，从而大幅度缩短世代间隔，加快群体遗传进展，并显著降低育种成本。虽然在某些畜种中（如奶牛），基因组选择取得了成功，群体也获得较大遗传进展，但仍无法满足快速增长的需求。因此，亟需寻找能够进一步加快遗传进展的方法。研究表明，在SNP标记数据中加入目标性状的已知功能基因信息，可以提高基因组育种值预测的准确性，进而加快遗传进展。而挖掘更多基因组信息的同时，开发更优化的分析方法可以更有助于目标的实现。文章总结了主要畜禽物种的可用基因组数据，包括牛、绵羊、山羊、猪和鸡以及这些数据是如何有助于鉴定影响重要性状的遗传标记和基因，从而进一步提高基因组选择的准确性。     

分子标记株鸭圆环病毒感染性核酸的构建

以临床分离的鸭圆环病毒（duck circovirus）（GenBank登录号：GU168779）阳性病料为材料,根据GenBank中所登录的鸭圆环病毒基困序列设计引物并对设计的引物5′末端进行磷酸化处理,通过引物设计替换碱基,以突变形成EcoRⅠ酶切位点。利用PCR方法扩增鸭圆环病毒的基因,经胶回收后,用T4 DNA连接酶进行环化,以获得鸭圆环病毒具有感染性的核酸。在含有分子标记的两端设计引物,进行PCR扩增,对PCR产物进行胶回收,连接T载体后测序,对胶回收产物进行EcoRⅠ酶切鉴定,均证明在第587位成功插入EcoRⅠ酶切位点。结果表明,本试验已成功构建带有分子标记的鸭圆环病毒的感染性核酸,为进一步开展该病毒的分子调控机制、致病性和开发基因工程疫苗研究奠定基础。