miRNA与其靶mRNA的相互作用：绑定位点的质量与数量特征的整合计算分析

miRNAs通过完全或不完全的碱基互补绑定到信使RNA(mRNA)上,通过抑制翻译或者直接导致mRNA降解的方式来调节靶基因的表达．为了研究miRNAs在转录水平上面的调控作用,两种人类基因组中组织特异的miRNAs(miR-1和miR-124)被转染到HeLa细胞中,微阵列(microarray)分析转染前后细胞中各基因mRNA表达水平变化情况的结果表明：动物基因组中靶基因与miRNAs不完全的碱基互补也会导致mRNA的直接降解．通过分析实验得到的mRNA表达水平变化数据,发现这相同miRNA的不同靶基因mRNA表达水平的下调倍数有着明显的差别,推测这些靶基因mRNA序列本身存在某些影响其受调节程度的因素．为此,提取和分析这些靶基因mRNA的序列特征,通过对这些序列特征与mRNA表达水平下调数据进行统计相关分析,最终发现,miRNA靶基因受调节的程度与以下几个因素相关联：mRNA序列中miRNA靶位点的个数,靶位点与miRNA序列碱基互补的程度,以及绑定后形成二级结构的稳定程度(即最低自由能的大小)．在此基础上,初步建立起一个多因子作用下的miRNA 靶基因mRNA表达水平下调程度模型,分析表明：该模型在一定程度上可以反映了部分序列特征对于miRNA靶基因mRNA表达水平下调程度的影响．     

鸡基因组差异表达基因翻译起始密码子上下游序列的比较研究

翻译起始调控是基因表达调控的一个关键步骤之一。本文以鸡为研究材料,比较研究了鸡基因组高表达基因和低表达基因翻译起始密码子上下游的碱基序列差异,旨在寻找影响鸡基因表达水平的特异性调控位点。全部3 020个单剪接基因完整的mRNA序列及有详细注释的5＇UTRs序列从Ensembl下载。编写计算机程序,读取每个基因mRNA起始密码子上下游各位点的碱基。研究发现,起始密码子上游-3、-2位点可能是鸡基因组基因表达起始密码子正确识别的关键位点。起始密码子上下游的碱基组成分析发现,高表达基因和低表达基因起始密码子的上游均倾向使用（G＋C）,高表达基因的使用偏倚尤为强烈。序列差异比较发现,高表达基因在-9、-6、-3、＋4位点显著偏向G,在-1、-2、-4、-5位点显著偏向C。低表达基因起始密码子上游使用A、U的频率显著高于低表达基因。在-19位点强烈偏向A,在＋1、＋11、＋14位点强烈偏向U。     

家畜转基因育种研究进展

转基因技术可以将外源基因导入家畜基因组,使其获得新的可遗传性状,为培育优良家畜品种提供了革命性途径。DNA显微注射法和体细胞克隆法是制备转基因家畜最常用的方法。应用转基因技术可以进行家畜抗病育种(抗病毒、抗菌和抗寄生虫),改良家畜的生产性状(胴体组成、奶品质、产毛、繁殖力和生长速度),培育环保型家畜新品种。文章从动物转基因技术入手,阐明其在家畜品种改良方面的研究现状和策略,并探讨家畜转基因育种面临的问题和应用前景。     

基因网络中基因关系图谱的构建及其应用

尽快破解基因组所包含基因的功能是一项费力但又很重要的工作。一个基因功能的实现依赖于该基因与其它基因间的相互作用。基因网络是一组基因的集合体,这些基因通过相互协作来控制生物体重要的生命过程。通过基因敲除、RNA干扰或其它方法改变基因网络中某个基因的表达水平,将会引起该网络中其它基因表达水平的变化。而这种变化可以方便地通过基因表达差异显示技术检测相应mRNA含量变化来反映。因此,将这两类方法组合在一起,可以在基因组水平上有效地检测出基因网络中的基因关系。这种策略对基因功能研究方法是一个重要补充。     

影响鼻疽伯克霍尔德氏菌基因组密码子用法的因素分析

鼻疽伯克霍尔德氏菌(Burkholderia mallei ATCC 23344)的基因组密码子使用受多种因素的影响,本研究根据该菌的完整基因组序列,运用多元统计分析和对应分析的方法,探讨了鼻疽伯克霍尔德氏菌全基因组序列密码子的使用模式和影响密码子使用的因素。结果表明基因表达水平的高低是影响密码子使用的主要因素;基因组中编码区的碱基组成、蛋白质的疏水性和基因的长度对密码子的使用也有一定的影响,但影响力不及基因的表达水平。同时,通过比较高表达的基因、低表达的基因密码子使用情况,GCG 和 CUC 等 21 个密码子被确定为鼻疽伯克霍尔德氏菌的主要偏爱密码子。以上结果对鼻疽伯克霍尔德氏菌的密码子用法研究、在分子水平上研究物种进化、基因组中未知基因的预测、开放阅读框的判断、功能基因的表达以及鼻疽病疫苗的研发等工作都提供了理论基础,具有较强的指导作用。     

遗传基因组学(Genetical genomics)的研究进展

遗传基因组学(geneticalgenomics)是将微阵列技术和数量性状座位(QTL)分析结合起来,全基因组水平上定位基因表达的QTL(eQTL).它为研究复杂性状的分子机理和调控网络提供全新的手段.遗传基因组这个概念和研究策略在2001年由Janson和Nap首先提出,到目前为止,遗传基因组学已应用于酵母、老鼠、人以及玉米等植物.研究结果表明:基因表达水平的差异是可遗传的复杂性状;eQTL可以分为顺式作用eQTL和反式作用eQTL,顺式作用eQTL就是某个基因的eQTL定位到该基因所在的基因组区域,表明可能是该基因本身的差别引起mRNA水平的差别,反式作用就是eQTL定位到其他基因组区域,表明其他基因的差别控制该基因mRNA水平的差异.将eQTL结果、基因功能注解以及多种统计分析方法相结合,不仅能更准确地鉴别控制复杂性状及其相关基因表达的候选基因,而且能构建相应的基因调控网络.     

褐飞虱卵巢特异性基因Nl10847的功能初探

褐飞虱Nilaparvata lugens(Stl)是水稻最主要的害虫,其繁殖力高低是影响水稻产量损失最直接的因素。本文旨在研究褐飞虱卵巢特异性基因Nl10847对褐飞虱繁殖力的影响。首先通过克隆验证了Nl10847的开放阅读框(ORF)为552 bp,编码184个氨基酸;然后利用q-PCR技术分析其时空表达模式,发现其在卵巢组织和成虫期高表达;最后通过RNAi的方法,发现下调Nl10847基因的表达后会抑制全虫和卵巢中NlVgR的mRNA表达水平,并严重阻碍卵巢的发育。这些结果表明Nl10847在卵巢发育成熟过程中承担重要的功能。     

应用RGS双荧光替代性报告载体提高CRISPR/Cas9对猪BMP15基因的打靶效率

我国是家猪养殖和消费大国,提高母猪的繁殖力对于促进我国生猪产业的发展具有重要的作用。排卵率和产仔数是影响家畜繁殖力的关键因素,其中BMP15 (bone morphogenetic protein 15)基因已被鉴定是控制绵羊排卵数和多胎性状的一个主效基因,然而目前在家猪BMP15基因中尚未发现类似绵羊多胎品系的天然突变。基于高等哺乳动物基因功能的保守性和CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)等基因组编辑技术对动物基因组定点修饰的高效性,应用CRISPR/Cas9技术对家猪BMP15基因进行精确的遗传修饰,使家猪获得类似多胎绵羊的天然突变,对于研究该基因对家猪繁殖力的影响以及培育高繁殖力家猪新品系具有重要的意义。本研究通过CRISPR/Cas9对长白猪胎儿成纤维(porcine embryonic fibroblasts, PEF)细胞中BMP15基因进行打靶,T7E1分析显示打靶效率仅有5%。随后通过共转染RGS双荧光替代性报告载体(RFP-GFP surrogate reporter),并应用流式细胞术分选出双荧光细胞,富集到基因组被CRISPR/Cas9修饰的细胞,使基因打靶效率提高至18%。本研究结果表明,应用RGS双荧光替代性报告载体可以有效提高CRISPR/Cas9在PEF细胞中对BMP15基因的打靶效率,为今后通过体细胞核移植技术培育BMP15基因编辑猪进行了有效的探索。     

大肠杆菌mRNA编码区长度、形成二级结构倾向与密码子偏好性的关系

从GenBank获得大肠杆菌K-12MG1655株的全基因组序列,计算了与基因密码子偏好性相关的多个参数(Nc、CAI、GC、GC3s),对其mRNA编码区长度、形成二级结构倾向与密码子偏好性之间的关系进行了统计学分析,发现虽然翻译效率(包括翻译速度和翻译精度)是制约大肠杆菌高表达基因的密码子偏好性的主要因素,同时,mRNA编码区长度及其形成二级结构的倾向也是形成这种偏好性的不可忽略的原因,而且对偏好性有一定程度的削弱。另外对mRNA编码区形成二级结构倾向的生物学意义进行了讨论分析。     

SNP功能活性研究方法进展

SNP位点是目前基因多态性研究的主要内容,包括检测分型和功能活性研究两个层次,已经建立了高度自动化和高通量的SNP检测分型技术.本文系统介绍了在性状功能、蛋白质表达、mRNA转录、基因组结构功能等不同层次上进行SNP功能活性研究的方法,并对相关研究结果进行分析,通过对各种研究方法及结果的比较,对SNP位点功能活性研究的前景进行了展望.     

Advanced technologies for genomic analysis in farm animals and its application for QTL mapping

Rapid progress in farm animal breeding has been made in the last few decades. Advanced technologies for genomic analysis in molecular genetics have led to the identification of genes or markers associated with genes that affect economic traits. Molecular markers, large-insert libraries and RH panels have been used to build the genetic linkage maps, physical maps and comparative maps in different farm animals. Moreover, EST sequencing, genome sequencing and SNPs maps are helping us to understand how genomes function in various organisms and further areas will be studied by DNA microarray technologies and proteomics methods. Because most economically important traits in farm animals are controlled by multiple genes and the environment, the main goal of genome research in farm animals is to map and characterize genes determining QTL. There are two main strategies to identify trait loci, candidate gene association tests and genome scan approaches. In recent years, some new concepts, such as RNAi, miRNA and eQTL, have been introduced into farm animal research, especially for QTL mapping and finding QTN. Several genes that influence important traits have already been identified or are close to being identified, and some of them have been applied in farm animal breeding programs by marker-assisted selection.     

影响二花脸猪高产仔性能的生理及遗传机制研究进展

二花脸猪是我国太湖流域以产仔数高而闻名于世界的优质猪种。已有的研究发现,二花脸猪排卵数高、胚胎死亡率低和子宫容积大是其高产性能的重要生理特征,通过候选基因法鉴别到其高产的重要候选基因——促卵泡素β亚基(follicular-stimulating hormone beta, FSH?),采用QTL定位方法在猪第2、6、7、8、12、13和15号染色体上定位到影响其产仔性状的QTL。但是,目前影响二花脸猪产仔数的主效基因尚不清楚。本文主要介绍了二花脸猪的高排卵数、低胚胎死亡率和高子宫容积特性对高产仔性能形成的生理作用;概括了基于传统方法鉴别出的影响二花脸猪高产仔性能的候选基因和QTL,以及基于组学方法鉴别出的二花脸猪高产候选基因;探讨了如何利用基因组、转录组、蛋白质组和功能组等多组学手段深入研究二花脸猪高产性能的成因,以期为深入解析二花脸猪高产性能的分子遗传机制提供参考。     

Differential gene expression in ovaries of pregnant pigs with high and low prolificacy levels and identification of candidate genes for litter size

Previous results from a genome scan in an F(2) Iberian × Meishan pig intercross showed several chromosome regions associated with litter size traits in this species. In order to identify candidate genes underlying these quantitative trait loci (QTL), we performed an ovary gene expression analysis during the sow's pregnancy. F(2) sows were ranked by their estimated breeding values for prolificacy: six sows with the highest estimated breeding value (EBV) (i.e., high prolificacy) and six sows with the lowest EBV (low prolificacy) were selected. Samples were hybridized using an Affymetrix GeneChip porcine genome array. Statistical analysis with a mixed model approach identified 221 differentially expressed probes, representing 189 genes. These genes were functionally annotated in order to identify genetic pathways overrepresented in this list. Among the functional groups most represented was, in first position, immune system response activation against external stimulus. The second group consisted of integrated genes that regulate maternal homeostasis by complement and coagulation cascades. A third group was involved in lipid and fatty acid enzymes of metabolic processes, which participate in the steroidogenesis pathway. In order to identify powerful candidate genes for prolificacy, the second approach of this study was to merge microarray data with the QTL positional information affecting litter size, previously detected in the same experimental cross. As a result, we have identified 27 differentially expressed genes colocalizing with QTL for litter size traits, which fulfill the biological, positional, and functional criteria.     

Segregation of regulatory polymorphisms with effects on the gluteus medius transcriptome in a purebred pig population

Background

The main goal of the present study was to analyse the genetic architecture of mRNA expression in muscle, a tissue with an outmost economic importance for pig breeders. Previous studies have used F₂ crosses to detect porcine expression QTL (eQTL), so they contributed with data that mostly represents the between-breed component of eQTL variation. Herewith, we have analysed eQTL segregation in an outbred Duroc population using two groups of animals with divergent fatness profiles. This approach is particularly suitable to analyse the within-breed component of eQTL variation, with a special emphasis on loci involved in lipid metabolism.

Methodology/Principal Findings

GeneChip Porcine Genome arrays (Affymetrix) were used to determine the mRNA expression levels of gluteus medius samples from 105 Duroc barrows. A whole-genome eQTL scan was carried out with a panel of 116 microsatellites. Results allowed us to detect 613 genome-wide significant eQTL unevenly distributed across the pig genome. A clear predominance of trans- over cis-eQTL, was observed. Moreover, 11 trans-regulatory hotspots affecting the expression levels of four to 16 genes were identified. A Gene Ontology study showed that regulatory polymorphisms affected the expression of muscle development and lipid metabolism genes. A number of positional concordances between eQTL and lipid trait QTL were also found, whereas limited evidence of a linear relationship between muscle fat deposition and mRNA levels of eQTL regulated genes was obtained.

Conclusions/Significance

Our data provide substantial evidence that there is a remarkable amount of within-breed genetic variation affecting muscle mRNA expression. Most of this variation acts in trans and influences biological processes related with muscle development, lipid deposition and energy balance. The identification of the underlying causal mutations and the ascertainment of their effects on phenotypes would allow gaining a fundamental perspective about how complex traits are built at the molecular level.     

Molecular approaches in pig breeding to improve meat quality.

This article reviews the advances in molecular genetics that have led to the identification of genes and markers associated with meat quality in pig. The development of a considerable number of annotated livestock genome sequences represents an incredibly rich source of information that can be used to identify candidate genes responsible for complex traits and quantitative trait loci effects. In pig, the huge amount of information emerging from the study of the genome has helped in the acquisition of new knowledge concerning biological systems and it is opening new opportunities for the genetic selection of this specie. Among the new fields of genomics recently developed, functional genomics and proteomics that allow considering many genes and proteins at the same time are very useful tools for a better understanding of the function and regulation of genes, and how these participate in complex networks controlling the phenotypic characteristics of a trait. In particular, global gene expression profiling at the mRNA and protein level can provide a better understanding of gene regulation that underlies biological functions and physiology related to the delivery of a better pig meat quality. Moreover, the possibility to realize an integrated approach of genomics and proteomics with bioinformatics tools is essential to obtain a complete exploitation of the available molecular genetics information. The development of this knowledge will benefit scientists, industry and breeders considering that the efficiency and accuracy of the traditional pig selection schemes will be improved by the implementation of molecular data into breeding programs.     

基因表达系列分析技术在真菌功能基因组学中的应用

基因表达系列分析( SAGE)是一种在mRNA水平上高通量、快速、灵敏分析细胞或组织基因表达信息,并在基因组学研究中广泛应用的技术.该技术不仅能够全面地分析特定组织或细胞表达的基因,比较不同时空条件下基因表达的差异,还可以在全基因组范围内获得基因的表达谱,从而发现新基因.综述基因表达系列分析技术在材料用量、标签长度、技术流程和标签测序等方面的研究进展及该技术在病原真菌、工业真菌和食用真菌功能基因组学中的应用.     

Transgenic technology and applications in swine.

The introduction of foreign DNA into the genome of livestock and its stable integration into the germ line has been a major technical advance in agriculture. Production of transgenic livestock provides a method to rapidly introduce "new" genes into cattle, swine, sheep and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Several recent developments will profoundly impact the use of transgenic technology in livestock production. These developments are: 1) the ability to isolate and maintain in vitro embryonic stem (ES) cells from preimplantation embryos, embryonic germ (EG) and somatic cells from fetuses; and somatic cells from adults, and 2) the ability to use these embryonic and somatic cells as nuclei donors in nuclear transfer or "cloning" strategies. Cell based (ES, EG, and somatic cells) strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. There are many potential applications of transgenic methodology to develop new and improved strains of livestock. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition and increased disease resistance. Development of transgenic farm animals will allow more flexibility in direct genetic manipulation of livestock.     

Genome imprinting and carcinogenesis

The preferential retention of paternal tumor suppressor alleles in sporadic tumors and the failure to demonstrate genetic linkage between disease predisposition and tumor suppressor loci in familial cases indicates that genome imprinting may be involved in the genesis of some pediatric cancers. A genetic model that invokes the activity of modifier loci (imprinting genes) on alleles to be modified (imprinted genes) is able to account for these data. Genome imprinting may be viewed as a special case of dominance modification, differing from other examples only in that the modification of dominance is dependent on gamete-of-origin. Data from human pediatric tumors, transgenes in the mouse and variegating position-effects in Drosophila, indicate that the net effect of modifier loci is the inactivation of alleles at affected loci. Polymorphism at the level of the modifier loci will result in different degrees of modification between individuals. With respect to tumors, the most important mechanism by which these differences are manifested is cellular mosaicism for the expression of a modified allele. Such characteristics are reminiscent of the behavior of variegating position-effects in Drosophila and the application of this paradigm to human disease phenotypes provides both a mechanism by which differential genome imprinting may be accomplished as well as genetic models that may explain the clinical association of syntenic diseases, the association between tumor progression and specific chromosomal aneuploidy and the unusual inheritance characteristics of many diseases.     

From genotype to phenotype in bovine functional genomics

In past 5 years, the promise that came with genome sequencing has revolutionized the functional genomics research field at unprecedented manner. It would soon know what all known genes do, particularly genes involved in genetic improvement of animal health and increase food animal production. With the availability of full bovine genomic sequence, yet we still have a lot of daunting tasks on 'genotype-to-phenotype problem' particularly about the phenotypic variations and trying to predict what genes are likely to be involved, and improved integrated interactive database. This article outlined and discussed about the current status of bovine functional genomics, recent development in bovine genome databases particularly in annotation of bovine genome, bovine quantitative trait loci database and its potential impact to unveil the from genotype-to-phenotype problem.